table of contentsmedia.journals.elsevier.com/content/files/part1-20144615.pdf · a lifetime passion...

3REFLECTIONS IN MUTATION RESEARCH: 1999 – 20192 ELSEVIER

Preface: Reflections in Mutation Research - George R. Hoffmann 5

Prologue: Twenty Years of Reflections in Mutation Research - George R. Hoffmann 6

Reflections in mutation research: an introductory essay - George R. Hoffmann, Donald G. MacPhee 22

Spontaneous mutation in man - James F. Crow 30

Frameshift mutation, microsatellites and mismatch repair - Bernard S. Strauss 35

The origins of the back-mutation assay method: a personal recollection - Geoffrey W. Grigg 44

Somatic mutations and ageing - Robin Holliday 56

Lysenko and Stalin: Commemorating the 50th anniversary of the August 1948 LAAAS Conference and the 100th anniversary of T.D. Lysenko’s birth, September 29, 1898 - Zhores A. Medvedev 62

Editorial : Reflections of Zhores Medvedev - George R. Hoffmann, Donald G. MacPhee 71

The Environmental Mutagen Society and the emergence of genetic toxicology: a sociological perspective - Scott Frickel 73

HPRT mutations in humans: biomarkers for mechanistic studies - Richard J. Albertini 81

The Discovery of mRNA - Elliot Volkin 95

Forging the links between metabolism and carcinogenesis - F. Peter Guengerich 100

Reflections and meditations upon complex chromosomal exchanges - John R.K. Savage 115

Shedding light on proteins, nucleic acids, cells, humans and fish - Richard B. Setlow 132

Hormesis: changing view of the dose-response, a personal account of the history and current status - Edward J. Calabrese 146

Observations at the interface of mutation research and regulatory policy - W. Gary Flamm 155

Mutation research at ABCC/RERF: cytogenetic studies of atomic bomb exposed populations - Akio Awa 162

Pitfalls of enzyme-based molecular anticancer dietary manipulations: food for thought - Moreno Paolini, Marion Nestle 177

Early days of mammalian somatic cell genetics: the beginning of experimental mutagenesis - Ernest H.Y. Chu 186

Incorporation of mammalian metabolism into mutagenicity testing - Heinrich V. Malling 193

The importance of mutation, then and now: studies with yeast cytochrome c - Fred Sherman 200

Extrapolations are the Achilles Heel of Risk Assessment - R.J. Preston 216

Isolation of plasmid pKM101 in the Stocker laboratory - Kristien Mortelmans 221

Mutation Research: The origin - A.T. Natarajan 235

Personal reflections on the life and legacy of Alexander Hollaender - Mary Esther Gaulden, John Jagger, Virginia P. White 237

Reflections on the impact of advances in the assessment of genetic risks of exposure to ionizing radiation on international radiation protection recommendations between the mid-1950s and the present - K. Sankaranarayanan, J.S. Wassom 253

TABLE OF CONTENTS

Electric light causes cancer? Surely you’re joking, Mr. Stevens - Richard G. Stevens 279

A lifetime passion for micronucleus cytome assays—Reflections from Down Under - Michael Fenech 285

DNA methylation and mutator genes in Escherichia coli K-12 - Martin G. Marinus 292

Exploring the processes of DNA repair and homologous integration in Neurospora - Hirokazu Inoue 298

Reflections on a lifetime in cytogenetics - Adayapalam T. Natarajan 309

The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses - Urbain Weyemi, Corinne Dupuy 315

Changing paradigms in radiobiology - Carmel Mothersill , Colin Seymour 320

How fruit flies came to launch the chromosome theory of heredity - Elof Axel Carlson 331

H.J. Muller’s contributions to mutation research - Elof Axel Carlson 337

The rise and fall of photomutagenesis - Lutz Müller, Elmar Gocke 342

The Mouse House: A brief history of the ORNL mouse-genetics program, 1947–2009 - Liane B. Russell 347

Memories of a friend and mentor – Charlotte Auerbach - Delbert M. Shankel 369

J.B.S. Haldane as I knew him, with a brief account of his contribution to mutation research - Krishna Dronamraju 374

Genome-based, mechanism-driven computational modeling of risks of ionizing radiation: The next frontier in genetic risk estimation? - K. Sankaranarayanan, H. Nikjoo 380

Reflections on the development and application of FISH whole chromosome painting - James D. Tucker 395

The comet assay: Reflections on its development, evolution and applications - Narendra P. Singh 408

Reflections on a career and on the history of genetic toxicity testing in the National Toxicology Program - Errol Zeiger 416

Scientific feuds, polemics, and ad hominem arguments in basic and special-interest genetics - Elof Axel Carlson 427

Mutagenesis: Interactions with a parallel universe - Jeffrey H. Miller 433

Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications - Micheline Kirsch-Volders, Francesca Pacchierotti, Elizabeth M. Parry, Antonella Russo, Ursula Eichenlaub-Ritter, Ilse-Dore Adler 437

“Reflections in Mutation Research” is a special feature of Mutation Research Reviews devoted to historical and philosophical themes related to mutation, mutagenesis, radiation research and genetic toxicology. This volume is a compilation of the papers published in the Reflections series since its initiation in 1999.

Reflections papers give insight into fundamental questions in modern mutation research, viewed in a historical context. The series considers mutation broadly, including its toxicological, medical, evolutionary, statisti-cal, and public policy dimensions, as well as the basic genetics and molecular biology that are central to mutation research. David DeMarini and Michael Waters, as the coeditors of Mutation Research Reviews, and I believe that Reflections articles have appealed to a broad readership through their presentation of the perspectives of people who have contributed greatly to our field, along with citations to historically important and recent literature. For these reasons, we thought it fitting to prepare a compilation of all the Reflections papers to mark the twentieth anniversary of the series. We are especially pleased that the Publisher has offered to make this collection of Reflections articles freely available as an e-book through links in the website of Mutation Research Reviews and in a continuing archive of Reflections articles on the Elsevier website.

George R. Hoffmann Editor, Reflections in Mutation Research

Department of Biology College of the Holy Cross Worcester, MA 01610, USA

5REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019

PREFACE

Reflections in Mutation ResearchGeorge R. Hoffmann

© Septermber 2019 by Elsevier. All rights reserved.

4 ELSEVIER


errors were identified, we expected them to be corrected, and if sub-stantive criticisms were raised, we expected that the authors wouldreconcile them. Though less structured than formal peer review, thisstyle has served us and the authors well, and we have never heard acomplaint from a reader who thought that criticisms were not properlyconsidered.

Running Reflections with autonomy meant that Donald and I sentaccepted manuscripts directly to issue managers or production editorsat Elsevier. This spared authors the administrative process after theirpaper was complete, and it gave us the ability to work collaborativelywith the publisher on special considerations in the formatting or style ofthe articles. Proofs were sent to the authors and to us, and the authors,of course, would approve the final version. The name and purposes ofReflections suggest some bias toward older authors who look back on ahistory. This bias has not been extreme, however, and there have beenexceptions where the reflections of a young scientist fit the purposes ofReflections perfectly. In a few cases, the misfortunes of time meant thataccepted invitations could not be completed, and I would like to say aword of appreciation for some valued colleagues who fall into this ca-tegory: Tom Cebula, Mary Lyon, Barry Margolin, Bill Morgan, JimParry, Herb Rosenkranz, Jack Von Borstel and John Wassom.

3. The first Reflections articles

3.1. An introductory essay

To initiate the Reflections series in Mutation Research Reviews,Donald and I submitted an introductory essay on our plans for the newseries [4]. We included a table of papers that we saw as being seminalworks in the formative years of the field of mutation research. We setthe pattern that all submissions to Reflections would be peer reviewed,and we made no exception here. I had heard from several of HermanBrockman's students at Illinois State University that when they sub-mitted drafts of papers to him for review, their manuscript was"Brockmanized." So, we chose Herman Brockman as a reviewer of ourpaper, along with John Wassom and Bill Healy, and we received threevaluable reviews, including good suggestions for the table. I might addthat the manuscript had been properly "Brockmanized."

3.2. James Crow on mutation in human germ cells

Reflections had an auspicious beginning when James F. Crow ac-cepted my invitation to write the first Reflections article after the in-troductory essay. Professor Crow, known to his friends worldwide as"Jim," was one of the truly great geneticists of the twentieth century andmade many contributions to theoretical population genetics and ap-plied topics in human genetics, including risks associated with ionizingradiation and chemical mutagens. He joined the faculty of theUniversity of Wisconsin in 1948 and worked there for 64 years, until hisdeath at age 95 in 2012. He is remembered not only for his scientificachievements but also for his fine qualities as a person and teacher,many contributions to the local, national and international scientificcommunity, skill as a musician and extraordinary generosity of spirit[5,6].

In Jim's case, we did not suggest a topic, as we knew that whateverhe chose would be good. As was typical of him, a well-written manu-script arrived on time, and it dealt with a historical theme that was, atthe same time, completely relevant to major questions in modern mu-tation research and genetic toxicology. Jim entitled his article"Spontaneous mutation in man" [7] and began with a clever introduc-tion, noting that using "man" generically to refer to the species Homosapiens was no longer quite acceptable. However, for him, it was okay,because he was making the point that the problem is indeed "man," inthat mutations disproportionately arise in the continuing meiotic divi-sions of male meiosis. He then traced the age effect in mutagenesis toWilhelm Weinberg, now most familiar because of the Hardy-Weinberg

Principle in population genetics, who had noted in 1912 an age effect inthe occurrence of genetic diseases. This was a perfect start for Reflec-tions, in that Jim traced his topic to the early days of genetics, linked itto a central question in modern mutation research –the origin of newmutations – and speculated about its current importance.

3.3. Bernard Strauss on frameshift mutagenesis and mismatch repair

DNA repair already comprised a thriving subdiscipline of mutationresearch when Reflections began, and we wanted it to be representedearly in the series. We were in luck when Bernard S. Strauss, who hadbeen active in the field of DNA damage, mutagenesis and repair sincearound 1960, accepted Donald MacPhee's invitation to contribute. Hewrote his reflections on the growing understanding of frameshift mu-tagenesis and genomic instability in eukaryotic cells, drawing togetherrepetitive sequences, slippage, and mismatch repair [8]. The articlebegins with comments on differences between the genomes of eu-karyotes and bacteria, notably including extensive repetitive sequencesand introns in eukaryotes. He reviews early studies elucidating thenature of base-pair substitutions and frameshift mutations, and hediscusses models of frameshift mutagenesis, including the widelyknown Streisinger slippage model [9,10]. While slippage remains animportant mechanism, it is not the only mechanism, and he points outthat frameshifts also arise, even at repeated sequences, by mechanismsnot associated with slippage. He notes the association between frame-shift mutagenesis and replication, and he relates deficiencies in mis-match repair to genetic instability. He suggests that the attributes ofeukaryotic genomes necessitated highly effective surveillance to protectagainst frameshifts and that the eukaryotic mismatch repair system,having differences from that in prokaryotes, is a key element in thatsurveillance. Professor Strauss is still professionally active, and readersmay like to see a commentary that he wrote recently on the double-strandedness of DNA and the history of DNA repair studies [11].

3.4. Zhores Medvedev on Lysenkoism in the Soviet Union

The Lysenko period in the Soviet Union, extending from the 1930suntil 1964, was a bizarre and troubled time in the history of science. AsTrofim Lysenko gained influence with the Soviet government andpromoted views incompatible with modern genetics and evolutionarybiology, legitimate geneticists and others who argued against the anti-scientific trend were removed from their positions and sometimes im-prisoned. The biologist Zhores Medvedev courageously spoke outagainst this trend and later managed to get a book manuscript to I.Michael Lerner, a geneticist at the University of California, Berkeley.The manuscript told the history of Lysenkoism in a direct way that wasnot permitted in the Soviet Union even after the fall of Lysenko. Lernertranslated Medvedev's manuscript from Russian, and it was published in1969 as The Rise and Fall of T.D. Lysenko [12]. The book received strongpraise from the eminent evolutionary biologist Theodosius Dobzhansky,both for its courage and its thorough documentation of the history [13].

In 1998, Donald MacPhee and I were able to contact Dr. Medvedev,who was now living in England, and we invited him to write an articlefor Reflections. We expressed interest in the recovery from Lysenkoismand lingering effects, but we gave him the latitude to define his specifictopic. We quickly received a courteous response that he was reluctant toaccept our invitation. A short time later, I received another letter fromhim, in which he explained that he found it increasingly difficult towrite text in English, and he asked whether we could consider amanuscript in Russian. I immediately thought of Charles ("Chuck")Severens, a former Russian professor and my colleague at Holy Cross.Chuck asked to see the text before committing himself to translating it.We took the gamble and agreed to Dr. Medvedev's proposal. When themanuscript arrived, Chuck looked it over and, fortunately, agreed. Hewanted nothing more than a footnote indicating "Translated fromRussian by Charles Severens." Dr. Medvedev's English turned out to be

G.R. Hoffmann Mutation Research-Reviews in Mutation Research xxx (xxxx) xxxx

2

Contents lists available at ScienceDirect

Mutation Research-Reviews in Mutation Research

journal homepage: www.elsevier.com/locate/mutrev

Reflections in Mutation Research

Twenty Years of Reflections in Mutation Research☆

George R. Hoffmann⁎

Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610, USA

A R T I C L E I N F O

Keywords:ReflectionsHistory of scienceMutagenesisGenetic toxicologyEnvironmental mutagenesis

A B S T R A C T

Reflections is a component of Mutation Research Reviews devoted to historical and philosophical themes per-taining to the subject of mutation. Reflections was initiated in 1999 and has included a broad array of topicscentered on mutation research, but overlapping other scientific fields and touching upon history, sociology,politics, philosophy and ethics. This commentary offers an editor's reflections on the 44 papers in the Reflectionsseries, including the people who contributed to the series and the topics that they discussed.

1. Establishment of Mutation Research Reviews and Reflections

The journal Mutation Research was founded in 1964 by ProfessorF.H. Sobels at the University of Leiden [1]. Professor Sobels, known tohis many friends as "Frits," established the reviews section of the journala decade later [2]. The reviews section flourished, and through theyears it operated under several similar titles, initiallyMutation Research:Reviews in Genetic Toxicology and most recently Mutation Research Re-views. After Prof. Sobels' death in 1993, Frederick J. de Serres and JohnS. Wassom succeeded him as the managing editors of the reviews sec-tion, serving from 1994 until Fred de Serres' retirement in 1998. Thepresent editors, David M. DeMarini and Michael D. Waters, assumed theeditorship at that time.

Reflections was established shortly after the appointment ofDeMarini and Waters. As I recall the history, David DeMarini tele-phoned me to discuss his idea for a new feature in Mutation ResearchReviews in the style of the "Perspectives" feature in the Genetics Societyof America's journal Genetics, edited by James F. Crow and William F.Dove. Perspectives was then a decade old and was well established as anappealing and extremely valuable component of Genetics [3].

David wondered what I thought of his idea. I liked it. He suggestedthat it would be a good idea to have two editors in different countries,befitting the international character of Mutation Research. He askedwhether I would be interested in serving as coeditor. I was certainlyinterested, but the thought of undertaking a feature modeled onPerspectives in Genetics was daunting, as it set a very high standard. Oneof the first people that I spoke to about this idea was James Crow,whom I had come to know through the National Academy of Sciences'

Committee on Chemical Environmental Mutagens. He was encouraging,and I moved closer to taking on the new project. David and I discussedthe particulars of how Reflections could be organized and managed. Hisplan was to give the coeditors complete autonomy in runningReflections. I asked whether he had someone in mind as the secondeditor, and he did. It was Donald G. MacPhee at LaTrobe University inAustralia. I was delighted, and no further discussion was needed frommy perspective. Fortunately, Donald agreed, and we were soon inregular email contact about Reflections.

2. Management of Reflections

We intended for Reflections to be comprised largely of invited papersfrom people who had made major contributions to the field of muta-genesis or related subjects. In extending invitations, we suggested topicsthat struck us as fitting, based on the prospective author's work, but wemade it clear that we were receptive to alternatives. We would also bereceptive to spontaneously submitted manuscripts, and several suchunsolicited submissions became valuable contributions to the series.

We decided at the outset that all Reflections articles would be subjectto careful peer review. We made a fine distinction, however, fromconventional peer review, so we referred to those who commented onthe manuscripts as "Readers" rather than "Reviewers." Readers wereasked to offer suggestions for the author's consideration. The maindifference from a formal review is that we gave Reflections authors greatlatitude in expressing their viewpoints. This process fits the purpose ofReflections, as we specifically wanted the personal perspectives ofpeople who had contributed much to the field. At the same time, if

https://doi.org/10.1016/j.mrrev.2019.05.004Accepted 25 April 2019

☆ This article is part of the Reflections in Mutation Research series. To suggest topics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann ([email protected]).

⁎ Corresponding author.E-mail address: [email protected].

Mutation Research-Reviews in Mutation Research xxx (xxxx) xxxx

1383-5742/ © 2019 Published by Elsevier B.V.

Please cite this article as: George R. Hoffmann, Mutation Research-Reviews in Mutation Research, https://doi.org/10.1016/j.mrrev.2019.05.004

Contents lists available at ScienceDirect

Mutation Research-Reviews in Mutation Research

journal homepage: www.elsevier.com/locate/mutrev


Twenty Years of Reflections in Mutation Research☆

George R. Hoffmann⁎


A R T I C L E I N F O

Keywords:ReflectionsHistory of scienceMutagenesisGenetic toxicologyEnvironmental mutagenesis

A B S T R A C T

Reflections is a component of Mutation Research Reviews devoted to historical and philosophical themes per-taining to the subject of mutation. Reflections was initiated in 1999 and has included a broad array of topicscentered on mutation research, but overlapping other scientific fields and touching upon history, sociology,politics, philosophy and ethics. This commentary offers an editor's reflections on the 44 papers in the Reflectionsseries, including the people who contributed to the series and the topics that they discussed.

1. Establishment of Mutation Research Reviews and Reflections

The journal Mutation Research was founded in 1964 by ProfessorF.H. Sobels at the University of Leiden [1]. Professor Sobels, known tohis many friends as "Frits," established the reviews section of the journala decade later [2]. The reviews section flourished, and through theyears it operated under several similar titles, initiallyMutation Research:Reviews in Genetic Toxicology and most recently Mutation Research Re-views. After Prof. Sobels' death in 1993, Frederick J. de Serres and JohnS. Wassom succeeded him as the managing editors of the reviews sec-tion, serving from 1994 until Fred de Serres' retirement in 1998. Thepresent editors, David M. DeMarini and Michael D. Waters, assumed theeditorship at that time.

Reflections was established shortly after the appointment ofDeMarini and Waters. As I recall the history, David DeMarini tele-phoned me to discuss his idea for a new feature in Mutation ResearchReviews in the style of the "Perspectives" feature in the Genetics Societyof America's journal Genetics, edited by James F. Crow and William F.Dove. Perspectives was then a decade old and was well established as anappealing and extremely valuable component of Genetics [3].

David wondered what I thought of his idea. I liked it. He suggestedthat it would be a good idea to have two editors in different countries,befitting the international character of Mutation Research. He askedwhether I would be interested in serving as coeditor. I was certainlyinterested, but the thought of undertaking a feature modeled onPerspectives in Genetics was daunting, as it set a very high standard. Oneof the first people that I spoke to about this idea was James Crow,whom I had come to know through the National Academy of Sciences'

Committee on Chemical Environmental Mutagens. He was encouraging,and I moved closer to taking on the new project. David and I discussedthe particulars of how Reflections could be organized and managed. Hisplan was to give the coeditors complete autonomy in runningReflections. I asked whether he had someone in mind as the secondeditor, and he did. It was Donald G. MacPhee at LaTrobe University inAustralia. I was delighted, and no further discussion was needed frommy perspective. Fortunately, Donald agreed, and we were soon inregular email contact about Reflections.

2. Management of Reflections

We intended for Reflections to be comprised largely of invited papersfrom people who had made major contributions to the field of muta-genesis or related subjects. In extending invitations, we suggested topicsthat struck us as fitting, based on the prospective author's work, but wemade it clear that we were receptive to alternatives. We would also bereceptive to spontaneously submitted manuscripts, and several suchunsolicited submissions became valuable contributions to the series.

We decided at the outset that all Reflections articles would be subjectto careful peer review. We made a fine distinction, however, fromconventional peer review, so we referred to those who commented onthe manuscripts as "Readers" rather than "Reviewers." Readers wereasked to offer suggestions for the author's consideration. The maindifference from a formal review is that we gave Reflections authors greatlatitude in expressing their viewpoints. This process fits the purpose ofReflections, as we specifically wanted the personal perspectives ofpeople who had contributed much to the field. At the same time, if

https://doi.org/10.1016/j.mrrev.2019.05.004Accepted 25 April 2019

☆ This article is part of the Reflections in Mutation Research series. To suggest topics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann ([email protected]).

⁎ Corresponding author.E-mail address: [email protected].

Mutation Research-Reviews in Mutation Research xxx (xxxx) xxxx

1383-5742/ © 2019 Published by Elsevier B.V.

Please cite this article as: George R. Hoffmann, Mutation Research-Reviews in Mutation Research, https://doi.org/10.1016/j.mrrev.2019.05.004

PROLOGUE

Mutation Research 780 2019. 106–120

Twenty Years of Reflections in Mutation Research ☆George R. Hoffmann *



induction, and polymorphisms. He notes how the bacterial umu assay,which measures the induction of the umu operon as part of the SOSresponse to DNA damage, was used to monitor genotoxicity. Thissimple assay was useful in guiding his attempts to identify genotoxicmetabolites and his work on the purification of human P450 enzymes.The elucidation of the roles of different P450 enzymes was also aided bythe development of transgenic microbial and mammalian-cell systemsthat express human P450's. A review of "Phase 1" reactions and the"Phase 2" enzymes that catalyze conjugation reactions is followed by adetailed look at a specific case that illustrates broad principles – me-tabolism of the mycotoxin aflatoxin B1. Fred then addresses the evo-lution of xenobiotic-metabolizing enzymes and provides perspective onconceptual advances in the understanding of carcinogen metabolism[31]. These insights and his concluding remarks are well-worth readingfor specialists and nonspecialists alike.

4.4. Richard Albertini on mutations in human cells in vivo

Clear positive selection techniques for forward mutations led to afew genes being predominant in early mutation tests in mammaliancells. Among these is the X-linked HPRT gene encoding hypoxanthine-guanine phosphoribosyltransferase. Mutants that cannot convert thepurine base analog 6-thioguanine or 8-azaguanine to its nucleotideform do not experience its toxicity and are selected by their growth inmedium containing the analog. A single mutational event is sufficient toconfer the mutant phenotype in wild-type cells because there is onlyone copy of this X-linked gene in cells from males and because femalesare functionally hemizygous due to X-inactivation. For this reason,HPRT was a prime candidate for detecting point mutations in humancells in vivo. Richard ("Dick") Albertini led the way in developing HPRTinto a functional in vivo mutation system in human peripheral T-lym-phocytes. An elevation in mutant frequencies was readily detected insuch exposed populations as patients who had received mutagenicchemotherapy. An astute observer of human immunology and phy-siology, Dick was able to uncover mechanistic complexities in the in-duction, proliferation, frequencies and molecular mechanisms under-lying the mutants detected in vivo. In his Reflections article [33] hedescribes this history and some of the surprises along the way. Being aclinician, as well as a researcher, he ends his reflections with specula-tion about the possibility of long-term transmissible effects of HPRTmutations in patients receiving therapy with purine analogs.

4.5. Richard B. Setlow on UV light, DNA damage and repair

Richard B. ("Dick") Setlow is remembered for his important dis-coveries on the biological effects of ultraviolet light (UV), character-ization of pyrimidine dimers, and the process of nucleotide excisionrepair. In a Festschrift celebrating his eightieth birthday in 2001, hewas described as "the Father of DNA Repair" [34]. I clearly rememberthe first time I met Dick Setlow. As a graduate student at the Universityof Tennessee, I did my graduate research at ORNL. Around 1970 was agood time to be at ORNL because the Biology Division had flourishedunder the leadership of Alexander Hollaender as its director from 1946-1966. When I arrived in Oak Ridge, my fellow graduate student, MikeShelby, told me that Dr. Hollaender met with Oak Ridge researchers,visitors and students on Sunday mornings by the railroad trestle inOliver Springs for hikes in the nearby Cumberland Mountains. TheCumberlands were of interest for their scenic beauty and natural his-tory, and also for the abundant fossils of Carboniferous plants found indisturbed sites near strip mines. On my first such hike, I found myselftalking to Dick Setlow and hearing first-hand about the groundbreakingresearch in his lab on effects of UV and the repair of DNA damage. I wastherefore pleased many years later when he accepted my invitation towrite an article for Reflections [35].

Dick's Reflections paper begins with his days as a physics studentand, later, a faculty member at Yale University from 1941 to 1960. His

interests evolved from physics to biophysics, and he describes his earlywork "shedding light" on simple biological systems. He found new waysto use UV action spectra to elucidate the properties of proteins, nucleicacids and viruses. After joining the Biology Division of ORNL in 1960,he extended this work to the formation of cyclobutane pyrimidine di-mers in DNA by UV and their detrimental effects in bacteria. This wasfollowed by his pioneering work on the elimination of dimers by pho-torepair, and ultimately the discovery of excision repair in 1963-1964.His interests soon expanded to include repair-deficient mutants, mu-tagenesis and carcinogenesis. In 1974, Dick moved to BrookhavenNational Laboratory, where he spent the remainder of his career. Hiscreative research continued, but his focus expanded to complex multi-cellular systems, including fish. The Amazon molly permitted in-formative comparisons with mammalian cells, because fish have anactive photorepair system, which nonmarsupial mammals lack. Dickbegan with basic biophysics and through his professional life increas-ingly concerned himself with the implications of his field for carcino-genesis, genetic effects, aging and health. I would refer readers to an inmemoriam tribute to Dick Setlow by Phil Hanawalt and colleagues [36].

4.6. Edward J. Calabrese on hormesis

Dose-response relationships had received much attentionthroughout the history of mutation research, and various models withand without thresholds have been considered. A linear model with nothreshold was, and to a great extent remains, a leading model for ge-netic effects of ionizing radiation and direct-acting chemical mutagens.Edward ("Ed") Calabrese, a toxicologist at the University ofMassachusetts, has been a leading advocate of an alternative modelbased on hormesis, which is a process whereby effects at low doses areopposite to those caused by high doses of the same agent for the samebiological endpoint. The notion that hormesis is broadly applicable tobiological processes is considered eccentric by many, especially insuggesting that low doses of radiation and toxicants may be beneficial.However, the idea was receiving growing attention, so we invited EdCalabrese to offer his reflections on the subject. We anticipated that itwould be controversial and so-noted in our introductory editorial [37].Ed Calabrese, open to suggestions and to contrary views of reviewers,enjoyed the debate and refined his text, holding to his views whileexercising restraint about overstatement. He offered a fascinating viewof a changing landscape with respect to thinking about hormesis [38].

4.7. John Savage on complex chromosomal rearrangements

The use of fluorescence in situ hybridization (FISH) to study chro-mosomal aberrations had become common in the 1990's, and JohnSavage was devising means to classify the richer array of alterationsthat could be seen with FISH than had been readily detectable byconventional staining. "Reflections and Meditations" in the title of hisReflections paper [39] is exactly right, as he thought deeply aboutchromosome structure and the mechanisms underlying chromosomalalterations. The previously unseen array of aberrations now apparentwith FISH led him to write "these strange, unexpected patterns requirednew descriptive methods, and two complementary scoring schemeswere developed, 'S&S' and 'PAINT'" [39]. The initials of John Savageand his colleague Paul Simpson became the name of the "S&S" no-menclature system, which they devised to classify complex aberrationson the basis of the possible interchanges formed by chromosome breaks[40,41]. His Reflections paper elucidates the underlying theory and itshistory [39]. I will return to the "PAINT" nomenclature system whencommenting on a later Reflections paper by Jim Tucker.

In 2002, we were still receiving printed manuscripts by conven-tional mail when John sent me his manuscript. The stationery that heused for his cover letter had a unique heading – his home address inReading, UK, to the left of which were his initials – JS – large and in twocolors. The "morphology" of the initials was that of chromosomes


4

very good, and he identified a few fine differences in meaning fromwhat he had intended. He and Chuck easily worked this out. When Dr.Medvedev's manuscript on Lysenko and Stalin was published [14],Donald MacPhee and I preceded the article with an "editorial," whichwas actually a brief biography and tribute to Zhores Medvedev, in-cluding his photograph in his apartment in the U.K. [15]. Dr. Medvedevdied in November 2018, and his obituaries give a good account of hisremarkable life [16,17].

3.5. Geoffrey Grigg on the "Grigg effect" and the history of reverse mutationassays

Those who think of the classical Ames test as the "good old days" ofenvironmental mutagenesis should enjoy reading Geoff Grigg's reflec-tions on questions surrounding the methods and first applications ofreverse mutation assays [18]. His studies of back mutations from aux-otrophy to prototrophy in microorganisms, principally Neurosporacrassa, include a critical paper in 1952 demonstrating that large num-bers of auxotrophs in the background can suppress the expression anddetection of new revertants [19]. This discovery, which is now wellknown but came as a surprise to many, became known as "the GriggEffect." It was our good fortune that Donald MacPhee had come to knowGeoff Grigg in Australia, and Donald's invitation to write for Reflectionswas readily accepted.

In his Reflections paper [18], Geoff Grigg describes the discovery ofthe Grigg Effect and the ability to detect it by means of reconstructionexperiments that simulate the conditions of the mutation experiment,including cell concentration, selective medium and growth conditions.He points out that after he reported the effect, he discovered that asimilar phenomenon had been reported earlier in bacteria, and hecredits Ryan and Schneider for their discovery in E. coli [20]. Grigg alsoexplores other sources of error in back-mutation experiments, and heraises the question whether the Grigg Effect fundamentally limits theapplicability of reverse mutation assays. He concludes that it does not ifone is careful to use appropriate strains and have proper controls todetect it, and history has borne this out. Besides the "Grigg Effect," di-verse other discoveries and applications bear his imprint, as nicelysummarized in a tribute by Robin Holliday and colleagues [21].

3.6. Robin Holliday on somatic mutations and aging

Another Reflections article from "Down Under" followed shortly afterthat of Geoff Grigg from another distinguished colleague and friend ofDonald MacPhee – Robin Holliday. Given the diversity of his scientificcontributions, notably including recombinational mechanisms (and theeponymous Holliday junction), there were many possible topics onwhich he might have written. His choice was his longstanding interestin the biology of aging. His Reflections article is a theoretical discussionof the many ways in which genetic and epigenetic alterations cancontribute to the aging process and the difficulty of sorting out theirrelative importance [22]. Those interested in the breadth of scholarlyworks of this noted polymath, ranging from molecular biology andaging to sculpture, will appreciate a tribute to Robin Holliday by Leo-nard Hayflick [23].

4. Reflections through the years

4.1. Scott Frickel on sociological analysis of the Environmental MutagenSociety

We had been thinking of Reflections as being centered on historicaland philosophical themes when interesting presentations at scientificmeetings led us to think about an occasional sociological or politicalcommentary. The first of these was by Scott Frickel, a sociologist whostudied the growth and mission of the Environmental Mutagen Society(EMS), now renamed the Environmental Mutagenesis and Genomics

Society (EMGS). He offered a sociological analysis of how the EMS,founded in 1969, had functioned at the boundary between environ-mental science and environmental politics, and he explored how itthrived in the early 1970s despite a variety of institutional challenges.His reflections bring sociological theory to bear on the historical de-velopment of genetic toxicology [24]. A book by Scott Frickel, now aprofessor of sociology at Brown University, gives a complete account ofhis sociological study of genetic toxicology and the EMS [25].

4.2. Elliot Volkin on the discovery of mRNA

John Wassom, the director of the Environmental MutagenInformation Center (EMIC) in Oak Ridge, was deeply interested in thehistory of environmental mutagenesis, and he contributed historicalreviews on the subject to the EMS's journal Environmental and MolecularMutagenesis [26,27]. He also took an interest in our plans for Reflectionsand wrote to me that Elliot Volkin, who was then retired from the re-search staff at Oak Ridge National Laboratory (ORNL), still came toORNL regularly and that John had "adopted" him at EMIC. They dis-cussed the history of genetics and mutation research often, and Johnthought that Dr. Volkin might be interested in writing an article forReflections. John wrote, "He is a jewel and an encyclopedia of knowl-edge. I think an article by him on the events leading up to and theactual discovery of mRNA would be of great interest to the MR read-ership." As usual, John's advice was good.

I contacted Dr. Volkin and invited him to write a paper forReflections, and he agreed. Working with him was a real pleasure. I wasstruck by his modesty in discussing his studies, working in collaborationwith Lazarus Astrachan, that were so close to one of the premierachievements of the "classical phase" of molecular biology – the dis-covery of mRNA. His Reflections paper is a perspective on this discovery[28]. Those interested in an independent appraisal of the substantialimportance of the experiments of Elliot Volkin and Lazarus Astrachan inthe history of molecular biology might refer to The Eighth Day ofCreation, a highly regarded history by Horace Freeland Judson [29] andthe autobiography of the Nobel laureate François Jacob, who viewedtheir research as a contemporary researcher pursuing closely relatedproblems [30].

4.3. F. Peter Guengerich on metabolism and carcinogenesis

F. Peter ("Fred") Guengerich is a member of the Department ofBiochemistry at Vanderbilt University School of Medicine with a re-markable record of research on carcinogen metabolism. Yet, when Iinvited him to write some reflections on the growing understanding ofthe role of metabolism in carcinogenesis and mutagenesis, he seemedsurprised. He first joked that he was too young to write reflections, andthen he came to his point directly: Professor James Miller, though atadvanced age, was professionally active, and I should really ask him towrite it. As Professor Miller's health was fragile, Fred graciously agreedto write the article, and he gave us a great paper on "forging the links"between metabolism and carcinogenesis [31]. James Miller died in late2000 as Fred's manuscript was being completed, and Fred dedicated thepaper to the memory of James and Elizabeth Miller. This Reflectionspaper thus became the first of several dedicated to mentors and leadersin the field. I would refer readers who would like more information onthe lives and scientific legacy of James and Elizabeth Miller to a tributeby Fred Kadlubar [32].

Fred Guengerich starts his reflections with the history of epide-miologic associations of tumors with carcinogens. He carries the storyforward into the 20th century by addressing animal studies, en-dogenous carcinogens, mutagenesis, and research on the metabolism ofcarcinogens in the 1930s and 1940s, which includes the early studies ofthe Millers. He reviews the cytochrome P450 monooxygenases andother enzymes of xenobiotic metabolism, the metabolic activation ofprocarcinogens into electrophiles, detoxication reactions, enzyme


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this argument was that such clear carcinogens as nitrosamines andpolycyclic hydrocarbons gave negative results in tests for mutagenicity.The lack of correlation was later explained by the lack of mammalianmetabolic activation in the mutation assays.

Heinrich reviews the history of metabolic-activation studies thatresolved this problem [47]. The early stages were the isolation of re-active carcinogen metabolites by James and Elizabeth Miller in 1960,host-mediated assays in the late 1960s, and his own studies on in vitrochemical activation in 1966. In a host-mediated assay, an indicatororganism (e.g., bacteria or Neurospora) was injected into an animal(e.g., rat or mouse) that was then treated with a test chemical. Themicroorganism was later recovered from the animal to measure muta-tions that had been induced by the chemical and those metabolites thatreached the target organism in the animal. A major breakthrough camein 1971 with Heinrich's reporting the metabolic activation of the pro-mutagen dimethylnitrosamine into a mutagen by a tissue homogenatefrom mouse liver [48]. This was followed by Bruce Ames's assimilationof liver homogenates into the newly developed Ames test in 1973 [49].Heinrich carries this history forward in a discussion of transgenic mu-tation assays in mammals in vivo, of which he was one of the earlydevelopers. He also reviews bacteria and mammalian cell lines en-gineered to express human cytochrome P450 enzymes and the detec-tion of induced mutations in endogenous mammalian genes in vivo [47].

On a personal note, Heinrich was my major professor as a graduatestudent, and I surely wanted to include him in the Reflections series. Iinvited him at the outset of Reflections, and he delayed a bit. When Ireminded him, I was pleased to receive the response that I wanted: "Yes!I should write the reflections paper, and I have a bad conscience that Ihave not done it yet." His explanation was that he always liked to lookforward rather than back, and this was true. Heinrich was always fo-cused on the future. His Reflections paper was completed in 2004 [47],and it was well worth the wait. Those interested in reading of the lifeand work of Heinrich Malling are referred to tributes written in hismemory [50,51].

4.13. Fred Sherman on molecular analysis of mutagenesis in yeast

Fred Sherman was a brilliant geneticist who designed the iso-1-cy-tochrome c (cyc1) mutation system in yeast. He was able to classifymutations at the molecular level by sequencing. While this may seemordinary to a modern reader, you need to remember that he and hiscolleague John Stewart did this work in the 1960s and 1970s, beforethe era of automated DNA sequencing. What they sequenced was theiso-1-cytochrome c protein, and from the amino acid sequences theydeduced the nucleic acid sequence changes in the cyc1 gene. While fewwould be tempted to do such work today when DNA sequencing is fastand commonplace, it was an important part of the foundation for un-derstanding mutagenesis at an increasingly molecular level. In hisReflections article [52], Fred tells the story with an interesting, ap-pealing style. Besides his brilliance in science, Fred will be rememberedby many as a man with a great sense of humor and far-reaching crea-tivity [53].

4.14. R.J. Preston on extrapolations in risk assessment

R. J. "Julian" Preston had an illustrious career in cytogenetics andradiation biology, which increasingly moved him into the area of riskassessment, working for years at the Chemical Industry Institute ofToxicology (later renamed the Hamner Institutes) and more recently atthe U.S. Environmental Protection Agency prior to his retirement. Notsurprisingly, his reflections are devoted to a hazard, but in this case it isneither chemical nor radiation but rather, the hazard of extrapolations.Julian entitled his Reflections essay "Extrapolations are the Achilles Heel ofRisk Assessment" [54], and he comments on extrapolations among spe-cies, among doses and among life stages, with an emphasis on carci-nogen risk assessment and a call for greater reliance on mechanistic

studies. Thus, he points the way to a trend that has been dominant intoxicology and risk assessment in recent years.

4.15. Kristien Mortelmans on plasmid pKM101 in mutation assays

Anyone who has worked with the Ames assay, more formally calledthe Salmonella/mammalian-microsome mutagenicity test, knows thatseveral of the key strains rely on plasmids that markedly enhance thesensitivity of the bacteria to mutagenesis. Among these, the mostknown and widely used is plasmid pKM101. What may be less knownnowadays is that the "KM" designates Kristien Mortelmans, and shewrote a fascinating Reflections article on how pKM101 came to have itsimportant role in the most widely used bacterial mutation assay [55].Kristien describes not only the history of pKM101 but also its me-chanisms of action, the Ames test more broadly, and the atmosphere ofthe bacterial genetics laboratory of Bruce Stocker, where she was agraduate student in the early 1970s. Kristien isolated pKM101 as adeletion derivative of the resistance-transfer plasmid R46 (also called R-Brighton). The strain constructions involved in the origin of pKM101were far more elaborate than one might have guessed by saying simplythat pKM101 is a deletion derivative of R46. Plasmid pKM101 hasgreatly enhanced the effectiveness of the Ames tester strains, includingthe well-known strains TA100, TA98, TA97, TA102 and TA104, and ithas also been incorporated into other strains of Salmonella and E. colifor studies of mutagenesis.

While substantively commenting on the bacterial genetics, Kristienalso includes a tribute to her mentor, Bruce A.D. Stocker. It includes asense of the laboratory of the times and photographs of ProfessorStocker, the vials that he used for his large Salmonella culture collec-tion, and the prong replicator that he designed to screen colonies foraltered antibiotic resistance. Thus, this became the first of severalReflections articles centered around a tribute to a fine mentor.

4.16. Mary Esther Gaulden, John Jagger and Virginia White on the legacyof Alexander Hollaender

As a broad, international field, environmental mutagenesis hasseveral founders. In the United States, Alexander Hollaender(1898–1986) stands out as the leader of the first generation of scientistsin the field. Dr. Hollaender became the director of the newly organizedBiology Division of ORNL in 1946. He would develop it into a leadingresearch institution and a center of activity in the new field of en-vironmental mutagenesis. His influence extended far beyond ORNL, ashe is the acknowledged founder of the Environmental Mutagen Society,which was formed in 1969. When I was thinking about a prospectiveauthor to write on Dr. Hollaender's legacy, Mary Esther Gaulden im-mediately came to mind. Mary Esther met Dr. Hollaender in 1942, wasa Senior Radiation Biologist at ORNL from 1949 to 1960, and remainedassociated with him for the next 26 years while she was a professor ofradiology at the University of Texas Southwestern Medical Center atDallas. Mary Esther accepted my invitation and began work on itpromptly. Known for her great productivity and generous service, Ithought that this would proceed quickly to a fine article. It did indeedbecome a fine article [56], but the process became complicated.

Mary Esther submitted a beautiful draft, with substantive detailsand many personal reflections, but the story was incomplete when ourcorrespondence lagged. Mary Esther was apologetic about this, but thesituation was largely out of her hands, as she was suffering from de-clining health. It seemed likely that the story would not be completedwhen her husband John Jagger and her friend Virginia P. White steppedin to complete the story. John was a biophysicist, photobiologist, andhighly regarded teacher at the University of Texas at Dallas. He knewDr. Hollaender since 1955 and joined the staff of ORNL in 1956. TheOak Ridge period was eventful in the lives of Mary Esther and John inmany respects, and the Reflections article [56] includes a photograph oftheir wedding in 1956 at the home of Alexander and Henrietta


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stained by FISH – an isochromatid / chromatid interchange.

4.8. Akio Awa on cytogenetic analysis of atomic bomb survivors

Donald MacPhee took a temporary leave from his academic positionat LaTrobe University to serve from 1999 to 2004 as Chief of theDepartment of Radiobiology at the Radiation Effects ResearchFoundation (RERF), in Hiroshima, Japan. Early in his stay, he invitedAkio Awa to write a paper for Reflections on the extensive cytogeneticstudies of survivors of the atomic bombings of Hiroshima and Nagasaki.From 1968 to 1993, Dr. Awa ran the cytogenetics program of ABCC/RERF (Atomic Bomb Casualty Commission, established in 1947 andreplaced by the Radiation Effects Research Foundation in 1975), andhis article gives a personal account of his experience in this unique andimportant effort [42].

Dr. Awa begins with the history of cytogenetics, noting that thehuman chromosome number was not correctly identified until the1950s, and with the technical challenges that characterized the earlyperiod at ABCC/RERF. He describes technical advances and efforts toidentify not only the easily observed unstable chromosome aberrations(i.e., asymmetrical exchanges – dicentrics, acentric fragments, andrings), but also the stable aberrations (i.e., symmetrical exchanges –reciprocal translocations and inversions) that would persist in bloodlong after the exposure in 1945. Dosimetry was a complex problem forseveral reasons, including distance from the epicenter, shielding indifferent locations, differences between the two bombs in relativeproportions of gamma rays and neutrons, and even the atmospherichumidity.

Characterizing the cytogenetic damage was a formidable challengein the days before chromosome banding and FISH. Despite the diffi-culty, they measured higher frequencies of unstable and stable chro-mosome aberrations in exposed survivors than in controls. Lower fre-quencies of unstable than stable aberrations reflected the loss of cellsbearing dicentrics, fragments and rings. An important goal was the useof aberration frequencies to estimate doses received by individualsurvivors, and Dr. Awa chose to pursue the more difficult, but neces-sary, quantification of stable aberrations. The quality control effort wasmeticulous. The frequency of cells with aberrations increased with ex-posure, and the data permitted an evaluation of not only dose-responserelationships, but also differences between Hiroshima and Nagasaki andratios of stable and unstable aberrations (initially a theoretical 1:1)reflecting loss of the latter over time. They observed a few individualswho had clones of many cells with identical aberrations, probably de-rived from aberrant lymphopoietic stem cells [42].

Through the years, methodology was updated to include chromo-some banding and, later, FISH. These methods led to somewhat higherfrequencies of aberrations detected, and FISH improved the ease ofscoring, but they supported the conclusions that Awa and colleagueshad reached through their excellent work with the earlier technology.As teeth of survivors became available, the enamel was analyzed byelectron spin resonance (ESR) to estimate gamma-ray doses received,and these estimates were consistent with the cytogenetic dosimetry. Dr.Awa summarizes results with conventional staining, banding, FISH andESR in Section 3 of his reflections, and I call your attention to hispersonal commentary at the end of this section. He met with manychildren and parent groups, and he offers his perspective on the ethicalissues surrounding the fears of the descendants of survivors and theneed to explain the science and its uncertainties [42]. The fine char-acter of Akio Awa is reflected in his Reflections article, and his ac-knowledgments include "the citizens of Hiroshima and Nagasaki."

4.9. Moreno Paolini and Marion Nestle on diet, cancer and public health

We were pleasantly surprised to receive a detailed proposal fromMoreno Paolini for a Reflections article on chemoprevention as astrategy to minimize risks of dietary carcinogens. A professor of

pharmacology and toxicology at the University of Bologna, he wouldwork together with Marion Nestle, a professor of nutrition, food studiesand public health at New York University, to give a shared perspective.We were pleased to have such distinguished scholars of nutrition andfood toxicology seek out the young Reflections feature to present theirviews of this complex and important problem. In their Reflections article[43], they explore prospects for chemoprevention and express skepti-cism about oversimplification in the growing enthusiasm for far-reaching benefits conferred by single dietary supplements or phyto-chemicals. Their article offers reasoned warning about risks of simplesolutions to complex problems, and they call for integrative studies ofthe complexity of diet and public health.

4.10. W. Gary Flamm on mutation research in relation to regulatory policy

In his Reflections article, Gary Flamm recalls the field of environ-mental mutagenesis seeming to "explode into existence like somecosmic big bang" [44]. The time was roughly 1970; the EMS had re-cently been founded; and EMIC, which was later assimilated into theNational Library of Medicine's TOXNET databases, was being estab-lished by Heinrich Malling and John Wassom in Oak Ridge. The Foodand Drug Administration in Washington, DC, was a center of activity forresearch on mutagenesis, and Dr. Flamm was its Branch Chief of Ge-netic Toxicology from 1972 to 1974. His reflections give a perspectiveon these specific organizations but, even more so, on the integration ofenvironmental mutagenesis into the field of toxicology and work at theinterface of basic science and policy that led to the development ofregulatory standards in this field [44]. A tribute by Gary's former col-leagues gives a clear sense of his remarkable accomplishments workingat the intersection of research, governmental agencies, industry andscientific societies [45].

4.11. Ernest H.Y. Chu on mammalian somatic cell mutagenesis

The next two Reflections articles came from Ernest H.Y. Chu andHeinrich Malling, two colleagues in the Biology Division of ORNL in thelate 1960s and early '70 s. They were good friends, conferring with eachother often, while "Ernie" worked on the detection of point mutations inmammalian cells and Heinrich worked on the analysis of mutagenesis,principally but not exclusively in Neurospora crassa. In his reflections[46], Ernie traces the unequivocal demonstration of experimentallyinduced gene mutations in mammalian cells to three laboratoriesworking independently of one another in the 1960's. He then describeshis own scientific development, leading to his lab being one of thethree. His research on the induction of azaguanine-resistant mutants inV79 Chinese hamster cells was groundbreaking work, contributing tothe foundation of the widely used hprt assays in mammalian cells bothin vitro and in vivo. His descriptions give a sense of the atmosphere ofmutation research at the time and his excitement at new insights thatarose in discussion with colleagues, notably including his talks withHeinrich Malling at the blackboard in Heinrich's tiny office. The way inwhich he credits Heinrich for a specific insight that contributed to hisresearch also quietly reflects Ernie Chu's well-deserved reputation asone of the great gentlemen of our field.

4.12. Heinrich Malling on metabolic activation in mutagenesis

Heinrich Malling was responsible for one of the seminal findings inthe history of environmental mutagenesis and genetic toxicology testing– the use of an exogenous metabolic activation system based on amammalian tissue homogenate to activate promutagens into mutagensin in vitro mutation assays. It is not an exaggeration to say that his workled to methodological changes that revolutionized the testing of che-micals for mutagenic activity. Heinrich begins his Reflections paper[47] by citing W.J. Burdette's argument in the 1950's that mutagenicitydoes not correlate with carcinogenicity. An important consideration in


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being an editor of Reflections. When we were invited to be co-editors, Ihad met him several times at scientific meetings, and we had on a fewoccasions exchanged bacterial strains through the mail. I held him inhigh regard, knowing him to be a first-rate microbial geneticist whoseexpertise extended to radiation biology and radiation protection, aswell as mutational mechanisms, DNA repair and chemical mutagenesis.He was also a deep thinker, a skilled writer and an enthusiastic con-tributor to the vitality of the scientific community. I soon came to ap-preciate how these qualities were complemented by dedication, sense ofhumor and generosity of spirit.

Our co-editing of Reflections kept us in regular contact, while he wasin the School of Microbiology at LaTrobe University in Melbourne,Australia, or at the Radiation Effects Research Foundation inHiroshima, Japan, from 1999 to 2004 during a reorganization in theRadiobiology Department. Our collaboration on Reflections soon grewfrom a cordial professional relationship into a real friendship, forgedthrough electrons zipping around the world in frequent email messages.In the early days, we often added a few stray comments on the weatheror whatever was going on in the world. This evolved into discussions ofmany things, and we came to know each other's families (whom we hadnever met), adventures and misadventures, health problems, pleasuresand problems of work, Massachusetts ice and snow storms or Australianbrush fires, and whatever else may have been on our minds at the time.While email had become the norm, it still impressed those of us comingfrom the era of typewriters, carbon paper, and awaiting conventionalmail, and so we enjoyed joking remarks about the impending Y2Kdisaster, when there were unnecessary worries about the entire cyberworld collapsing at the turn of the millennium, or the curiosity of timezones in such remarks as "G'day, I was pleased to receive the email thatyou sent me tomorrow."

The saddest day in the history of Reflections came with Donald'sdeath on September 16, 2009 [68,69]. I think often about our 11 yearsof regular correspondence, and I am glad to have had the chance for usto meet at the time of the International Conference on EnvironmentalMutagens in Shizuoka, Japan, in 2001. A fine scholar and a true gen-tleman, it is no wonder that Donald MacPhee is missed by friends andcolleagues around the world.

6. The second decade of Reflections

6.1. Martin Marinus on DNA methylation and mutator genes in bacteria

Martin Marinus worked for decades on DNA methylation and mu-tators in E. coli – studies that have contributed significantly to a modernunderstanding of mutagenesis and DNA repair. His Reflections articlegives a personal perspective on this history, including his discovery ofthe first DNA adenine methyltransferase (dam) mutants in E. coli in theearly 1970s and his many years of research at the University ofMassachusetts Medical School elucidating mechanisms underlyingbacterial DNA methylation, its functions, and associated mutator phe-notypes [70]. Later research revealed the role of 6-methyladenine instrand discrimination in mismatch repair in E. coli. The content ofMartin's commentary is broad, including an overview of DNA methy-lation mutants, mismatch repair, effects on recombination and mutatorphenotypes. He ends his commentary with the interesting observationthat the frequency of mutators in pathogenic or commensal strains of E.coli is much higher than that in standard laboratory strains that are notgrown under the intense selective pressures of new habitat colonization[70].

6.2. Hirokazu Inoue on DNA repair and homologous integration inNeurospora

Hirokazu Inoue was introduced to the fungus Neurospora crassa as amodel organism in 1969, when he became a graduate student of TatsuoIshikawa at the University of Tokyo. In addition to Dr. Ishikawa, he

considered the distinguished Neurospora geneticist David Perkins ofStanford University as an important mentor early in his career. Hisexperience with Neurospora was enriched by a postdoctoral fellowshipwith Fred de Serres at the National Institute of Environmental HealthSciences (NIEHS) in North Carolina, where I came to know him as afriend in the 1970s. Hirokazu never abandoned Neurospora, but headopted the methodology of molecular biology early, making majorcontributions that led to his being recognized with the MetzenbergAward for his lifetime achievements in Neurospora research, given bythe Neurospora research community at the Fungal Genetics Conferencein Asilomar, California, in 2009. His Reflections article combines apersonal perspective with a review of the early history of Neurosporaresearch, the transition to molecular analysis, and an emphasis oncurrent understanding of DNA repair, recombination, and homologousintegration in Neurospora [71]. His research over many years at Sai-tama University in Japan is an important component of this history, andhis account of it includes detailed tables. He concludes with thoughts onpromising new directions for research on DNA repair and mutagensensitivity in Neurospora.

6.3. Carmel Mothersill and Colin Seymour on unforeseen discoveries inradiation biology

Carmel Mothersill is perhaps best known for her creative researchon nontargeted effects of ionizing radiation, especially bystander ef-fects, in which cells that did not experience the direct energy depositionare affected by signals received directly or indirectly from irradiatedcells. A Reflections article that she wrote together with her husband andlongtime research collaborator Colin Seymour comments on diverseaspects of radiation biology [72]. They focus on unforeseen discoveriesand lingering uncertainties that led to changes in longstanding viewsabout effects of radiation, which had been derived from target theory.The changes stem from findings on nonlinear dose responses, dose-rateeffects, indirect effects of radiation mediated by radicals, inducible DNArepair, adaptive responses, and bystander effects. An incomplete un-derstanding of such phenomena remains an obstacle to defining the bestpractices for radioprotection and radiotherapy. Besides a fascinatingcommentary on unresolved questions in radiation biology, Carmel andColin include a tribute to the pioneering radiobiologist Tikvah Alper(1909–1995), and Carmel gives us a view of her own original abstractpaintings on the theme of bystander effects of radiation [72].

6.4. A.T. Natarajan on a lifetime of studies in cytogenetics

A.T. Natarajan returned to Reflections a few years after his initialcommentary [57] to reflect on his long career in cytogenetics [73]. It isa great personal story. Nat begins autobiographically with his earlyyears in India. His research career began in 1948, and he offers a fas-cinating look at a time and place when the challenges that a youngscientist encountered were very different from those in modern Indiatoday or in prosperous countries then. He began in botany, because thatwas the most practical choice, but from the beginning he longed to getinto genetics and work with chromosomes. He recounts his leavingIndia to pursue opportunities in the U.S. and Sweden, followed by aninterlude in India, then Vienna, and ultimately at Leiden University inthe Netherlands, where he spent the rest of his professional life. Herecalls specific events with great style and humor, but he never loses hisfocus on science.

Nat summarizes some of his major scientific accomplishments andthe revolutionary changes that occurred in cytogenetics during his ca-reer, including the molecular analysis of alterations and FISH. Appliedresearch was important to Nat, and this is reflected in his researchcontributions in radiation cytogenetics and chemical mutagenesis,complemented by his contribution to social and political efforts tominimize the impact of radiation accidents and chemical exposures. Hecomments with appreciation on his mentors along the way, including


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Hollaender in Oak Ridge. John tells the story in his reflections withgreat style. Virginia White had been an administrator at Fisk Universityin Nashville and joined the staff of ORNL in 1955, ultimately becomingthe Assistant to the Director of the Biology Division, where she man-aged such matters as personnel, budgeting, purchasing and publica-tions. Virginia includes interesting observations about the location andtimes that extend those of Mary Esther and John. In the end, this is"Reflections in Three Parts" that complement one another perfectly.

4.17. A.T. Natarajan on the origin of the journal Mutation Research

The article on the life and legacy of Alexander Hollaender, a founderof our field, was followed by our shortest, yet very fitting, Reflectionsarticle on another foundational event. A.T. ("Nat") Natarajan submitteda reflection on the origin of the journal Mutation Research [57]. It is abrief commentary with a photograph from an international symposiumon DNA repair, which Nat thought may have been the first ever on thistopic. It was held in Leiden, The Netherlands, in August 1962. At themeeting, attended by such luminaries of our field as Charlotte Auer-bach, Hermann Muller, Evelyn Witkin and Tikvah Alper, Professor F.H.("Frits") Sobels proposed the establishment of a new journal, and theinaugural issue of Mutation Research appeared in January 1964. Natcomments that he was early in his scientific career at the time, and hefound the conference inspirational. A half-century after the conference,in 2012, Nat would be writing reflections on his own illustrious cyto-genetics career.

4.18. K. Sankaranarayanan and John Wassom on radiation risk assessment

K. Sankaranarayanan ("Sankar") studied radiation-induced geneticdamage for decades and combined his scientific expertise with publicspiritedness through service to international radiation protection or-ganizations and scientific journals. He has served on committees andtask forces for the United Nations Scientific Committee on the Effects ofAtomic Radiation (UNSCEAR), which was established in 1955, and forthe International Commission on Radiological Protection (ICRP), whichwas formed 1928. He wrote chapters of UNSCEAR reports from 1970 to2001. In 2008, he teamed up with John Wassom to write reflections onthe history of radiation protection [58]. This was a good combination ofskills, as John was the Director of EMIC and had access to all publishedarticles in the field.

The emphasis of their article is on genetic (i.e., transmissible) effectsof radiation and the work of UNSCEAR and ICRP, but they also considercarcinogenic effects and the work of the U.S. National Academy ofSciences' Committee on Biological Effects of Atomic Radiation (BEAR)and its more recent successor, the Committee on Biological Effects ofIonizing Radiation (BEIR). Their analysis spans the history of radiationprotection with substantive, well-documented discussion. Its tablesdefine the specialized terminology of the field; list the reports ofUNSCEAR, ICRP and BEAR/BEIR; identify major developments in theearly decades of radiation protection; and compile estimates of geneticdisease frequencies, genetic risks, and dose limits. The detailed scien-tific content is nicely complemented by personal reflections on theevents and the people who played an important role.

4.19. Michael Fenech on cytokinesis-block micronucleus assays

The development of the cytokinesis-block micronucleus (CBMN)assay in human lymphocytes radically changed the cytogenetic detec-tion of chromosomal damage induced by radiation and chemicals. TheCBMN assay was first reported in the 1980s by Michael Fenech andAlexander Morley of Flinders University in South Australia [59,60]. Inthis new method, cytochalasin B was added to cultures of mitogen-sti-mulated lymphocytes to block cytokinesis after enough time hadelapsed for the cells to be entering their first post-stimulation mitoticdivision. At an appropriate time, slides were made, and cells that had

undergone a single mitotic division were easily recognized because theyhad two nuclei. Micronuclei – small chromatin-containing bodies –were counted only in binucleated cells, thereby normalizing the fre-quency to those cells that had undergone a single mitotic division.Dose-dependent increases in micronucleus frequencies after mutagenexposure were quantified, and the scoring of large numbers of cells wasmuch faster and required less skill than classical metaphase analysis[59,60]. Within a few years, Fenech and Morley added kinetochorestaining to the assay to distinguish micronuclei containing wholechromosomes from those containing fragments [61].

In the years that followed, the assay became widely used around theworld, and Michael Fenech attentively cultivated it, unraveled its me-chanisms and ingeniously expanded its possible applications throughhis research at the Commonwealth Scientific and Industrial ResearchOrganisation (CSIRO) in Adelaide. His Reflections article offers an ap-pealing blend of scientific advances and personal memories [62]. Mi-chael traces his scientific development to his native Malta, where hebecame interested in marine ecology, an interest that extended to whatis now called ecotoxicology. His pursuit of advanced studies broughthim to Australia, which he considered unlikely at the time but provedfortuitous as his scientific life blossomed "Down Under," and it is nowover 35 years later. He gives much credit to Alexander ("Alec") Morley,his mentor at Flinders University Medical School, where his experienceslaunched him on an illustrious research career combined with a sub-stantial commitment to environmental mutagenesis, nutrition andpublic health.

Michael describes the origin of the cytokinesis-block micronucleusassay – his "eureka moment" – with great style and then moves to thedevelopments that led to worldwide use of the assay and its refinementand broadening applications. He credits earlier studies of the inhibitoryaction of cytochalasin mycotoxins [63] and of micronuclei without thecytokinesis block [64]. He then describes the great advances of the later"Cytome" version of his CBMN assay, supported by photographs anddrawings, including binucleated and multinucleated cells, micronuclei,nucleoplasmic bridges, nuclear buds, and necrotic cells, giving distin-guishable evidence of chromosome breakage, aneuploidy, dicentricchromosomes, gene amplification, mitotic inhibition, necrosis, andapoptosis. His "Reflections from Down Under" on his "Lifetime passionfor micronucleus cytome assays" is a great story told well [62].

4.20. Richard Stevens on circadian disruption and cancer incidence

I had the good fortune to meet Richard Stevens, an epidemiologistfrom the University of Connecticut School of Medicine, at the meetingof the European Environmental Mutagen Society in Cavtat, Croatia,where he made a presentation on "Breast cancer and circadian disrup-tion from electric lighting." I enjoyed the presentation, discussed it withhim, and invited him to submit an article for Reflections. He agreed. Iknew that I was about to read something interesting and unconven-tional when I received his manuscript, entitled "Electric light causescancer? Surely you’re joking, Mr. Stevens," adapting the title from apopular book by the Nobel laureate physicist Richard Feynman [65]and starting with a fitting Feynman quotation. The manuscript did notdisappoint. Richard describes the association between exposure to lightat night and cancer incidence [66]. His text shows the power of epi-demiologic studies in revealing an unanticipated association andleading to an understanding of the association when combined withmechanistic studies. I won't comment further, so as not to detract fromhis compelling story [66], except to note that the International Agencyfor Research on Cancer (IARC) now classifies "shiftwork that involvescircadian disruption" in its Group 2A, designating "Probably carcino-genic to humans" [67].

5. Reflections on Donald MacPhee

Working with Donald MacPhee ranks high among the pleasures of


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"the Fall" applies to hopes for a simple solution to a complex problemand not to the value of basic research on photomutagenesis or to thegoal of avoiding phototoxicity and photocarcinogenesis. To support theconclusion that photomutagenicity screening is not practical in safetyassessment, they draw upon collaborative European studies; problemsof evaluating UVB, UVA or a mixture of wavelengths; phototoxicity offluoroquinolone antibiotics; micronucleus and comet assays in skin; andthe problem of "pseudo-photoclastogens" – apparent photoclastogensthat do not even absorb light. In 2012, the International Conference onHarmonisation (ICH) guideline stated that "Testing of photo-genotoxicity is not recommended as a part of the standard photosafetytesting programme,’’ but a phototoxicity assay would remain in place.

6.9. Liane B. Russell on genetics research in the "Mouse House" in OakRidge

One of the pleasures of being the editor of Reflections was workingwith Liane ("Lee") Russell as she wrote her reflections on the history ofthe mouse genetics facility at ORNL, known as the Mouse House [82].The rationale for its establishment in 1946 was the need to understandthe effects of ionizing radiation in the context of the bombings of Hir-oshima and Nagasaki, nuclear testing, and prospects for peacetime usesof atomic energy. When Alexander Hollaender was developing theBiology Division of ORNL, he recruited William L. ("Bill") Russell(1910–2003), a mammalian geneticist at the Jackson Laboratory inMaine, to develop and direct the program. Liane Brauch ("Lee") Russell,Bill's wife and also a mammalian geneticist, would be an independentresearcher at ORNL. She became the director of the Mouse House uponBill's retirement in 1975. In fact, Bill and Lee worked as a team bothbefore and after his official retirement.

In her Reflections article [82], Lee gives a thorough review of theresearch in the Mouse House, a fascinating historical account, and herpersonal perspective on the times, people and events. There are pho-tographs of Bill and Lee in the 1950s, other Mouse-House people, andthe specialized equipment of the time. There are also cartoons of micewith personalities reflecting their genetic attributes, drawn by the an-imal-care supervisor Louis Wickham. Lee praises her young techniciansand comments on rewarding international collaborations and visitingdignitaries. She includes such interesting anecdotes as that of a con-gressman getting stuck between floors on a lab visit to ORNL, and Billdriving mice to Nevada to oversee their exposure in one of the lastabove-ground nuclear bomb tests.

The Mouse House is probably most known for studies of germ-cellmutagenesis, the mouse specific-locus test, and the use of mouse datafor estimating risks of ionizing radiation. However, the contributionswere much broader than that. Over six decades, the Mouse Housegenerated a wealth of knowledge on mammalian genetics, molecularbiology, cytogenetics, reproductive biology and teratology. Lee sum-marizes an impressive list of accomplishments spanning such diversetopics as germ-cell development and stage sensitivity, dosimetry,spontaneous mutations in the perigametic period, the supermutagen N-ethyl-N-nitrosourea (ENU), DNA repair in vivo, fine structure genomicmapping, molecular analysis of mutations, radiation-induced ter-atogenesis, critical periods in embryonic development, homeotic shifts,mammalian sex determination, heritable translocations, dominant le-thals, aneuploidy, mosaicism, and mouse models for human disorders[82]. Lee's story ends with changing priorities in funding, the gradualdecline of the Mouse House and, ultimately, the closing of the facility.This is told with some sadness, but also with an appreciation for itsremarkable legacy.

I would like to point out an interview that Lee gave in Oak Ridge inApril 2018 for "Voices of the Manhattan Project." The website [83]shows Lee on camera responding to questions about many aspects ofher life, including her family history, their having to leave her nativeAustria because of its annexation by Nazi Germany in her teen years,and the events that brought her to Oak Ridge. She describes the work of

the Mouse House for a general audience, and she comments on otheraspects of her experience in Oak Ridge. Those who know Lee as a mousegeneticist may be unaware that she and Bill became involved in theenvironmental movement in the 1960's, working to protect Tennesseerivers and wilderness. They founded Tennessee Citizens for WildernessPlanning (TCWP) in 1966, and she is still the editor of the TCWPnewsletter in 2019. TCWP has had remarkable influence in protectingthe Obed River and the Big South Fork of the Cumberland River and inmany other conservation projects. It is a tribute to Lee and Bill that theyhave had such a profound impact in two very different areas.

6.10. Delbert Shankel in remembrance of Charlotte Auerbach

Delbert ("Del") Shankel's Reflections article is a tribute to CharlotteAuerbach (1899–1994), who obtained the first unequivocal evidence ofchemical mutagenesis. Del describes her scientific contributions, whichgo far beyond the discovery of chemical mutagenesis in the 1940s, andhis narrative is enriched by his memories of her based on a sabbaticalthat he spent with her in Edinburgh in 1967 [84]. Del gives both adetailed biographical account of Charlotte Auerbach, called "Lotte" byher friends, and a sense of her vibrant personality and humanitarianvalues.

Sadly, Del Shankel died in July 2018. Del was soft-spoken and notinclined to boast of his accomplishments. I knew him for years as amicrobiologist and an EMS colleague working on antimutagens before Irealized the scale of his service and leadership over 50 years at theUniversity of Kansas. Beginning as an Assistant Professor ofMicrobiology in 1959, he became Professor of Microbiology, Chairmanof the Department of Microbiology, and a Fellow of the AmericanAcademy of Microbiology. He later served as Assistant Dean, AssociateDean, Acting Dean of the College, Executive Vice Chancellor, andActing Chancellor. Whenever the University of Kansas called upon him,Del served with distinction, and in 1995 he was named as the 15thChancellor of the University. Those who know him may want to see atribute to him from the University of Kansas Alumni Association, whichincludes his picture standing by the Delbert M. Shankel StructuralBiology Center, named in his honor [85]. I have a specific memory fromworking with Del on his Reflections paper. He had an original photo-graph of Charlotte Auerbach, and he was unsure about how to prepareit for publication, so I offered to take care of it if he would send thephoto to me. I suggested sending it registered and insured. A few dayslater, the photo arrived by regular mail. Del said that he mailed his firstletter in ordinary U.S. mail when he was 5 years old in 1932, has beendoing so ever since, and never lost a single item.

6.11. James D. Tucker on FISH and chromosome painting

James D. ("Jim") Tucker played a leading role in the development ofwhole-chromosome painting by means of fluorescence in situ hy-bridization (FISH). In a Reflections article [86], he reviews key elementsof his scientific work and describes the obstacles that he encounteredand how they were resolved. He offers a rich personal commentary onmentors, students, the upheavals and uncertainties that young scientistsoften face, and the struggles and pleasures of a research career, viewedthrough his experience at Lawrence Livermore National Laboratory(LLNL) in California and later at Wayne State University in Detroit. Jimbegins his story with thoughts on the seminal role that a key paper canplay in stimulating the interests of a young scientist and suggesting apath for research. In his case the paper was by Mary Lou Pardue andJoseph Gall on the first localization of nucleic acids within metaphasechromosomes by in situ hybridization using radioactive label in the daysbefore FISH [87]. Mentors were another source of inspiration, and heacknowledges Joginder Nath at West Virginia University and Tong-manOng at the National Institute for Occupational Safety and Health in thiscontext. A dedicated teacher himself in later years, he comments on hishopes for students.


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M.S. Swaminathan, who is known as the Father of India's GreenRevolution, Lars Ehrenberg in Sweden, and later F.H. "Frits" Sobels inLeiden. A gentle man with great intellect and wit, he describes how hevalued the many friends who enriched his professional and personallife. This was reciprocal, as Nat was highly regarded and is missed bymany former students and colleagues around the world. For those whowould like to read more of Nat's life and scientific contributions, Iwould refer you to a beautiful tribute by Awadhesh Jha of theUniversity of Plymouth, who knew Nat as his mentor both in India andlater in Leiden [74], and to a special issue of Mutation Research in Nat'shonor, nicely introduced by Adayabalam Balajee and colleagues [75].

6.5. Urbain Weyemi and Corinne Dupuy on reactive oxygen species, NOX4and DNA damage

Rather than reflecting on events of years gone by, our nextReflections authors, Urbain Weyemi and Corinne Dupuy, reflected onthe implications of a new area of investigation – the role of a specificoxidase in the generation of reactive oxygen species (ROS) involved incellular oxidative stress [76]. The NADPH oxidase NOX4 is localized inthe perinuclear region of cells, and the ROS that it generates whenactivated are implicated in genetic damage and genomic instability butalso play a role in normal redox processes, signal transduction and re-pair. Two informative figures nicely illustrate their hypothesis: oneintroducing the generation of ROS by NOX enzymes, and the otherpresenting the hypothesized actions of NOX4 in modulating oxidativestress. Weyemi and Dupuy suggest that dysregulation of NOX4 is im-plicated in inflammation, cancer, and such other disorders as diabetes,fibrosis and kidney dysfunction. They speculate that NOX4 (and per-haps other NOX enzymes) can be considered as a potential target fortherapeutic applications [76]. At the end of 2018, a PubMed search for"NOX4" yielded 2101 citations, including 1527 for "NOX4 and reactiveoxygen species," reflecting the fact that NOX4 remains a topic of intenseinterest.

6.6. Elof Axel Carlson on the legacy of Hermann J. Muller

Hermann J. Muller is one of the giants of 20th century genetics andmutation research. The history of Muller's life and work was toldbeautifully by Elof Axel Carlson in a biography written in 1981 [77].When Professor Sobels established the journalMutation Research [1], hecommented that he was "particularly honored" that the first paper in thenew journal was by the distinguished geneticist H.J. Muller. In thispaper on the relationship between recombination and the accumulationof mutations in populations, Muller discussed what later came to beknown as "Muller's Ratchet" [78]. Given this background, I contactedDr. Carlson, who is now a Visiting Scholar at the University of Indiana,where he continues his work with the Muller archives, in hopes that hewould consider submitting an article to Reflections. I was delighted tofind that he was receptive to my invitation, and that began an enjoyableassociation with him that has spanned three Reflections articles. His firstwas on H.J. Muller's contributions to mutation research and includespersonal reflections on having been a student of H.J. Muller in the1950s [79].

The article offers both a substantive commentary on Muller's far-reaching scientific achievements and a nice personal touch, including aphotograph of several pages of his own class notes when he tookMuller's course, called "Mutation and the Gene,’’ at Indiana Universityin 1955. Elof's Reflections article comments on many aspects of H.J.Muller: his personality and qualities as a teacher and mentor; his early,deep understanding of the role of the gene in life and evolutionarytheory; his formative years in the fabled "Fly Lab" of Thomas HuntMorgan at Columbia University; his extraordinary ability to designgenetic stocks to solve important problems experimentally; his dis-covery of x-ray mutagenesis in 1927, followed by his Nobel Prize in1946; his commitment to understanding dose-response relationships;

his advocacy for radiation safety; his distinction among gene mutations,changes in chromosome structure, and changes in chromosomenumber; his recognition of genetic interactions and environmental in-fluences on gene expression; his explanation for the rarity of polyploidyin animals relative to plants; and his views on various social and poli-tical controversies, including eugenics and Lysenkoism.

6.7. Elof Axel Carlson on the introduction of Drosophila into mutationresearch

Shortly after writing his reflections on H.J. Muller [79], includingMuller's experience in Morgan's Fly Lab, Prof. Carlson began to writesome reflections on the fly. How did Drosophila come to its centralposition in twentieth century genetics? Elof weaves the history ofDrosophila's arriving in the Fly Lab at Columbia into a fascinating storywith surprising elements, such as studies in Indiana's limestone cavesand committee meetings involving mirror tricks at Indiana University,complete with photographs dating from 1902 to roughly 1914 [80].Fruit flies were chosen early for studies of experimental evolution, andthe route to Morgan's lab involved William Castle at Harvard Uni-versity, Frank Lutz at Cold Spring Harbor, and a critical role played byresearchers at Indiana University. The Indiana biologists Carl Eigen-mann, William Moenkhaus, and Fernandus Payne were interested in theevolution of blind cave fish and other blind cave fauna. Moenkhausintroduced fruit fly research to Indiana University, and Payne was in-trigued by the prospect of using them to study experimental evolution.It is likely that Castle, who was conducting studies with Drosophila atHarvard, influenced Morgan to consider using flies. The actual source ofthe flies, however, was probably Fernandus Payne. Payne moved fromIndiana to Columbia University for graduate work, and he used fliesthere for his studies of experimental evolution and for teaching. ElofCarlson had met Payne at Indiana University in 1957 and later inter-viewed him when he was writing his biography of H.J. Muller. Paynetold Elof that he caught his flies on a windowsill at Columbia, and whenMorgan later asked him for some, he obliged. This led eventually toMorgan's isolation of the famous white-eye mutation and to the be-ginnings of classical genetic analysis, involving such luminaries amongMorgan's students as Alfred Sturtevant, Calvin Bridges and H.J. Muller.

6.8. Lutz Müller and Elmar Gocke on photomutagenesis

The history of photochemical mutagenesis has had its ups anddowns, and the story is nicely told by Lutz Müller and Elmar Gocke whowere in the thick of the action at Hoffmann-La Roche, Ltd., in Basel,Switzerland [81]. They called their reflections "The Rise and Fall ofPhotomutagenesis." Concerns about possible phototoxicity associatedwith exposure to sunscreens, cosmetics, and pharmaceuticals includedphotomutagenic, photocarcinogenic, and photoallergenic effects. Sub-stantial efforts were made to develop simple screening methods forphotomutagenicity, especially using bacterial reversion assays andmammalian cell cytogenetics. Despite the initial enthusiasm, it becameincreasingly evident that photomutagenesis entails complexity withinteractions at several levels and technical obstacles that were hard toresolve. This led to a realization that simple screening assays would notsuffice and that a more laborious case-by-case evaluation was needed.The Reflections paper includes interesting anecdotes, such as Elmar'sobservation that holding an Ames-test plate of strain TA100 in thesunlight outside the lab for 15 s led to a doubling of the revertant fre-quency. In another, Elmar noticed that a puzzling, apparently irrepro-ducible, positive response to compound "RO19-8022" in strain TA102occurred only when the test was performed in the afternoon. It turnedout that the cause was the increased ambient light when the "sun camearound the building." Compound RO19-8022 turned out to be highlyphototoxic and photomutagenic; it was terminated as a drug candidatebut was made available for research purposes.

In commenting on the "Rise and Fall," Müller and Gocke stated that


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and chromosomes, along with his wife, who copied journal articles byhand to help him. Singh's interest from the outset was to study aging bymeans of cytogenetics and molecular biology. Ultimately, he soughtopportunities abroad, and this brought him to Ohio State University(OSU). He describes the adventure of this trip with funny stories, suchas carrying a suitcase of cashews and raisins, as a cautious vegetariannot knowing what to expect. He was pleased with OSU, supportivementors and a "kindly grandmother" who rented him a room, but therewere also many trips to the immigration office in Cincinnati seekingapproval to stay in the U.S. He later moved to various institutions, withcritical periods at the National Institutes of Health (NIH) and theUniversity of Washington.

Singh acknowledges a book chapter by B. Rydberg and Karl-JohanJohanson for ideas leading him to what would become the comet assayand also a chance meeting with Ray Tice at a scientific conference asthe origin of their fruitful collaboration. A critical opportunity offeredto him at the NIH Institute on Aging (NIA) was training with PeterCerutti in Lausanne, Switzerland, where he learned to do alkaline elu-tion of DNA. He describes Cerutti as an inspiring teacher, and in a funnystory he notes that everything there was perfect, except that at a dinner,he unknowingly ate caviar and was aghast when he learned a fewminutes later what it was. Back at NIA in 1986, everything was in placefor his development of the comet assay. Thinking back to Rydberg andJohanson, he had recognized three elements of the problem – isolationof living cells, embedding of cells, and lysis of cells – and the idea cameto him to electrophorese the cells in order to move small DNA frag-ments.

He describes the problems encountered (e.g., getting rid of RNA andfinding good stains) and how they were resolved. It turns out thatOstling and Johanson had independently decided on electrophoresisand published a paper using similar methods a few years earlier [95].Singh identified a shortcoming of the assay as described, and he mod-ified it by using alkaline electrophoresis. He describes his excitement atfirst observing "comets" and running to tell his colleagues Mike McCoy,Ray Tice and Ed Schneider, who became his coauthors in publishing themethod that is the basis for today's comet assay [96]. The name of themethod also evolved from “microelectrophoresis” [95], to "microgelelectrophoresis" [96], to "single-cell gel electrophoresis," and ultimatelythe "comet assay," a name introduced by Peggy Olive and colleaguesbased on the appearance of the affected cells and their DNA whenviewed through a microscope. Singh comments that the name "cometassay" has "rightly stuck for the last 25 years." Singh devotes the re-mainder of his reflections to his subsequent studies and applications ofthe assay [94]. These include the influence of aging on DNA damage,detection of exposure to low doses of ionizing radiation, effects ofradiofrequency radiation, genotoxicity of chemicals, apoptosis, DNAdamage in various cell types, and refinements of equipment and com-puter analysis. With A.T. Natarajan, he combined the comet assay withFISH for visualizing specific gene sequences and centromeres. He notesthat the comet assay is now used internationally for such varied pur-poses as human biomonitoring of DNA damage, ecotoxicological ap-plications in diverse species, and genotoxicology assessment.

6.15. Elof Axel Carlson on ad hominem arguments in the scientific literature

An issue of publication policy and ethics that occasionally arises inscience is the use of vitriolic arguments and ad hominem attacks topromote or denigrate viewpoints. Elof Carlson has been deeply con-cerned about personal attacks being made against H.J. Muller becauseof Muller's historic influence in the process of radiation risk assessment.This led Elof to reflect on ad hominem attacks in the scientific literature,both with respect to current and historical instances [97]. Whileworking on his book, The Gene: A Critical History [98], Elof had theimpression that disputes among respected geneticists were especiallyintense shortly after the turn of the 20th century. Then, around 1919,the vitriolic arguments seemed to disappear, perhaps owing to changes

in editorial policies. He briefly traces the history of ad hominem argu-ments in science to antiquity, discusses Isaac Newton and Robert Hookein the 17th to 18th century, and concentrates on the time after thehistoric work of Mendel and Darwin in the 19th century. While Darwinpreferred to stay clear of disputes, his detractors and defenders did not.After the rediscovery of Mendel's work in 1900, the disputes in the newfield of genetics became intense, and Elof gives a vivid account of theantagonism between William Bateson, who tended to think of Mende-lian heredity in terms of discontinuous phenotypes, and the biometricschool of Francis Galton, Karl Pearson and Raphael Weldon. Thesedisputes spilled over to T.H. Morgan's fly lab, including H.J. Muller, andultimately, they were largely reconciled through the merging of clas-sical Mendelian genetics and evolutionary biology. Other cases in Elof'sreview are the very consequential attacks of T.D. Lysenko on the agri-cultural geneticist N.I. Vavilov in the Soviet Union and the less con-sequential, yet illustrative, public disdain of Erwin Chargaff for JamesWatson and Francis Crick in the early days of molecular biology.

With this background, Elof takes up the resurgence of ad hominemattacks in the case of H.J. Muller, and he comments on the degradingeffect of such polemics on scientific discourse and truth [97]. He citesthe attacks by E.J. Calabrese on Muller and others involved in NationalAcademy of Sciences studies of ionizing radiation, and he offers anevidence-based defense of their reputations, but there is still concernthat clarity can be lost in the repetitive attacks. He makes re-commendations on how the structure of scientific literature offers thehope of avoiding personal attacks and the distortion of historical events.They include a role for journal editors and reviewers, instructions toauthors, and more referrals of such manuscripts to journals that focuson (and fairly review) the history, sociology and philosophy of science.In his Reflections article on "scientific feuds, polemics, and ad hominemarguments," Elof Carlson makes important points about the integrity ofthe scientific process that, in my view, deserve careful consideration.

6.16. Errol Zeiger on the history of genetic toxicology testing

The last three decades of the 20th century were the heyday of large-scale systematic screening of chemicals for mutagenicity. The testingprogram of the U.S. National Toxicology Program (NTP) at the NationalInstitute of Environmental Health Sciences (NIEHS) was central to thiseffort, and Errol Zeiger was central to the NTP program. His involve-ment in genetic toxicology testing and evaluation preceded the NTPprogram. In his Reflections article [99], he gives a fine historical reviewof this period, combined with personal anecdotes. Errol begins with thecircumstances by which he came to the field that would become genetictoxicology. In 1968, he was looking for part-time laboratory work whilecontinuing his studies toward a Ph.D. By a circuitous route, describedwith some funny elements, he was hired by Marvin Legator at the Foodand Drug Administration (FDA) in 1969 to work on the metabolic ac-tivation of chemicals into mutagens in Salmonella in the host-mediatedassay. He was able to continue his graduate work for his Ph.D. at theFDA. At that time, Marvin was involved in organizational work as partof the group that formed the EMS in 1969, led by Alexander Hollaender.This group was urging the FDA and other agencies to take up the testingof food additives, drugs, pesticides and other chemicals for mutageni-city. Errol worked with the key players in this movement, includingGary Flamm, Heinrich Malling and Fred de Serres. Gary succeededMarvin as FDA Branch Chief, and in 1976 Heinrich and Fred recruitedErrol to NIEHS, where he ran the NIEHS (later NTP) genetic toxicologytesting program. He also came to know Bruce Ames, who was devel-oping the Ames test and sent him the Salmonella strains.

In 1971 Marvin Legator persuaded the FDA to award two contractsto test food additives for mutagenicity in vitro and in vivo, and Errol wasresponsible for test protocols for the host-mediated assay, Salmonellaspot tests (which preceded the Ames assay) and yeast recombinationtests. In the next few years, Ames and colleagues reported on theSalmonella/mammalian-microsome mutagenicity test, and Errol


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Jim had his first experience in molecular cytogenetics at LLNL,leading to his pioneering work on chromosome painting. His text con-tains 7 figures of fluorescent human and mouse chromosomes illus-trating specific advances in the methodology, one photo of fluorescentkinetochores in the CBMN assay, and one of a coffee cup. Why a coffeecup? I'll let you read Jim's Reflections article for the story. Early in itsdevelopment, chromosome painting worked well with human chro-mosome 4, and it permitted the detection of translocations that werenot readily detected with conventional staining. Simultaneouslystaining chromosomes 1, 2 and 4 greatly increased the portion of thegenome covered. By 1998, Jim and his colleagues were simultaneouslypainting chromosomes 1, 2 and 4 in one color and chromosomes 3, 5,and 6 in another. Ultimately, painting probes would be available for theentire genome [86]. The aim now was to use chromosome painting toexplore basic scientific problems and applications.

The development of painting probes for mouse and rat chromo-somes took a high priority, as methods in model organisms would beuseful for comparative studies and controlled exposures. Jim commentson the use of chromosome painting to study chemical mutagens (e.g.,heterocyclic amines in foods), radiation-exposed cleanup workers atChernobyl, and effects of age on spontaneous aberration frequencies.He explores diverse topics where painting has been useful, includingcomplex aberrations, the persistence of translocations in vivo and theirdecline over time, the distribution of breakpoints in chromosomes,clones bearing translocations in vivo, deviations from the theoretical 1:1ratio of translocations to dicentrics, and the use of translocations inradiation dosimetry [86]. The ability to see many more aberrationswith FISH than with conventional staining, including small slivers ofcolor in the unpainted chromosomes, led to controversy about no-menclature for classifying the aberrations. Jim describes a meeting ofnine cytogeneticists in 1993 devising a nomenclature system calledPAINT (Protocol for Aberration Identification and Nomenclature Ter-minology) [88]. What may have appeared to be competitive nomen-clature systems, "PAINT" and "S&S," were resolved by two of theirprincipal proponents. Jim Tucker and John Savage explained the uti-lity, strengths, weaknesses, and somewhat different purposes of Sa-vage's S&S system and the PAINT system in which Jim had a centralrole [89].

6.12. K. Sankaranarayanan and H. Nikjoo on computational modeling ofradiation risks

K. Sankaranarayanan ("Sankar") had long been interested in pro-spects for strengthening radiation risk assessment. After retiring fromLeiden University in 1998, he became a visiting scientist at theKarolinska Institute in Stockholm where he joined Hooshang Nikjoo, aprofessor in the Radiation Biophysics Group, to pursue this subject. Intheir Reflections article [90], Sankar and Hooshang assert that the timehas come for strategies that draw more substantively on genomic in-formation, mechanistic studies on radiation effects, and sophisticatedcomputational modeling. They focus on germ-cell effects, and the paperbegins with a history of radiation protection, supported by a table ofestimates of genetic risks of ionizing radiation and a glossary of tech-nical terminology and abbreviations. They identify uncertainties thatremain despite decades of effort, including questions about the use of adoubling-dose method in risk assessment, insufficient information ongerm-cell radiosensitivity in human females, and the absence of evi-dence on radiation-induced genetic disease.

Sankar and Hooshang review computational modeling related togenomic data, mechanisms of mutagenesis, responses to DNA damage,and repair mechanisms. They point out a critical need for modelingstudies of deletions. To relate recent findings to historical radiationprotection, they briefly review the linear no-threshold (LNT) model thathas been the longstanding default assumption in genetic risk assess-ment. They point out alternative views, but they endorse the continueduse of LNT, as recommended by ICRP and the U.S. National Academy's

most recent BEIR Committee. They also comment on computationalmodeling as it relates to LNT. They conclude that computationalmodeling is a promising approach for resolving important questionsand strengthening risk assessment in the years ahead [90].

6.13. Krishna Dronamraju on J.B.S. Haldane as a scholar and a mentor

We are privileged to have personal reflections on the great geneti-cist, evolutionary biologist, mathematician, statistician and generalscholar J.B.S. Haldane (1892–1964), written by a distinguished ge-neticist and historian of science who knew him personally – KrishnaDronamraju. Dr. Dronamraju was a student when Haldane moved toIndia in 1957. He wrote to Haldane expressing interest in working withhim, and he became Haldane's student at the Indian Statistical Institutein Calcutta. He admired Haldane greatly and received his Ph.D. asHaldane's student in 1964, in the same year that Haldane died. Throughthe years, he has written several books on Haldane's life and work, thefirst in 1968 [91] and the most recent in 2017 [92].

Dr. Dronamraju begins his Reflections article with comments onHaldane's brilliance, his colorful personality and his remarkable scien-tific productivity, including being one of the founders of populationgenetics [93]. He notes Haldane's observations on interspecific crossesin animals that came to be known as "Haldane's Rule" and Haldane'shypothesis about the origin of life dating to 1929. In discussing Hal-dane's life, he includes interesting stories of his childhood in a scientificfamily, his preference for a simple way of living, his adoption of anIndian life style, and his great generosity. His interest in mutation de-veloped early, and it included the first estimate of a mutation rate for ahuman gene – the sex-linked hemophilia gene – in 1932. Haldane ex-plored many topics related to mutation and evolution, including re-lationships between mutation and selection, the evolutionary selectionof mutation rates, genetic load, the impact of mutation, effects of io-nizing radiation, and the relationship between frequencies of geneticpolymorphisms and diseases. Much of evolutionary genetics bearsHaldane's imprint, including relationships among mutation, naturalselection, inbreeding, linkage, population distributions, and environ-mental factors. Dr. Dronamraju ends his reflections with thoughts aboutthe last years of Haldane's life in India, where he became a citizen,guided students toward research on Indian plants and animals, andadopted an Indian perspective on many things, including evolution. It isa fine tribute to the extraordinary person that Dronamraju knew well,and to the geneticist / evolutionary biologist who left an indelible markon our field.

6.14. Narendra Singh on the comet assay

The comet assay has had a large impact on genetic toxicology overseveral decades. It has the advantage of being a simple, direct indicatorof DNA damage that can be measured in diverse tissues and in manydifferent organisms, not restricted to defined genotypes as are manyassays. I thought of Ray Tice, a longtime friend and colleague, as apossible author for Reflections on the assay, given his extensive workwith it. Ray advised me that the person who was the creative forcebehind the assay and should write the article is his colleague NarendraP. Singh. I invited Dr. Singh, and he was receptive. His article covers notonly the development of the comet assay but also gives a view of hisearly life and scientific development in India, followed by his years as ayoung scientist living abroad, and work with the comet assay over threedecades [94].

Singh describes his experience at King George’s Medical College atLucknow, India, with fond memories, having been there as a student,then a graduate student, and finally a faculty member. He gives a senseof the limitations of the times through anecdotes about isolating phy-tohemagglutinin from beans to culture lymphocytes, bringing a pres-sure cooker from home to use as an autoclave, and suffering throughpower outages. He also recalls hours in the library learning about DNA


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acknowledge the editors of Mutation Research Reviews, David DeMariniand Mike Waters, for giving Donald and me autonomy in runningReflections and for giving us their continuing support whenever needed.Finally, I thank the superb scientists and authors who made Reflectionswhat it is.

Conflicts of interest

None.

Acknowledgements

I thank Malcolm Lippert, Mike Shelby, and Errol Zeiger for helpfulcomments on this manuscript. I thank the Elsevier journal managerswho worked with Reflections manuscripts through the years andAssociate Publisher Hélène Hodak for her support in bringing thistwentieth-anniversary collection of Reflections papers to fruition.

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14

oversaw bacterial mutagenicity testing at FDA. An interagency advisorycommittee was formed in 1975 with members from FDA, NIEHS and theNational Cancer Institute (NCI), chaired by Virginia Dunkel of NCI, toestablish a validation program for mutagenicity tests. Contracts wereawarded to perform testing in Salmonella and in mouse lymphomacells, the protocols for which were later refined and adopted for in-ternational test guidelines.

A little-remembered event had a big impact: Bruce Ames and col-leagues reported the mutagenicity of hair dye components. This createdconcern in some and alarm in others, including congressmen, and re-commendations were made at NIH budget hearings for a major muta-genicity testing program. Funding and staffing requests were approvedfor a large program centered at NIEHS. Errol designed and managed theprogram and describes in detail how it evolved and grew, beginningwith Salmonella, chromosome aberrations and sister chromatid ex-changes (SCE) in cultured mammalian cells, and the mouse lymphomaTK mutation assay. Staff members were recruited to the NIEHS, andErrol describes the intricate planning and staffing needed to make themassive effort work. He also gives a sense of the conditions at the time –analyzing, managing, organizing and maintaining the program anddatabase in the days before the computer capabilities that we nowconsider routine. As results were obtained, the new information led tochanges in assays, protocols, and contractors doing the laboratorywork. The data handling evolved to take advantage of newer computertechnology, and the information and test results from the program weremade publicly available and published in Environmental Mutagenesis[99].

Quality control was critical, and it included coded samples, appro-priate positive and negative controls, monitoring of strains, and checkson intra- and inter-laboratory reproducibility and on the frequency ofmislabeling of chemical samples. The NIEHS project officers were notonly administrators but also researchers having first-hand experiencewith the assays and methods. A highly qualified statistician, BarryMargolin, provided the expertise to evaluate statistical methods. Thenumber of chemicals for systematic screening gradually declined in the1990s, and the emphasis shifted to comparative studies, concordancewith carcinogenicity data, and prospects for alternative assays andmethods. Errol retired from NIEHS in 2000 but remains active in ge-netic toxicology and gives a sense of the current program at NIEHS. Heends his reflections with thoughts on the role of chance in determiningthe course of events, and satisfaction that he had the good fortune to be"in the right place at the right time."

6.17. Jeffrey Miller on unexpected interactions between mutagens

Reflections articles have taken us to many disciplines and havespanned the globe. In a new twist, Jeffrey H. Miller's article carries us toa parallel universe [100]. After a distinguished career in bacterial ge-netics and mutagenesis, Jeffrey was nonetheless taken aback by resultsthat emerged from his studies of mutagen-induced mutation spectra.One might have anticipated that combined treatments with differentmutagens would give responses that were additive, or perhaps antag-onistic or synergistic, as interactions of these types are known to occurin toxicology, including mutagenesis. When determining mutationspectra after a combined treatment, one might expect to see molecularmechanisms that reflect the two individual agents, perhaps augmentedor diminished by each other. Jeffrey's point is that some combinationsof treatments with base- and nucleoside-analogs give unique mutationspectra and hotspots of mutagenesis with no obvious relatedness to thespectra of the individual agents, as though the mutagenesis were hap-pening in another universe. He documents his argument with data onmutation spectra induced by the purine analog 2-aminopurine and thecytidine analog zebularine in the rpoB gene of E. coli. He speculates onmechanisms, and he notes that there is now evidence that such inter-actions also occur with compounds other than base analogs. Jeffreydevelops this analogy in an amusing way, referring back to Douglas

Adams's comedy and science fiction series “Hitchhiker’s Guide to theGalaxy.”

6.18. Micheline Kirsch-Volders and colleagues on European collaborativeefforts on aneuploidy

The most recent Reflections article reviews a large European pro-gram on aneuploidy, including both the science and implications forpublic policy [101]. This article differs from its predecessors in being aneffort by 6 authors in 4 countries who reflect on an international col-laborative effort that has had a lasting legacy and influence. MichelineKirsch-Volders (Belgium), Francesca Pacchierotti (Italy), E.M. ("Liz")Parry (UK), Antonella Russo (Italy), Ursula Eichenlaub-Ritter (Ger-many) and Ilse-Dore Adler (Germany) worked intensively on bringingtogether a text that is simultaneously a scientific review, a history, anda reflective commentary fully documented with literature citations,tables and figures. The organizational structure that they describe ranfrom 1981 through 2004. This was a period of remarkable accom-plishment with respect to the understanding of mechanisms underlyinganeuploidy and their implications for human health. Among the topicsdiscussed are historical studies on abnormal chromosome numbers,mechanisms and consequences of aneuploidy in somatic cells and germcells, cellular and molecular targets for aneuploidy induction, the roleof aneuploidy in cancer, relationships to apoptosis and chromothripsis,aneugenic chemicals, assays for aneuploidy, adaptation of micronucleusassays to detect aneuploidy, applications of FISH, environmental in-fluences on aneuploidy, metabolic activation of aneugens, dose-re-sponse relationships, the likelihood of thresholds, data requirements foreffective regulation, and efforts to formulate regulatory guidelines. Asin any area of science, there are still many unresolved questions, but theperspectives of the authors provide valuable suggestions for chartingthe route ahead so as to protect public health.

The authors dedicated their Reflections article to the memory of J.M.("Jim") Parry of Swansea University who inspired and coordinatedmany of the studies discussed and who was a major presence in thegenetic toxicology community. He was a strong advocate for the in-clusion of aneuploidy as an endpoint of concern, along with gene mu-tations and chromosomal aberrations. Like the authors, I remember Jimfor his incisive thinking, productivity, dynamic personality, and senseof humor. I feel confident that he would be complimented to have thisgroup of authors dedicate their efforts to his memory. I would referreaders to a remembrance of Jim in the journal Mutagenesis, of whichhe was the founding editor [102].

7. Final reflections on Reflections

Since its outset in 1999, the Reflections series in Mutation ResearchReviews has been centered on historical themes related to mutation,while simultaneously offering insight into current mutation research. Indoing so, the focus has frequently been on the genetics and molecularbiology that are at the heart of mutation research, but we have viewedmutation broadly, incorporating toxicological, radiological, evolu-tionary, medical, statistical, and public policy aspects of the field. Assuch, there have been interesting extensions into sociology, philosophy,politics, ethics and, of course, the history of science. I was pleased tolearn that the publisher, Elsevier, would like to prepare a compilation ofall the Reflections articles in time for the 50th anniversary of theEnvironmental Mutagenesis and Genomics Society (EMGS), founded in1969 as the Environmental Mutagen Society (EMS). We are also lookingahead to the International Conference on Environmental Mutagens(ICEM) that will take place in Ottawa in 2021.

For me, Reflections has been a pleasure. I have enjoyed a greatpartnership with Donald MacPhee, my coeditor for the first 10 years,and the interaction with a fine group of scientists who have contributedmuch to our field. I am indebted to many excellent "readers" (i.e., re-viewers), who offered a great service to me and to the authors. I also


13


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Mutagenesis 32 (2017) 545–546.[75] A.S. Balajee, F. Palitti, J. Surrallés, M.P. Hande, In Memory of Professor

Adayapalam T Natarajan, Mutat. Res. 836 (Pt A) (2018) 1–2.[76] U. Weyemi, C. Dupuy, The emerging role of ROS-generating NADPH oxidase

NOX4 in DNA-damage responses, Mutat. Res. 751 (2012) 77–81.[77] E.A. Carlson, Genes, Radiation, and Society: The Life and Work of H.J. Muller,

Cornell University Press, Ithaca, 1981.[78] H.J. Muller, The relation of recombination to mutational advance, Mutat. Res. 1

(1964) 2–9.[79] E.A. Carlson, H.J. Muller’s contributions to mutation research, Mutat. Res. 752

(2013) 1–5.[80] E.A. Carlson, How fruit flies came to launch the chromosome theory of heredity,

Mutat. Res. 753 (2013) 1–6.[81] L. Müller, E. Gocke, The rise and fall of photomutagenesis, Mutat. Res. 752 (2013)

67–71.[82] L.B. Russell, The Mouse House: a brief history of the ORNL mouse genetics pro-

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2018, Atomic Heritage Foundation, Oak Ridge, TN, 2018 Accessed Dec. 10, 2018https://www.manhattanprojectvoices.org/oral-histories/liane-russells-interview.

[84] D.M. Shankel, Memories of a friend and mentor – Charlotte Auerbach, Mutat. Res.761 (2014) 1–5.

[85] University of Kansas Alumni Association, Remembering Chancellor Shankel,Alumni News, 2018 July 12 (http://www.kualumni.org/news/remembering-chancellor-shankel/ ).

[86] J.D. Tucker, Reflections on the development and application of FISH wholechromosome painting, Mutat. Res. 763 (2015) 2–14.

[87] M.L. Pardue, J.G. Gall, Chromosomal localization of mouse satellite DNA, Science168 (1970) 1356–1358.

[88] J.D. Tucker, W.F. Morgan, A.A. Awa, M. Bauchinger, D. Blakey, M.N. Cornforth,L.G. Littlefield, A.T. Natarajan, C. Shasserre, PAINT: a proposed nomenclature forstructural aberrations detected by whole chromosome painting, Mutat. Res. 347(#1) (1995) 21–24.

[89] J.R. Savage, J.D. Tucker, Nomenclature systems for FISH-painted chromosomeaberrations, Mutat. Res. 366 (#2) (1996) 153–161.

[90] K. Sankaranarayanan, H. Nikjoo, Genome-based, mechanism-driven computa-tional modeling of genetic risks of ionizing radiation: the next frontier in geneticrisk estimation? Mutat. Res. 764 (2015) 1–15.

[91] K. Dronamraju, Haldane and Modern Biology, The Johns Hopkins University Press,Baltimore, 1968.

[92] K. Dronamraju, Popularizing Science: The Life and Work of JBS Haldane, OxfordUniversity Press, New York, 2017.

[93] K. Dronamraju, J.B.S. Haldane as I knew him, with a brief account of his con-tribution to mutation research, Mutat. Res. 765 (2015) 1–6.

[94] N.P. Singh, The comet assay: reflections on its development, evolution and ap-plications, Mutat. Res. 767 (2016) 23–30.

[95] O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation-induced DNAdamages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123(1984) 291–298.

[96] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quan-titation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988)184–191.

[97] E.A. Carlson, Scientific feuds, polemics, and ad hominem arguments in basic andspecial-interest genetics, Mutat. Res. 771 (2017) 128–133.

[98] E.A. Carlson, The Gene: A Critical History, W.B. Saunders, Philadelphia, 1966.[99] E. Zeiger, Reflections on a career and on the history of genetic toxicity testing in

the National Toxicology Program, Mutat. Res. 773 (2017) 282–292.[100] J.H. Miller, Mutagenesis: interactions with a parallel universe, Mutat. Res. 776

(2018) 78–81.[101] M. Kirsch-Volders, F. Pacchierotti, E.M. Parry, A. Russo, U. Eichenlaub-Ritter, I.D.

Adler. Risks of aneuploidy induction from chemical exposure: twenty years ofcollaborative research in Europe from basic science to regulatory implications,Mutat. Res. 779 (2019) 126–147.

[102] R. Waters, M. Kirsch-Volders, Obituary: James M. Parry (1940–2010),Mutagenesis 26 (#1) (2011) 1–2.


15


( )G.R. Hoffmann, D.G. MacPheerMutation Research 436 1999 123–130124

Table 1Mutation research chronology

1859 Charles Darwin:Origin of Species formulates the evolutionary theory that has become the central paradigm of modern biology and calls for anunderstanding of the genetic basis of biological variation.

1865 Gregor Mendel:Mendel’s discoveries on the fundamental basis for the inheritance of characteristics mark the beginnings of modern genetics.

1869 Friedrich Miescher:Miescher’s isolation of DNA, which he called nuclein, presages the 20th-century characterization of the chemical basis of heredity.

1873 A. Schneider, Otto Butschli, and Hermann Fol:¨Description of the nuclear changes associated with mitosis.

1876 Oskar Hertwig:Observation and description of chromosomes.

1882 Walther Flemming:Clear formulation of the modern concept of mitosis.

1880s Edouard van Beneden, Theodor Boveri, and Oskar Hertwig:Characterization of meiosis.

1885 O. Hertwig, Edouard Strasburger, Rudolf Kolliker, and August Weismann:¨Independently concluded that the chromosomes are the physical basis for inheritance.

1887 August Weismann:Hypothesized how meiosis and fertilization provide for constancy in the amount of genetic material.

1900 Hugo de Vries, Carl Correns, and Erich von Tschermak:Rediscovery of Mendel’s principles.

1901 Hugo de Vries:de Vries’ ‘Mutation Theory’ notes the occurrence of sudden changes in organisms through hereditary mechanisms.

1903 Walter Sutton and Theodor Boveri:Clear formulation of the Chromosome Theory of Inheritance; recognition that the Mendelian units are carried on chromosomesestablished the essential link between cellular processes and transmission genetics.

1905 William Bateson and R.C. Punnett:Demonstration of genetic linkage.

1908 Godfrey Hardy and Wilhelm Weinberg:The foundation of population genetics is formed by the independent studies of Hardy and Weinberg.

1908 Archibald Garrod:Explanation of human genetic disease as inborn errors of metabolism.

1910 Thomas Hunt Morgan:Elucidation of sex linked inheritance in Drosophila, explanation of the chromosomal basis for genetic linkage, and the firstobservation of a newly arisen mutant in the laboratory.

1913 Alfred H. Sturtevant:Exploration of linkage among genes in Drosophila as a basis for chromosome mapping.

1917 Sewall Wright:Explanation of coat color inheritance in mammals explores gene function.

1925 Calvin Bridges:Understanding of mechanisms of sex-determination in Drosophila.

1925 Alfred H. Sturtevant:First mechanistic analysis of a genetic alteration: unequal crossing over in the Bar locus of Drosophila.

1927 Hermann J. Muller:Unequivocal demonstration of the induction of mutations by X-rays in Drosophila.

1930 Ronald A. Fisher:Exposition of theoretical linkages between genetics and evolution.

1931 Harriet Creighton and Barbara McClintock:Conclusive evidence that genetic crossing over in corn involves a physical exchange of chromatids between homologues.

1931 Curt Stern:Association of crossing over and chromatid exchange in Drosophila.

1936 F. Macfarlane Burnet and D. Lush:First study of phage mutations.

Ž .Mutation Research 436 1999 123–130

Series: Reflections in Mutation Research, No. 1

Reflections in mutation research: an introductory essay 1

George R. Hoffmann a,), Donald G. MacPhee b

a Department of Biology, College of the Holy Cross, Worcester, MA 01610, USAb Department of Microbiology, La Trobe UniÕersity, Bundoora, Victoria 3083, Australia

Accepted 25 October 1998

Keywords: Reflections; History of science; Philosophy of science; Mutation research; Genetic toxicology; Mutagenesis

In this article, we announce a new feature thatwill appear regularly in ReÕiews in Mutation Re-search. The feature is to be called Reflections, and itwill be devoted to topics in mutation research viewedfrom a historical, philosophical, or integrative per-spective. The rich history of mutation research lendsitself naturally to such themes. Unlike the geneticcode, there is no AUG codon marking the beginningsof human interest in mutation. The roots of ourinterest in the origins, uses, and transmission ofbiological variation go back through centuries. Nev-ertheless, modern mutation research is almost whollya 20th century science that has experienced an explo-sion of activity in the last few decades. Because itsorigins overlap those of its parent sciences of genet-ics, evolutionary biology, and toxicology, Mendel,Darwin, and Paracelsus mark convenient beginnings.

) Corresponding author. Tel.: q1-508-793-2655; Fax: q1-508-793-3530; E-mail: [email protected]

1 To suggest topics or authors for Reflections, readers shouldcontact either of the authors by mail at the addresses shown above

Ž .or by e-mail: G.R. Hoffmann [email protected] D.G.Ž .MacPhee [email protected] .

In Table 1 we have listed key events as a Muta-tion Research Chronology. We decided to stop thetimeline 20 years ago to avoid the pitfalls of judgingthe importance of recent discoveries. We also apolo-gize for the inevitable omissions, and we acknowl-edge that there would be important differences ifothers were to make the choices. Our chronologydoes, however, highlight the central theme of muta-tion research—a unification of transmission genet-ics, cell biology, biochemistry, population genetics,and toxicology that provides insight into the natureand implications of the mutation process. This themewas actually recognized early in the history of ourfield, as is clearly reflected in the following prophetic

w xquotation from Muller 8 : ‘‘Hence we cannot cate-gorically deny that perhaps we may be able to grindgenes in a mortar and cook them in a beaker after all.Must we geneticists become bacteriologists, physio-logical chemists and physicists, simultaneously withbeing zoologists and botanists? Let us hope so.’’ Ourgoal in the Reflections series is to draw on diverseelements of the mutation research synthesis and, indoing so, to present the personal perspectives ofresearchers who have contributed to the fieldthroughout the second half of this century.

1383-5742r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S1383-5742 98 00026-X


Reflections in mutation research: an introductory essay 1George R. Hoffmann a *, Donald G. MacPhee b

a Department of Biology, College of the Holy Cross, Worcester, MA 01610, USAb Department of Microbiology, La Trobe UniÍersity, Bundoora, Victoria 3083, AustraliaAccepted 25 October 1998



Ž .Table 1 continued

1961 Sydney Brenner, Leslie Barnett, Francis Crick, and Alice Orgel:Characterization of frameshift mutations.

1961 Sydney Brenner, Francois Jacob, and Matthew Meselson:Role of mRNA as an intermediate in protein synthesis.

1961 Francois Jacob and Jacques Monod:Operon model for genetic regulation.

1964 Richard B. Setlow and William L. CarrierDiscovery of excision repair.

1964 Charles Yanofsky:Colinearity of DNA and protein.

1966 Marshall Nirenberg, Philip Leder, and Har Ghobind Khorana:Elucidation of the genetic code.

1966 George Streisinger:The slippage model: a mechanism of frameshift mutagenesis.

1967 Evelyn Witkin:Genetics of DNA repair-deficient mutants in bacteria.

1968 James CleaverDiscovery of a human DNA repair defect in xeroderma pigmentosum.

1969 EMS Founders:The Environmental Mutagen Society is founded by Alexander Hollaender and colleagues to stimulate research on mutagenesis andto address concerns about chemical mutagens.

1970 Hamilton O. Smith and Daniel Nathans:Discovery of restriction enzymes, providing the basis for a revolution in molecular biology.

1971 Bruce Ames:Development of the Ames test, the most widely used of all assays for the detection of chemical mutagens.

1971 Heinrich Malling:Ž .Incorporation of mammalian metabolism as tissue homogenates S9 into microbial assays for mutagenesis.

1970’s Genetic toxicologists:Initiation of the widespread testing of hundreds of chemicals for mutagenicity.

1972 Paul Berg:Artificial construction of recombinant DNA molecules.

1973 Stanley Cohen and Herbert Boyer:Cloning of DNA in a plasmid.

1974 Miroslav Radman:The SOS repair hypothesis, revealing the role in mutagenesis of errors in the processing of DNA damage.

1977 Walter Gilbert and Frederick Sanger:Development of methods of DNA sequencing, making possible the direct molecular characterization of mutations.

Some of the articles will be direct outgrowths ofthe scientific contributions listed in our chronologyŽ .Table 1 . They will not be exclusively historical,however; often they will provide useful insights intocurrent science. There is a tendency in reviewing thehistory of science to see it as a succession of bigevents rather than an accumulation of small contribu-tions. However, instances of ‘‘Eureka!’’ foretelling achange in the world are few and far between, andgradualism is too simplistic a viewpoint. Scientificprogress is perhaps better described as a matter ofpunctuated equilibrium. Even Mendel, whose prede-cessors may seem obscure, worked in the scientificcontext of his time. We hope that Reflections will

give young scientists a sense of the context in whichthe mutation researchers of yesteryear were working,at the same time providing them with a sense of thequestions that were being tackled in the past in waysthat still bear scrutiny.

Ideas on mechanisms of heredity reach back wellbeyond the geneticists’ enlightenment, which almostcertainly dates from the mid-19th century insights ofGregor Mendel. Thus, one can find in such historical

w x w xworks as those of Sturtevant 17 , Stubbe 16 , andw xMoore 7 fascinating accounts of thought on the

determination of biological characteristics extendingfrom prehistoric times to ancient Greece and Rome,through the rejuvenated scientific inquiry and experi-

( )G.R. Hoffmann, D.G. MacPheerMutation Research 436 1999 123–130 125

Ž .Table 1 continued

1937 Milislav Demerec:Discovery of a mutator gene in Drosophila.

1938-41 Karl Sax:Characterization of the induction of chromosome aberrations by X-rays and the cellular consequences of chromosomal damage.

1941 George W. Beadle and Edward L. Tatum:Elucidation of relationships between genes and biochemical pathways in Neurospora; induction and selection of biochemical

Ž .mutants i.e., auxotrophs .1942 Charlotte Auerbach and J.M. Robson:

First unequivocal demonstration of chemical mutagenesis.1943 Salvador E. Luria and Max Delbruck:

Demonstrated that mutant bacteria were present in a population before exposure to the selective agent necessary to reveal them,thus providing evidence that mutational variation is due to random spontaneous mutation rather than to direct environmentalpressure.

1944 Oswald T. Avery:Through studies of bacterial transformation, Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrate that DNA is thehereditary material.

1946 Joshua Lederberg and Edward L. Tatum:Discovery of conjugation in bacteria.

1948 Albert Kelner:Discovery of photoreactivation.

1948 Norman Giles and Esther Lederberg:Use of reversions as an assay to detect induced mutation.

1949 Linus Pauling:The molecular basis of a human genetic disease revealed through studies of sickle cell anemia.

1950 Erwin Chargaff:Characterization of the chemical composition of nucleic acids.

1950 Barbara McClintock:Discovery of genetic instabilities and transposable genetic elements in corn.

1951 Aaron Novick and Leo Szilard:Development and use of the chemostat for studying spontaneous and chemically induced mutation.

1951 William L. Russell:Quantification of germ-cell mutations in mammals.

1952 Norton Zinder and Joshua Lederberg:Transduction in bacteria.

1952 Alfred D. Hershey and Martha Chase:Demonstration that DNA is the hereditary material in phage.

1953 James D. Watson and Francis Crick:Proposal of the B-DNA model for the structure of DNA, which has led to the transformation of modern biology; explorationof the implications of the model for modes of replication and mutation.

1950’s National and International Commissions:ŽRecommendation of standards limiting human exposure to ionizing radiation e.g., United Nations Scientific Committee on the

Effects of Atomic Radiation; International Commission on Radiological Protection; U.S. National Research Council Committee.on Biological Effects of Atomic Radiation; British Medical Research Council .

1955 Seymour Benzer:Fine structure genetic mapping of mutational sites in the rII region of bacteriophage T4 in E. coli; recognition of site specificityof mutation and hotspots within a gene.

1956 Milislav Demerec and Philip E. HartmanFine structure mapping of the genome using transduction.

1958 Matthew Meselson and Franklin Stahl:Semiconservative replication of DNA.

1959 Ernst Freese:Recognition of the transition and transversion classes of base-pair-substitution mutations and postulation of mutationalmechanisms.

1960 Arthur Kornberg:DNA polymerase and its role in DNA synthesis.



and he recognized that such changes could be impor-tant in evolution. Though later work showed that themutants that de Vries observed in evening primrosesactually stemmed from the behavior of pre-existingchromosomal alterations and ploidy differences ratherthan being de novo mutations, the concept of suddenchanges in the units of heredity had been established.

The notion that mutations could be produced byenvironmental agents also arose early. De Vries sug-gested the potential utility of the induction of di-rected mutations, and in 1904 he proposed that X-rays, discovered in the preceding decade, might be

w xable to modify the units of heredity 19 . Interest inthe possibility that chemicals can induce mutationsfollowed shortly, with the first claims of chemicalmutagenesis reported by Franz Wolff and ElizabethSchliemann, two students of Erwin Baur, in 1909

w xand 1912, respectively 19 . The unequivocal demon-stration of the induction of mutations, however, had

w xto await the classic study of Muller 9 , who reportedthe induction of mutations in Drosophila by X-rays.To do so, Muller used a marked chromosome calledClB in an ingenious technique that he devised fordetecting recessive lethal mutations at many loci onthe X chromosome. The method, as well as theobservation of mutagenesis, proved important, as itprovided a rationale for later mutation assays inDrosophila.

Muller’s study marks the beginning of the modernera of mutation research. One year later, Stadlerw x14,15 reported that X-rays are mutagenic in plants,and the mutagenicity of ultraviolet light was demon-

w xstrated a few years later 1 . Interest in the possibilityof chemical mutagenesis persisted, and reports on theinduction of mutations by chemicals appeared in the

w xscientific literature 18 . However, the first indis-putable evidence of chemical mutagenesis was ob-tained in Scotland in 1942 by Auerbach and Robson,who demonstrated the mutagenicity of mustard gasin Drosophila and reported their results a few years

w xlater when wartime censorship was lifted 3,4 . Dur-w xing the war, Oehlkers 10 in Germany had reported

that urethane induces chromosome aberrations, andw xRapoport 11 shortly thereafter reported in the So-

viet Union that ethylene oxide, ethylenimine,epichlorohydrin, diazomethane, diethyl sulfate, gly-cidol, and several other chemicals are mutagenic.Thus, by the end of the 1940s, chemical mutagenesis

was a well-established and growing area of interestin genetics.

Mutagens generated great interest because of theirpotential for revealing the nature of mutation and forobtaining mutants for use in genetic studies, agricul-ture, and industry. Muller suggested in his 1927paper and during the 1930s that mutagenesis insomatic cells could cause cancer, but it was not untilthe late 1950s and early 1960s that the health hazardof mutagenesis was generally recognized. Thus be-gan the component of mutation research that hascome to be called environmental mutagenesis or

w xgenetic toxicology 6,19 . Its objectives and methodsare closely aligned with other aspects of toxicology,in that its focus is the characterization of the adverseeffects of chemicals on biological systems.

Like genetics, toxicology has its origins in antiq-uity and enjoys a colorful history extending throughclassical Greece and Rome, the middle ages, and the

w xRenaissance to modern times 5 . However, its exis-tence as a scientific discipline owes much to Paracel-

w xsus 5 . Philippus Aureolus Theophrastus BombastusŽ .von Hohenheim-Paracelsus 1493–1541 was a

physician–alchemist who developed the ideas thatexperimentation is essential in evaluating responsesto chemicals, that the therapeutic and toxic propertiesof substances are sometimes indistinguishable exceptby dose, and that one can find specificity in the toxiceffects of chemicals. His most notable legacy is theclear articulation of the concept of dose–responserelationships, as reflected in his quotation that ‘‘Allsubstances are poisons; the right dose differentiates a

w xpoison from a remedy’’ 5 . Although their originsare early, the problems of interpreting dose-responserelationships and kinetics remain an area of activestudy and controversy in mutation research.

Occupational hazards of chemical exposure wererecognized in miners and smiths in the 15th and 16thcenturies. The industrial revolution led to many newoccupational exposures and the recognition of occu-pational diseases, a much cited case being PercivalPott’s observation in 1775 that soot causes scrotalcancer in chimney sweeps. Organic chemistry was ayoung science in the 19th century, and its practition-ers generated many new compounds whose toxicitywould soon come to be appreciated. Toxicologyblossomed after World War II in a time of expandedproduction of industrial chemicals, drugs, pesticides,


mentation of the 17th and 18th centuries, to theseminal discoveries of the 19th century. The devel-opment of experimentalism merges with an invigo-rated philosophical approach to science, perhaps most

Ž .closely associated with Francis Bacon 1561–1626 ,and it provides a basis for appreciating the intellec-tual tradition and historical setting in which Darwinand Mendel worked.

Contrary to popular belief, the approaches of clas-sical genetics did not originate de novo in the 19thcentury but were actively pursued earlier, as in thestudies of Pierre Louis Moreau de MaupertuisŽ .1698–1759 , who explored inheritance through ex-perimental crosses in animals and analyzed humanpedigrees quantitatively. Perceptions of biologicalchange, though intermingled with such concepts asthe inheritance of acquired characteristics, were wellrepresented in the work of such creative thinkers as

Ž .Jean Baptiste Lamarck 1744–1829 . These founda-tions provided a general context in which to contem-plate the evolution of life before it was crystallizedso very coherently by Charles Darwin and AlfredWallace.

We sometimes think of the last decades of the19th century as a void with respect to genetics, asMendel’s discoveries on the basis for inheritancewere ‘lost’. There was hardly a void. The CellTheory, a concept clearly articulated in the 1830s byMatthias Jakob Schleiden and Theodor Schwann andextended to encompass the principle that all cellscome from pre-existing cells by Rudolph LudwigVirchow in the 1850s, was ready to be built upon.The understanding of mitosis and meiosis achievedin the last decades of the 19th century was essentialfor the 20th-century linkage between cellular pro-cesses and the transmission of genetic characteristics.Thus, the synthesis linking the transmission of char-acteristics to cellular, molecular, and population as-pects of biology has diverse roots, and its cytologicalelements flourished in the 1870s to 1890s. Our

Ž .chronology Table 1 shows these decades to be atime of foundation-building, leading to the renais-sance of genetics in the early 20th century. DNAmade its entry onto the stage at roughly the sametime as the details of chromosome structure, mitosis,meiosis, and fertilization were being elucidated bynineteenth century biologists. These biologists, inturn, were building upon and renewing the earlier

foundations laid by the biologists and microscopistsof the preceding two centuries, including such lumi-

Ž .naries as Antonie van Leeuwenhoek 1632–1723 ,Ž .Regnier de Graaf 1641–1673 , Lazzaro Spallanzani

Ž . Ž1727–1799 , and Caspar Friedrich Wolff 1733–.1794 .

Perhaps the most fabled event in the history ofgenetics is the independent rediscovery of Mendel’s

Ž .principles by Hugo de Vries 1848–1935 in Hol-Ž .land, Carl Correns 1864–1933 in Germany, and

Ž .Erich von Tschermak-Seysenegg 1871–1962 inAustria. All undertook plant hybridization studies,probably unaware of Mendel’s findings, becameaware of Mendel’s work well into the course of theirstudies, and published clear statements of principles

w xgoverning inheritance in 1900 16 . The new science,which was widely disseminated by William BatesonŽ .1861–1926 and came to be called ‘genetics’ in theterminology that he introduced, was well establishedin the first decade of the 20th century. Most notably,its cellular and population components were soonintegrated into the discipline.

In the first few years after the rediscovery, cytolo-gists, zoologists, and botanists linked the behavior ofchromosomes to inheritance, and the proposal thatthe heredity particles of Mendel are carried on chro-mosomes, which came to be known as the Sutton–Boveri Hypothesis after Walter Sutton and TheodorBoveri, became a foundation of the relationship be-tween cellular processes and the transmission of

w xcharacteristics 12 . Godfrey Hardy and WilhelmWeinberg independently explored relationships be-tween genotypes and allele frequencies, establishingin 1908 the foundation of population genetics that isnow called the Hardy–Weinberg Principle. Thechronology in Table 1 summarizes subsequent eventsin the development of genetics; the integration of itscellular, molecular, and population components; andthe development of the aspect of genetics focusing

w xon mutation 12,13,16,17 .Although mutations were used for many centuries

in animal and plant domestication, and later in scien-tific exploration through hybridization, the modernconcept of mutation has relatively recent origins.Hugo de Vries proposed his ‘Mutation Theory’ in

w x1901 2 . He observed that organisms occasionallyproduce new types of offspring through sudden

Ž .changes mutations in the hereditary mechanism,



Casarett and Doull’s Toxicology: The Basic Science of Poi-sons, 5th edn., McGraw-Hill, New York, 1996, pp. 269–300.

w x7 J.A. Moore, Science as a way of knowing—genetics, Am.Ž .Zool. 26 1986 583–747.

w x8 H.J. Muller, Variation due to change in the individual gene,Ž .Am. Naturalist 56 1922 32–50.

w x9 H.J. Muller, Artificial transmutation of the gene, Science 66Ž .1927 84–87.

w x10 F. Oehlkers, Die Auslosung von Chromosomenmutationsenin der Meiosis durch Einwirkung von Chemikalien, Z. Ind.

Ž .Abst. u. Vererbungsl. 81 1943 313–341.w x11 I.A. Rapoport, Carbonyl compounds and the chemical mech-

Ž .anism of mutations, C.R. Dokl. Acad. Sci. URSS, NS 54Ž .1946 65–67.

w x12 J.A. Peters, Classic Papers in Genetics, Prentice-Hall, Engle-wood Cliffs, NJ, 1959.

w x13 P.J. Russell, Genetics, 5th edn., BenjaminrCummings Publ.,Menlo Park, CA, 1998, pp. 10–11.

w x14 L.J. Stadler, Genetic effects of X-rays in maize, Proc. Natl.Ž .Acad. Sci. U.S.A. 14 1928 69–75.

w x15 L.J. Stadler, Mutations in barley induced by X-rays andŽ .radium, Science 68 1928 186–187.

w x16 H. Stubbe, History of Genetics. Massachusetts Institute ofTechnology Press, Cambridge, MA and London, England,

Ž1972 Originally published by VEB Gustav Fischer Verlag,Jena, Germany, as Kurze Geschichte der Genetik bis zur

.Wiederentdeckung der Vererbungsregeln Gregor Mendels .w x17 A.H. Sturtevant, A History of Genetics, Harper and Row,

New York, 1965.w x18 C. Thom, R.A. Steinberg, The chemical induction of genetic

Ž .changes in fungi, Proc. Natl. Acad. Sci. U.S.A. 25 1939329–335.

w x19 J.S. Wassom, Origins of genetic toxicology and the Environ-Ž .mental Mutagen Society, Environ. Mol. Mutagen. 14 1989

1–6, Suppl. 16.


and munitions. It is here that we see the merging ofmutagenesis and toxicology. Safety concerns and thedevelopment of health, occupational, and environ-mental regulations have been a motivation for con-tinuing development both in general toxicology andin genetic toxicology, and they continue to be instru-mental in applied aspects of our field.

Besides Muller, other biologists voiced early con-cerns about chemical mutagenesis, including AlfredBarthelmess, Charlotte Auerbach, Joshua Lederberg,Avram Goldstein, Frits Sobels, and James F. Croww x19 . Though worry about carcinogenesis had beenraised earlier by Muller, the focus now includedgerm-cell mutagenesis and genetic disease. The ideathat mutagenicity should be included in the toxico-logic evaluation of chemicals became widespread inthis period. Alexander Hollaender took the initiativeto mobilize efforts in the new field of environmentalmutagenesis in the 1960s, and his efforts, along withthose of Frederick de Serres, Heinrich Malling, Mar-vin Legator, Ernst Freese, Samuel Epstein, MatthewMeselson, and others, led to the founding of the

Ž . w xEnvironmental Mutagen Society EMS in 1969 19 .The EMS and related organizations now includethousands of geneticists and toxicologists involved inresearch on mutation or in public policy related to

w xmutagenesis 6 .w xAs suggested by Auerbach 2 , the history of

mutation research can be viewed as consisting of thefollowing phases:

Ž .1 1900–1927: Origin of the concepts of muta-tion and mutation rates, formulation of basic ques-tions about the nature of mutation, and the develop-ment of methods for measuring mutation.

Ž .2 1927–f1940: Development of a refined con-cept of the nature of mutation and target theory fromthe discovery of X-ray mutagenesis.

Ž .3 1941–1953: Growing knowledge of chemicalmutagenesis and the development of new methods ofmutational analysis through the use of microorgan-isms.

Ž .4 . 1953–f1965: Discovery of the structure ofDNA, integration of new knowledge of biochemistryinto the mutation concept, and growing awareness ofthe importance of cellular processes in mutagenesis.

Ž .5 f1965–present: Application of diverse phys-ical and chemical means to expand the molecularcharacterization of mutation, to acquire greater un-

derstanding of mutation as a biological process, andto reveal its diverse implications.

For obvious reasons, most contributions to Re-flections in Mutation Research will focus on theactivities and thinking of participants in the last ofthese broadly defined phases, but this will usually bepossible only through the lens of the first four peri-ods. Thus, Reflections articles are intended to offerinsight into fundamental questions in modern muta-tion research where a historical, philosophical, orintegrative viewpoint can shed light on current sci-ence. Reflections will strive to cover mutation asbroadly as possible, including its evolutionary, toxi-cological, medical, statistical, and public policy di-mensions, as well as the basic genetics and molecu-lar biology that form the core of mutation research.The series will begin in the next issue of ReÕiews inMutation Research with an article by James F. Crowon the origins and implications of human sponta-neous mutation. Articles on a broad array of topics inmutation research will then follow at regular inter-vals. We welcome an active dialogue with the read-ership and encourage comments, discussion, andsuggestions of topics and authors for Reflections.

Acknowledgements

We thank Herman Brockman, William R. Healy,and John S. Wassom for helpful suggestions on thiscommentary.

References

w x1 E. Altenburg, The artificial production of mutations by ultra-Ž .violet light, Am. Naturalist 68 1934 491–507.

w x2 C. Auerbach, 1976. Mutation Research: Problems, Results,and Perspectives. Chapman & Hall, London.

w x3 C. Auerbach, J.M. Robson, Chemical production of muta-Ž .tions, Nature 157 1946 302.

w x4 C. Auerbach, J.M. Robson, The production of mutations byŽ . Ž .chemical substances, Proc. R. Soc. Edinburgh B 62 1947

271–283.w x5 M.A. Gallo, History and Scope of Toxicology, in: C.D.

Ž .Klaassen Ed. , Casarett and Doull’s Toxicology: The BasicScience of Poisons, 5th edn., McGraw-Hill, New York, 1996,pp. 3–11.

w x Ž .6 G.R. Hoffmann, Genetic Toxicology, in: C.D. Klaassen Ed. ,


Ž .Mutation Research 437 1999 5–9www.elsevier.comrlocaterreviewsmr

Community address: www.elsevier.comrlocatermutres


Spontaneous mutation in man 1

James F. Crow )

Genetics Department, UniÕersity of Wisconsin, 1400 UniÕersity AÕenue, Madison, WI 53706-1599, USA

Received 28 April 1998; accepted 11 May 1998

I realize that in some circles using the word‘‘man’’ generically is no longer considered proper.So, to stake out my claim to political correctness, Iquickly assert my belief that human spontaneousmutation is disproportionately a male problem andmore specifically one of older males. Much of what Isay here was suspected long ago, but recent evidenceis far more convincing.

The first to recognize the possibility of an ageeffect on mutation was Wilhelm Weinberg, the greatidea man of early human genetics. As early as 1912,he noted that children with achondroplasia whoseparents were normal tended to be born late in thesibship. He wrote that if this is confirmed it wouldsuggest a mutational origin, a remarkably insightful

w xobservation at that time in the history of genetics 1 .Weinberg is a hero of mine. He is best known,

along with Hardy, for the Hardy–Weinberg rule.But, although it plays a central role in populationgenetics, this simple application of the binomialtheorem could hardly tax either of their intellects.Hardy was one of Britain’s greatest mathematicians,capable of plumbing the deepest recesses of numbertheory. Weinberg, even before he knew of Mendel’swork, suggested that fraternal twins come from two

) Tel.: q1-608-233-6709; fax: q1-608-262-2976; E-mail:[email protected]

1 To suggest topics or authors for Reflections, readers shouldcontact either of the authors by mail at the addresses shown above

Ž .or by e-mail: G.R. Hoffman [email protected] D.G.Ž .MacPhee [email protected] .

eggs and identical from one, and computed the pro-portion of the two types from sex ratio data. Hedeveloped the ‘‘proband’’ and ‘‘a priori’’ methodsfor correcting ascertainment bias in Mendelian ratios.He offered explanations for the greater severity ofinherited diseases in children than in their parents,and of ‘‘anticipation’’. And, he invented variousclever ways to deal with mortality and epidemiologi-cal statistics. Altogether, he did more than anyoneelse in the period before World War I to devisemethods for studying genetics in that peculiarly in-tractable species, Homo sapiens. Moreover, he didall this in addition to being a busy and sociallyconscious physician, who practised medicine for 42

w xyears and delivered more than 3500 births 2 . Whatwas perhaps his deepest work was for a long timealmost entirely unrecognized. He derived correla-tions and covariances between relatives, thereby an-ticipating by several years the work of Fisher andWright, and actually, by considering both domi-nance and environment, doing something neither of

w w x xthem did Ref. 3 , pp. 11–15 . Weinberg usuallyworked alone and got little attention even in his ownland. And, since he wrote in German and his mathe-matical methods were abstruse, he was overlookedby English-speaking geneticists, even though hispublications were in influential international jour-nals. Like Mendel, his discoveries were made at atime when his contemporaries were unable to appre-

w xciate them 2 .Three decades elapsed before his mutation hy-

w xpothesis was confirmed. In 1941, Mørch 4 showed,

1383-5742r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S1383-5742 99 00063-0


Spontaneous mutation in man 1James F. Crow *

Genetics Department, University of Wisconsin, 1400 University Avenue, Madison, WI 53706-1599, USAReceived 28 April 1998; accepted 11 May 1998

( )J.F. CrowrMutation Research 437 1999 5–96

also for achondroplasia, that the increased incidencewas due to parental age rather than birth order. And

w xas late as 1955, Penrose 5 noted the greater numberof cell divisions between zygote and sperm thanbetween zygote and egg and argued that mutationwas associated with cell division. This would alsoaccount for the increased incidence of new mutations

w xwith paternal age. Earlier, Haldane 6 had calculatedfrom hemophilia pedigrees that the male mutationrate per generation is an order of magnitude higherthan that of females. Haldane’s methods were indi-rect and the data uncertain, but he managed to reachthe right conclusion.

We can compute the expected increase in paternalage from knowledge of the number of cell divisionsprior to the formation of a sperm. Estimates are

w w x xgiven by Vogel and Motulsky Ref. 7 , pp. 402–407 .These are about 30 cell divisions from zygote topuberty, 23 per year thereafter, and six for prolifera-tion and meiosis. Letting x stand for age and x forpage at puberty, the expected number of germ-linecell divisions at age x is thus:

N s30q23 xyx q6s36q23 xyx .Ž . Ž .x p p

Letting x s15, this gives 36 divisions at age 15,p151 at age 20, 381 at age 30, 611 at age 40, and 841at age 50. For comparison, the corresponding num-

Ž w xber for females is 24. In earlier publications 8–10 ,I took the age of puberty as 13, the value assumed by

w xVogel and Rathenberg 11 . But the value, 15, fromw xthe latest edition of Vogel and Motulsky 7 seems

preferable. This accounts for the somewhat different.numbers in my earlier papers. Similar formulae are

w xgiven by Drost and Lee 12 .Although the Vogel–Rathenberg formula predicts

a large increase in mutation rate with age, the actualincrease is still greater. The expected increase in theage of fathers of newborns with de novo dominant

Žmutations is about 3 years. For an explanation, seew x .Ref. 8 , but use 15 instead of 13 for age of puberty.

The observed age of fathers of affected children wasabout 6 years older than those of unaffected children,so the linear hypothesis is clearly inadequate. Directobservation of graphs plotting relative incidence atdifferent ages for achondroplasia and Apert’s syn-drome shows an accelerating rather than a linear

w xincrease with age 10 . The graphs suggest that evena quadratic relationship is insufficient and a cubic

relationship fits better, although the accuracy of thedata does not merit detailed curve-fitting.

That the mutation rate per cell division increaseswith age comes as no surprise. Almost everythinggets worse with age, and I would expect the fidelityof transcription, the accuracy of editing, and theefficiency of various repair mechanisms also to dete-riorate along with ability to play tennis. There isroom for considerable uncertainty in the estimates ofthe number of cell divisions, with the complicationsof stem-cell divisions and possible turnover in thegerminal epithelium. But, despite these uncertaintiesof detail, the monotone increase of cell divisionswith age and the nonlinear increase of mutationfrequency with age are clear. The observations onwhich the estimates were made are quite old andthere is room for additional research with the im-proved techniques now available.

Since there are so many more cell divisions in themale germ line than in that of a female, a differencein the mutation rate per generation in the two sexesis expected. As I mentioned earlier, this was argued

w xby Haldane 6 from hemophilia data. Results forw xX-linked Lesch–Nyhan syndrome are similar 10 . In

classical genetics, it was not possible to determinethe parental origin of a new mutation, so all theinferences were indirect. That has changed radicallywith molecular techniques, which often make such adetermination possible by observing linked markers.

Earlier I reported three conditions, multiple en-Ž .docrine neoplasia Type B MEN2B , Type A

Ž . ŽMEN2A , and acrocephalosyndactyly Apert’s Syn-.drome in which the parental origin of a number of

w xde novo mutations was determined 10 . There were,respectively, 25, 10, and 57 new mutations for a totalof 92, and all were paternal. Weinberg’s classicalcondition, achondroplasia, is now understood inmolecular terms. The mutations are in the fibroblast

Ž .growth factor receptor 3 FGFR3 and all involvechanges of a specific glycine to arginine. My earlier

w x w xreview 10 included 16 cases. Szabo et al. 13 haveadded 37 more, for a total of 53. Again, all werepaternal. This makes a total of 145 paternally derivedmutations and no maternal, too many for the celldivision hypothesis?

There are exceptions to this pattern, however.Two conspicuous ones are Duchenne muscular dys-trophy and neurofibromatosis. Neither shows a sig-


( )J.F. CrowrMutation Research 437 1999 5–98

w xgren and Fridolfsson 19 studied several bird speciesand found a lower rate for the W chromosome,which is female-limited in birds. The estimatedmalerfemale mutation ratio is 3.9 for synonymoussites and 6.5 for introns. So birds have come to therescue of the hypothesis of predominantly male-driven neutral evolution.

Two other items provide additional evidence for acell division-dependent mutation rate. Research in

w xJapan 20 in connection with study of survivors ofŽthe atomic bombs have shown among those not

.receiving radiation a substantial age-effect in thefrequency of somatic mutations of the glycophorin A

Ž .gene the MN blood group locus . The proportion ofmutant cells increased at about 3% per year in thecombined data from both sexes. Similar evidencecomes from long-lived mangrove trees. Klekowski

w xand Godfrey 21 found 25 times as many chloro-phyll-deficient mutants as in annual plants. Studiesof the cell-cycle kinetics and the age-dependence ofsuch things as DNA repair mechanisms could add agreat deal to our understanding of this subject.

Returning to achondroplasia and Apert’s syn-drome, we find that in achondroplasia all the muta-

w xtions were at a CpG dinucleotide 13 . They wereeither TAC GGG™TAC AGG or TAC GGG™

TAC CGG; in either case, the change was fromglycine to arginine. The results for Apert syndrome

w xwere similar 22 . Mutations were either TCG™

TGG or CCT™CGT. The first, the more common,is at a CpG dinucleotide, but the second is not. Themutation rate at the CpG hot spot is about twice ashigh as the other. Yet both mutations are C™Gtransversions. Taylor’s analysis of mouse mutationsshowed that almost half of the point mutations wereat CpG sites.

The studies of achondroplasia and Apert’s syn-drome between them included 110 new mutations,all paternally derived. The fathers probably averagedabout 36 years of age. On the formula given earlier,there would be 519 divisions compared to 24 in thefemale, a ratio of 21.6. Finding no female mutationswhen 110r21.6s5.1 are expected would happen bychance about six times per thousand. But the rela-tionship is not linear, as I have emphasized earlier.Methylation is more extensive in sperm than in eggs,and may be in pre-meiotic male germ cells. It will beinteresting to find if CpG-mediated mutation is a

male phenomenon and whether CCT is a male-specific warm spot. In any case, whether at CpGsites or elsewhere, point mutations show a largepaternal age effect and a much greater rate in malesthan in females. It should be possible soon, if it isnot already, to detect point mutations directly insperm.

How could such a large age-dependency comeabout. There must surely be selection to keep themutation rate in bounds. But, I suspect that amongour hunter–gatherer ancestors males hardly ever livedlong enough to reach the sharply ascending part ofthe mutation–paternal age curve. A high mutationrate may be a price we pay for living in an environ-ment in which reproduction is possible at higher andhigher ages.

Their high mutation rate suggests that a majormutational risk, perhaps the major mutational risk,comes from fertile old males. This is likely to beparticularly true for mutations with very minor ef-fects that are more likely to be base substitutions.There is, of course, a solution: collect sperm atpuberty and freeze until needed. Needless to say, Iam not suggesting that such a socially disruptivepolicy be implemented. For one thing, I may beover-generalizing from a small number of diseases.But the data make one wonder whether spontaneousmutations in males might not be a larger factor thanenvironmental chemicals in creating a mutation load.

I, for one, would be content to have a mutationrate of zero, if this could be achieved. We probablywould not notice it for centuries, except for theabsence of some highly undesirable dominant muta-tions. We certainly have enough genetic variabilityto satisfy the most wild-eyed eugenicist; you haveonly to look around. And if we ever need mutations,we certainly know how to produce them. But this isutopian, so let me consider a real problem.

We live in a time of continually improving livingconditions. Traits that would have been weeded outby selection in the past are being preserved. This canonly mean a greater rate of mutation accumulation.We do not notice any ill effects of this because ofthe improved living environment. But can we keepimproving living conditions forever, or will there bea day of reckoning and we find ourselves devotingan increasing fraction of our resources to taking careof each other’s genetic defects?

( )J.F. CrowrMutation Research 437 1999 5–9 7

Žnificant sex difference or a striking paternal or inthe cases of X-linked muscular dystrophy, grandpa-

.ternal age effect. The data on neurofibromatosis areextensive, and there is a slight paternal age increase,but nothing like the large increases found for achon-droplasia and Apert’s syndrome.

The explanation seems to lie in the nature of themutations. Both Duchenne dystrophy and neurofibro-matosis are caused by very large genes with manyexons, and deletions or duplications of a few hun-dred or thousand bases are less likely to be lethalthan if they occurred in a smaller gene. Grimm et al.w x14 reported on 198 mutations to muscular dystro-phy in which the parental origin could be deter-mined. Of these, 114 were deletions, eight wereduplications, and 76 were not detectable by deletionscreening — presumably point mutations. The greatmajority of the latter were paternal, as expected, butmore than half of the duplications and deletions werematernal. Although the maternalrpaternal ratio isnot significantly different from 1:1, the data at facevalue suggest that the rate of deletions may be higherin females. In any case, there is nothing like thepaternal excess found for base substitutions. The data

w xfor neurofibromatosis 15 are similar. Of 11 pointmutations, nine were paternal; of 21 deletions, 16were maternal.

The hypothesis, then, is that point mutations oc-cur much more often in males and there is a largepaternal-age effect. These are presumably producedin connection with chromosome replication. In con-trast, deletions and duplications do not show an ageeffect and the rate, if it is indeed different in thesexes, is greater in females. Perhaps the event canhappen only once in the life cycle, not at every celldivision. Possibly the pachytene arrest in oogenesismay be favorable for chromosome breakage. Thepresence of both point mutations and deletions ex-plains the weak paternal age-dependence for neurofi-

w x w xbromatosis. For a graph, see Ref. 10 or Ref. 16 .w xRisch et al. 16 did a thorough review of paternal

age studies and presented several graphs showing theage distribution for various conditions. They classi-fied 12 traits as having a large paternal age effect

Žand five with little or no effect. The 12 syndromeswith large paternal age effect were acrodysostosis,achondroplasia, ossificans progressive, Marfan,oculo-dental-digital, Pfeiffer, progeria and Waarden-

burg; the other five were multiple exostoses, neurofi-bromatosis, retinoblastoma, Sotos, and Treacher-Col-

.lins. I would suggest that the latter group include asubstantial component of deletions and duplications.In fact I anticipate that with more data we shall finda continuous range of paternal age effects rangingfrom very little with mainly deletions to a very largeeffect with a preponderance of point mutations. Atpresent, the latter group appear to be the majority.

The human data are too sparse for any securegeneralization at this stage, but we can obtain someguidance from mouse studies. A large number ofmorphological and behavioral mutants have beenidentified over the years and Ben Taylor has pro-

Ž .vided an analysis personal communication . A roughclassification of the lesions involved, based on a totalof about 150 mutants, revealed that some 2r3 arepoint mutations, about 1r5 deletions, with the re-mainder being mainly due to retroposons along witha few duplications. If we are like mice in this regard,we can expect a large fraction of human mutations tobe predominantly paternal and paternal-age depen-dent.

Studies of molecular evolution provide indepen-dent evidence of a greater rate in males than infemales for base-substitution mutations. Neutral sub-stitutions are driven by mutation and random drift, sothe long-time evolution rate should be proportionalto the mutation rate. This has provided a rationale forthe popular molecular clock assumption. Severalstudies have suggested that Y-chromosome DNAevolves faster than X-chromosome DNA. This isexpected since a Y chromosome is found only inmales, whereas an X chromosome is carried twice asoften in females as in males. The most careful andextensive study leads to an estimate of 6 for the ratio

w w x xof the male to female rate Ref. 17 , pp. 225–228 .The ratio is much less than that for base substitutionsin the human population, but remember that thecurrent human life span is much longer than that ofour remote ancestors and primate relatives.

A revisionist interpretation of the low rate ofX-chromosome evolution was promulgated by

w xMcVean and Hurst 18 . They argued that the X-chromosome has an intrinsically lower mutation rate,and invoked a standard selectionist argument. Thecrucial test is provided by birds, where the heteroga-metic sex is reversed compared to mammals. Elle-





Frameshift mutation, microsatellites and mismatch repair 1

Bernard S. Strauss )

Department of Molecular Genetics and Cell Biology, UniÕersity of Chicago, 920 East 58th Street, Chicago, IL 60637 USA

Accepted 18 August 1998

Abstract

The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to themaintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cellmust be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognizeframeshifts or which hold the DNA in place during replication is likely to result in genomic instability. q 1999 ElsevierScience B.V. All rights reserved.

Keywords: Frameshift mutation; Mismatch repair; Homopolymer runs; DNA polymerase; Genetic stability

1. Introduction

Frameshift mutations are base additions or dele-tions within the coding region of a gene disturbingthe reading frame so that the entire set of tripletsdownstream of the addition or deletion is altered. Inmany cases, the addition or deletion results in theexposure of in-frame termination sequences whichtruncate the product. Frameshift mutations are there-fore likely to result in more severe phenotypic ef-fects than do many of the base changes which resultin either silent or conservative changes in proteinproducts. Since there is nothing at the nucleic acidlevel to distinguish frameshifts within coding regions

) Tel.: q1-773-702-1628; fax: q1-773-702-3172; E-mail:[email protected]

1 This article is part of the Reflections in Mutation Researchseries. To suggest topics and authors for Reflections, readers

Žshould contact the series editors, G.R. Hoffmann ghoffmann. Ž [email protected] or D.G. MacPhee [email protected] .

from other base additions or deletions it is conve-Ž .nient to refer to all simple one or two base addi-

tions or deletions as ‘‘frameshifts’’. 2

2. Base substitutions and frameshifts

The recognition that there is a class of mutationcharacterized by the addition or loss of one or a fewnucleotides depends on the demonstration that muta-

Ž .tions can be and often are the result of singlenucleotide changes within the DNA. The evidencethat this is so derives in large part from the experi-

w xments of Benzer and Freese 1 on the rII locus ofbacteriophage T4. Benzer’s experiments are cited in

2 As pointed out by Dr. Gordenin, this convenience can beillusory when dealing with triplet repeats in which addition ordeletion of a repeating unit occur with maintenance of the readingframe.


( )J.F. CrowrMutation Research 437 1999 5–9 9

How great is the genetic risk and how soon will itbecome manifest? I do not know of course, exceptthat the time scale is considerably longer than wecustomarily take into account in societal decisions.What I do know, however, is that there are otherproblems with a much shorter waiting period. Unlessthe world-wide birth rate can be brought into somekind of balance with our economic constrictions anddwindling resources, we will not have the luxury ofworrying about the mutation rate.

Acknowledgements

I should like to thank Bill Dove for a carefulreading of the manuscript and many helpful sugges-tions and Ben Taylor for permission to use some ofhis unpublished mouse data.

References

w x1 W. Weinberg, Sur Vererbung des Zwergwuchses, Arch. Rass.Ž .Ges. Biol. 9 1912 710–718.

w x Ž .2 C. Stern, Wilhelm Weinberg, 1862–1937, Genetics 47 19621–6.

w x3 W.G. Hill, Quantitative Genetics: Part 1. Explanation andAnalysis of Continuous Variation, Van Nostrand Reinhold,New York, 1984. This volume includes a translation byKarin Meyer of the first part of Weinberg’s article, WeitereBeitrage zur Theorie de Vererbung, Arch. Rass. Ges. Biol., 7¨Ž .1910 35–49.

w x4 E.T. Mørch, Chondrodystrophic dwarfs in Denmark,Munkesgaard, Copenhagen, 1941.

w x Ž .5 L.S. Penrose, Parental age and mutation, Lancet 2 1955312.

w x6 J.B.S. Haldane, The mutation rate of the gene for hemophilia,and its segregation ratios in males and females, Ann. Eugen.

Ž .13 1947 262–271.w x7 F. Vogel, A.G. Motulsky, Human Genetics; Problems and

Approaches, Springer-Verlag, Berlin, 1997.

w x8 J.F. Crow, How much do we know about spontaneous humanŽ .mutation rates?, Environ. Mol. Mutagen. 21 1993 122–129.

w x9 J.F. Crow, Spontaneous mutation as a risk factor, Exp. Clin.Ž .Immunogenet. 12 1995 121–128.

w x10 J.F. Crow, The high spontaneous mutation rate: is it a healthŽ .risk?, Proc. Natl. Acad. Sci. U.S.A. 94 1997 8380–8386.

w x11 F. Vogel, R. Rathenberg, Spontaneous mutation in man, Adv.Ž .Hum. Genet. 5 1975 223–318.

w x12 J.B. Drost, W.R. Lee, Biological basis of germline mutation,Ž .Environ. Mol. Mutagen. 25 1995 48–64, Suppl. 26.

w x13 J.K. Szabo, D.J. Wilkin, R. Cameron, S. Henderson, G.Bellus, I. Kaitila, J. Loughlin, A. Munnich, B. Sykes, J.Bonaventure, C.A. Francomano, The achondroplasia muta-tion occurs exclusively n the paternally derived fibroblast

Ž .growth receptor 3 FGFR3 allele, Am. J. Hum. Genet. 61Ž .1997 A348.

w x14 T. Grimm, G. Meng, S. Liechti-Gallati, T. Ettecken, C.R.Muller, B. Muller, On the origin of deletions and point¨ ¨mutations in Duchenne muscular dystrophy: most deletionsarise in oogenesis and most point mutations result from

Ž .events in spermatogenesis, J. Med. Genet. 31 1994 183–186.

w x15 C. Lazaro, A. Gaona, P. Ainsworth, R. Tenconi, D. Vidaud,H. Kruyer, E. Ars, V. Folpini, X. Estivill, Sex differences inthe mutational rate and mutational mechanism in the NFgene in neurofibromatosis type 1 patients, Hum. Genet. 98Ž .1996 696–699.

w x16 N. Risch, E.W. Reich, M.M. Wishnick, J.G. McCarthy,Spontaneous mutation and parental age in humans, Am. J.

Ž .Hum. Genet. 41 1987 218–248.w x17 W.-H. Li, Molecular Evolution, Sinauer Associates, Sunder-

land, MA, 1997.w x18 G.T. McVean, L.D. Hurst, Evidence for a selectively

favourable reduction in the mutation rate of the X-chro-Ž .mosome, Nature 386 1997 388–392.

w x19 H. Ellegren, A.-K. Fridolfsson, Male-driven evolution ofŽ .DNA sequences in birds, Nat. Genet. 17 1997 182–184.

w x20 M. Akiyama, S. Kyoizumi, Y. Hirai, Y. Kusunoki, K.S.Iwamoto, N. Nakamura, Mutation frequency in human blood

Ž .cells increases with age, Mutat. Res. 338 1995 141–149.w x21 E.J. Klekowski, P.J. Godfrey, Ageing and mutation in plants,

Ž .Nature 340 1989 389–391.w x22 D.M. Moloney, S.F. Slaney, M. Oldridge, S.A. Wall, P.

Sahlin, G. Stenman, A.O.M. Wilkie, Exclusive paternal ori-gin of new mutations in Apert syndrome, Nat. Genet. 13Ž .1996 48–53.


Frameshift mutation, microsatellites and mismatch repair 1Bernard S. Strauss *

Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637 USAAccepted 18 August 1998


( )B.S. StraussrMutation Research 437 1999 195–203 197

and that diploidy for the gene to be mutated wasnecessary. However, at about the same time it was

Žshown that a group of acridine derivatives the ICR.compounds that covalently bound to DNA were

highly efficient frameshift mutagens for Escherichiaw xcoli 9 . In addition it was observed that frameshift

mutation induced in E. coli by ICR-191 or 5-Žaminoacridine occurred at the time of replication as

.determined with synchronized cultures , was in-creased in nucleotide excision repair defective cul-tures and was not decreased in recAy and recBy

w x w x Žstrains 10 . Newton et al. 10 concluded that in-.duced frameshift mutation was somehow associated

with replication. In fact, this is where we are today.Given the right conditions, susceptibility to frameshiftmutagens is found in all organisms and recombina-tion is not required. There is likely more than one

w xmechanism of frameshift mutation 11 but all areassociated with replication at some stage. Not allmechanisms need be associated with slippage, evenat repeated sequences. Some of the sites foracridine-induced mutation of T4 bacteriophage cor-respond to the sites at which topoisomerase breaks

w xDNA in the presence of acridine 12 , and althoughthe site is repetitive, slippage does not seem to be

w xinvolved 13 . Slippage does occur in the replicationof DNA with repeated elements by polymerases invitro but such experiments need not invariably re-

w xflect the in vivo situation 11 .The Streisinger model does focus on repeated

nucleotide sequences in DNA. It had been recog-nized from the earliest studies on E. coli DNApolymerase I that large oligonucleotide polymerscould be synthesized without template after a longlag period. It was presumed that this synthesis wasbased on polynucleotide contaminants in the enzyme

w xpreparations that could serve as templates 14 . Addedtemplate-primers as small as six base pairs could

w xgenerate large polymers by what Kornberg 15 calledreiterative replication. A ‘‘scheme of reiteration andslippage’’ was provided to account for these resultswhich require the 5X™3X exonuclease activity of theenzyme. These early observations have been re-peated and their possible biological role in the syn-thesis of repeated elements has been commented

w xupon 16 . At the time the early studies were done,the only repetitive sequence known in nature was the

Ž .poly-d A–T which makes up as much as a fourth of

the total DNA in some species of crab and sedimentsin a CsCl as a satellite band separable from the main

Ž w x.component of higher density see Ref. 15 . It wastherefore not appreciated by biologists that theseexperiments illustrated an important property of thepolymerases: that without associated protein theywere permissive in allowing their substrates to slip.An estimate of the amount of slippage that occurs ineach replication event in organisms can be obtainedusing mutants that are unable to repair slippage

w xmistakes 17 . In E. coli, a mutant with no mismatchrepair and with minimal proofreading makes aboutone frameshift per 100 cycles when replicating a run

Ž . w xof 14 CA s 18 . The rate is proportional to thelength of the sequence being replicated in an experi-mental system in which repeats are inserted into the

w xgenome of single stranded M13 bacteriophage 19 .w x w xIn yeast 20 and in phage T4 21 , there is an

exponential dependence on the length of the run.

3. The nature of eukaryotic DNA

By the early 1970s our understanding of molecu-lar genetics had become fairly sophisticated and therelationships between DNA, RNA and the protein

w x 7products seemed clear 22,23 . The bacterial sys-tems were viewed as good models for what went onin eukaryotes. A major premise of molecular genet-ics as it was understood then was that there shouldbe an absolute collinearity between the nucleotidesequence of a gene and the amino acids of the

w xprotein product 24 . This premise was coupled withthe implicit assumption that since the genetic mate-rial is DNA, all DNA carries genetic information.There was the ‘‘scandal’’ of the very large amountof DNA contained in the nucleus of some amphib-ians but this DNA was considered a special case of‘‘junk’’ DNA, left over from evolutionary experi-

w xments 25 . A gradual shift from bacteria to eukary-otes as experimental organisms coincided with agrowing understanding of the complexity of eukary-otic DNAs. In 1977, the astounding discovery wasmade that large chunks of DNA were interspersed

7 Crick called this relationship the ‘‘Central Dogma’’ of molec-ular biology.

( )B.S. StraussrMutation Research 437 1999 195–203196

standard textbooks of genetics as defining the natureof the gene. His work showed that recombinationcould separate mutations which his calculations im-plied were separated by only single nucleotides. Heand his colleagues also used a variety of chemicalmutagens to show that the sites of mutation inducedby chemicals differed from the spontaneous locationand that certain regions of the genome were more

w xmutable than others, the so-called ‘‘hot spots’’ 1 .Then, Ernst Freese 3 who had worked with Benzer

w xon the phage T4 system 1 found that mutationsinduced by base analogs were revertible only byother base analogs and not by mutagens of theacridine family.

w xFreese 2 concluded that there were two kinds ofmutations, both characterized by base changes: tran-sitions in which pyrimidines were replaced by pyrim-

Ž .idines and purines by purines and transversions inwhich purines were replaced by pyrimidines orpyrimidines by purines. He supposed that bro-mouracil and aminopurine induced transitions, acri-dine induced transversions and that spontaneous mu-tations were a mixture of both. A few years later,

w xBrenner et al. 3 at the MRC reinterpreted this dataand argued on the basis of their failure to obtain‘‘leaky’’ mutants with proflavin that the changesFreese had interpreted as transversions were actuallyframeshift mutations. An additional argument wasthe finding that none of the supposed ‘‘transver-sions’’ mapped at exactly the same site as any of thetransitions — which was not understandable if theywere both simple base substitutions 4. Understandingframeshift mutations permitted the MRC group todemonstrate by purely genetic techniques that the

3 Freese had been trained as a solid state physicist. He objectedto the need for physicists to work in large teams and he also feltthat new discoveries were hard to come by in physics. In contrast,he told me after a seminar in Chicago, biology in the 1950sseemed to promise unlimited opportunity!

4 Benzer was highly annoyed — at least at first. He visited theUniversity of Chicago in 1961 to receive the Ricketts award. Iknew him slightly and talked to him after the lecture. He wasindignant at the idea that his former collaborator had been cor-rected and at that time was not ready to accept the deductions ofthe Cambridge group which were also based on indirect evidence.Of course both groups were leading the genetics community to amore molecular understanding of what happened in mutation.Brenner also happened to be correct.

w xcodons needed to be triplets 4 and they did this byŽshowing that the third mutation in a series of q1 or

.y1 mutation restored genetic function. The evi-dence that acridine induced mutations were mostlyframeshifts came from the demonstration that muta-

Ž . Ž .tions could be arbitrarily put into two groups: q1Ž . Ž . Ž .and y1 and that combination of q1 and y1

mutations gave a suppressed strain that was almostw x 5normal 5 . Biochemical evidence for this hypothe-

w xsis came when Streisinger et al. 6 isolated anddetermined the amino acid sequence of bacterio-phage lysozyme mutants. The suppressed mutantshad a normal sequence except for a group of fiveamino acids. Given the original and substituted aminoacid sequence, the sequence of nucleotides involvedcould be uniquely deduced. 6 Streisinger et al. thensuggested the following: ‘‘frameshift mutation wouldinvolve the insertion of a base or a base doublet,identical to an adjacent one already present in thewild-type DNA. The insertion would be most likelyto occur in a region of repeating bases or basedoublets through the pairing of a set of bases in onechain of the DNA molecule with the wrong, butcomplementary set in the other chain.’’ This is the‘‘slippage’’ model for frameshifts althoughStreisinger et al. does not use the term in the 1966paper.

It was not immediately clear what aberrant bio-chemical process produced frameshift mutation norwhat the role of the acridine mutagens might be. Forsome years it appeared that acridine mutagenesis wasrestricted to the bacteriophage and that bacteria were

w ximmune. On structural grounds, Lerman 7 had sug-gested that frameshifts should occur by recombina-tion and this remained a reasonable hypothesis dur-ing the 1960s. The role of recombination was sup-ported by the observation from Adelberg’s labora-

w xtory 8 that proflavin mutagenesis could be observedin a mating mixture of bacteria but not in haploids

5 Although the experiments were announced in 1961, the fullpaper giving the details of how the frameshift experiments were

w xdone was not published until 1967 5 !6 It is hard to remember but there was a period in which amino

acid sequences in protein could be determined — with difficulty— but nucleotide sequences were just not experimentally accessi-ble!



ization in the different mutS analogs MSH2, MSH3and MSH6 which act as heterodimers so that theMSH2rMSH3 dimer is more efficient at the recogni-tion of loops caused by additions or deletions whereasthe MSH2rMSH6 complex is most efficient at the

w xrecognition of base mismatches 39–41 . The mis-match repair system is limiting in E. coli even inexponential growth and the amount of mismatchrepair falls precipitously as cells enter stationary

w xphase 42 . In particular there is a 10-fold drop in theamount of mutS protein. E. coli deficient in proof-reading are also deficient in mismatch repair becausethe system is saturated by the onslaught of errorsw x43 .

The mononucleotide repeat sequences in prokary-otes are limited in size to about eight or less and are

w xmostly found in coding regions 44,45 , probablybecause prokaryotes contain relatively little non-cod-ing DNA. Much of the correction of replicationerrors is therefore due to the proofreading exonucle-ase which recognizes both single base mismatches

w xand frameshifts in non-repeated 17 or in low repeatw xsequences 18,46 . In sharp contrast, the higher eu-

karyotes have many repeated sequences and in yeastŽ .the majority of the long sequences )8 are found

w xin the non-coding regions 45 . It is an obviousspeculation that the differentiation of the mismatchrepair system in eukaryotes is due to the greaterstructural complexity of this DNA with one part ofthe mismatch repair apparatus being dedicated tokeeping slippage in repeated sequences under con-trol. Although both exonucleolytic proofreading andmismatch repair correct errors in newly synthesizedDNA, only mismatch repair deficiencies have beenconvincingly associated with the instability of re-peats. The mismatch repair system is uniquelyadapted to the repair of additions or deletions in longrepeated sequences since it has been shown both in

w xvitro 46 and in vivo that proofreading plays a minorrole in the prevention of mutations in the longer runsw x18,20,32 .

5. The mechanism of slippage

Mismatch repair and, depending on the size of therepair tract, exonucleolytic proofreading correctframeshift errors once made. The errors need to be

made before being corrected. One can therefore askwhat factors regulate the production of frameshifts?Actually, the question is, what are the factors whichprevent slippage and frameshifting? Slippage isclearly a feature of all simple polymerization pro-

w xcesses as discussed above 15 . Replicating systemsin real organisms need to have some way of prevent-ing the massive slippage that occurs in these simpleexperimental systems. A priori, both nucleic acid andprotein structure must be involved. Consider whatmust happen for a frameshift mutation to occurduring DNA synthesis across a region of repeatedelements. First, there must be slippage. Elongationcontinues. However, the slipped intermediate canalso slip back. If this reverse slippage occurs whileDNA synthesis is still traversing the repeated tract,then no mutation will occur. However, if the bulgeproduced by slippage is stabilized long enough forDNA synthesis to proceed past the repeated regionand into a region of unique sequence, then the bulgewill be fixed since the growing point is now‘‘anchored’’ by the unique sequences. Frameshiftingis therefore the resultant of a competition betweenthe rate of elongation and the stability of the DNAintermediate. One might therefore expect structuresin which the extrahelical nucleotide structure is par-ticularly stable to frameshift more readily than struc-tures with an unstable extrahelical loop.

A characteristic of the repeated sequences in yeast,Žhumans, and C. elegans but not E. coli or M.

. Žleprae is the large contribution of runs of A’s or. w xT’s 44 . The overall composition of the genome is

not sufficient to account for this overrepresentationw xof A and T 45 . The distribution of runs in coding

regions is restricted by the amino acid code — a runŽ .of glycines GGG might not be tolerated. However,

in non-coding regions the genome should be morepermissive. Why should there be an excess of Aruns? One possibility is that poly-A tracts play regu-latory roles. Alternatively, A-enriched sequencesmight be parts of mobile DNAs or the tendency ofpolymerases to insert A’s at the site of damaged

w xbases 47 might lead to an accumulation of thisnucleotide. Yet an additional alternative is that runsof A:T are more likely to slip leading to expansionsor that runs of A or T, once formed, are more stable.In E. coli, repeats of G’s and C’s were observed toframeshift more frequently than repeats of A’s in a


within the coding regions and that these interveningsequences or introns had to be spliced out of the

w xRNA transcript to make functional message 26 . Therest of the eukaryotic genome contains multiplecopies of a variety of DNA sequences, some appar-ently the remnants of retroviruses or retrotransposons

w xthat had inserted themselves into the genome 27 . Inaddition, the ability to sequence DNA led to thediscovery that there were extensive runs of mononu-cleotides, dinucleotides, trinucleotides and even te-tranucleotides scattered throughout the genome.There are more than 5000 dinucleotide repeat se-

Žquences in the human genome where the repeat is.six or more interspersed throughout the genome.

Since these sequences are polymorphic they haveserved as the basis for mapping the mammalian

w xgenome 28 . To be used as markers they need to bestable. Nonetheless, they should be subject to slip-page during replication and so be less stable thanunique sequences. An average germinal mutationrate of 1.2=10y3 per locus per gamete per genera-tion was reported for dinucleotide repeats but theauthors cautioned that the figure might be inflated by

w xsomatic events 29 . Even this rate is quite stableconsidering the expected rates of slippage. Somemechanism must keep the variability of the repeatedsequences within bounds since as indicated above,without such correction a replication of a run of 14dinucleotide repeats leads to an error at least 1% of

w xthe time 18 !

4. The role of mismatch repair

Recognition of how this is accomplished in eu-karyotes came with the discovery that the instabilityof dinucleotide repeats is greatly enhanced in thecolon carcinomas of individuals with an inherited

w xsusceptibility to non-polyposis colon carcinoma 30 .This phenomenon was termed microsatellite instabil-ity and was initially supposed to be due to anaberrant form of DNA replication in these tumors. Itwas, of course, an example of extensive frameshiftmutation. It had been realized from the earliest inves-tigations on the generalized mismatch repair system

Žin bacteria involving the mutS, mutH, mutL and.dam genes that the system recognized and corrected

w xframeshift mutations 31 . In particular, it had been

shown that mutants of yeast, deficient in mismatchrepair, made numerous frameshifts in repeated se-

w xquences 32 . One of the investigators who hadworked out the detailed biochemistry of mismatch

w xrepair in bacteria, Paul Modrich 33 , heard aboutthese experiments and recognized that a deficiencyin mismatch repair was likely to account for the

w xmicrosatellite instability in tumors 30 . A simultane-ous 8 discovery of the role of mismatch repair camefrom the laboratory of Richard Kolodner who hadbeen working on mismatch repair homologs in yeastw x34 . They had sequenced a yeast repair gene andfound that a published sequence in the human genomeincluded a homolog of their yeast repair genes andmapped at the same position as the gene responsible

w xfor the microsatellite instability 35 . It was thendiscovered that the tumors of such individuals weredeficient in their ability to carry out mismatch repairbecause of a deficiency in one or the other of the

w xmismatch repair proteins 34,36 .Although the mismatch repair systems in prokary-

otes and eukaryotes are homologous, there are im-w xportant differences 37 . Mismatch repair in bacteria

is generally considered as happening after replica-tion, during a window of time in which the parentstrand can be distinguished from the newly synthe-sized daughter. In a very few prokaryotes such as E.coli the signal that distinguishes old from new strandsis hemimethylation of adenines. Adenines in thenewly synthesized strand at GATC sites are notmethylated by the dam-encoded methylase until some

Žtime after replication. In eukaryotes and in fact.many bacteria methylation is not used and the signal

is most likely a single strand break. In fact, whereasreplication and mismatch repair are at least concep-tually separable in prokaryotes, replication and mis-

w xmatch repair may be linked in eukaryotes 38 .A second distinction in the mismatch repair sys-

tems of eukaryotes and prokaryotes is the occurrenceof several mutS and mutL homologs in the eukary-otes, whereas single proteins carry out all the mis-match repair functions in bacteria. There is special-

8 The Fishel paper was received by Cell on November 8,revised on November 18 and published in the December 3, 1993issue. The Parsons paper was received by Cell on November 29thand published in the issue of December 17, 1993.



One of the earliest papers on rapid mutagen iden-w xtification by Ames et al. 72 is entitled: ‘‘Carcino-

gens as frameshift mutagens’’ as though there weresomething particularly carcinogenic in frameshiftmutagens. It appears that he had it right! Several ofthe important tumor suppressor genes containmononucleotide runs within the coding sequencesw x Ž .73–76 . These constitute ‘‘at-risk-motifs’’ ARMsw x77 which are targets for frameshift mutagenesis andare peculiarly sensitive to mismatch repair defi-ciency. One of the interesting illustrations is thefinding of a mutation which predisposes to cancer byaltering a base in the midst of a homonucleotide run

w xthereby creating an ultrasensitive ARM 78 .Frameshift mutations, particularly frameshift muta-tions in repeated sequences appear as a major factorof eukaryotic life and the control of these mutationshas probably resulted in the refinement of the mis-match repair systems of higher organisms.

Acknowledgements

The work from the authors laboratory was per-formed, in part, with support from the National

ŽCancer Institute, National Institutes of Health CA.32436 . I would like to thank Dr. Louise Prakash,

Dr. Satya Prakash and Dr. Dmitry Gordenin for theircomments on a draft of the manuscript.

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w x1 S. Benzer, E. Freese, Induction of specific mutations withŽ .5-bromouracil, Proc. Natl. Acad. Sci. USA 44 1958 112–

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w xmismatch repair deficient strain 48,49 . Runs of Gand C may be less stable because the frameshiftintermediate with extrahelical G’s and C’s is morestable. There is physical data to support this vieww x50 .

Other kinetic properties of the elongating DNAare also likely to be involved in the production offrameshifts. For example, treatments which delayDNA chain progression are likely to result in dele-tions or additions of bases. One of the methods ofdelaying DNA synthesis is by the insertion of anincorrect base at some position in the DNA. Thisdelays synthesis and, as suggested by Bebenek et al.w x51 , a misalignment of the initially misinserted basecould result in the generation of correct terminalbase pairs which were then elongated. The alterationof a nucleotide by formation of an adduct whichreduces the rate of elongation would have somewhatthe same effect and in fact, misinsertions followedby frameshifts have been observed both in vitro and

w xin vivo 52 . One of the most efficient frameshiftingagents is acetyl aminofluorene which inhibits DNAsynthesis and also seems to stabilize the frameshifted

w xintermediate 53 . In fact, acetylaminofluorene andits action has become a paradigm for a frameshiftmutagen. An aminofluorene adduct with the acetylgroup removed is no longer a major block to DNA

w xsynthesis 54,55 and results in base substitutionw xrather than frameshift mutations 56 . This cannot be

the whole story since different polymerases behavedifferently in their ability to misinsert bases as com-

w xpared to making errors by base dislocation 57 . Inaddition, the frequency with which a series of mutantT7 DNA polymerases produced UV-inducedframeshifts in vitro correlated with the velocity withwhich they replicated a M13 template but not with

w xtheir exonuclease activity 58 .Ž .The structure of the polymerase protein s itself

might be expected to be important, particularly in thecase of frameshifts at repeated sequences since onemight expect the amino acids of the polymerase tointeract with the individual bases to prevent themovements of the primer or template that result inslippage. This interaction would not necessarily beexpected at the catalytic center. For example, aspointed out above, the longer the run of repeated

w xunits, the less the effect of proofreading 46 indicat-ing that the slippage events need not occur at the

Žgrowing point or that the slippage ‘‘bulge’’ mi-.grates away from the growing point . Site directed

mutagenesis studies with single protein polymerasesillustrate these concepts. Substitution of alanine forother amino acids in the thumb domain of both HIV

w x w xreverse transcriptase 59 and pol I 60 results in anenzyme which makes increased numbers offrameshifts. In contrast to the ‘‘simpler’’ poly-merases which have been studied, most replicativepolymerases of free living organisms are made ofnumerous subunits and the three dimensional struc-ture and mode of interactions of these subunits isstill being investigated. Mutations of the catalyticsubunit of E.coli pol III leading to a frameshift

w xmutator effect have been isolated 61,62 but it is stillunknown how the mutator effect comes about. Forexample, a mutation leading to a decreased ability tobind and activate the proofreading subunit wouldlead to decreased proofreading and a mutator effect.The various domains of the E. coli polymerase are

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w xrepair mutants 38,64 . Since the boundaries of repli-cation and mismatch repair in eukaryotes are not

w xsharply defined 38 , this conclusion may yet bemodified.

6. Biological role of DNA frameshifts

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w xhypermutable state 69–71 .



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The origins of the back-mutation assay method:a personal recollection 1

Geoffrey W. GriggCSIRO Laboratory of Molecular Science, PO Box 184, North Ryde, 2113 Sydney, Australia

Accepted 7 September 1999

Abstract

The back-mutation assay method for determining the mutagenicity of various treatments was first developed a little over50 years ago and has been in continuous use ever since. Shortly after the method was first used it became evident that certainfactors of cell density, composition of media, etc., had to be carefully controlled to preserve an acceptable reliability of themethod. A factor of particular importance was the suppression of growth of back-mutant prototrophic cells by the largenumber of auxotrophic cells present, a phenomenon which later became known as the ‘‘Grigg Effect.’’ This review describesthe origins of the back-mutation method and of the confounding competitive suppression phenomenon, the cause ofcompetitive suppression, methods of diagnosing whether it is likely to bias the interpretation of a particular back-mutationexperiment, and an experimental design which removes it entirely as a possible source of error. A number of otherphenomena, such as phenotypic lag and coincident mutation associated with back-mutation, are also discussed as possiblesources of error. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Back-mutation assay; Grigg Effect; Mutation

1. Introduction

The way in which mutations arise in living organ-isms has been of considerable interest to geneticistsin particular and to biologists in general for the past100 years. More recently, the recognition that someimportant diseases, most notably cancer, are caused

Ž w x.by somatic mutation and epimutation 1 has fo-cused an interest on minimizing exposure to environ-

1 This article is part of the Reflections in Mutation Re-search series. To suggest topics and authors for Reflections,

Žreaders should contact the series editors, G.R. Hoffmann ghoff-. Ž [email protected] or D.G. MacPhee [email protected] .

Ž .mental mutagens and epimutagens . Much of whatwe know about the mutagenicity of chemicals andelectromagnetic radiation has come from the use ofthe microbial back-mutation assay method whichwas developed in the late 1940s to permit the effi-cient identification of the few individuals that hadundergone mutation in a large population. Themethod made it possible to measure easily and accu-rately the frequency of mutation of an altered charac-ter, commonly a requirement for a growth factorsuch as a particular amino acid, back to the wild type

Ž .condition non-requirement . Back-mutation assaysare still used as a primary screen in testing themutagenicity of various radiations and chemicals. Inthis paper I revisit some of the earliest work on the



The origins of the back-mutation assay method:a personal recollection 1Geoffrey W. Grigg

CSIRO Laboratory of Molecular Science, PO Box 184, North Ryde, 2113 Sydney, AustraliaAccepted 7 September 1999

( )G.W. GriggrMutation Research 463 2000 1–122

development of this method and discuss factors af-fecting its accuracy, including the phenomenon thatwas originally called competitive suppression and

w xlater came to be known as the ‘‘Grigg Effect’’ 2,3 .In 1950, the gene was defined only as a functional

unit, a phenotypic character, which mapped at aspecific site on a chromosome — it was not thedefined chemical entity that it is today. Mutationswithin one gene were recognised as such by theirco-segregational qualities. Well before DNA se-quencing was invented, fine structure analysis beganto shake the model of the gene as a simple bead on astring, with studies on bacteriophage, Aspergillusnidulans, Neurospora crassa and Schizosacharo-myces pombe leading the way. But in the late 1940’sand early 1950’s the gene was still recognised, de-scribed and analysed as a unit. Geneticists debatedwhether the process causing deletion of genetic ma-terial was qualitatively different from that leading to‘‘point’’ mutations. Since the large deletions thatcould be detected by cytological methods did notback-mutate, one definition of a ‘‘point’’ mutation

Žwas a mutation which was reversible i.e., subject to.back-mutation . There was an interest in determining

whether back-mutation was induced by X-rays —since this would test the notion that X-rays produce‘‘point’’ mutations as well as deletions. Today wecan pick holes in this sort of logic, but 50 years agoit did not seem at all unreasonable.

Of the possible uses of the back-mutation assaysystem, the most important was to learn about themechanism of mutation and the nature of the gene.Another was to describe the genetic toxicity of ourenvironment.

The back-mutation method was not the methodoriginally used to establish that mutation frequenciescould be enhanced by environmental factors, how-ever. The first factor identified as a mutagen —some 70 years ago — was ionising radiation, andfruit flies and barley were the genetic organisms

w xused 4–6 . By mid-century, objective methods ofmeasuring the mutagenicity of various treatmentssuch as Muller’s sex-linked lethal test in Drosophila,and plant assays based on pigmentation, were used

Žwidely. These methods were tedious, and some such.as the sex-linked lethal test suffered from the con-

cern that they might detect only certain types ofgenetic change such as deletions. Development of

the back-mutation method in microorganisms solvedmany of these problems.

The interest engendered by the demonstration thatw xchemicals could be mutagenic 7–10 happened to

coincide with the development of the biochemicalgenetics of microorganisms — principally at theCalifornia Institute of Technology in the 1940s.Strains of N. crassa were isolated that were unableto carry out specific metabolic functions, and conse-quently had a growth requirement for an amino acidor a vitamin, a purine or a pyrimidine. The character-isation of such mutants allowed the detailed descrip-tion of biosynthetic pathways, and led to the isola-tion of the enzymes involved and the ‘‘one gene–oneenzyme’’ theory — a seminal development in micro-bial genetics. It was observed that many of themutant strains could back-mutate spontaneously tothe wild type phenotype at low frequency — anevent readily detected by spreading N. crassa coni-

Ž .dia asexual spores from a mutant strain on agarmedium lacking the specific growth factor required.Hence, if the mutant strain for example requiredhistidine for normal growth, a basal medium lackinghistidine could be used to select for back-mutationsto a non-histidine requiring state.

Earlier it had been found that treatment of wildtype N. crassa with X-rays, nitrogen mustard orultraviolet irradiation increased the yield of biochem-

w xical mutants 10–12 . At best this was a laboriousmethod of assaying mutagenicity, and it was onlywith the development of simple methods of selectingback-mutants that the use of microorganisms in mu-tation studies became widespread. Back-mutationmethods were rapid and objective, and they enabledlarge cell populations to be screened for the presenceof rare variants. They had an advantage over meth-ods in complex eukaryotic organisms in that singlecells containing one or more nuclei could be bathedin a solution of the chemical under test. Of course,this did not ensure that the chemical would diffuseinto the cell and reach the nucleus. Often the type ofsuspension fluid used played a major part in theprocess, but at least the chemical in known concen-tration could be applied to the outer surface of thecell whose genetic material was the target.

An objective method of assaying putative muta-gens in microorganisms was developed first in thebacteria Staphylococcus aureus and Escherichia coli


( )G.W. GriggrMutation Research 463 2000 1–12 3

w x w xby Demerec et al. 13–15 and Witkin 16 , usingforward mutation to drug resistance as the selectivecharacter. Already in 1946, Demerec had noted thatthe yield of mutations induced by UV-irradiationincreased as the population of irradiated bacteria was

w x 2allowed to multiply 13,14 . It was not long beforew xseveral groups of Neurospora workers 18,19

recognised the potential of using their biochemicalmutants as the basis of a powerful system to examinethe mutagenicity of various chemicals or radiations.As N. crassa was a ‘‘respectable’’ genetic organism,the mutation assays built around it were regarded asmore reliable than the bacterial systems whose genet-ics had yet to be worked out.

1950 was an interesting time for a young biologistto be alive, particularly one who had just arrived inCambridge to do a PhD. Although there was littlemoney for research in Universities such as Mel-

Ž .bourne where I had graduated or even at Cam-bridge with its great tradition in science, a series ofmajor discoveries in biology around this time initi-ated one of the greatest periods of intellectualachievement of the 20th century, culminating in thediscovery of the structure and function of the geneticcode. One of the first signposts along this intellectualhighway was the discovery of transformation ofserotypes in Pneumococcus, which had been reportedsome 20 years previously by the English pathologist

w xGriffith 20 . He clearly recognised the significanceand importance of his observation. 3 Transformationwas linked to the transfer of DNA from one cell toanother by Avery and his group at the Rockefeller

w xInstitute in New York in 1944 22 . The significanceof DNA as genetic material was reinforced by theisotope experiments of Hershey and Chase at Cold

w xSpring Harbour a few years later 23 . Yeasts, bacte-ria and bacteriophages were just emerging in genetic

2 Subsequently this phenomenon was reexamined by Witkin asan important part of her definitive studies on UV mutagenesis. By

w x1956 17 she had established that the amount of protein synthesisin the target cells after mutagenic treatment was the critical factorin determining the yield of mutations, not simply cell division.

3 Ž .Many years later, Griffith’s nephew John Griffith , then achemistry student in Cambridge, played a significant part in aidingWatson and Crick to devise their model of DNA and latersuggested how proteins could be infective agents in diseases such

w xas scrapie 21 .

research, and a plethora of genetic tools was beingassembled to explore the intricacies of geneticrecombination, mutagenesis, and a few years later,gene regulation and the genetic code. The back-mu-tation assay offered an obvious approach to theinvestigation of some of these important questions.

2. Back-mutation assays in fungi

The back-mutation method was based on the ob-Žservation that asexual spores uninucleate microconi-

.dia or multinucleate macroconidia from a biochemi-Ž .cal auxotrophic mutant of Neurospora may occa-

sionally back-mutate to the wild type or prototrophiccondition and form visible colonies on the minimalmedium. This medium consisted of a mixture ofinorganic salts, a carbohydrate, biotin, and if used inpetri dishes, agar to solidify it. It was assumed thatthe conidia containing a prototrophic nucleus wouldproduce a visible colony. In order to verify thatprototrophic colonies did, in fact, represent back-mu-tations and not suppressor mutations at other loci,some of the prototrophic colonies were crossed witha wild type strain and the resultant ordered asciexamined. An 8:0 ratio of prototrophs to auxotrophsin the ascospore cultures from each ascus suggestedthat back-mutation had occurred, or that, if a sup-pressor mutant were involved, it was linked with thegene it suppressed. A 6:2 ratio indicated that muta-tion at another locus was responsible for the growthon minimal medium, as one-fourth of the haploidspores would contain both the original mutation andthe suppressor if the two genes were unlinked. As-sumptions of the method were that the prototrophiccolonies arising after mutagenic treatment were theconsequence of stable and real genetic changes, andthat prototrophic back-mutants would grow into visi-ble colonies independently of the presence of a largepopulation of non-growing auxotrophic cells. Thislatter assumption had not been adequately tested bythe Neurospora workers.

3. Are the assumptions valid?

Ryan and Schneider first explored the secondassumption in a classic study of mutation in E. coli


w x24 . They commented on an inhibitory effect ofnon-growing histidine-requiring bacteria on thegrowth of wild type back-mutants and suggested thatthis effect could bias mutation experiments. Thesignificance of their discovery was missed by mostworkers in the field including, for a while, myself. In1949–1950, the details of the genetics of bacteriawere meagre and confusing so that many establishedgeneticists were uncertain whether bacterial geneswere similar to those of organisms with more con-ventional genetic systems. The nature of mutationand of the genetic material itself was the subject ofcontroversy. To make matters worse the E. coli-15strain used by Ryan and Schneider could not begenetically analysed to check the nature of the ge-netic change involved in ‘mutation’. It was for simi-lar reasons that Demerec’s pioneering studies onforward mutation to drug resistance in S. aureus,Salmonella typhimurium and E. coli received lessrecognition than their priority might have indicated.‘‘Establishment’’ geneticists were uncertain whetherthe mutations which Demerec et al. were scoringwere similar to the mutations recognised in organ-isms with well-characterised genetics such as thefungi. Of course, views changed with the passage oftime and an understanding of the details of bacterialgenetics, so that today bacteria have largely replacedfungi as the preferred organisms for use in mutationassays — but that was not the situation 50 years agowhen it all began.

Early in 1952, my paper describing some prob-w xlems in the back-mutation assay in N. crassa 2 was

published and attracted immediate attention. Someyears later, when writing up my PhD thesis, I came

w xacross Ryan and Schneider’s observations 24 andacknowledged their prior claim to the discovery of aphenomenon in E. coli which seemed similar to the

w xone I had discovered in N. crassa 3 .My entry into the mutation field came by a cir-

cuitous route. After graduating from Melbourne Uni-versity in 1948 in Zoology and Chemistry, I hadspent a couple of years doing a Masters Degreestudying the morphology and physiology of chickensperm. I selected this field for the basest of motives— the availability of financial support! My ambitionwas to study biochemical genetics, but there were nolaboratories working in this new field in Australia in1948. Following my MSc course work, I won a

Ž .scholarship to Cambridge UK and in so doing wasable to satisfy a long held passion to get into bio-chemical genetics. However, the research on chickensperm proved quite interesting. Alan Hodge ofCSIRO and I developed novel methods which en-

Ž .abled us to demonstrate for the first time a biologi-Ž .cal constant, the 9q2 structure of flagella and cilia

and produce a first description of the microtubulew x25–27 . A better understanding of the mechanics ofsperm movement in the reproductive tract of the hen,to which I contributed, led to improvements in theefficiency of artificial insemination in poultry and tonovel ideas on how infertility might be remedied.

I was very excited to have the chance of studyingin Cambridge, but first I had to be admitted as agraduate student by the University and by a Cam-bridge college. I had the good fortune to be acceptedby Kings College, with its famous Chapel and choirand a bit of the pomp and performance which ac-companied it. In addition to classicists andeconomists in the college, there were a few scientistsof note — Malcolm Dixon was a Fellow and FredSanger had just been elected to the Fellowship;Sydney Brenner and John Griffith joined them alittle later. The buildings and traditions of Cambridgewere ancient, but the ideas with which we as stu-dents were surrounded were not.

We had rented a small rose-covered cottage inGirton village 2 miles out. Next door lived ourlandlady who was the charming, kindly yet form-idable H. Wilfrieder Leakey, retired archeologist andfirst wife of Louis Leakey of East African fame, who‘ran’ the village. Each Sunday she held court, andwe were always invited to join in and meet a crowdof interesting people, many of whom remainedfriends long after we left Cambridge.

I came to Dr. D.G. Catcheside’s laboratory at theBotany School in Cambridge in the autumn of 1950,knowing virtually no genetics or microbiology butwith a few papers published from my MSc work.Catcheside suggested that I pick a topic of my ownchoosing to work on. At the time he had manystudents; I was the 13th in the laboratory. On hearingof my interest in mutagenesis, he threw me somereprints from the Giles and the Westergaard labora-tories and told me to ‘go to it’. He also gave mesome excellent advice — don’t take for grantedanything spoken or written until you have checked it



for yourself. So I started on my PhD topic to studymutagenesis in N. crassa with this advice ringing inmy ears — advice which I remembered especiallyperhaps because of my general ignorance of thefield.

The Botany School in Cambridge was remarkablysimilar in ‘feel’ to the Zoology Department in Mel-bourne that I had just left. Both buildings were old,neither had significant funds for research, and therewas a paucity of modern equipment. Centrifugeswere manually operated — by arm or leg. For a longcentrifuge run you mounted a bicycle hooked to thecentrifuge and peddled! For a fast run you peddledfast. There was no quantitative glassware available inthe store — you made and calibrated your own. Theincubators were home-made too — from scraps ofcast-off plywood, timber, old vacuum cleaner mo-tors, etc. The incubators had mercury–toluene ther-mostats, hand-made by the lab technician. The year Iarrived, the Department acquired its first electricallyoperated centrifuge — an MSE Minor! The samefinancial strictures pertained to other departments inthis famous University, but this did not stop peoplelike Sanger or Perutz or Wigglesworth, who workedaround the corner from the Botany laboratory, fromperforming experimental miracles. The cost of theequipment may have been trivial, but the ideas thesepeople developed and the ingenuity they displayed intesting them were not.

So my first couple of days in the Botany Schoolwere spent with some glass tubing and the glassblower’s torch making and calibrating pipettes. Thenthere was agar to be washed free of growth factorsand petri dishes to be washed and sterilised. And Ihad to learn something about genetics and N. crassa— how to set up crosses and analyse asci and so on.By Christmas of 1950, I was ready to check theback-mutation assay method and then hopefully togo on and use it to do something really interesting.But by March I had a few results and it seemed thatthe back-mutation method might have some prob-lems. I remember that at the time this discoverydismayed rather than elated me. Also in March,Catcheside had me invited to the first of three Micro-bial Genetics Symposia funded by the RockefellerFoundation which was to be held in Copenhagen andorganised by Professor Mogens Westergaard. I wasthe most recent arrival in the laboratory, and I was

more than a little surprised to be the one selected toattend, but going to Copenhagen was exciting. 4

By June of 1951 I had enough data on the prob-lems of the back-mutation method to give a paper onmy work at the annual meeting of the GeneticsSociety held in Glasgow and hosted by Prof. GuidoPontecorvo. The latter, who had been one of the fourwho raised the funds for the first Rockefeller Sym-posium, was particularly kind to me, a small thingbut something I remember with pleasure. Later, both

Ž .he and Charlotte Lottie Auerbach wrote to Wester-gaard about the ‘threat’ my discovery created to thelatter’s work on mutation assay. Shortly after return-ing to Cambridge I received a charming letter fromWestergaard who mentioned that he had had lettersfrom two Scots, ‘‘Lottie Mac-Auerbach’’ and ‘‘PontyMac-Pontecorvo’’ with some alarming news on theeffect of my results on his studies and inquired aboutthe basis for their alarm.

Very shortly afterwards Catcheside, who earlier inthe year had been elected to the Royal Society,announced that he had accepted an invitation to thenewly created Chair of Genetics at Adelaide Univer-sity — the first in Australia — and would be leavingCambridge in the autumn. At the time he gave methis news he also invited me to a lectureship in hisnew department in Australia, which was very flatter-

Žing for a young man I had just turned 25 at the.time halfway through his PhD. By the time of this

w xdiscussion, my paper 2 had gone off to Nature, andI knew the mechanism of what later became knownas the ‘‘Grigg Effect.’’

In the first half of 1952, having explored themechanism of the competitive suppression effect Ihad discovered in N. crassa, I turned my attention tothe genetics and physiology of some interestingNeurospora conidial phenotypes that had turned up

4 Everyone who was anyone in microbial genetics in EuropeŽ .was there about 25 in all , plus a handful of young scientists and

a few Americans, including F.J. Ryan and his wife Elizabeth fromColumbia University on sabbatical at the Pasteur in 1950. Wester-gaard was our host for the meeting, and at the official dinner NilsBohr gave the after dinner talk and we all chatted with the greatman. At this dinner I tasted my first aquavit, and my first cigar. Ialso had my first tramp through snow when we walked from theUniversity back to our hotel near the Nyhavn — a good distancebut we were well insulated against discomfort. The meeting wasexciting for me and I learnt a lot.


in the course of my project. In the middle of the yearI was invited to the second Rockefeller MicrobialGenetics symposium, 5 this time to be held at Pal-lanza-Verbania on Lake Maggiore in Italy. It was atthis meeting that the term ‘‘Grigg Effect’’ was firstused. Pontecorvo asked a speaker at the meeting,whose name I cannot recollect, if he had taken the‘‘Grigg Effect’’ into consideration in interpreting hisresults. The speaker said ‘‘What is the Grigg Effect?’’and Pontecorvo replied, ‘‘Grigg is here, why don’tyou ask him?’’

5 Pontecorvo and Luca Cavalli-Sforza were the organisers ofthe Second Rockefeller Microbial. Genetics Symposium. As be-fore, the 48 invitees, who had their expenses paid, consisted ofestablished workers plus a handful of young post-docs and one

Ž .student me . The meeting lasted a week and what a wonderfulweek it was. The rationing and general frugality of life as astudent in England contrasted sharply with a week in the GrandeHotel Majestic on the edge of the beautiful Lake Maggiore,plentiful and delicious food and wine, and the company of greatbiologists such as Ephrussi and his wife Harriet, Monod together

Žwith wife and mistress, Brachet, Emerson from Cal. Tech, on.sabbatical in Cambridge , Hammerling and entourage, Magni,¨

Lwoff, Hayes with wife Nora, Leupold, Westergaard, Beale,Pollock, Weigle, Fries, Buzzati-Traverso, Chain, Sermonti, Vis-conti, and of course Pontecorvo and Cavalli-Sforza. The youngergroup included John Fincham, Francois Jacob, Barbara WrightŽ . Ž .later Wright-Kalckar , Alan Roberts from Ponty’s lab , PiotrSlonimski, Walter Harm, H. Marcovich, G. Rizet, E. Wollman, R.Kaplan, H. Stich, G. Kolmark, N. Nyborn and a gaggle of brightyoung Americans working in Europe, who included Jim Watson,Seymour Benzer, Herb Hirsch and Arnold Ravin. Slonimski hadno passport and had to elude the border guards by walking intoItaly over the mountains. The agenda for the meeting limitedformal presentation of papers to a couple of hours in the morningsand another couple of hours in the afternoons with eveninglectures by Andre Lwoff and Francois Jacob. This left plenty of´time for discussion and for swimming in the clear waters of thelake and across to a small island occupied by Arturo Toscaniniand family. By prior agreement, Xphage genetics was excludedfrom discussion — much to Jim Watson’s chagrin. However, inpartial compensation a greeting telegram was sent to Max Del-bruck at Cal Tech expressing everyone’s good wishes. But from 8am to midnight on each day we talked and talked — and talked.The main controversy was the proper interpretation of crossingexperiments in E. coli. The Lederberg camp was represented by

Ž .Cavalli-Sforza. The other camp of one was Bill Hayes, who hadjust published his experiment showing the one-way transfer ofgenetic material in E. coli. I believe that this was the firstinternational scientific meeting Hayes had attended; he acquittedhimself modestly but convincingly at the session on bacterialgenetics and clearly won the day.

4. Competitive suppression or the ‘‘Grigg Effect’’

The central assumption of the back-mutation as-say method, that the germination of prototrophicback-mutant cells and their growth to visible colonieswas not affected by the presence of large numbers ofauxotrophs lacking the ability to grow on the selec-tive medium, could be tested in reconstruction exper-iments. In these experiments, a small number of

Ž .prototrophic cells conidia were either spread on theselective medium without auxotrophs or mixed witha large population of auxotrophic cells used in muta-tion assay experiments before plating. If equal num-bers of prototrophic colonies appeared on the twoseries of plates one could conclude that the presenceof the auxotrophs did not affect the growth of singleprototrophic conidia into colonies. When I performedsuch experiments, using densities of auxotrophic cellscommonly used in mutation assays, a deficiency ofprototrophs was observed consistently in the groupcontaining the auxotrophs. The result of a typicalexperiment is illustrated in Fig. 1, taken from Ref.w x2 . Similar results were obtained with a variety ofauxotrophic strains, using either microconidia ormacroconidia. Despite the advantage of having onlyone nucleus, microconidia were not much used formutation assay because their viability was lower andsomewhat variable by comparison with that ofmacroconidia.

The results of these reconstruction experimentssuggested that the few prototrophic conidia that hadarisen by back-mutation in a population of aux-otrophs might not grow to visible colonies on thecontrol plates in mutation experiments if the numberof auxotrophs exceeded a particular limit. Whenvarious numbers of auxotrophic conidia were plated

Ž . Ž .on or in a selective medium on or in whichprototrophs should be able to grow the truth of thisprediction was verified. Above a certain concentra-tion of auxotrophic conidia no prototrophic coloniesarose; but when serial dilutions of these auxotrophiccells were plated the presence of cryptic prototrophsin the original population was revealed.

This phenomenon, which soon received confirma-Ž w x.tion from other laboratories e.g., Ref. 28 , affected

the interpretation of mutation experiments. Sincemost mutagenic treatments are also toxic, one had tobe certain that the prototrophs that were scored



Fig. 1. Inhibition of prototrophic microconidia by W40 conidia. All plates contain the same number of prototrophic conidia in sorboseŽ . Ž . 5 Ž . 6 Ž . 7minimal medium. The following numbers of W40 conidia were added: a none; b 3=10 ; c 3=10 ; d 3=10 . Note the paucity of

prototrophic colonies on the petri dishes containing non-growing W40 conidia.

resulted from a mutagenic event, rather than from thekilling of a suppressing auxotrophic population. Aquantitative example of how the lethal effects of UVradiation could mimic a mutagenic effect was dis-

w xcussed in Ref. 2 . In this experiment mutation fromŽ y.leucine requirement leu to non-requirement

Ž q.leu was scored. The population of auxotrophicleuy conidia used in this study contained pro-totrophic conidia that had arisen by back-mutation.However, when 5=107 of these leuy conidia wereadded to petri dishes containing the selective mediumŽ .lacking leucine no prototrophic colonies appeared.Plating a one-tenth dilution, i.e., 5=106 conidia,yielded 22.6"3.5 prototrophic colonies; a one hun-

Ž 5 .dredth dilution 5=10 conidia resulted in morethan 200 prototrophic colonies; and 5=104 gave17.5"0.8 colonies. Growth of the cryptic pro-

totrophic conidia was evidently being suppressed byŽ 5. ythe presence of more than 10 leu conidia. When

an aliquot of the original cell population was irradi-ated with UV-light and spread on selective medium,the observed yield of prototrophic colonies could beexplained simply by the lethality of the UV treat-ment, which reduced the number of viable conidia onthe petri dishes, thus allowing previously suppressedcryptic prototrophs to form visible colonies. In thiscase the competitive suppression completely con-founded the detection of the mutagenicity of theUV-radiation. Thus, competitive suppression is afactor to be considered in performing microbial mu-tation experiments or in assessing the reliability ofpublished mutation experiments. An analysis of theresults of many back-mutation experiments carriedout in the late 1940’s and early 1950’s suggested that


competitive suppression was biasing their proper in-w xterpretation 3 .

(5. Detection of competitive suppression ‘‘Grigg)Effect’’

To test for an inhibitory effect by auxotrophiccells, one can set up reconstruction experiments thatreproduce the conditions of cell concentration, selec-tive medium, etc., closely. It is sometimes difficult,however, to detect competitive suppression — par-ticularly when evaluating the reliability of resultsfrom other laboratories. An indication that competi-tive suppression is operating can emerge from check-ing the frequency of prototrophic colonies in control

Ž .plates against the concentration of cells cellsrplateused in each experiment. Such correlations are mostinformative if they are calculated from a single cellpopulation as I did when plating various numbers ofleuy Neurospora conidia. A deficiency in pro-totrophs in plates containing larger numbers of aux-otrophs is suspicious. Unfortunately it is often notpossible to extract such information from publishedpapers these days because journal editors usuallydemand that the experimental data be condensed andin some cases even summarised.

w xAs I mentioned earlier, Ryan and Schneider 24noted that prototrophic cells of E. coli did notmultiply in basal medium if there were 7=109 hisybacteria or more per 5 mg of glucose. The effect issimilar to what I found in Neurospora. Such caseshave also been described with other bacterial strainsw x29,30 . Observations of an apparent negative corre-lation between frequency of spontaneous revertantsand concentration of cells plated in experiments with

w x w xE. coli 31,32 and Pseudomonas fluorescens 33are readily explicable by competitive suppression.

6. Mechanism of competitive suppression

In both N. crassa and E. coli the inhibition ofprototrophic cells by large numbers of auxotrophsproved to be a starvation effect. The non-growingcells remove and utilise the carbohydrate in themedium, leaving insufficient energy source to sup-

Ž .port the growth of the few prototrophs into visiblew xcolonies 3 . The extent of suppression in my experi-

ments reflected the concentration of the available

energy source, the concentration of auxotrophic cells,and the rate of germination and growth of the pro-totrophs. More rapid growth meant less suppression.

I had investigated the mechanism of competitivesuppression while still in Cambridge and a few yearslater returned to the topic in Australia to clear upsome details. I moved to Adelaide in 1953 to join myold PhD supervisor Prof. D.G. Catcheside at theDepartment of Genetics, where I was employed byCSIRO. There I extended my studies of the mecha-nism of the effect by measuring glucose uptake byauxotrophic Neurospora conidia. It was no surpriseto confirm that the auxotrophs removed and utilisedglucose from the medium, but to find that glucoseuptake by auxotrophic conidia for the first 2 daysafter plating equalled that by growing prototrophicones in the minimal medium was unexpected. 6

6 To determine rates of utilisation of glucose by the auxotrophicNeurospora conidia, I used a glucose-specific manometric methodbased on the oxidation of glucose catalysed by glucose oxidase.This study almost cost me my life. The Warburg equipment onwhich the manometric experiments were performed was housed ina basement room in the Biochemistry Department of AdelaideUniversity. The machine I used had just been acquired — in fact Iwas its first real user, after a technician from Biochemistry hadcalibrated the manometers. The Warburg machines were neverswitched off at the main switch by the staff of the BiochemistryDepartment. Being a new boy to manometry I was more conserva-tive than the others and carefully turned the ‘‘mains’’ switch toOFF on the new machine before setting up the experiments,grinding in the manometer cups, etc. It turned out subsequentlythat my machine had been manufactured with a potentially lethalwiring fault. When it was switched on, the unshielded wireleading down to the mercury reservoir of the mercury toluenethermostat carried only 10 V — as it was designed to do. When itwas switched off, however, this bare unshielded wire carried thefull mains voltage, viz 240 V AC. On accidentally touching thebare ‘‘live’’ wire my arm flexed violently in an involuntarymovement knocking the wire into the water bath where my handswere immersed up to the wrist. The floor was bare concrete andwet;, the water was a conducting fluid and ‘‘live’’, and I was incomplete tetany, vibrating at 60 times per second in time with the60 cycle AC current. I should have been electrocuted, but Iwasn’t. After what seemed minutes but must have been onlyseconds, I managed to collapse away from the electrified waterbath. Before doing so, however, I smashed some six or sevenmanometers with my bare hands. All in all it was an expensivemorning’s work, since my hands needed minor surgery. Undoubt-edly my old and well worn rubber-sole shoes saved my life. I havebeen an advocate of the use of rubber soled shoes in laboratoriesever since. The offending Warburg machine was returned to itsmaker for modification, but I escaped this indignity.



While increasing the concentration of the energysource in the selective medium diminishes the risk ofcompetitive suppression, other components of themedium can play a role too. For example, in bacte-rial experiments the use of a ‘‘semi-enriched’’

Ž .medium enriched with a small amount of broth ,instead of a synthetic basal medium enriched with aspecific growth factor, to allow several post-platingcell doublings on the agar plate, resulted in signifi-cantly increased risk of competitive suppression bias-

w xing the results 29,34 , even though the amount ofextra growth on the two types of media was identi-cal. The reasons for this phenomenon are not known.

ŽSince the numbers of auxotrophic bacteria and pro-.totrophic revertants were comparable in basal and

semi-enriched media, numbers alone cannot be thecause. Presumably one has to look at altered physio-logical states of the auxotrophs, the prototrophs, orboth, for an explanation.

7. Does competitive suppression invalidate allback-mutation experiments?

w xClearly this is not the case 3 . With propercontrols and the use of appropriate mutant strainsthat have low background frequencies of prototrophs,together with media that minimise the suppressiveeffect, good quality data can be collected. However,ignoring the possibility of competitive suppression isrisky.

8. Prevention of competitive suppression

Ž .Since competition for a limiting nutrient a sugarbetween auxotrophs and newly arisen prototrophs isthe cause of the suppression effect, use of a highersugar concentration in the medium should ameliorateits magnitude. But there are limits to the sugarconcentration tolerated by both Neurospora and E.coli. There is another approach that seems to workand which I shall describe shortly.

By the mid 1950’s a better understanding of thegenetics of E. coli and related bacteria and of thechemical nature of genes led to greater acceptance ofbacteria as appropriate organisms for studying DNAdamage and mutagenic events. I became converted to

the usefulness of E. coli as an experimental toolduring an extended visit to Francis J. Ryan’s labora-tory at the Zoology Department of Columbia Univer-sity, NY, in 1960r1961. At this time Ryan’s stu-dents were devising biological methods of determin-ing the nature of base substitutions in a series ofmutant strains. One student was working on themechanism of stationary phase mutation — a subjecton which Ryan made some important contributionsw x35–38 . It was not until I returned to Sydney that Iwas able to suggest a connection between DNArepair-related DNA turnover and stationary phase

w xmutation 39,40 . Meanwhile at Columbia I foundthat X-irradiation induced back-mutation of transi-

w xtion mutations in E. coli 41 . It was during thisperiod that I came to the idea that the problem ofcompetitive suppression in mutation assays would besolved if one could prevent auxotrophic cells fromutilising the available energy source in the mediumwithout interfering with the potential of prototrophicback-mutants to use this energy source for growth.

This goal could be achieved by growing an E.coli amino-acid requiring strain having a Lacq geno-

Ž .type e.g., WP-2 in glucose medium to exhaustionŽof the specific required growth factor tryptophan in

.the case of WP-2 . The lac operon in the bacteriawould then be in a repressed state. This bacterial

Ž .population after treating with a mutagen could bespread on a selective minimal medium having lac-tose as the energy source. In the absence of proteinsynthesis, b-galactosidase would not be induced inthe repressed bacteria, so the auxotrophs would beunable to utilise lactose. Back-mutant prototrophscould synthesise proteins, however; so that b-galactosidase could be induced, lactose could beutilised for growth and the newly-arising prototrophswould form visible colonies. When put to the test,this simple protocol worked well and allowed the useof very high bacterial numbers per petri plate with-out generating problems of competitive suppressionw x42 . Another advantage of using lactose as the en-ergy source in E. coli is that the lac-repressedbacteria survive for long periods on the lactose agarplates. This means that background prototrophs in anauxotrophic population spread on such plates andallowed to grow for a short period can be identifiedprior to performing mutation experiments with theauxotrophs. This is particularly useful in identifying


mutagenic effects that are small in relation to thefrequency of background prototrophs in the aux-

w xotrophic population 43 .

9. Other sources of error in back-mutation exper-iments

Phenotypically prototrophic colonies that arise inback-mutation experiments do not always prove tobe stable when tested further.

Forty to fifty years ago when mutation experi-ments using microorganisms were in their infancy,the stability of revertant prototrophic colonies was

Žchecked routinely by re-streaking them on test me-.dia to verify that they were still prototrophic . Some

prototrophs would also be crossed back to the parentalgenotype to check that each was the result of aback-mutation rather than a suppressor mutation atanother locus. Unfortunately such tests are rarelyused today. Unexpected complexities in the back-mutation process suggest that it would be wise toadopt a more conservative strategy in planning suchexperiments. If scientists restrict their experimentalmethods to detecting only the results that they ex-pect, they may well miss some fundamental truths. Ishall give a few examples of observations made inmy laboratory in conducting back-mutation experi-ments, which would not have been expected if con-ventional criteria had been applied. No doubt manyother workers have had similar experiences.

9.1. Example 1: cytoplasmic effects

The ability of a genotypically prototrophic cell togerminate and grow into a colony on minimalmedium can be affected by the presence of otherauxotrophic components of its cytoplasm. In separateexperiments when microconidia and macroconidiafrom heterocaryons of composition hisyrhisq of N.crassa were plated on minimal medium and onhistidine-supplemented medium a substantial deficitof colonies was observed on the minimal agar platesw x3,44 . The difference could not be explained by thenumber of histidine-requiring colonies scored. More-over, cryptic prototrophic conidia that did not germi-nate on the minimal plates could be rescued bysubsequent supplementation with histidine. The exis-

tence of such cryptic prototrophs could be a sourceof error in mutation experiments. Most mutagens aretoxic and cell-killing may be accompanied by therelease of growth factors such as histidine into themedium, permitting growth of this cryptic cell popu-lation. Unfortunately, the cryptic prototrophic popu-lation in the control group would remain unidenti-fied.

9.2. Example 2: unstable prototrophs

In some studies a high enough proportion ofprototrophic revertants in Neurospora and E. coliare unstable to ensure that the interpretation of re-

Žsults can be affected Ryan personal communication;w x.Refs. 41,45 .

9.3. Example 3: heterogeneity of the ‘‘back-muta-tional product’’

In some early studies of UV-induced mutationwith a uninucleate hisy strain of N. crassa most ofthe prototrophic colonies which appeared were hete-

w xrocaryons 46 . When the homocaryotic componentsof these heterocaryons were isolated and tested theyproved to have neither a prototrophic nor a mutantcharacter identical to that of the parental strain or ofthe original prototroph. Each pair of genotypes, how-ever, complemented each other so that the hetero-caryon gave a normal prototrophic phenotype. Itseemed as if these back-mutant phenotypes were dueto two coincidental and complementary events insister DNA strands or sister half strands. Superfi-cially at least, these results had similarities to those

w x w xreported in Serratia 47 and in E. coli 41 . Thus,the prototrophic colonies were not due to a simpleback-mutation at a particular site in the gene. Theseresults are not easily explicable by conventionalprocesses, and warrant further investigation.

10. Epilogue

Ž .The ‘‘Grigg Effect’’ competitive suppression isstill remembered in some labs, but many now ignoreit when performing mutation experiments, despitethe fact that it is as pertinent now as it was 50 years



ago. Unfortunately, it is no longer easy to deducefrom published data whether the ‘‘Effect’’ is biasinga mutation experiment or not. Fifty years ago it wascommon practice to publish detailed experimentaldata so that readers could further analyse them bysuch means as plotting the frequency of back-mutantcolonies as a function of cell density to see if theexpected relationship was actually observed. Todayjournal editors do not usually allow such a ‘‘waste ofspace.’’ As a consequence, the quality of the infor-mation is often difficult to assess, and phenomenanot expected by the authors cannot now be dug outof the published data by an imaginative reader.

Acknowledgements

I wish to thank Drs. George Hoffmann, RobinHolliday and Donald MacPhee for their helpful edi-torial assistance, Ms. Jenny Young for her skills andpatience in typing the manuscript, and the Editor ofNature for permission to reproduce the photograph inFig. 1.

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Mutation Research 463 (2000) 173–178


Somatic mutations and ageing1

Robin Holliday∗

12 Roma Court, West Pennant Hills, NSW 2125, Australia

Received 26 July 1999; accepted 27 September 1999

Keywords: Ageing; Multiple theories of ageing; Free radical theory; Somatic mutation theory; Senescence

Early discussion of the relationship betweenchanges in genes and ageing did not always dis-tinguish three fundamental possibilities. The first isdamage to genes, which we now know only occasion-ally leads to mutations. The second is chromosomalabnormality, such as a re-arrangement, deletion, orchange in chromosome number. The third is mutationitself, which is a stable heritable change in gene struc-ture. There was also the theoretical proposal, whicharose largely from discussion of the genetic effects ofionising radiation, that the genetic “hit” might be theimportant event in ageing. This was developed to ahigh level of mathematical sophistication by Szilard[1], who concluded that the gene was too small a tar-get for hits, but instead the whole chromosome mightbe the important target.

Discussion of the importance of genetic damage inageing received support from many studies on the ef-fects of ionising radiation in shortening the lifespan ofmice (reviewed in Ref. [2]). There is not much doubtthat this premature ageing is very similar to, if not

∗Tel.: +61-2-9873-3476; fax: +61-2-9871-2159.E-mail address: [email protected] (R. Holliday).1 This article is part of the Reflections in Mutation Researchseries. To suggest topics and authors for Reflections, readersshould contact the series editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]).

identical with, the natural ageing of untreated animals[3]. The facts are not in doubt, but the experimentsprovide little information about the nature of the ge-netic damage, assuming that the important target is infact DNA. One of the early supporters of the somaticmutation theory was Curtis [4], but his experimen-tal observations were on chromosome aberrations. Hefound that their rate of accumulation in liver cells wascorrelated with lifespan. It was faster in short-lived in-bred strains of mice than long-lived ones, and slowerin dogs. Harman [5] was the first to draw attention tothe possible importance of oxygen free radicals as acause of ageing. This was also compatible with thelife-shortening effects of ionising radiation, becausesuch radiation has its effects through the formation ofactive radicals. Until recent years, the free radical the-ory of ageing did not receive much attention.

My own involvement in the study of mutations andageing came from two directions. The first was thediscovery by Hayflick and Moorhead [6,7] that nor-mal human diploid cells have finite lifespan in cul-ture. What was the cause of the ultimate senescenceof these cultured cells? The second was an interest inOrgel’s protein error theory of ageing [8]. As origi-nally formulated, this had nothing whatsoever to dowith genetic damage or gene mutation. It was basedon the supposition that protein synthesis is unlikely to

1383-5742/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S1383 - 574 2 (00 )00048 -X


Somatic mutations and ageing 1Robin Holliday *

12 Roma Court, West Pennant Hills, NSW 2125, AustraliaReceived 26 July 1999; accepted 27 September 1999

174 R. Holliday / Mutation Research 463 (2000) 173–178

be completely accurate, so it is possible that primaryerrors in the synthesis of protein molecules could feedback into the processes of transcription or translation,and thereby cause further errors. In this way, the levelof errors might gradually increase to a lethal “errorcatastrophe” in protein synthesis. In its original form,this was a cytoplasmic theory of ageing, but it wassoon realised that a general breakdown in the accuracyof information transfer would also affect DNA syn-thesis itself, so that mutations would be expected toincrease during ageing as well [9–11]. However, thespecific prediction was that mutations would increaseexponentially rather than linearly with time. With re-gard to the senescence of human cells, Hayflick [7]had already suggested that this might be due to theaccumulation of multiple events or hits, and Sakselaand Moorhead [12] had demonstrated a very signifi-cant increase in chromosome abnormalities in the finalsenescent phase of growth. Early attempts to test theprotein error theory of ageing were carried out withDrosophila and fungi [9,13,14]. Two strains of Neu-rospora were used which had finite growth in culture,nd (natural death) and leu-5, which was a temperaturesensitive leucine auxotroph with a finite lifespan atthe restrictive temperature (35◦C). It was shown thatleu-S became a mutator strain when grown at 35◦C[9]: both back- and forward-mutation rates were seento be very significantly increased when conidia wereplated at the permissive temperature (25◦C).

The measurement of mutations normally dependson plating cells on a medium or under conditionswhere only mutants can grow, or alternatively, whererare mutant colonies can be distinguished from a largebackground of non-mutant ones. In the case of culturedhuman cells, this cannot be done, because senescentcells have run out of growth potential. This problemcan be circumvented if mutations could be detected insingle cells, rather than as whole colonies. To this end,a histochemical assay for mutations was developed bymy student Stephen Fulder, specifically for use withhuman fibroblasts of different age [15]. It was basedon the observation that certain variants in the enzymeglucose-6-phosphate dehydrogenase (G6PD) coulduse the analogue substrate deoxyglucose-6-phosphate(dG6P) more efficiently than the normal substrate.Therefore, a histochemical assay for G6PD activitywas developed with only dG6P as substrate. Sureenough, rare cells were detected with strong staining

amongst a background of very weakly stained cells.However, it was subsequently shown that these cellssimply had an enhanced level of G6PD activity, asthey could also be detected using a low level of thenormal substrate (G6P) in the reaction mixture. Alarge number of observations showed that the numberof these cell variants increased exponentially duringthe growth and senescence of human diploid cells[15]. This is what would be expected if the cells werelosing their fidelity in information transfer, since anyfeedback model necessarily gives an exponential in-crease in defects or errors. It was also shown that thephenotype of increased G6PD activity was heritablein young cultures, but it could not be proved that thevariants seen were actually mutations. These resultswere certainly compatible with the possibility thatregulatory mutations occurred during ageing whichvery significantly increased the level of G6PD in thecell. One of the most widely used assays for mutationin animal or human cells is resistance to 8-azaguanineand 6-thioguanine, which is due to loss of activityof hypoxanthine phosphoribosyl transferase (HPRT).Gupta [16] applied this assay to human fibroblasts,and found no increase in frequency during serial pas-saging. Since the assay depends on the growth ofcolonies in the presence of one or other analogue, itis not possible to apply it to senescent cells. Thus,Gupta’s results are not in fact incompatible with thoseobtained in my laboratory by Fulder.Another approach was to actually look at the fidelity

of DNA polymerase in cell free extracts obtainedfrom cells of different age. An assay for fidelity wasavailable, in which the misincorporation of a labellednucleotide triphosphate is measured using an oligonu-cleotide template with no complementary base. Thefirst studies were carried out by Stuart Linn fromthe Department of Biochemistry, Berkeley, who wasvisiting my laboratory for a year. The results werevery promising, and were submitted for publicationin Nature. Our manuscript was rejected on groundswhich Linn, the senior author and a world expert inDNA enzymology, found incomprehensible. He askedBruce Ames to communicate the paper to the Pro-ceedings of the National Academy of Sciences, USA,where it was published [17]. At that time, Nature andThe Times newspaper ran a Nature Times ScienceNews column which summarised important currentadvances. I was greatly amused to see in The Times


R. Holliday / Mutation Research 463 (2000) 173–178 175

a report of our studies on the loss of fidelity of DNApolymerase during the senescence of human cells.This work was followed up by a much more detailedstudy by my student Vincent Murray [18]. A largeproportion of Vincent’s experimental work compriseda series of important controls to rule out various pos-sible experimental artefacts. These two studies cer-tainly do not demonstrate that DNA polymerases insenescent cells have alterations in primary structure.Moreover, other experiments in Stuart Linn’s ownlaboratory in Berkeley showed that young cells heldconfluent for long periods contain inaccurate forms ofDNA polymerases �, �, and � [19]. The interpretationof all these results has never been resolved.

When Szilard [1] published his theoretical paper onthe possibility that genetic “hits” may cause ageing,it was strongly criticised by Maynard Smith [20,21].He assembled evidence against the somatic mutationtheory, and particularly telling was the absence ofstrong differences in longevity in animals with dif-ferent ploidy levels. Recessive mutations should beexpressed in haploid animals, and not in diploid ones.In the case cited, the wasp Habrobracon, the differ-ences in longevity of animals with different ploidywere much less than would be expected from thesomatic mutation theory of ageing. However, adultinsects consist very largely of non-dividing cells andthey are highly resistant to ionising radiation. Theyare therefore a poor model for what may be occurringin vertebrates during ageing.My colleague Katherine Thompson confirmed the

earlier study of increased chromosome abnormalitiesduring the senescence of human cells [22]. During thecourse of this work, she discovered that populationsof young cells which survived colchicine treatmentcontained a substantial proportion of tetraploid cells.These grew at the same rate as the normal ones. Thequestion therefore arose as to whether these cells withfour copies of the genome would have a longer life-span than the normal diploid cells. The experimentswere carried out and it became clear that diploid andtetraploid cells had the same lifespan [23]. This wascertainly strong evidence against the possibility thatthe accumulation of recessive mutations was importantduring ageing.

At about the same time, my colleague Tom Kirk-wood and I thought that a theoretical study wouldbe very worthwhile. We were also stimulated to un-

dertake the study by the “mortalisation” theory ofsomatic cell ageing proposed by Shall and Stein [24].We set up a plausible model [25] which assumed thatthe targets for mutational hits are single indispensablegenes. We assumed that there are 103–104 such genesper haploid genome, and that hits on both homo-logues would be necessary to inactivate a cell, or stopit growing. We also took into account the fact that theX chromosome, which comprises 5% of the genome,is essentially haploid (since X inactivation occurs infemales). A single mutation in an X linked indispens-able gene would kill or inactivate the cell. We foundthat to explain the “Hayflick limit” to cell growth, thesomatic mutation rate would have to be unacceptablyhigh, in fact, in the region of 10−3–10−4 mutations pergene per generation. We also found that the numberof non-cycling cells which would be seen in popula-tions which were still growing would be much higherthan had actually been observed (using 3H thymidinelabeling). Finally, our conclusions were not compati-ble with the mortalisation theory of aging. This statedthat during the finite lifespan of human fibroblasts,the probability of a daughter cell never dividing againincreases as a function of the generation number i.e.,the number of population doublings the culture hasachieved. The predictions of the somatic mutation andmortalisation theories are in fact quite similar with re-gard to the rate of increase of non-dividing cells [25].

In all the work that I have so far summarised,whether experimental or theoretical, none relates di-rectly to the actual measurement of somatic mutationsduring the ageing of real organisms. In fact, up tothe early 1980s, no such data were available in man,mouse, or any other animal. The best available dataconcerned chromosomes, since there had been exten-sive documentation of chromosome abnormalities inhuman lymphocytes, and it had been demonstratedthat these increased with age [26]. These resultshave been supported by more recent studies, whichmeasured the frequencies of micronuclei during thehuman lifespan [27]. Such micronuclei are formedby single chromosomes, or chromosome fragments,which become enclosed within a nuclear membrane.

Alec Morley came from the Department of Haema-tology, Flinders Medical Centre, to work in my lab-oratory for a year. He wanted to study real cells in areal organism, rather than cultured human fibroblasts.Preliminary experiments by Strauss and Albertini


[28] had shown that stimulated human lymphocyteswere killed by 6-thioguanine, or at least did not enterS phase. Cells which lack HPRT are resistant andbecome labeled with 3H thymidine. Alec refined theearlier technique and applied it to samples of lympho-cytes taken from individuals of different age. Sincehe was a clinician he was able to take blood fromindividuals in the laboratory, as well as from thosein a home for the elderly, and a few children. For thevery first time, the frequency of bona fide mutationswas being measured in individuals of different age.The results supported the somatic mutation theory;at least, they showed that mutations were increasingwith age. The increase was closer to exponential thanlinear, but it was not statistically incompatible with thelatter. The results were submitted to Nature, and therewas every reason to believe that they were suitablefor publication. After all, they documented for thevery first time a highly significant increase in somaticmutations with age. Time went by, and no editorialdecision was made. Our manuscript was either putaside or lost, and results by Evans and Vijayalaxmi[29], using the same method but submitted after ours,were published instead. Moreover, their results werea good deal less clear cut than ours. Our paper waseventually withdrawn and published in Mechanismsin Ageing and Development [30].

Alec Morley and his colleagues have continuedtheir work on mutations in human lymphocytes tothis day. They have developed a new assay, which isbased on the loss of histocompatibility (HLA) alleles[31]. Antibodies to an allele at the A locus specifi-cally select cells which have lost that particular allele.This means that cells are selected which have eitherbecome homozygous by recombination, or have lostan allele by mutation. Further molecular analysis candistinguish between these possibilities. The increasein mutations with age has been confirmed [31]. Inaddition, HPRT− mutations in mouse lymphocyteshave also been measured and shown to increase withage [32]. Remarkably, it was also found that calorierestriction, which is well known to increase lifespan,reduced the age-related accumulation of mutations.This provides some of the best evidence for a rela-tionship between ageing and somatic mutation.

The frequencies of mutations in mouse and humanlymphocytes were similar, so it was first thought thatthe rates are also comparable. But this is not the

case, because the observed increase in the incidenceof lymphocyte mutation in mouse occurs over a farshorter time span than in man. When this is taken intoaccount, it can be calculated that the rate of mutationis very significantly higher in the shorter-lived species[33]. This is a very satisfying result, because thereis very good evidence that the efficiency of severalDNA maintenance functions, including the repair ofUV or UV light induced lesions, is correlated withlongevity [34,35].

Some investigators have believed that modernmolecular methods for detecting mutations in cellscan be applied to studies of cells during the ageingof organisms. The problem is that although each cellmay have many mutations, these are randomly dis-tributed in the genome. Therefore a probe, or PCR,which studies only one gene is not very sensitive. Itmust be capable of detecting, say, one mutation in105 cells, and current procedures are not really sensi-tive enough to detect this frequency. Nevertheless, wecan expect new molecular methods to be developed,which will monitor mutation rates in specific genesduring ageing.Specific theories of ageing are often based on the

supposition that there is one major cause of ageing.The somatic mutation theory proposes that changesin DNA are responsible for the changes that bringabout senescence and death [36,37]. These changescan include damage to DNA, heritable mutations, andchromosome abnormalities. Although the frequencyof mutation per gene is low, the number per genomemay be fairly high, perhaps around one mutation percell generation. An adult is the cumulative end resultof 40–50 cell divisions, so it would be expected thatevery cell has a significant number of gene mutations.These will affect, in one way or another, proteins, reg-ulation, membranes, organelles, the immune responseand so on.

In addition to mutations, it can be argued that sometypes of DNA damage are not repaired. Lindahl [38]has pointed out that damage which is rare may simplybe tolerated by organisms when there has not beensufficient selective pressure for the evolution of a re-pair pathway. In long lived organisms such as man, theaccumulation of such damage may be significant, andit will give rise to background “noise” which couldcontribute to ageing. Such noise could take the formof disruption to transcription, and possibly the forma-


R. Holliday / Mutation Research 463 (2000) 173–178 177

tion of aberrant protein molecules. The age-dependentDNA modifications known as I-compounds may bean example of DNA damage which is not attributableto mutation. It could be particularly important innon-dividing cells such as neurons.

Another type of DNA change is likely to be of con-siderable importance. It is now well established that5-methyl cytosine in DNA has a very significant rolein the control of gene activity. The abnormal loss orgain of such methylation in the promoters of genes, orin other genetic contexts, is very likely to be an im-portant age-related change in DNA. These alterationsare now known as epimutations because they are her-itable, but not due to changes in DNA sequence [39].It must be admitted, however, that the evidence thatepimutations increase during ageing is so far mainlyindirect (reviewed in Ref. [34]).

The mutation theory has received support frommany studies of reactive oxygen species [40]. Theseinteract with DNA and can cause mutation. Indeed,there is considerable overlap between the free rad-ical theory of ageing, which is currently in favour,and the somatic mutation theory. Also, it has beenshown without doubt that mutations accumulate inmitochondrial DNA, and it is known that the ratesof such mutation are very significantly higher thanin chromosomal DNA [41]. Not surprisingly, thereare adherents of the mitochondrial theory of ageing.Enough is now known about the many changes thatoccur during senescence and ageing to be sure thatthe overall process, or set of processes, is multicausal.It is hard to see how mutations influence long-termchanges in proteins such as collagen, and crystallin inthe eye lens. These proteins can last a lifetime. It iswell established that collagen becomes progressivelycross-linked with age, and crystallin suffers from anumber of post-synthetic changes, including deami-dation, glycosylation, oxidation, and so on. Also, itis unlikely that the many changes which occur in thewalls of major arteries as we age are due to mutations.The same can be said of the wearing out of joints, andof several other changes during ageing. If ageing isindeed multicausal, then it is likely that DNA damageand mutation contribute to ageing, but it is extremelyunlikely that all the changes seen are due to mutations.The somatic mutation theory is just one of severaltheories which have to be taken seriously. I have ar-gued elsewhere that all major theories of ageing are

likely to be correct, and that we should adopt a globalview, which encompasses features of all of them [34].

So what is the situation at the end of the century withregard to the status of the somatic mutation theory?We know that mutations and chromosome changesaccumulate with age, and no doubt further documen-tation will become available (see, for example Ref.[42]). It is not yet clear whether DNA damage otherthan mutation accumulates, and this needs furtherstudy. There is much evidence that many mutationsoccur in mitochondrial DNA, but given the number ofmitochondria per cell, it is not yet clear whether respi-ratory function is significantly affected. Future workwill better establish the relative importance of changeat the DNA level, post-synthetic changes in proteins,and general “noise” which could include a breakdownin the accuracy of pathways of information trans-fer, or abnormalities in regulation and homeostaticmechanisms. This research should have high priority,because it will enable us to better understand all thesechanges which give rise to the many age associateddiseases, which in toto consume an ever-increasingproportion of health care costs in developed nations.

Acknowledgements

I thank Alec A. Morley for reading and commentingon the manuscript.

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[31] S.A. Grist, M. McCarron, A. Kutlaca, D.R. Turner, A.A.Morley, In vivo human somatic mutation: frequency andspectrum with age, Mutat. Res. 266 (1992) 189–196.

[32] J.L. Dempsey, M. Pfeiffer, A.A. Morley, Effect of dietaryrestriction on in vivo somatic mutation in mice, Mutat. Res.291 (1993) 141–145.

[33] A.A. Morley, Somatic mutation and ageing, Ann. N. Y. Acad.Sci. 854 (1998) 20–22.

[34] R. Holliday, Understanding Ageing, Cambridge Univ. Press,Cambridge, 1995.

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[36] F.M. Burnett, Intrinsic Mutagenesis: A Genetic Approach toAging, Wiley, New York, 1974.

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Lysenko and Stalin: Commemorating the 50th anniversary of theAugust 1948 LAAAS Conference and the 100th anniversary of

T.D. Lysenko’s birth, September 29, 1898 1

Zhores A. Medvedev4 Osborn Gardens, Mill Hill, London NW7 1DY, UK

Ž .Translated from Russian by Charles Severens

Keywords: Reflections; Lysenko; Stalin; Vavilov; Lamarckism; Soviet science

On July 27, 1948, after a 10-day interruptioncaused by an unknown malady and late in the eveningas was his habit, Stalin made an appearance in hisKremlin office. At 10 min past 10:00 that eveningtwo other people arrived at Stalin’s office: Malenkov

w xand T.D. Lysenko 1 . They anticipated that Stalinwould turn over to them with his stamp of approval areport by Lysenko entitled ‘‘On the Situation inBiological Science.’’ Malenkov had sent this reportto Stalin’s country home in Kuntsevo on July 23rd.Malenkov had already read Lysenko’s paper and hadmade no comments. He and Lysenko were startled tofind that Stalin had made a number of changes andcorrections as well as critical comments in the mar-gins of the pages. In the course of 1 h, as Lysenko

w xhimself later noted 2 , Stalin ‘‘gave me a detailedexplanation of his corrections and instructions onhow better to present particular parts of my report.’’An hour later, at 11:10 p.m., they were joinedby Beria, Bulganin, Mikoyan, Voznesensky and

1 To suggest topics or authors for Reflections, readers shouldcontact either of the editors by mail at the addresses shown on theinside front cover or by e-mail:

Ž .G.R. Hoffmann [email protected] , D.G. MacPheeŽ [email protected] .

Kaganovitch. There ensued an hour-long discussionof certain problems. Lysenko, in particular, was toldby Stalin to announce at the final assembly of theconference that the report had been examined andapproved by the Central Committee of the All-Union

Ž .Communist Party Bolshevik , in other words he wasto make an announcement about something which infact had not taken place.

The status of Lysenko as the President of theLenin All-Union Academy of Agricultural SciencesŽ .LAAAS grew steadily more unstable after the endof WWII. This crisis came to a head in April of 1948at a seminar for the regional party cadre when YuriZhdanov openly criticized Lysenko and his

Ž‘‘Michurin biology.’’ Michurin biology takes itsname from the Russian horticulturalist I.V. Michurin,whose thoughts on plant breeding ran counter to

.Mendelian genetics. Yuri Zhdanov was the son ofA.A. Zhdanov, a member of the Politburo. Theyoung Zhdanov, although only 29 then, had an ad-vanced degree in chemistry thanks to the connectionsof his father and of Stalin himself. Yuri was, afterall, Stalin’s son-in-law by virtue of his marriage toSvetlana. Not surprisingly, Yuri held the impressivepost of head of the Central Committee’s ScienceDivision which neither his scientific nor Party expe-



Lysenko and Stalin: Commemorating the 50th anniversary of the August 1948 LAAAS Conference and the 100th anniversary ofT.D. Lysenko’s birth, September 29, 1898 1Zhores A. Medvedev

4 Osborn Gardens, Mill Hill, London NW7 1DY, UKTranslated from Russian by Charles Severens.

( )Z.A. MedÕedeÕrMutation Research 462 2000 3–114

rience warranted. Yuri Zhdanov criticized Lysenkoon many points and inadvertently revealed that hewas expressing his own opinion and not a new lineof the Party. However, the regional party cadreaccepted the report unconditionally as if it were adirective. Lysenko was not present in the auditoriumbut listened to Zhdanov’s speech through a hook-upin the office of M. Mitin, a professor of philosophy.Lysenko was very alarmed. On April 17th, he sent aletter of protest to Stalin, Chairman of the USSRCouncil of Ministers, and to A.A. Zhdanov, Secre-tary of the Communist Party’s Central Committee.Failing to get an answer after a month, Lysenko senta formal letter of resignation from his post as Presi-dent of LAAAS to the Minister of Agriculture, I.A.

w xBenediktov 3 .Inasmuch as Benediktov could not resolve such a

problem independently, Stalin’s intervention in oneform or another was inevitable. The opportunity forthis arose on May 31st when the Politburo met toconsider candidates for Stalin prizes in science andinvention. Traditionally, Stalin himself would an-nounce the final recommendations for recipients ofthe ‘‘first rank’’ of awards. Present at this session inaddition to members of the Politburo were Vyach-

Ž .eslav Malyshev Minister of Shipbuilding , S.V. Kaf-Ž .tanov Minister of Higher Education , and Academi-

Žcian Alexander Nesmeyanov Chairman of the Stalin.Prize Awards committee .

V. Malyshev noted the next day in his diaryrecently uncovered in party archives: ‘‘Before re-

Ž .viewing these questions about the awards , ComradeStalin brought everyone’s attention to the fact that

Ž .Yuri Zhdanov the son of A.A. Zhdanov had deliv-ered a lecture condemning Lysenko and, in so doing,stated himself that he was expressing his own per-sonal opinions. Comrade Stalin said that personalopinions and personal points of view had no place inthe Party, and that only the Party could have anopinion. Yuri Zhdanov had set as his goal Lysenko’sdestruction and annihilation. This is wrong.’’ ‘‘Onemust not forget,’’ said Comrade Stalin, ‘‘that Ly-senko today is the Michurin of agrotechnology. Ly-senko has his faults and errors as a scientist and man,and he has to be controlled, but to set as his goal thedestruction of Lysenko as a scientist is like pouring

w x Žoil on the fires of the Zhebrakians’’ 4 . Prof. AntonRomanovich Zhebrak was then the head of the De-

partment of Genetics and Plant Selection at Moscow’sŽ .Timiryazev Academy of Agriculture TAA . In 1947,

Zhebrak was also elected President of the Belorus-sian Academy of Sciences. Zhebrak was an energeticopponent of Lysenko, and it was thought that he was

.the person who advised Yuri Zhdanov on genetics.

1. Malenkov, Zhdanov and Lysenko

Stalin’s remarks meant that some decisions wouldhave to be made. The job of laying the groundworkfor these decisions fell to A.A. Zhdanov since hewas the one answerable to the Secretariat of theCentral Committee and to the Politburo for ideologi-cal, scientific and cultural correctness. He wouldhave to come up with the necessary initiative. Thejob of producing a proposal for the Central Commit-tee’s consideration fell to D.T. Shepilov, editor ofthe newspaper PraÕda and to M.B. Mitin, academi-cian in charge of party philosophy. This report wasfinished on July 7th, edited by A.A. Zhdanov and

w xsent to Stalin 5 . Stalin, however, was against sim-ple directives. He considered it more appropriate toorganize an ostensibly open discussion of Lysenko’soriginal report. Plans for holding an LAAAS confer-ence to elect new academicians had been in theworks since 1947 but postponed several times due tothe fact that Lysenko saw as too slim his chances ofgetting his own cohorts elected to the Academy.Suddenly, there was now an urgent need to preparefor an LAAAS conference which was to start on July31st. Malenkov, rather than Zhdanov, handled thepreparations for the conference.

Instead of electing new members to the academy,the Council of Ministers of the USSR simply an-nounced the appointment of 35 new academicianswhom they had chosen on July 15th from a list of

Žnames submitted by Lysenko. This decision of theCouncil of Ministers was not published in the centralpress until July 28th, after Stalin’s Kremlin meeting

.with Lysenko. Before July 30th, the date on whichStalin got Lysenko’s paper edited by Malenkov andincorporating Stalin’s suggestions, any communica-tion between Lysenko and Stalin was accomplishedwith Malenkov acting as go-between.

Malenkov’s interest in organizing the LAAASconference has led several historians to suggest that


( )Z.A. MedÕedeÕrMutation Research 462 2000 3–11 5

Malenkov was concerned not only in saving Lysenkobut in compromising A.A. Zhdanov who was at thattime Malenkov’s primary rival. Strictly speaking,Zhdanov was ranked second in the party hierarchyafter Stalin. However, while Zhdanov was located inLeningrad during the war, Malenkov was in Moscowtaking charge of all party matters even though hewas not a member of the Politburo but only acandidate for a seat in it. A significant number ofimportant documents being sent by various agenciesto Stalin for his own information or for approvalwere sent also to Molotov and Malenkov. The threeformed a unique triumvirate which ran the countryw x6 .

In 1946, Zhdanov relocated in Moscow and as-sumed the roles of head Party ideologue and over-seer of the activities of foreign Communist Parties.

Ž .The Communist Information Bureau Cominformwas created to replace the earlier disbanded Com-intern. Zhdanov was an extremely conservative Stal-inist whose sphere of activities in Moscow includedamong other things, companies which tried to free

ŽSoviet culture from foreign influences the struggleagainst ‘‘cosmopolitanism’’ and ‘‘reverence for any-thing foreign,’’ the persecution of certain writers and

.composers and the introduction of myriad restric-tions in science. Although Malenkov was elected to

Ž .the Politburo in March of 1946 along with Beria ,Zhdanov managed to squeeze him out of operationalcontrol of the country. Ever since May of 1946,memoranda, especially those from the MVDŽ .Ministry of Internal Affairs , did not get toMalenkov; his name had been crossed off the distri-bution list. Among the names on the distribution list,in addition to Stalin and Molotov, one saw withincreasing frequency those of Beria, Zhdanov, andN.A. Voznesensky. Malenkov was, as is well known,put in charge of the Central Committee of the Com-munist Party of Uzbekistan. However, Malenkovspent precious little time in Tashkent and continuedas before to meet three or four times a week with theother Politburo members in evening and night ses-sions in Stalin’s office.

Zhdanov, despite his anti-Western ideology, wasnevertheless not an advocate of Lysenko and hispromises. This is why he was unsuitable for organiz-ing the LAAAS conference. Almost immediatelyafter the last session of the conference, Malenkov

was returned to the group in charge of running thecountry and on August 19th he became once againeligible to see and receive secret memos and reportsfrom the MVD. Meanwhile, on July 10th Zhdanovwas crossed off the distribution list; this was animmediate consequence of the decision to hold theLAAAS conference and not to convene before theCentral Committee of the All-Union CommunistParty. The latter choice would unquestionably haveprovided good cover for Lysenko from his critics.However, the LAAAS conference with Lysenko’sown report, approved by the Central Committee aswell as by Stalin, made Lysenko the unlimited dicta-tor of science. The LAAAS became an even moreinfluential center of the natural sciences than theUSSR Academy of Sciences itself.

Late in August, A.A. Zhdanov took a vacation atthe Central Committee’s resort in Valdai. Once there,he suffered two heart attacks and died. In 4 years,during the Fall of 1952, it was precisely Zhdanov’sheart attacks which initiated the infamous case of theKremlin physicians which Stalin had conceived as ameans of removing Beria and Malenkov from theinner ruling circle.

2. Stalin’s corrections

Lysenko kept his report with Stalin’s own hand-written suggestions on it in his office and would attimes show it to visitors. After Stalin’s death Ly-senko turned over the original paper with Stalin’scorrections to the Party’s central archives, keepingonly a copy for himself. In 1993 K.O. Rossianov, aresearcher from the Institute of Natural Science His-tory and Technology of the Russian Academy ofSciences, was studying the proceedings of the Au-gust session of the LAAAS and found in the Party’sarchive the original document and was thus able tobe the first to comment on the nature of those

w xchanges and corrections which Stalin had added 7 .Stalin, contrary to expectations, did not use his

usual heavy hand but took it easy on Lysenko. Forinstance he removed all mention of ‘‘bourgeois biol-ogy’’ from the report. Stalin crossed out the sectionentitled ‘‘The false basis of bourgeois biology.’’ Inthe margin next to Lysenko’s statement that ‘‘anyscience is based on class’’ Stalin wrote, ‘‘Ha-ha-ha!!


and what about mathematics? Or Darwinism?’’ Inanother section Stalin added an entire paragraphwhich bore witness to the fact that Stalin had pre-served the neo-Lamarckian convictions of his youthŽwhich one sees in his essay ‘‘Anarchism or social-

.ism’’ of 1906 : ‘‘One cannot deny,’’ adds Stalin,‘‘that in the debate which heated up in the firstquarter of the 20th century between the Weisman-nists and the Lamarckians, the latter were closer tothe truth for they upheld the interests of sciencewhereas the Weismannists abandoned science andbecame addicted to mysticism.’’

Stalin’s remarks showed a decisive departure fromthe theme of the class nature of science in the 1920sand ’30s. Stalin’s world view was clearly influencedby the large advances made in the U.S. and GreatBritain in nuclear physics and in the subsequentcreation of the atomic bomb. By the end of the warStalin had come to realize that progress in scienceand technology was less a matter of ideology thanone of healthy financial support for the scientists.Not everyone recognized this even after the speechthat Stalin gave on February 9, 1946, at a meeting ofMoscow’s Stalin District Electorate at the Bolshoi

w xTheatre. In particular, Stalin said that day 8 , ‘‘ . . . Iam confident that if we give our scientists the helpthey need, they will in the very near future not onlycatch up with but go beyond the achievements ofscience in other parts of the world.’’ This statementwas not just an empty declaration. By March of1946, the allocation for science in the national bud-get had tripled. Scientists and technicians in allbranches of scientific activity received very healthypay increases. However, rejection of the obsoletetheme on the class nature of all sciences, includingthe natural sciences, was not to be taken as Stalin’srecognition of a world science community. Still pre-served was the division of scientific direction andtheory into ‘‘materialistic’’ and ‘‘idealistic’’ camps.The notion of ‘‘Soviet science’’ came now to mean‘‘science of the fatherland’’ in order to emphasizethe succession between the Soviet and the Russian,pre-revolutionary, periods. This broadened the rangeof activities subject to criticism and punishment. Notonly acts which could be classified as ‘‘anti-Soviet’’but also those which would be called ‘‘anti-patriotic’’were now lumped together. Scientists were strictlyforbidden to publish the results of their work abroad.

3. Stalin as a Lamarckian

In many articles about Lysenko in both the Sovietand Western press, the opinion was expressed that hepossessed the special psychological or hypnotic pow-ers of Grigory Rasputin and was thus able to thrustupon Soviet leaders, first Stalin and then Khrushchev,his completely unfounded and false ideas. In factLysenko had no such ‘‘Rasputin-like’’ talents. Hedid not really try to persuade the leaders by over-powering them with his own views. Rather, he gotcaught up in the game of trying to make sense of thesometimes absurd ideas expressed by Stalin and laterby Khrushchev and then creating from thempseudo-scientific assertions. Stalin and Khrushchevwere essentially Lamarckians, which was only natu-ral for the Bolsheviks who were convinced thatanything could be re-made by establishing the rightconditions. ‘‘Existence defines consciousness’’ wasa formula which could be extended to apply to otherqualities and characteristics.

Many people still remember how Khrushchev triedto promote the idea of growing corn in theArchangelsk and Leningrad regions and even‘‘adapting’’ it for Siberia. However, very few peopleremember that the genetics debate which ultimatelyled to the 1948 LAAAS conference did not begin asa scientific quarrel between Lysenko and NikolaiVavilov. Rather, it was the result of decisions madeby Party and government leaders in August of 1931w x9 . The conclusion drawn by these leaders was thatthe nature of agricultural crops could be redesignedin a time frame so short as to contradict everyprinciple of genetic selection. With the intention ofsupporting the collectivization process by introduc-ing new high yield seed varieties, the Soviet govern-ment in the guise of the Central Control Commissionof the Communist Party and the Workers and Peas-ants Inspection Commissariat issued a resolution ‘‘On

w xselection and seed growing’’ 9 . According to thisresolution, the full range of cultivated low-yieldcrops was to be replaced by high-yield varieties overthe entire country in the course of 2 years. Theresolution demanded that new varieties of wheat becreated which could replace rye in the northern andeastern parts of the country. The southern regionswere to get newly created varieties of the potato.Simultaneously, the resolution called for reducing



the time for producing new varieties from 10 or 12years to 4 or 5 years. It was expected that after 4 or 5years Soviet wheat could be high-yield, resilient,with high protein content, non-shedding, cold resis-tant, drought resistant, pest resistant and blight resis-tant. Nikolai Vavilov and the majority of Sovietgeneticists and selection specialists found these goalsto be the products of wishful thinking and quiteunrealistic. Lysenko and his still small group offollowers promised that they would meet these goals.When they were subsequently unable to meet theirpromises, Lysenko et al. explained away their failureby blaming it on the lack of cooperation from thosewho sided with ‘‘bourgeois’’ genetics. The latterwere gradually liquidated during the Terror of the1930s.

Even after the war, Stalin continued to believethat the problems of Soviet agriculture could only besolved by ‘‘re-makes’’ and ‘‘miracle varieties’’ ofone kind or another. In 1947, Lysenko began boast-ing about the unusual prospects of a so-called‘‘branched wheat,’’ seed samples of which he hadreceived from Stalin during their brief meeting on

Ž .December 30, 1946 Fig. 1 . Spikes of wheat hadŽ .been sent to Stalin from Soviet Georgia Gruzia .

However, despite the large spikes that could beproduced, but only by severely thinning out thesowing, this particular variety of wheat — alreadyknown in ancient Egypt — was not only low-yieldbut showed poor resistance to disease and producedflour with a low protein content. The very fact thatLysenko promoted this wheat so widely in 1947already proved that he, worried about the stability ofhis position, used the promotion as a means ofstressing his close relations with Stalin. In fact, therewas no real closeness between Stalin and Lysenko.They were never together in any circumstances otherthan official.

Stalin repeatedly revealed his own initiatives rela-tive to plant ‘‘re-makes.’’ Plants, especially flowersand fruits, were one of Stalin’s hobbies. Stalin’ssummer homes near Moscow and in the south hadgreenhouses which were so situated that he couldenter them alone directly from the house both dayand night. He attempted to grow exotic plants anddid his own pruning.

In his novel, Happiness, the well known Sovietwriter Peter Pavlenko, who lived in Yalta and was

invited to see Stalin whenever Stalin visited theCrimea, put together a dialogue between Stalin and agardener. This conversation was not entirely fic-tional; it reflected Stalin’s actual musings uttered atvarious times. The novel reflects the events of 1945in the Crimea when Stalin went there in the winter toparticipate in the Yalta conference of the leaders ofthe three powers. One of the novel’s heroes, a formersoldier in the frontlines, Voropaev, was invited toStalin’s home. In a light-colored spring tunic and ina light-colored service cap, Stalin stood next to theold gardener by the gravevines. Glancing atVoropaev, he was finishing up showing the gardenersomething in which obviously they both had a seri-ous interest. ‘‘Go ahead and try this method, don’tbe afraid,’’ said Stalin, ‘‘I have checked it myself; itwon’t let you down.’’ But the gardener, confusedlyand at the same time with childlike admiration,glanced at his conversational partner and made ahelpless gesture: ‘‘It’s a little scary to go againstscience, Iosif Vissarionovich. In the days of the tsarthere were some specialists here, but they didn’t sayanything.’’ ‘‘They had plenty of reason to keepquiet,’’ — replied Stalin. ‘‘Under the tsar peoplegrew up in ignorance, but what’s that got to do withus today. Experiment away! We need grapes andlemons in other regions besides here.’’ ‘‘The cli-mate, Iosif Vissarionovich, puts a halt to everything.Look how fragile, how delicate they are — how canthey survive a frost?’’ the gardener pointed to thegrapevines. ‘‘Train them to accept harsh conditions,don’t be afraid! You and I are southerners yet wehave learned how to handle the north,’’ Stalin fin-ished speaking and took several steps towardVoropaev; ‘‘Here is a gardener . . . he’s been at itforty-five years but it still afraid of science. This, hesays, won’t work, and that, he says, won’t either. InPushkin’s time eggplants were imported to Odessafrom Greece as a rarity, and now only fifteen yearsago we started growing tomatoes in Murmansk. Ifwe wanted it to work — it did. Grapes, lemons, figsneed to be taught to grow in the north. We were toldthat cotton wouldn’t grow in the Kuban region, inthe Ukraine, but now it does. If you want somethingbadly enough, you can achieve it — that’s the main

w xpoint.’’ 10 .Attempts to grow cotton in the Ukraine and in the

Northern Caucasus were made in fact during the


Fig. 1. Bronze monument of Stalin and Lysenko. Stalin is holding a sheaf of branched wheat.

1930s. However, these efforts were subsequentlyabandoned. More successful was the introduction oftea in Georgia, Azerbaijan, and the Krasnodar re-gion, and likewise the introduction of peanuts in thesouthern part of the Ukraine. All of these were

initiated by Stalin. However, Stalin’s plan to turnTurkmenia into a country of olive plantations wasunsuccessful. The attempt to cultivate wild field

Ž .rubber plants kok-sagyz ended in failure. Not farfrom Stalin’s summer complex near Ritza Lake,



Fig. 2. A 1949 poster: And We Shall Defeat Drought. Using forest belts to alleviate the persistent problem of wind erosion in the prairieŽ .steppe regions of the Northern Caucasus, in the Rostov and Voronezh regions, and in the Ukraine was a method that could give positiveresults; however, state efforts to create broad forest belts from the North to the Caspian Sea ended in futility. Here, all the plants andseedlings died in the course of 1 or 2 years.

greenhouses had been built where scientists tried to‘‘re-make’’ cacao and coffee trees. That was notsuccessful nor was the attempt to grow lemons in theCrimea.

Stalin was a firm believer in the principle thatacquired traits could be inherited. He viewed theconnection between heredity and some kind of genesor another to be sheer mysticism. Based also onStalin’s Lamarckian convictions was the famous‘‘Nature Transformation Plan’’ announced in 1948.The confidence that forest zones of oak, pine andother central belt cultures could flourish in the dryZavolga steppes and in the salty, semi-arid areas nearthe Caspian Sea was not based on any experimentaldata but rather on the expectation that the newlyintroduced trees and plants would adapt to their new

environment. Lysenko had no direct involvement inŽ .the details of this plan Fig. 2 .

4. Stalin, Lysenko and Sergei Vavilov

During the 1930s, genetics and geneticists in-volved in agriculture were almost completely liqui-dated. Nikolai Vavilov, arrested in August of 1940,was the last victim. Only his international fameallowed him to survive as long as he did. Indeed,there was no lack of denunciations against him; quitethe contrary, he had a multitude of detractors whohad denounced him. His arrest had to have beensanctioned at the very highest level because of hisinternational reputation. By 1940, the main surge ofthe terror had nearly played itself out. At the same


time, however, the war engulfing Europe made theinternational reputation of any given scientist an itemof secondary interest. Nikolai Vavilov was arrestedon August 6, 1940, after a complicated processwhich included a business trip to the Western Ukrainewhich had been ceded to the Soviet Union under theterms of the Molotov–Ribbentrop pact. His arresttook place out in the country away from witnesses. Agroup of NKVD agents who had arrived in hastefrom Moscow had as their mission to arrest Vavilovas if he had been caught in the act of crossing theborder from occupied Poland.

The complex arrangements were evidence thatVavilov’s arrest had been worked out in great detail.In the 1930s, only ordinary people were arrestedduring the night. Important people, generals andmarshalls, were arrested according to carefullyscripted scenarios in order to ward off publicity andthe possibility of resistance. Those who plannedVavilov’s arrest did a good job. The arrest washardly noted if at all, and there was no internationalreaction. A few scientists and scholars, mostly inEngland and the U.S., started asking questions aboutthe fate of Nicolai Vavilov, but that wasn’t until1944. In 1945, the number of inquiries about Vav-ilov’s fate increased abruptly. A particularly largenumber of letters to the USSR Academy of Sciencesand to various diplomatic channels came from theRoyal Society of Great Britain of which Vavilov hadbeen a member since being elected in 1942, bywhich time he was already dying in a Saratov prison.

In June of 1945, the Academy of Sciences of theUSSR triumphantly observed its 220th anniversaryand to commemorate the occasion more than a hun-dred scientists from abroad had been invited. Duringthe anniversary session, Vavilov’s foreign friendsfound out the basic facts about his fate. Present atthese meetings was Nikolai Vavilov’s youngerbrother, Sergei, who also had an international reputa-tion as a physicist who specialized in light, fluores-cence and optics. Lysenko did not attend the anniver-sary session. The president of the Academy of Sci-ences of the USSR was the botanist, V.L. Komarov.He was very old, and July 17, 1945, had beendesignated as the day on which to elect a newpresident. Sergei Vavilov, the younger brother of thedead geneticist, was chosen for that post. His elec-tion was received very enthusiastically. It was viewed

as a sign that persecution and repression directed atthe field of genetics was over. Vavilov’s electionwas a serious blow to Lysenko, whose influence hadalready previously been declining. His opponents inthe Academy and in the universities promoted a new,far-ranging discussion which would have an impacton several agricultural institutes. Yuri Zhdanov’sspeech in April of 1948 was part of that discussionwhich now threatened the existence of the entireschool of ‘‘Michurin biology.’’

Incidentally, the election of Sergei Vavilov wasmost certainly not an indication of the improvedstatus of genetics or of the end of repression. Every-body understood that the final choice from amongthe short list of candidates was Stalin’s alone. Thisfact is confirmed by recently published documents

w xfrom the archives 11 . By choosing S.I. Vavilovfrom a list of 22 candidates, Stalin was indicatingthat he had nothing to do with Nikolai Vavilov’sarrest.

Each of the candidates’ names on the list wasaccompanied by a brief biography put together bythe NKGB — the People’s Commissariat of StateSecurity. Stalin deflected from himself any blame forthe death of the great scientist whose enormousinternational prestige was only now becoming clearto him. The NKGB biography of Sergei Vavilovgave Stalin no grounds to deny Sergei the postexcept for the fact of a brother who was arrested andwho died in prison. The NKGB document stated thatSergei Vavilov was ‘‘politically loyal’’ and furthernoted his enormous authority in the sciences as wellas his organizational abilities. ‘‘His manner is sim-ple, his daily life modest,’’ added the authors ofSergei Vavilov’s brief which was signed by theChief of the Second Section of the NKGB, Lt.General N.V. Fedotov. Molotov and Malenkov alsoreceived copies of the candidates’ biographical pro-files. Other outstanding scientists on the list did notfare so well in the NKGB’s evaluation of theirpersonal characteristics. Ivan Bardin, the Vice Presi-dent of the Academy of Sciences ‘‘does not associate

Ž .with other scientists due to the extreme greedinessof his wife.’’ The academician Aleksander Zavarit-sky is ‘‘by nature cantankerous, and leads a closedlife.’’ The academician and mathematician IvanVinogradov is ‘‘unsociable and ignorant of otherfields of science . . . single, a heavy drinker.’’ Even





Editorial : Reflections of Zhores Medvedev 1

George R. Hoffmann a, Donald G. MacPhee b

a Department of Biology, College of the Holy Cross, Worcester, MA 01610, USAb Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732-0815, Japan

It is a privilege to introduce the following contri-bution to Reflections in Mutation Research by Dr.Zhores Medvedev. This article on Lysenko and Stalintranscends the subject of mutation research; its im-port spans the whole of science. The science ofgenetics suffered a severe setback in the SovietUnion under Joseph Stalin, when fine scientists werepersecuted, Mendelian genetics fell into disfavor,and the ill-founded notions of the agronomist T.D.Lysenko gained acceptance for political reasons. Dr.Medvedev offers his reflections on that period fromfirst-hand experience.

Ž .Dr. Medvedev was born in 1925 in Tiflis Tbilisi ,then the capital of Soviet Georgia. He grew up inLeningrad, was educated there and in Moscow, andreceived advanced degrees in plant physiology andbiochemistry. In the 1950s, he achieved an interna-tional reputation for his distinguished work on bio-chemical genetics and the biochemistry of aging. Hisscientific integrity brought him into increasing disfa-vor with Soviet authorities, as he championed freecommunications between Soviet scientists and theircolleagues in other countries. Moreover, he becamean outspoken opponent of doctrines of T.D. Ly-senko, which he correctly perceived as a seriousthreat to Russian science. After Lysenko’s fall from

1 To suggest topics or authors for Reflections, readers shouldcontact either of the editors by mail at the addresses shown on theinside front cover or by e-mail:

Ž .G.R. Hoffmann [email protected] , D.G. MacPheeŽ [email protected] .

influence in 1964, the political climate in the SovietUnion still did not allow Dr. Medvedev’s frankdiscussion of the Lysenko period and open explo-ration of its broader implications. His writings werecirculated in samizdat, the Soviet literary under-ground. In 1969, he courageously published his in-fluential book ‘‘The Rise and Fall of T.D. Lysenko’’

Ž .in English Columbia University Press , givingworld-wide coverage to the events surrounding theLysenko affair.

Dr. Medvedev’s honest discussion of controver-sial issues in the politics of science led to his furtherestrangement from Soviet authorities and, ultimately,to his dismissal from his scientific post and a briefperiod of confinement in 1970. A protest organizedby his brother, the historian Roy Medvedev, wasjoined by such notable Soviet dissidents as AndreiSakharov and was effectively supported by academi-cians Petr Kapitsa and Boris Astaurov, as well as bymany foreign scientists. His release was secured, buthis relationship with Soviet officials remained for-ever strained.

Resuming his scientific work, he secured permis-sion to work in England on the biochemistry ofaging with Dr. Robin Holliday at the National Insti-tute for Medical Research in Mill Hill, London. Hecontinued to be an outspoken critic of the persecu-tion of scientists and scholars by Soviet authorities,and his forceful opinions led to his being stripped ofhis Soviet citizenship in 1973. Today, Dr. Medvedevlives in London and writes on scientific and politicalissues relating to the Soviet Union and modern



Igor Kurchatov, a favorite of Stalin, was not withoutsin: ‘‘by nature reserved, cautious, sly and a realdiplomat.’’ But for secret atomic projects these de-fects were, of course, virtues.

5. State pseudoscience

In the summer of 1948, I was still a student atTAA. I spent that summer in the Crimea and workedin the Nikitsky Botanical Garden near Yalta complet-ing a scientific project as part of my degree require-ments. I followed the proceedings at the LAAASconference by reading about it in PraÕda. I was gladto see that my scientific advisor, Petr MikhailovichZhukovsky, an academician at LAAAS and Chair ofthe Botany Department at TAA, gave a very strongand ironic speech on August 3rd in which he criti-cized Lysenko’s basic theories. But at the final meet-ing of the conference after Lysenko let it be knownthat his paper had been approved by the CentralCommittee of the Communist Party of the USSR,Zhukovsky’s address was filled with apologies andself-criticism. The organizers of the conferenceneeded participants who had not only been defeated,but who also admitted the error of their ways. P.M.Zhukovsky arrived at the Nikitsky Botanical Gardenin mid-August. There he ran a few projects in orderto recuperate from everything that had gone on at theconference. ‘‘I concluded a Brest–Litovsk peace withLysenko,’’ he told me as soon as we were alone. PetrPavlenko arrived at Zhukovsky’s place that eveningas a guest. Zhukovsky and Pavlenko had becomegood friends in Tiflis before the revolution. In earlySeptember, having buried Zhdanov, Stalin arrived inthe Crimea for a vacation. Stalin usually took thefirst part of his long vacation in the Crimea and thensailed along the coast of the Caucasus in October onone of the cruisers under the Black Sea Naval Com-mand.

When I returned to Moscow in early October, thewar being waged successfully throughout the countryagainst genetics had already been completed. Ly-senko and his principal cohorts who had been given

emergency powers, worked relentlessly. A chain re-action began in October. Acting upon the example ofthe LAAAS conference, pseudoscientific conceptsand tendencies gained preeminence in other spheresof knowledge as well. Physiology, microbiology,chemistry and cybernetics were all pushed decadesbackwards. The ‘‘Brest–Litovsk peace’’ with Ly-senko continued too long, until 1965 and into 1966.

The negative consequences of this long reign ofpseudoscience in the USSR spread for an even longerperiod. A full ‘‘recovery from these consequences’’has yet to be achieved even today. Indeed, thereduced authority of Soviet science, the delayeddevelopment of biotechnology and the hypertrophyof far too expensive and complex projects in thefields of atomic physics and space — all madeSoviet science too dependent on government cofferswhich are now almost empty. Science in the USSRhas not become the primary mover of technologicaland economic progress. Science was continually re-vitalizing itself, but the development of technologyand the economy were basically copies of whateverhad already been done in other countries.

References

w x Ž .1 Guests of Stalin’s Kremlin Office 1947–49 , Historicalarchive, No. 5r6, 1996, p. 41.

w x2 Pravda, March 8, 1953.w x Ž .3 V. Soifer, Bitter fruit, OGONEK 1r2 1988 .w x4 Archive of the President of the RF. FZ, op. 62, D. 131, p.

2–94. Manuscript. In 1997, V. Malyshev’s diary was pub-lished in the journal Istochnik.

w x5 K.O. Rossijanov, Stalin as Lysenko’s editor, Voprosy filosofiiŽ .2 1993 56–69.

w x Ž .6 V.A. Kozlov, S.V. Mironenko Eds. , Archive of the RecentHistory of Russia, Vol. I, ‘‘Special file’’ of I.V. Stalin,Catalog of documents, Blagovest, Moscow, 1994.

w x7 K.O. Rossijanov, Stalin as Lysenko’s editor, Voprosy filosofiiŽ .2 1993 56–69.

w x8 Pravda, Feb. 10, 1946.w x9 Pravda, Aug. 3, 1931.w x10 P. Pavlenko. Happiness, Sovetski Pisatel, Moscow, 1947, pp.

164–165 and 167.w x11 Elected or selected? More on the story of the election of the

President of the Academy of Sciences of the USSR, July,Ž .1945, Istoricheskii Arkhiv., 2 1996 142–151.


Editorial : Reflections of Zhores Medvedev 1George R. Hoffmann a, Donald G. MacPhee b

a Department of Biology, College of the Holy Cross, Worcester, MA 01610, USAb Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,

Hiroshima 732-0815, Japan



The Environmental Mutagen Society and the emergence of genetic toxicology: a sociological perspective 1Scott Frickel *

Department of History and Sociology of Science, Logan Hall, University of Pennsylvania, Pennsylvania, PA 19104-6304, USAAccepted 24 August 2000

Editorial2

Zhores Medvedev in his home in London, England.

Russia. His books have documented and explainedthe events of Soviet science, agriculture, and nucleardisasters in the Urals and at Chernobyl. He nowtravels in Russia and writes in Russian for the popu-lar press. He has been recognized internationally forhis leadership in the fight for freedom of expression

and scientific inquiry, including being awarded theMendel Medal from the Gregor Mendel Museum in

Ž .Brno, Czechoslovakia now Czech Republic .We are indeed pleased to be able to present Dr.

Medvedev’s reflections on Lysenko and Stalin inReflections in Mutation Research.



The Environmental Mutagen Society and the emergence ofgenetic toxicology: a sociological perspective1

Scott Frickel∗

Department of History and Sociology of Science, Logan Hall, University of Pennsylvania,Pennsylvania, PA 19104-6304, USA

Accepted 24 August 2000

Keywords: Environmental Mutagen Society; Genetic toxicology; Mutagens; Environmental Mutagen Information Center; History of genetics

1. Introduction

In surveying the historical record of genetictoxicology’s institutional development in the UnitedStates, no feature stands out more clearly than thefield’s comparatively rapid growth during the early1970s. After nearly three decades of nominal increase,publication rates of the scientific literature on chemi-cal mutagenesis registered increases of between 200and 500 per year between 1968 and 1972 [1]. Simi-larly steep increases are found in the occurrence of“institutionalizing events” such as the organization ofsymposia, conferences, and training workshops, thecreation of new journals, the publication of textbooksand monographs, and the formation of professionalsocieties (Fig. 1). Consider the latter: The Environ-mental Mutagen Society (EMS) was established in theSpring of 1969 in order “to encourage interest in and

∗ Tel.: +1-215-898-8210.E-mail address: [email protected] (S. Frickel).

1This article is part of the Reflections in Mutation Researchseries. To suggest topics and authors for Reflections, readersshould contact the series editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]).

study of mutagens in the human environment, partic-ularly as these may be of concern to public health.” 2

That June, EMS membership was pegged at a modest87; 1 year later, the EMS claimed 452 dues-payingmembers — a more than five-fold increase [2]. Within2 years sister societies had been established in Japan(JEMS) and Europe (EEMS), and national sectionsof the EEMS had formed in Italy, West Germany,and Czechoslovakia. By 1976, the number of formalEMS-related societies had risen to nine. Within therelatively short space of 8 years, the major institu-tional structures of genetic toxicology — includingnewly created funding mechanisms and collaborativeinter-laboratory and inter-agency research programsand review panels — had been established.

This article examines the discipline-buildingprocess in genetic toxicology from a sociologicalperspective. My focus is on the boundary between en-vironmental science and the politics of environmentalprotection. At issue is the EMS’s role in regulatingthat boundary.

2 News Release (1 March 1969), Environmental Mutagen SocietyArchives, Genomics and Toxicology Group, Oak Ridge NationalLaboratory, Oak Ridge, TN (Henceforward, EMSA).

1383-5742/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S1383 -5742 (00 )00057 -0


S. Frickel / Mutation Research 488 (2001) 1–8 3

traditional institutional carriers of disciplinary au-thority, identity, and culture. They also serve a crit-ical economic function by reproducing expert labor(Ph.D.s) through graduate-level research training.Generally speaking, there were few, if any, organi-zational niches in universities and medical schoolsready-made to adopt genetic toxicology in 1970.Thus, the transmission of genetic toxicology’s cul-tural identity and the social reproduction of ge-netic toxicologists — work typically accomplishedin academic departments — required alternativesolutions.

Third, despite being centered in governmental in-stitutions, genetic toxicology was not pushed intoexistence “from above,” so to speak, by governmentalpatronage or decree. While federal support of environ-mental mutagenesis generally increased throughoutthe period in question, the drive to better understandand minimize the detrimental genetic effects of chem-ical mutagens received nowhere near the level offederal support that, for example, radiation geneticsreceived from the Atomic Energy Commission in the1950s and 1960s [7,8], or that systems ecology re-ceived from the National Science Foundation in the1970s [9]. Moreover, Congressional passage of theToxic Substances Control Act of 1976, the legisla-tion requiring mutagenicity testing of new chemicalsand thereby ensuring a consumer market for genetictoxicology knowledge [10], marks the end of the ini-tial phase of genetic toxicology’s development, notits beginning. That is, genetic toxicology coalescedas a new scientific field before federal regulatorypolicy created a national market for mutagenicitydata.Finally, as an interdisciplinary field that attracted

scientists from government, academics, and industry,the establishment of genetic toxicology, by definition,required considerable “boundary work”. As concep-tualized in sociology, this term describes scientists’rhetorical attempts to strengthen and preserve thesocial authority of science by guarding or — as inthe present case — dissolving scientific boundaries,be they methodological, disciplinary, or institutional[11,12]. In genetic toxicology, the historical recordis replete with boundary work of various sorts. Wefind, for example, EMS President Alexander Hollaen-der complaining that disciplinary ethnocentrism pre-vented many biologists’ appreciation for the broader,

ecological, implications of their own investigations. 3

We also see EMS Treasurer Marvin Legator urging “aclose alliance between the diverse sciences of toxicol-ogy and genetics” [13]. At the same time, however,EMS council members worried about losing controlof the “genetic thrust of the EMS” were the Societyto widen its scope to include “environmental effectsother than the purely genetic.” 4 Lively debate oversuch issues as the efficacy of testing methods, ap-propriate standards of genetic risk, and the relativeimportance of the distinctions between mutagenicity,carcinogenicity, and teratogenicity fill the pages ofthe EMS Newsletter and other outlets all suggestingthat, during the early 1970s, many people had a stakein how genetic toxicology came to be defined andcontrolled. Cross-cut with divergent and at times con-flicting interests, genetic toxicology emerged in partas the product of these ultimately political strugglesover cultural and professional boundaries.

In spite of these four challenges, the expansionof genetic toxicology continued apace such that, by1976, the defining intellectual, social, and economicfeatures of a scientific (sub)discipline were basicallyin place. These included (1) relative consensus amongpractitioners with respect to genetic toxicology’smain subjects and an established set of theories andstandardized research practices for attacking them,(2) the establishment of communication and rewardstructures manifest in professional societies, jour-nals, annual meetings, and the allocation of awards, 5

and (3) a system for producing a trained labor forcein order to supply the growing demand for genetictoxicology knowledge.

Therein arises genetic toxicology’s main sociolo-gical puzzle: given the various challenges enumer-ated above, what explains the rapid rate of genetictoxicology’s institutional development? Part of theanswer lies in understanding the EMS’s role in

3 Hollaender, Alexander. “Opening Remarks, Symposium on En-vironmental Pollutants.” Annual Meetings of the Radiation Re-search Society, Dallas, TX (2 March 1970). MS-1261, Box 3,Folder 16. Radiation Research Society Archives, Hoskins Library,University of Tennessee, Knoxville, TN (Henceforward, RRSA).

4 Minutes, EMS Council Meeting (18–19 September and 22March 1970), EMSA.

5 The EMS Award was first presented in 1972, to CharlotteAuerbach.

2 S. Frickel / Mutation Research 488 (2001) 1–8

Fig. 1. Institutionalizing events in genetic toxicology, 1964–1976. Note: The data for this table were collected through a systematic searchof announcements and news items in several key journals, newsletters, and annual reports. Sources: Mutation Research, 1–32 (1964–1976);Environmental Mutagen Society Newsletter, 1–6 (1969–1972); Environmental Mutagenesis and Related Subjects (1973–1976); Reviews inGenetic Toxicology (1975–1976); Genetic Toxicology Testing (1976); National Institute of Environmental Health Sciences, Annual Report“Environmental Mutagenesis Branch — Summary Statement” (1972–1976); Oak Ridge National Laboratory Biology Division, Annual andSemi-Annual Progress Reports (1965–1969).

2. Genetic toxicology’s sociological puzzle

The speed with which the institutional features ofgenetic toxicology coalesced is even more strikingwhen we consider four related institutional challengesthat shaped the new field’s developmental trajectory.First, in contrast to the standard academic settingsthat conditioned the birth of genetics, biology, andbiochemistry in the late-19th and early-20th centuries(e.g. private research universities, land grant universi-ties, and medical schools), genetic toxicology was, inmany respects, a child of the federal government. Twofederal science institutions, in particular, can be cred-ited with assuming the lion’s share of responsibilityfor promoting and organizing research on the geneticeffects of environmental chemicals during genetictoxicology’s infancy: Oak Ridge National LaboratoryBiology Division, beginning in the mid-1960s, and theNational Institute of Environmental Health Sciences,

beginning around 1972, served as dual anchors forenvironmental mutagenesis research and test develop-ment [3,4]. Moreover, as genetic toxicology expandedthroughout the 1970s, the field’s institutional moor-ings, mainly in the form of laboratories, coordinatingcommittees, and research support, remained largelywithin the federal science system — for example, atthe Environmental Protection Agency, the Food andDrug Administration, Lawrence Livermore NationalLaboratory, and the National Center for ToxicologicalResearch.

Second, while university scientists played vitallyimportant roles in genetic toxicology’s develop-ment from the very beginning, university depart-ments did not. This had important implications, for,as historians of science have long argued, univer-sity departments traditionally have been the institu-tional building blocks that “embody and perpetuate[scientific] disciplines” [5,6]. Departments are the



the EMS will, through scientific congresses, sym-posia, a journal and a newsletter, provide the tra-ditional forums through which scientists of similarprofessional interest have for generations com-municated with one another and with the public.Experimental data and new theories are shared andsubjected to the inspection and critical review ofinformed colleagues 6

Tax exemption placed definite constraints on thekinds of political activities the EMS could legallypursue; a lawyer cautioned EMS Secretary SamuelEpstein to “be wary of any participation in a publiccampaign during the adoption or rejection of specificlegislation.” 7 These legal constraints enhanced theorganization’s credibility as one whose main, andperhaps only, formal interest was in the “inspectionand critical review” of scientific knowledge.

On that basis, formal relations with environmentalgroups, for example, were roundly discouraged. In ref-erence to a letter that Joshua Lederberg received fromthe Natural Resources Defense Council, purportedlyrequesting information on environmental mutagenesisand that Lederberg brought to the attention of theEMS Executive Council, EMS President Hollaender“proposed to make it clear . . . that EMS would bewilling to function only as a resource facility, and notin the development of any action program.” 8 Fiveyears later, Hollaender complained again of frequentrequests for information on “chemical toxicology,”this time from the group Resources for the Future. 9

There is little evidence that, in the interim, the EMSentered into relationships — formal or otherwise —with environmental organizations.

Organizations that may be assumed to have har-bored political and economic interests biased in theopposite direction received similar rebuffs. A pro-posal that the Association of Analytical Chemists beinvited to review validity and reproducibility studiesof mutagenicity tests was struck down on the grounds

6 EMS report to IRS (draft, no date). MS 1261, Box 3, Folder9: “EMS Legal Correspondence (1969)”, RRSA.

7 Blinkoff to Epstein (2 October 1969), MS 1261, Box 3, Folder9: “EMS Legal Correspondence (1969)”, RRSA.

8 Minutes, EMS Council Meeting (27 July 1971), Malling Papers,EMSA.

9 Hollaender to Sobels (29 April 1976), MS 1261, Box 3, Folder13, RRSA.

that it “has no special expertise in this matter.” 10

The same attitude guided relationships with firmshaving a direct economic interest in the productionof genetic toxicology data. A report from an EMSCommittee on Methods advanced the position that“the EMS should avoid putting itself into a positionof certifying or providing an endorsement to anylaboratory or test method. It should serve only as anassembly of scientists willing to provide individualexpertise, upon request, to anyone requesting it.” 11

The Committee advocated this position as a meansof avoiding potential legal difficulties or conflict ofinterest charges. Such outcomes would threaten theEMS’s appearance of organizational neutrality andundercut efforts by the EMS leadership to institution-alize ideological purity. Those efforts are perhaps bestillustrated by considering the science/politics bound-ary as it came to be embodied in the EnvironmentalMutagen Information Center (EMIC).

3.2. Science in the public service

EMIC began formal operations in September 1970.Housed at Oak Ridge National Laboratory BiologyDivision, and initially directed by Heinrich Malling,EMIC served as an information clearinghouse for mu-tagenicity data. It employed a small technical staffcharged with collecting published literature on chem-ical mutagenesis, condensing the data presented inthose articles into uniform tabular abstracts, and build-ing a computer database from that information whichcould be accessed via one of a number of standardizedindex codes [20,21].

EMIC’s other primary task was disseminating thatconcentrated information. The main mechanism forthis was an annual literature survey that EMIC pro-duced and distributed, mostly to members of the var-ious EMS societies around the world. 12 EMIC staffalso published occasional “awareness lists” — shortbibliographies of important subclasses of chemicalcompounds — in the EMS Newsletter. Far more fre-quently, EMIC staff attended to the specific requests

10 Minutes, EMS Council Meeting (17 October 1972), MallingPapers, EMSA.11 Zeiger to Drake (5 November 1976), EMSA.12 EMIC Annual Report to EMS Council (22 March 1971), JohnWassom, personal files.


negotiating the cultural boundaries between environ-mental science and environmental politics.

3. The boundary work of EMS

Like most formal organizations, the EMS pres-cribed rules for membership, governance, and rela-tions with other organizations and individuals. Andlike most scientific societies [14], the EMS servedas an organ for institutionalizing scientific commu-nication through the publication of a newsletter andthe organization of annual meetings. The EMS alsoaccomplished a considerable amount of the pedagog-ical work for university departments by developingcurricula, training workshops, and symposia in thetheory and practice of genetic toxicology. Finally, inits organization and sponsorship of the EnvironmentalMutagen Information Center (EMIC) and the publica-tion of the 10-volume series on Chemical Mutagens:Principles and Methods for Their Detection [15], theEMS took an early lead in organizing data collectionand methods development in genetic toxicology — arole not unlike that often assumed by governmentalagencies.

Amidst these various roles and functions swirled anapparent contradiction. Building an interdisciplinaryresearch community, a stable funding base, and a mar-ket for genetic toxicology information and practices— all interrelated goals — required the transcendenceof partisan interest politics. Likewise, organizationalsuccess within the EMS depended largely on the levelof credibility the organization’s leadership was ableto foster among its members, patrons, and other con-sumers of genetic toxicology information. In order toelaborate a vision of genetic toxicology amenable todiverse and often competing interests that were boundto surface at the crossroads of university, governmentand industry science, the EMS itself had to remainabove politics.

At the same time, success in the substantive goalsset out by the EMS — which involved essentiallytransforming the meaning of mutation research andchanging how it was done, by whom, and for whatpurposes — relied heavily on overt political rhetoricand action. Scientists active in the campaign to estab-lish genetic toxicology sought to challenge the basisof federal chemical regulatory policy by refocusing

legislative attention on the genetic impacts of environ-mental chemicals. They did so in part by interpretinggenetic toxicology in moral and political terms as anissue of scientist, corporate, and government respon-sibility for the protection of the public health and thepreservation of genetic integrity [16–19]. How did theEMS successfully balance its scientific and politicalprojects without undermining either?

As one of the central mechanisms created to orga-nize and engage a systematic attack on the problem ofenvironmental mutagens, the EMS embodied not onekind of boundary, but many. And although overcom-ing taken-for-granted divisions and finding commonpurpose among disparate knowledge communitieswere arguably among the EMS’s most significantearly achievements, they did not come easily. Dis-solving the cultural and professional boundaries thatthreatened to impede the campaign to establish genetictoxicology required careful and constant regulationof the science/politics boundary.

3.1. The EMS’s public face

Scientists troubled by potential genetic hazardslittering the human environment did not have to jointhe EMS to address the problem in their own re-search; yet many did. The legitimacy enjoyed by theEMS and reflected in the steep rise in membershipwas not, however, derived solely from the nature ofthe threat to public health posed by environmentalmutagens. Organizational credibility also mattered,and that was a thing to be earned, not given. Thusdid the successful campaign to establish genetic tox-icology depend in part upon scientists’ generalizedperception that the EMS embodied a spirit of sci-entific neutrality. To attract members and financialbacking, the EMS was best served by presentingitself as a society committed first and foremost tothe production, rationalization, and dissemination ofobjective knowledge. In its official statements andin its routinized activities, the EMS kept environ-mental politics out of genetic toxicology data andinformation.

The public face of the EMS is perhaps best des-cribed in a “statement of activities” contained in an In-ternal Revenue Service application for tax-exemptionstatus filed on behalf of the EMS in 1969. “Like mostorganizations of scholars,” the report read,



tax-exempt status, reducing the young organization’seconomic burden even as it reinforced the science/politics boundary through restrictions on politicallobbying and partisan endorsements. The rhetoricalconstruction of “good” science in the interest of envi-ronmental health also attracted support from scientificand political elites. Inversely, the same spirit of neu-trality gave drug and chemical companies little roomto charge the EMS with environmentalist bias and,therefore, avoid taking some responsibility in fundingand participating in genetic toxicology’s development.It is difficult to imagine the same kind of support

coming from so many different quarters if the EMShad not made the focused efforts it did to draw thisboundary. By contrast, the experience of the JapaneseEnvironmental Mutagen Society (JEMS) illustratesthe potential organizational costs of not maintainingsome ideological distance from environmental poli-tics. A representative of that society reported to EMSExecutive Council members in 1972 that “a majorproblem at the development of (JEMS) had beenpolitical implications on environmental problems, as‘left-wing’ parties were using these issues to attackthe Japanese government.” 21 At the same time thatJEMS was hampered by their association with the en-vironmental movement in Japan, in the United Statesthe EMS was quickly gaining firmer financial and or-ganizational footing. After 2 years of very precariousbudgeting arrangements, in 1972, the funds commit-ted to support EMIC from both the FDA and NIEHSincreased considerably. With this support these fed-eral agencies gave the EMS and EMIC a stamp oflegitimacy and provided a public endorsement ofthe importance of genetic toxicology research. 22 By1977, NIEHS was funding EMIC to the tune of US$190,000 per year [22].

4. Conclusion: fusing science and politics

Concentrated outside the university system, andin the absence of either a ready-made labor forceor market-creating legislation, EMS members in the

21 Minutes, EMS Council Meeting (8 July 1972), Malling Papers,EMSA.22 Minutes, EMS Council Meeting (26 March 1972), MallingPapers, EMSA.

United States essentially built the institutional founda-tions of genetic toxicology from the ground up. Theydid so collectively, by creating mechanisms for recrui-ting and training scientists, coordinating research,standardizing research tools and practices, and un-dertaking public outreach and education [23]. Main-taining a strict distance from groups with clear-cutgovernment and/or economic interests representedan organizational strategy for balancing the compet-ing claims that research promoted by the EMS wasat once socially relevant and unblemished by socialbias. Policing that boundary, in turn, enhanced thelegitimacy of the EMS’s overall project — genetictoxicology — as well as its own influence and auton-omy. Keeping environmental politics out of genetictoxicology was ultimately a strategy for gaining andholding onto that authority.

It was also, paradoxically, a strategy for accomplish-ing the EMS’s own political work. At least for a time,the boundary between science and environmental ac-tivism blurred within the EMS. As the organization’scommitments to the production of accurate knowl-edge, to the integrity of the new discipline, and to thereduction of environmental genetic hazards converged,collecting and disseminating mutagenicity data, on theone hand, and building the political and moral caseagainst the indiscriminate use of mutagenic chemicals,on the other, came to be treated as complementary andmutually reinforcing projects.

The EMS was a central player in the campaign toinstitute the new order of environmental inquiry thatby 1976 genetic toxicology had come to represent[24]. Ironically, the new field’s rapid rise may betraced in part to the EMS’s effectiveness at main-taining a publicly visible boundary between envi-ronmental science and environmental politics whilesimultaneously subverting that same boundary withinits own organizational domain. In effect, the EMSfunctioned as two organizations in one — a profes-sional society of scientists and a scientific environ-mental movement organization.

Acknowledgements

Research support for this article was provided bya National Science Foundation Dissertation Grant(SBR-9710776). I would like to thank John Wassom


for data by “anyone who requested it” — literally. 13

Keeping scientists but also “the general public in-formed about highly technical data” was a centralconcern and explicit function of EMIC. 14 In a letterwritten in 1970, for example, Malling mentioned thathe had been answering questions on mutagenicity at“a rate of one per day.” 15 A year later EMIC staff re-ported receiving 222 individual requests for informa-tion. “The greatest proportion of these requests werefrom persons engaged in research, but some camefrom a variety of sources” the report noted. Theseincluded “city municipalities, high school students,free lance writers” and the occasional legislator. 16

While it is reasonable to assume that under condi-tions of resource scarcity, requests from high schoolstudents or citizens’ groups might not receive thesame level of attention as those coming from scientistsactive in the mutation research field, the historicalrecord makes clear that, in principle, EMIC — and,by direct extension, the EMS — was committed toserving the public interest as an impartial messengerof genetic toxicology information. That impartialityextended into the economic sphere as well; althoughthe EMS Executive Council several times consideredbilling industry and foreign researchers for chemicalmutagenicity data as a means of offsetting tight bud-gets, such a policy did not materialize; no one paidmoney for EMIC’s information services. 17

3.3. Institutionalizing impartiality

During its formative years, the EMS ExecutiveCouncil pursued policies of conduct, scientific review,and public service that depended on and reinforceda strict division between the EMS as a scientific re-search organization and politics of various stripes —

13 Minutes, EMS Council Meeting (26 March 1972), MallingPapers, EMSA.14 Answers to Study of Environmental Quality Information Pro-grams questionnaire (27 March 1970), John Wassom, personalfiles.15 Malling to Peters (4 June 1970), Malling Papers, EMSA.16 EMIC Annual Report to EMS Council (22 March 1971), JohnWassom, personal files.17 Minutes, EMIC Register Meeting (25 March 1970); Minutes,EMS Council Meeting (26 March 1972), Malling Papers, EMSA;and Minutes, EMIC Program Committee (18 December 1970),John Wassom, personal files.

from environmental protest to the endorsement ofparticular testing protocols. The organization’s pub-lic boundary work, embodied most explicitly in thesocial service functions of EMIC, can be understoodas an explicit attempt to establish a very distinct,unyielding, and publicly visible boundary around theEMS, EMIC, and genetic toxicology more generally.

It would be wrong, however, to interpret these polic-ing efforts as only or merely ideological in nature.They were also born of organizational necessity. At thetime, genetic toxicology could boast few if any stablesources of funding. As Heinrich Malling wrote to FritzSobels in the fall of 1970, “The money situation in theU.S. is very tight. There is essentially no money forscreening for the mutagenicity of harmful pollutants.Besides the standard mutagens such as EMS, MMS,etc., the only new compound with which research isin progress is cyclophosphamide.” 18 Much of thefunding at the time came in the form of federal budgetline items during a period of general decline in thefunding rate for basic research. Money for EMIC andsupport for other EMS projects were not at first easilyobtained or readily recommitted. Numerous federalagencies, various chemical and drug companies, pri-vate foundations, the National Laboratories, and fouror five of the National Institutes of Health contributedsmall sums to sponsor EMS conferences and work-shops and to support the work conducted at EMIC,usually on a year-to-year basis. 19 Thus, a budgetaryshortfall in 1971 forced EMIC to temporarily curtailmany of its data collection efforts and sent EMS of-ficers scrambling to locate additional “emergency”funds to keep the center running through the year. 20

Given the heterogeneity of EMIC’s patrons, and theresulting instability of the economic foundation under-lying research and development in genetic toxicology,the boundary work conducted by the EMS can be seenas an organizational survival strategy, and not merelyan ideological reaction to “politics”, environmentalor otherwise. What is more, the strategy worked.

Organizational impartiality served a number ofspecific practical purposes. It helped secure EMS’s

18 Malling to Sobels (1 October 1970), EMSA.19 Minutes, EMS Council Meeting (18–19 September 1970),Malling Papers, EMSA.20 Hollaender to Ruckelshaus (4 January 1971); Kissman to Davis(11 January 1971); Memo, Malling to EMIC Staff (11 February1971), all in John Wassom, personal files.



HPRT mutations in humans: biomarkers for mechanistic studies ☆Richard J. Albertini *

University of Vermont Genetic Toxicology Laboratory, 32 North Prospect Street, Burlington, VT 05401, USAAccepted 21 June 2001



HPRT mutations in humans: biomarkers for mechanistic studies�

Richard J. Albertini∗

University of Vermont Genetic Toxicology Laboratory, 32 North Prospect Street, Burlington, VT 05401, USA

Accepted 21 June 2001

Abstract

The X-chromosomal gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), first recognized through its hu-man germinal mutations, quickly became a useful target for studies of somatic mutations in vitro and in vivo in humans andanimals. In this role, HPRT serves as a simple reporter gene. The in vivo mutational studies have concentrated on peripheralblood lymphocytes, for obvious reasons. In vivo mutations in T cells are now used to monitor humans exposed to environ-mental mutagens with analyses of molecular mutational spectra serving as adjuncts for determining causation. Studies of thedistributions of HPRT mutants among T cell receptor (TCR) gene-defined T cell clones in vivo have revealed an unexpectedclonality, suggesting that HPRT mutations may be probes for fundamental cellular and biological processes. Use of HPRTin this way has allowed the analyses of V(D)J recombinase mediated mutations as markers of a mutational process withcarcinogenic potential, the use of somatic mutations as surrogate markers for the in vivo T cell proliferation that underliesimmunological processes, and the discovery and study of mutator phenotypes in non-malignant T cells. In this last application,the role of HPRT is related to its function, as well as to its utility as a reporter of mutation. Most recently, HPRT is findinguse in studies of in vivo selection for in vivo mutations arising in either somatic or germinal cells. © 2001 Elsevier ScienceB.V. All rights reserved.

Keywords: HPRT mutations; Biomarker; Humans; Genomic instability; Lesch–Nyhan syndrome

1. Introduction

This essay is a very personal reflection on HPRT.It is not a comprehensive review as it focuses mostlyon my own involvement with human studies. Overallreviews of HPRT are available elsewhere [1,2]. Here,I recall the many turns the story has taken and howthe questions have changed. Serendipity has been a

� This article is part of the Reflections in Mutation Researchseries. To suggest topics and authors for Reflections, readersshould contact the series editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]).

∗ Tel.: +1-802-656-8347; fax: +1-802-656-8333.E-mail address: [email protected] (R.J. Albertini).

major player; mentors and colleagues — especially PatO’Neill and Jan Nicklas — have provided the stimu-lus. Curiously, the HPRT story will be seen to developa symmetry: as it unfolds, it turns back on itself,returning to its beginnings.

2. The beginning

The human HPRT story began over 30 years agowith a report in “Science” that described an enzymedefect associated with a sex-linked human neurolo-gical disorder and excessive purine metabolism [3].The disorder, eventually called the Lesch–Nyhan

1383-5742/01/$ – see front matter. © 2001 Elsevier Science B.V. All rights reserved.PII: S1383 -5742 (01 )00064 -3


for sharing documents from his personal files, DavidDeMarini for reading and commenting on my disser-tation in its entirety, and Daniel Kleinman and JohnWassom for their comments on an earlier draft of themanuscript.

References

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[2] J.S. Wassom, Origins of genetic toxicology and the Environ-mental Mutagen Society, Environ. Mol. Mutagen. 14 (Suppl.16) (1989) 1–6.

[3] National Institute of Environmental Health Sciences, AnnualReport, National Institute of Environmental Health Sciences,Research Triangle Park, 1972.

[4] Oak Ridge National Laboratory Biology Division, BiologyDivision Semiannual Progress Report for Period Ending 31July 1965, Oak Ridge, 1965.

[5] R.E. Kohler, From Medical Chemistry to Biochemistry: TheMaking of a Biomedical Discipline, Cambridge UniversityPress, Cambridge, 1982.

[6] C.E. Rosenberg, Toward an ecology of knowledge: ondiscipline, context, and history, in: A. Olesen, J. Voss(Eds.), The Organization of Knowledge in Modern America,1860–1920, Johns Hopkins University Press, Baltimore, MD,1979, pp. 440–455.

[7] National Academy of Science — National Research Council,The Biological Effects of Atomic Radiation, SummaryReports, National Academy of Science — National ResearchCouncil, Washington, DC, 1956.

[8] National Academy of Sciences — National Research Council,The Biological Effects of Atomic Radiation, SummaryReports from a Study by the National Academy of Sciences,National Academy of Sciences — National Research Council,Washington, DC, 1960.

[9] C. Kwa, Representations of nature mediating between ecologyand science policy: the case of the International BiologicalProgramme, Soc. Stud. Sci. 17 (1987) 413–442.

[10] M.J. Prival, V.L. Dellarco, Evolution of social concerns andenvironmental policies for chemical mutagens, Environ. Mol.Mutagen. 14 (Suppl. 16) (1989) 46–50.

[11] T.F. Gieryn, Boundaries of science, in: S. Jasanoff, G.E.Markle, J.C. Petersen, T. Pinch (Eds.), Handbook of Scienceand Technology Studies, Sage, Thousand Oaks, CA, 1994,pp. 393–443.

[12] S. Shapin, Discipline and bounding: the history and sociologyof science as seen through the externalism–internalism debate,Hist. Sci. 30 (1992) 333–369.

[13] M.S. Legator, Chemical mutagenesis comes of age: environ-mental implications, J. Hered. 61 (1970) 239–242.

[14] R. Whitley, The Intellectual and Social Organization of theSciences, Clarendon Press, Oxford, 1984.

[15] A. Hollaender, F.J. de Serres (Eds.), Chemical Mutagens:Principles and Methods for their Detection, Vols. 1–10,Plenum Press, New York, 1971–1986.

[16] J.F. Crow, Chemical risk to future generations, Scientist andCitizen 10 (1968) 113–117.

[17] S.S. Epstein, Testimony of Dr. Samuel S. Epstein, in: Hearingson Chemicals and the Future of Man, United States Senate,Committee on Government Operations, US GPO, Washington,DC, 1971, pp. 6–26.

[18] H.V. Malling, Chemical mutagens as a possible genetic hazardin human populations, J. Am. Ind. Hyg. Assoc. 31 (1970)657–666.

[19] J.V. Neel, Evaluation of the effects of chemical mutagens onman: the long road ahead, Proc. Natl. Acad. Sci. 67 (1970)908–915.

[20] H.V. Malling, J.S. Wassom, Environmental Mutagen Informa-tion Center (EMIC) I. Initial organization, Environ. Mutagen.Soc. Newslett. 1 (1960) 16–18.

[21] H.V. Malling, Environmental Mutagen Information Center(EMIC) II. Development for the Future, Environ. Mutagen.Soc. Newslett. 4 (1971) 11–15.

[22] National Institute of Environmental Health Sciences, AnnualReport, National Institute of Environmental Health Sciences,Research Triangle Park, 1977.

[23] S. Frickel, Disciplining Environmentalism: OpportunityStructures, Scientist-Activism, and the Rise of GeneticToxicology, 1941–1976, Ph.D. Dissertation, Department ofSociology, University of Wisconsin — Madison, 2000.

[24] J.W. Drake, et al., Environmental mutagenic hazards, Science187 (1975) 503–514.


R.J. Albertini / Mutation Research 489 (2001) 1–16 3

vivo mutation frequency. First question — “whatwas the denominator in my little experiment?” Al-though I thought it obvious, I replied “the numberof cells plated”. Second question — “how about thenumerator?” Although I was becoming wary (andtentative), I answered that this should be the numberof cells cloning in selective medium. Finally, the coupde grace — “did I know how fibroblasts grew?” Somuch for kindness!

This was my first serious encounter with the issueof clonality. After totally ignoring the problem ofclonal distributions of mutant cells in skin, I now com-pensated by taking a second biopsy, then a third, thenmore. I became entangled in calculations of means ofPoisson distributions in front of a distinguished groupof increasingly skeptical people. Finally, I had to con-clude the obvious. My poor imaginary study subject,by now having undergone many skin biopsies, neededsurgery. Although I was getting close to determiningan in vivo mutation frequency, this was no way todo human biomonitoring. A usable assay for in vivosomatic mutations in humans could not sample skin.The problem of non-random distributions of mutantsin fixed tissues was too fundamental for simple solu-tion (I did pass my exam, due more to persistencethan performance).

This is how I came to consider peripheral blood lym-phocytes for studies of in vivo somatic mutations inhumans. Lymphocytes are freely circulating in blood.Issues of clonality should not be a concern (or so Ithought). Lymphocytes traverse the body, may be ex-posed to mutagens at many sites and, even though theydo not themselves have full activating capabilities, can“borrow” metabolism from the different tissues. Bloodsamples are easily obtained. Finally, even though mostlymphocytes in vivo are in an arrested G0 stage of thecell cycle, the T cells can be stimulated to divide andundergo short-term polyclonal proliferation in vitro.

I began to study minority in vivo populations ofHPRT mutant T cells shortly after my debacle withfibroblast clonality. I returned to the source of HPRTfor material — individuals carrying an inheritedLesch–Nyhan mutation. This time, however, I was in-terested in heterozygous females. Because of randomX-chromosome inactivation, females heterozygousfor mutations of X-linked genes have two populationsof somatic cells, those expressing the wild-type andthose expressing the mutant allele [29,30]. Systems

capable of discriminating between wild-type andmutant cells can therefore diagnose heterozygosityfor certain X-linked genes. This was the basis ofLesch–Nyhan heterozygote detection assays, then inuse that employed either skin fibroblasts or hair folliclecells [31–35]. Although earlier studies had suggestedthat Lesch–Nyhan heterozygous females had only thewild-type blood cells, the methods used would havemissed low levels of mosaicism, i.e. <10% [36–38].This, in fact, was the case. An assay based on PHAstimulation of peripheral blood T lymphocytes in se-lective medium, using scintillation spectroscopy of3H-thymidine incorporation into DNA as the read-out,showed minority populations of 8-azaguanine resis-tant T cells (<10%) in three of four heterozygousfemales [39]. Although far too insensitive to detectrare somatic HPRT mutations arising in vivo in nor-mal individuals, this was a start. It also showed thatlymphocytes could be used for heterozygote detection.

The obvious next step in developing an assay forde novo T cell mutations of HPRT arising in vivo innormal individuals was to convert scintillation count-ing, which measures 3H-thymidine incorporation ina population of cells, to autoradiography for detect-ing incorporation in single cells. This step was takenby Gary Strauss as a graduate student in Vermont.With meticulous attention to detail, Gary developed ashort-term assay for quantitating HPRT mutations inT cells that arose at low frequencies in normal indi-viduals [40,41]. Although met with initial skepticism,these early reports did serve to stimulate interest in“in vivo” mutagenicity monitoring.

Despite its influence, the originally describedmethod for measuring in vivo arising HPRT muta-tions was seriously flawed, illustrating one of theperils of in vivo mutagenicity testing using pheno-typic assays. When relying on phenotype alone, inthis case, 6-thioguanine resistance — it is difficult toknow if all cells with the variant phenotype actuallyhave a genetic (i.e. mutational) basis. Indeed, theydid not with the autoradiographic assay as originallydescribed. Although most T lymphocytes are in anarrested G0 stage in vivo, a small but variable mi-nority is activated and cycling. Exposures to evenhigh concentrations of purine analogues do not in-stantly inhibit the cycling cells. Some — those inG1, can progress to early DNA synthesis and becomelabeled, even though eventually killed. This resulted

2 R.J. Albertini / Mutation Research 489 (2001) 1–16

syndrome for its original authors [4], is a devastat-ing clinical condition with neurological, psychiatric,arthritic and metabolic disabilities including massiveurate overproduction [5–8]. Fortunately, it is rare, andthe manifestations are now recognized to be of varyingseverity [9–11]. The X-linked gene described in thisearly report was, of course, the hypoxanthine-guaninephosphoribosyltransferase (HPRT) gene [3,12–14].These early investigations dealt with germinal HPRTmutations and their global expression in the affectedmales who inherited them. HPRT was important be-cause of its function — not for its utility as a reporter.

Somatic cell genetics was an almost immediate ben-eficiary of these newly recognized mutations becausethey conferred a distinctive cellular as well as clinicalphenotype [15]. HPRT enzyme activity is requiredfor the phosphoribosylation of hypoxanthine andguanine, salvaging them for nucleic acid biosynthe-sis. It also phosphoribosylates purine analogues (e.g.8-azaguanine, 6-thioguanine and 6-mercaptopurine)— a necessary step for their cytotoxicity [16]. Resis-tance to these analogues provides a highly efficientselective system for HPRT mutant cells, allowingthem to grow while wild-type cells are killed [15].Conversely, HPRT mutant cells, lacking the salvagepathway, are dependent on de novo purine biosynthe-sis for synthesis of nucleic acids. They are, therefore,exquisitely sensitive to inhibitors of one-carbon trans-fer, being killed at concentrations that do not affectwild-type cells [15]. This is the basis of the HAT(hypoxanthine, aminopterin and thymine) reverseselection system.

3. HPRT mutations in vitro in human fibroblasts

These discoveries came at about the time that thenow classical papers appeared showing that genemutations could be recognized, induced and quan-tified in vitro in cultured mammalian cells [17,18].The modern era of mammalian and ultimately humansomatic cell genetics had begun. The HPRT gene wasan obvious target for further mutagenicity studies incultured cells. It could be put to use as a reporter.

It was my good fortune to be in Robert DeMars’laboratory at the University of Wisconsin during thisperiod. Bob’s group had defined the Lesch–Nyhancellular phenotype and had worked out optimal

conditions for culturing diploid human fibroblasts[15,19]. He suggested that for my Ph.D. project Iattempt to develop a quantitative system for studyinghuman somatic cell mutations in vitro using thesemethods. Human cells resistant to 8-azaguanine hadbeen selected from sensitive populations as early as1959 [20], but the cells used were genetically un-stable and metabolic cooperation — a process thatkills resistant cells in physical contact with sensi-tive cells in selective medium [21] was unknown.Therefore, resistant cells could be reliably induced orquantitated. Armed with an awareness of metaboliccooperation and having a source of stable diploidwild-type and Lesch–Nyhan mutant fibroblasts, Icould not fail [22,23]. Cells from Lesch–Nyhan boyswith inherited HPRT mutations were the prototypemutant cells serving to establish selection conditionsfor rare somatic mutations arising de novo.

HPRT is now an important component of thearmamentarium of genetic markers used for in vitromutagenesis studies in animal and human cells. It is asingle copy gene at position Xq26–27 in humans [24].The amino acid sequence of the enzyme [25] andthe nucleotide sequence of the HPRT coding region(654 bp) [26] are known. The gene in genomic DNA isapproximately 44 kb in length, includes nine relativelyshort exons and eight much larger introns and has beensequenced in its entirety [27]. Four non-functionalHPRT pseudogenes are located on chromosomes 5, 9and 11 [27,28]. It is important to my story that thisgene, so widely used in mutation research, came toour attention via its human germinal mutations.

4. HPRT mutations in vivo in humanlymphocytes

4.1. Developing the assays

I was asked during my oral genetics “prelim exam”at the University of Wisconsin how HPRT might beused for measuring in vivo mutations in humans. Ithought this to be a gesture of kindness — a “softball” question to put me at ease. The scenario wentsomething like this. I described how easily a skinbiopsy could be put into culture in selective andnon-selective media and the proportion of mutations(resistant cells) directly determined to define an in



being answered. Mutations are biomarkers of effect,i.e. they indicate in vivo genotoxicity. HPRT MFincreases associated with known mutagenic expo-sures indicate that the exposure is having an effect inthe setting being evaluated. Genotoxicity in humansexposed to a chemical that is a known genotoxic car-cinogen in animals adds to the weight of evidence thatthe agent is also carcinogenic in humans, making thisendpoint increasingly relevant for making cancer riskassessments. In vivo HPRT mutations or their sup-pression can indicate the efficacy of chemopreventionprograms designed to protect against the mutagenicconsequences of a particular environment or a can-cer treatment, or define “safe” levels of exposure toknown genotoxic agents. Finally, HPRT mutationscan be used as indicators of exposure when thereis no other biomarker for this purpose or, occasion-ally, to identify a specific exposure. The latter, how-ever, requires determination of molecular mutationalspectra.

Pat O’Neill joined me in Vermont in 1983, and JanNicklas came to the group shortly thereafter. Pat im-mediately began development of an in vitro assay forstudying HPRT mutations in human T cells [55]. Thisallowed manipulations of mutagenic treatments whichproved invaluable for interpreting and anticipatingresults of human in vivo studies. Jan became involvedin the molecular studies that were just beginning.

4.3. HPRT mutational spectra

Particularly rapid progress has been made in themolecular analysis of HPRT mutations arising inhuman T cells. Southern blots were used initially,followed by sequencing of polymerase chain reac-tion (PCR) products, both reverse transcriptase (RT)PCR for cDNA analyses and multiplex PCR forgenomic DNA analyses. Thousands of background(“spontaneous”) mutations arising in vivo in hu-mans have now been characterized at the molecularlevel. This background spectrum has been reportedin several reviews and as a computerized database ofpublished results [1,2,56–58]. HPRT mutations aris-ing in vitro in human cells or in vivo in animals havealso been characterized.

An unexpected early finding in these molecularstudies was that the adult in vivo background spec-trum differs markedly from that in newborns or in

young children. In adults, less than 15% of the HPRTmutations are due to gross structural alterations suchas deletions, insertions, etc. of large segments ofDNA, i.e. those visible on Southern blots [59]. Theremaining >85% are “point mutations” which includebase substitutions, frameshifts, smaller deletions andinsertions and complex changes revealed by sequenc-ing. Matt McGinniss, however, working as a graduatestudent with our group, found quite a different picturefor HPRT mutants isolated from placental blood thatrepresent in vivo mutational events in the fetus [60].He found the most frequent single kind of mutationto be a deletion of exons 2 and 3. In placental blood,75–85% of the mutations are due to gross alterations;the remaining are point mutations.

Jim Fuscoe, while still at the University of Connecti-cut, became interested in these exon 2,3 deletion muta-tions. As the deletions were all identical on Southernblots, he developed exon 1 and 3 primers based onfragment sizes to sequence across the breakpoints.In Matt’s original description of these deletions, wesuggested that they may represent errors made whilerearranging the T cell receptor (TCR) genes, so wehad some idea what to look for. Cells of the immunesystem recognize the universe of antigens via their sur-face receptors, i.e. surface immunoglobulins (Ig) on Blymphocytes and T cell receptors (TCR) on T lympho-cytes. There is an enormous diversity of these recep-tors generated by rearrangements of germline-encodedvariable (V), diversity (D), junctional (J) and constant(C) regions of the Ig and TCR genes [61–63]. Rear-rangements are mediated by an enzyme called V(D)Jrecombinase that is directed to highly conserved con-sensus sequences in DNA consisting of a heptamer(CACAGTG/A) and nonamer separated by 12 or 23bases for cleavage. V(D)J-mediated rearrangementsare characterized by certain hallmarks in the junctionalregions, i.e. nibbling back from the point of incision,the presence of P-nucleotides, and the insertion ofnon-germline templated bases, presumably by termi-nal deoxynucleotidyl transferase (TdT) activity [63].Normally, these processes occur only in the Ig or TCRgenes during the maturation stages of B and T cells,respectively. For T cells, this is within the thymus dur-ing fetal life and early childhood. If our hypothesis forthe origin of the HPRT deletion mutations was correct,the consensus cleavage sequences should be found atthe breakpoints, with the other hallmarks of V(D)J


in “phenocopies”, i.e. pseudo-6-thioguanine-resistantwild-type T cells that were scored as mutants. Cry-opreservation of the peripheral blood lymphocyteseliminated this problem, presumably by synchroniz-ing the cycling T cells so they did not appear in thelabeling window of the autoradiographic assay [42].With this modification, the short-term assay for invivo HPRT mutations remains in use today.

By the late 1970s, however, newer methods werebecoming available for the long-term culture of Tlymphocytes. T cell growth factor, later shown tobe the lymphokine IL-2, could support long-termgrowth of properly stimulated T cells. I was able totake sabbatical leave and return to Wisconsin, to theimmunology laboratory of Richard Hong, where ittook only a few months to develop a cloning assayfor in vivo HPRT mutations in human T cells [43].Mutant cells could be isolated, propagated in vitro,and characterized. This first report of a cloning as-say also showed that the mutant T cells were trulydeficient in HPRT enzyme activity — a step towardsdemonstrating their mutational basis.

The cloning assay gained much wider acceptancethan the autoradiographic assay as a method forstudying in vivo mutations because material was nowavailable for analyses. Alec Morley, who had earlieralso pursued autoradiographic studies of in vivo mu-tations, reported the development of a cloning assaythe following year [44]. Others adapted the assayto animals that could be manipulated to investigatedose-response characteristics of HPRT mutations in-duced in vivo [45]. Studies could now move forward inearnest.

4.2. Quantitative studies

As the assays for in vivo mutations were originallydeveloped as tools for human biomonitoring, the earlystudies by many groups were aimed at establishingbackground mutant frequencies (MFs) in human pop-ulations (these were termed variant frequencies [VFs]for the autoradiographic assay, remembering the phe-nocopy problem). Although there was considerablevariability among and even within individuals, meanMFs (and VFs) for groups were remarkable consis-tent, being ∼(5–10 × 10−6) for young adults (andsomewhat lower for VFs) [1,2,46]. Values were foundto increase with age, i.e. MFs in placental blood

are ∼10-fold lower than in adults, become higher inyoung children and increase at ∼2.5% per year af-ter adolescence [47]. Markedly higher VFs and MFswere found in individuals homozygous for the differ-ent rare genetic instability syndromes, i.e. xerodermapigmentosa, ataxia telangiectasia, Bloom syndrome,Werner syndrome and Fanconi anemia (although onestudy with the cloning assay failed to find an elevationin this last condition) [1,2,48].

Studies in individuals exposed to “model” envi-ronmental mutagens came next. Smoking has beenassociated with elevated MFs and VFs in most but notall studies [1,2]. Cancer patients receiving mutagenictherapies have usually shown the expected increasesin in vivo mutations. The issue of persistence of mu-tations is important for monitoring. Studies of atomicbomb survivors and Chernobyl workers showed that40 years is too long for useful recovery of mutantsbut that group elevations in MFs can be detected aslong as ten years after exposure to ionizing radiation[49,50]. For the autoradiographic assay, a massivechemical exposure gave the maximal VF elevationtwo weeks later while, for MFs determined by cloningassay, significant elevations were seen at 6 monthsin breast cancer patients receiving chemotherapy[51,52]. Optimal expression and persistence times,which are difficult to determine in humans, are notprecisely known even to this day.

Studies of populations exposed to environmentaland occupational mutagens came next, and continue.Elevations of VFs and MFs have been associated withionizing radiation and chemicals in several but notall studies [1,2]. In general, HPRT mutations are notthe most sensitive biomarkers for detecting mutagenexposures per se. Chromosome aberrations remainthe gold standard for acute ionizing radiation. Forchemicals, true biomarkers of exposure such as uri-nary metabolites or adducts in hemoglobin or DNAare certainly more sensitive. A study that comparedseveral biomarkers for their sensitivity in reflecting anexposure to an alkylating agent found them to be inthe order of hemoglobin adducts > sister chromatidexchanges (SCE) > chromosome aberrations >

micronuclei > HPRT mutations, even though eachgave a positive response [53].

If HPRT mutations are not sensitive measures ofexposure to genotoxic agents, why use them at all forhuman biomonitoring [54]? That question is slowly



5.2. TCR gene-defined clonalityof HPRT mutant T cells

As must be apparent, I have been concerned withquestions of in vivo mutant distributions since my“near-death” experience with fibroblast clonality. Iwas somewhat concerned that we interpreted T cellHPRT MFs, which are the proportions of mutantsto total lymphocytes, as HPRT mutation frequencies,which are the proportions of mutational events to totallymphocytes. Certainly, the frequency of mutant cellsis the best estimator of the frequency of mutationalevents in exponentially growing cell populations (bothmutant and wild-type cells undergoing clonal amplifi-cations). However, what is the best estimator when allbut a few of the cells in the population are in an ar-rested G0 stage, as is the case for T cells in vivo? Arethe mutational events randomly distributed amongdividing and non-dividing cells in such populations?Although feeling like a curmudgeon (and being so ac-cused), my concerns about clonality intensified as webegan to define mutational spectra. Initially we coulddefine HPRT mutational changes only by patternson Southern blots. Occasionally, we saw patterns fordifferent isolates from the same person that appearedto be identical, suggesting clonality. And, what aboutthe more than 85% of the mutants that showed nochanges on Southern blots? Did each represent a sin-gle mutation? Clonality does make a difference fordescribing mutational spectra, even for the restrictedspectra of deletion mutants that we could identify inthose days.

Shortly upon returning to Vermont after develop-ing the cloning assay in Wisconsin, I was complain-ing aloud about the lack of a marker of clonality in Tcells similar to that afforded to B cells by the Ig generearrangements. Jan Nicklas had recently finished apost-doctoral fellowship in immunogenetics. She in-formed us of the then new class of genes that appearedto control the long sought T cell receptor. Important forour purposes, these genes appeared to generate theirdiversity through somatic rearrangement, analogous tothe Ig genes (as described above), and could serve inthe same way as molecular markers of clonality. Ourstudies of these rearrangements date from that time,initially at the level of Southern blots. We expected todemonstrate that every HPRT mutant isolate from anindividual would show a different TCR gene rearrange-

ment, indicating its origin in a different in vivo matureT cell clone. In the absence of sequence data on HPRT,we would use TCR gene clonality as a surrogate forHPRT clonality assuming that, in adults, the mutationsarose in mature differentiated T cells (there was otherevidence for this, described below). HPRT mutationsarising in vivo in different TCR gene-defined cloneswould be considered independent.

Our first publication suggested that indeed all of theHPRT mutants did arise as independent in vivo events[82]. Soon, however, this delusion was destroyed.We began to notice “doublets” and “triplets” of TCRgene patterns among mutants isolated from individ-uals. Although subliminally troubling, we managedto overlook these aberrations. Then, we observedour first serious “outlier” — a woman with a MFof ∼500 × 10−6 in whom an excess of 90% of themutants derived from the same in vivo T cell clone[83]. This was hard to ignore. Clonality, lurking inthe shadows for over 15 years, was back.

We refined our analyses of the TCR genes, usinga two-step RT-PCR amplification and sequencing ofthe highly polymorphic CDR3/variable region to un-ambiguously identify specific rearranged TCR genesin the T cell isolates. We soon recognized that thesemolecular signatures, in addition to defining clonal-ity, provide points of reference for ordering temporalevents in an in vivo clone (such as HPRT mutation)that occurred before (in pre-thymic “stem” cells) orafter (in post-thymic mature T cells) the TCR generearrangement. This was how we could determinethat, in adults, almost all of the HPRT mutations arisein post-thymic mature T cells while, in the fetus andchildren, pre-thymic mutations are not infrequent.Four patterns of TCR gene rearrangement and HPRTmutational change define these clonal and temporalrelationships.

1. The same HPRT mutational change and TCR generearrangement in two or more mutant isolates fromthe same individual defines sibling HPRT mutantsoriginating from a single in vivo HPRT mutationalevent in a mature post-thymic T cell, with subse-quent clonal amplification.

2. Different HPRT mutational changes but the sameTCR gene rearrangement in two or more mutantisolates from the same individual defines indepen-dent in vivo HPRT mutational events originating


mediated rearrangements present in the junctionalregions. Jim was able to demonstrate these signaturechanges in the HPRT exon 2,3 deletion mutations [64].We now know that these particular HPRT mutationsare biomarkers for illegitimate V(D)J recombinaseactivity.

The characterization and exploitation of induced invivo and in vitro HPRT mutational spectra are justbeginning. Low LET ionizing radiation produces aspectrum that becomes increasingly dominated bylarge structural alterations such as deletions as ra-diation doses increase [65–67]. High LET ionizingradiation produces a somewhat different picture withsmaller deletions and even tandem mutations [68].Studies of chemical mutagen exposures in humanshave, thus far given mixed results, probably becauseof insufficient numbers of mutants analyzed [69].However, this area too is rapidly progressing.

Characterizations of HPRT mutational spectratherefore are providing databases for mutagenicitymonitoring, as well as insights into mutagenic mech-anisms. Although monitoring is the reason the assaywas developed in the first place, its application toinvestigations of mechanisms illustrates the changinguses of HPRT mutations.

5. HPRT mutations as mechanistic probes

Molecular studies have suggested that HPRT mu-tations reflect mutagenic and biologic processes thattranscend this locus. This use as probes for fundamen-tal processes is optimized when both the HPRT muta-tional changes and the TCR gene rearrangements arecharacterized in the same mutant isolates.

5.1. V(D)J recombinase-mediated HPRT deletions

These deletions in HPRT capture a mutagenicmechanism that appears to be ubiquitous in humanlymphoid malignancies, several of which are charac-terized by non-random chromosome rearrangementswith one breakpoint near an Ig or TCR gene [70–72].This breakpoint is in the heptamer–nonomer consen-sus sequence that directs canonical rearrangementsof these genes, while the other breakpoint is in acryptic consensus heptamer near an oncogene. Alter-natively, a submicroscopic deletion event may occur

with both breakpoints in V(D)J cryptic consensussequences, one being near a constitutively expressedgene and the other near an oncogene [73]. The junc-tional regions of the translocated chromosome or thedeletion frequently bear the hallmarks of a V(D)Jrecombinase-mediated event. Both serve to disregu-late the oncogene with carcinogenic consequences.Comparative analyses of the carcinogenic transloca-tions and deletions on the one hand and the reporterHPRT V(D)J recombinase mediated deletions onthe other have found striking similarities betweenthem [74].

V(D)J recombinase-mediated HPRT deletions arealso present in normal adults, but at much lowerfrequencies (∼1.6% of mutants) [75]. Have these mu-tants persisted since their induction decades earlierwhen the individual had a functioning thymus, or hasthe V(D)J recombinase activity somehow been reac-tivated in extra-thymic sites? Investigations of thesepossibilities can be undertaken using the HPRT dele-tion mutants as probes and have potential relevanceto both immunology and oncology. Regarding the lat-ter, Glen McGreggor, working with Veronica Maher,has shown induction of these deletions in vitro by1-nitropyrene in adult T lymphocytes [76]. Chen et al.have shown the same in a T cell line treated withetoposide [77]. Barry Finette in Vermont has recentlyshown that passive maternal exposure to cigarettesmoke during pregnancy increases the frequenciesof these mutations in placental blood [78]. This mayoffer an explanation for the observed relationship be-tween paternal cigarette smoking and early childhoodacute lymphocytic leukemia, the kind that originatesin utero. Paternal smoking results in passive maternalexposure to tobacco smoke. Bill Bigbee has recentlyreported results that are consistent with this associ-ation between maternal exposures to tobacco smokeand V(D)J recombinase-mediated HPRT deletions inthe fetus [79].

There are at least two PCR based direct assays fordetecting illegitimate V(D)J recombinase mediatedevents in vivo [80,81]. Both are easier and fasterthan the assay for HPRT mutations. However, onlythe cloning assay provides mutant cells for charac-terization and functional studies, the former perhapsallowing identification of permissive mechanisms andthe latter defining the immunological significance ofthese unusual mutants.



a heterogeneous population of cells (i.e. in this caseT cells) will also select for those cells in the popu-lation that are most likely to mutate (i.e. those rarepre-existing cells with genomic instability). There aremany possibilities for the occurrence in vivo of rarecells with genomic instability in large heterogeneouspopulations. One is an underlying constitutional het-erozygosity for DNA repair genes with the occurrenceof rare “null” cells arising from an earlier somaticmutation or recombination event — perhaps duringfetal development. Once an HPRT mutation occurs ina cell with pre-existing genomic instability, the pro-cess becomes progressive, resulting in non-selectedmutations, i.e. the secondary and tertiary HPRT muta-tions in single alleles that already contain the selectedmutation. This process of cell proliferation, selectionand mutation goes forward in apparently normal Tcells and may be an inherent biologic potential in allcells. This proposed model for the selective enrich-ment of HPRT mutants in children treated for ALLas a result of proliferation and genomic instabilityis shown in Fig. 1. We could recognize this processbecause we were studying a mutational event thatproduced an in vivo selective growth advantage. Eventhough the HPRT gene usually serves as a simple re-porter biomarker for population studies, its mutationsprovide a growth advantage in the context of purineanalogue chemotherapy. Mutant T cells, therefore,under the selective stimulus of restorative prolifera-tion following chemotherapy, have an in vivo growthadvantage similar to that in early pre-malignant cellswhere selection is also for mutations that confergrowth advantage. Further studies using this approachwill be useful to understand the progression fromearly initiating mutations to the generation of largeclones of cells that have lost genomic stability.

6. Back to the beginning

In our studies of genomic instability, HPRT changedfrom being purely a reporter gene to being one withfunctional significance. This significance, however,was in the growth advantage provided by mutations —there was no clinical component. As our studies pro-gressed, and as we began to look for HPRT mutationsin other patients receiving purine analogue therapies,we began to find alarming increases in HPRT MFs. In

the extreme, in individuals receiving the commonlyused immunosuppressive agent azathioprine, whichis metabolized to 6-mercaptopurine in vivo [103], wefound MFs as high as 2.0 × 10−1 ( i.e. 20%).

Concerns about clinical significance become hardto ignore when one out of five somatic cells is HPRTdeficient. Perhaps, such individuals should not receivefolate antagonists, which would be tantamount to re-ceiving in vivo HAT. What else might happen? Then,I had a truly frightening thought. What might happenif in vivo selection operated not only for somatic cellssuch as T cells, but also for germ cells? This returnsthe HPRT story to its beginnings [3,4].

This most recent concern about HPRT deficient cellsis not so much what is happening as a result of invivo mutations (although mutations must occur to givethe problem), but what is occurring as a result of invivo selection. With selection, there seems to be noa priori reason why HPRT mutant cells should notaccumulate in any cell population capable of sustainedin vivo proliferation. The enormous increases in T cellHPRT MFs probably are occurring in many cell typesin vivo, including the male germ cells. Potentially,these accumulations could reach several percent ofsperm. What might be the heritable consequences ofsuch accumulations?

If the HPRT mutant sperm are capable of fertilizingan ovum (and why wouldn’t they be, as all “Y”-bearingsperm are HPRT-deficient?), they will produce indi-viduals who carry the mutation. Therefore, selectionfor mutant sperm will be converted to selection fora heritable mutation. If fertilization does occur, andif the mutant sperm are at the frequencies suggestedby the T cells, one could predict an epidemic of indi-viduals with the Lesch–Nyhan syndrome. It might bethought that this can not be the case, as purine ana-logues have been used in medicine for decades withno such outcome. However, it must be rememberedthat the HPRT locus is on the X-chromosome. As thesituation of sustained germ-cell proliferation occursonly in males, and as males pass their X-chromosomesto their daughters, the mutant HPRT alleles will havegone to females in the first generation. This will haveno clinical consequences in that generation. However,in the next generation, these females, who will be car-riers, will pass half the mutant bearing chromosometo their sons. The manifestation of this epidemic willthen be in males born to mothers whose fathers were


had received combination chemotherapies. Althoughtreatment regimens differed, they were all completedin approximately 2.5 years and included mutagenicagents. More importantly, however, all the therapeuticregimens included long-term treatment with a purineanalogue such as 6-mercaptopurine or 6-thioguaninethat positively selected in vivo for cells deficient inHPRT, i.e. the precise cellular phenotype we werestudying.

It is useful to recall at this juncture that ourobservations for T cells in vivo have counterpartsin observations in both prokaryotic and eukaryoticcells in vitro [100–102]. In those systems, progres-sive rounds of mutation and selection for mutantphenotypes in populations of proliferating wild-typecells greatly enrich for cells with mutator phenotypes.Successive rounds of mutagenic chemotherapy cou-pled with in vivo selection for HPRT mutations in

Fig. 1. Selective clonal enrichment of HPRT mutants with and without genomic instability.

the ALL patients mimic these in vitro mutagenesisexperiments.

We believe that T cell clones with mutator pheno-types arise in the following manner. The ALL patientshad undergone several rounds of cytotoxic chemother-apy. Following each round, the T cell clones thatsurvived the extensive cell killing had to undergoseveral cycles of proliferation to restore cell numbers.Somatic mutations (including HPRT) will arise inthese proliferating clones in increased numbers (butnot at increased rates) as a result of the proliferation.However, although the mutational process itself is notspecific, the mutations affecting HPRT are selectedbecause of the long-term exposure to the purine ana-logues. The HPRT mutants themselves then are ableto proliferate more rapidly than the non-mutant Tcells and eventually to overgrow them. Importantly,selecting for any mutation (i.e. in this case HPRT) in



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taking a purine analogue medication at some timeprior to the mother’s conception. This is hardly anobvious or easily recognized clinical presentation!

It is perhaps stating the obvious that this sce-nario may be but one extreme example of somethingthat occurs more commonly that is realized. Mightselection by drugs or other agents (as opposed tomutations) be the driving force in increasing otherheritable mutations in human populations?

This may be pure fantasy. For once in my career, Ihope that a hypothesis of mine is wrong. However, Ihave been able to infect Dan Casciano at the NationalCenter for Toxicological Research (NCTR) with thisconcern. He is making it possible for Bob Heflich andme to investigate this possibility in mice. The exper-iments to be done are obvious. Others, including in-vestigators at NCTR, have adapted the HPRT cloningassay to rodents, and results are entirely analogous towhat is found in humans.

The HPRT cloning assay for mutations in vivo willnow be used to detect heterozygosity in females, inthis case, female mice. The precursor of the cloningassay began as a proof-of-principle demonstration thata small minority population of HPRT deficient T cellscould be found in known Lesch–Nyhan heterozygotes.The current concern arose because it became possiblethrough those proof-of-principle experiments to quan-tify HPRT mutant somatic cells in vivo in humans.The circle is complete.

7. Conclusion

I hope I’ve been able to show why HPRT haskept at least my attention for over three decades. Thestory began in somatic cell genetics, went to muta-genicity monitoring and then availed itself of the newtechnologies of molecular genetics. HPRT mutationshave become useful probes for studying mechanisms— some underlying the mutagenic process itself andothers underlying immunological responses. HPRTmutant T cell populations have also demonstrated thein vivo evolution of genomic instability — somethingthat may not be limited to malignant cells but may bea fundamental property of all cells. The only sour noteis that this wonderful target gene and its mutationsbecame known to us through a devastating humanheritable disease — the Lesch–Nyhan syndrome.

Perhaps our use of this target gene to prevent anincrease of affected individuals or, better yet, to con-vince ourselves that such an increase is not going tooccur, will be a partial pay-back.

I’m often asked if I’m still “doing” HPRT, or if Ihave moved on. Yes, I’m still doing HPRT.

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The Discovery of mRNA ☆Elliot Volkin * 1

Toxicology and Risk Analysis Section, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USAAccepted 24 October 2000



The Discovery of mRNA�

Elliot Volkin∗,1

Toxicology and Risk Analysis Section, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA

Accepted 24 October 2000

Keywords: mRNA; Oak Ridge; History of science; RNA; Nucleic acids

1. The Biology Division: early years

It was 1947, and our government was left with thelegacy of having dropped the atomic bombs that killedthousands but brought WWII to an end. The AtomicEnergy Commission (AEC) was then obligated tostudy thoroughly the effects of radiation on humans,as well as all living things. In addition, an opportu-nity existed for investigating the many peaceful usesof a variety of radioisotopes. That year, AlexanderHollaender was assigned the task of developing abiology unit at the Oak Ridge National Laboratory(ORNL) to carry out these missions. Hollaendercame from the experience of having investigated themutagenic effects of UV and X-ray irradiation at theUniversity of Wisconsin and then as lead biophysi-cist at the Washington Biophysics Institute. With thisbackground, and with his special knack for gettingfunding as well as the unique ability for anticipating

� This article is part of the Reflections in Mutation Researchseries. To suggest topics and authors for Reflections, readersshould contact the series editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]).

∗ Tel.: +1-865-241-0030; fax: +1-865-241-0397.1 Oak Ridge National Laboratory is managed by UT-Battelle,

LLC, for the US Department of Energy under Contract no.DE-AC0500OR22725.

the cutting edges of science, he was a perfect fit asDirector of the Biology Division at the Oak RidgeNational Laboratory (ORNL).

Although Hollaender was promised almost unlim-ited funding from the AEC, it was with the stipulationthat the Biology Division must occupy a buildingused previously to test the centrifugal process forthe separation of uranium isotopes. This process andthe building had quickly been abandoned in favor ofthe method of gaseous diffusion. The structure,with its factory-like appearance and totally inade-quate facilities, would eventually become a model ofbiological research.

Hollaender, even at that time, realized the vitalsignificance of genetics and made that his centraltheme in assembling a research staff. In the late 1940sand early 1950s, he hired an outstanding collection ofvery bright young geneticists whose interests covereda wide expanse of animal and plant life: Dan Linds-ley with Ed Novitsky and Larry Sandler (Drosophila,although Dan is also famous for his butterflycollection); Rhoda and Ed Grell (irradiation effects onDrosophila); Drew Schwartz (maize); Alan Conger(Tradescantia) and Sheldon Wolff (Vicia faba); JackVon Borstel (Hymenoptera); Kim Atwood (Neuro-spora) and Fred de Serres with Herman Brockman(Neurospora); Mary Esther Gaulden (grasshopper

1383-5742/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S1383 -5742 (00 )00061 -2


E. Volkin / Mutation Research 488 (2001) 87–91 89

As a result of these experiments, we were able toestablish the 3′- to 5′-internucleotide bonding forRNA, as in DNA. I am prompted to ask the ques-tion of whether honest-to-goodness biochemistry wetlabs exist in academia anymore. I am overwhelmedby the vast array of commercially available, highlypurified RNA, DNA, and protein preparations, thehuge selection of enzyme kits including PCR, DNAchips for all transcription needs, etc. In order to carryout the enzyme experiments described above, andespecially to determine the structure and sequenceof the various oligonucleotides, we had to purify avariety of exo- and endonuclease enzymes. Some ofthe starting materials were bones, potatoes, barley,and calf livers and intestines. On one occasion, aftera tiring day of stripping calf intestines and bones,my associates decided to hang the exhausted entrailsand bones on all of the overhead service pipes. Wehad no air conditioning or drop ceilings in thosedays. Alex Hollaender happened to come into our labthe next morning. He took a hard look at this uglysight, asked how everything was going, and walkedout.

3. Bacteriophage

After a few years, I was able to form my owngroup, and I was anxious to take a more biologicallyoriented direction. The bacteriophage had becomeattractive as a complete, but perhaps the least com-plicated, biological system for biochemical studies.I had the good fortune of being taught the basics ofhandling these materials by a very patient Gus Doer-mann. The big question that intrigued me about thesesystems was the fact that, in spite of highly activeDNA and protein synthesis following lytic phage in-fection of Escherichia coli, material RNA synthesisabruptly stopped. This was contrary to the case witha large number of actively growing, or dividing, bio-logical systems where RNA synthesis accompaniedthe synthesis of protein and DNA. Hershey, however,in his study of the uptake of 32P into DNA after T2infection, observed the incorporation of the label intoa fraction with the properties of RNA [4]. With theseobservations in mind, it occurred to me that we hadthe tools to unequivocally determine whether or notRNA synthesis existed after bacteriophage infection.

Our experience with the ion-exchange proceduresdeveloped by Cohn [5] made it clear that we couldclearly separate the 2′- and 3′-mononucleotides re-sulting from alkaline digestion of RNA from anyfree-standing 5′-nucleotides. Furthermore, DNA iscompletely untouched by the alkaline conditions usedto hydrolyze RNA. The experimental approach wasfairly direct. Shortly after infection of E. coli B with ahigh multiplicity of infection with bacteriophage T2,32P was added, and samples taken at various timesafterward. Total nucleic acid was extracted from thesamples, hydrolyzed with mild alkali to produce theRNA mononucleotides and these subjected to analy-sis by our ion-exchange process. It was clear from thevery first that a small but definite amount of isotopewas incorporated into RNA, but this amount quicklyleveled off with time after 32P addition. But whatreally struck me immediately was that the nucleotidecomposition was nothing like that of the host RNA,but instead mimicked the composition of the analo-gous nucleotide composition of the infecting phage’sDNA. That is, adenylic acid (A) was equal to uridylicacid (U); and cytidylic acid (C) was equal to guanylicacid (G); and the ratio of UA/CG was about 2/1, thesame as the AT/GhmC ratio, where T is thymidylicacid, G is guanylic acid, and hmC is hydroxymethyl-cytidylic acid in T2 DNA.

At this point, Larry Astrachan joined me in carry-ing out a number of more definitive experiments overthe next 3 years [6]. By heavily labeling the phageitself we ruled out the possibility that it containedeven a very small amount of RNA [7]. Subsequentexperiments showed that the labeling of RNA in in-fected cells was not a result of stoppage of RNAsynthesis, but was a result of highly active turnoverof this species of RNA, preventing any significantaccumulation [8]. These pulse-chase experimentswere accomplished by adding an excess of unlabeledinorganic phosphate at various times after the ad-dition of the radioactive compound. An interestingobservation from the pulse-chase experiments wasthat additional RNA species of varying compositionwere synthesized with later times after infection [9],an observation in line with the increasing reports ofso-called early and late gene function [10]. Additionalexperiments, with the unrelated T7 bacteriophage [11]and uninfected E. coli [12], confirmed that this minorspecies of RNA with its rapid turnover could be found

88 E. Volkin / Mutation Research 488 (2001) 87–91

neuroblast embryos); Larry Morse (bacterial genet-ics); Bill Welshons (Drosophila, notch locus); BillBaker (Drosophila), consultant to the Division onthe staff of the University of Tennessee; Dick Kim-ball (Paramecium); Bill and Liane Russell, Bill asdirector of the large mouse colony and Lee subse-quently as director after Bill’s retirement; and theone person to whom I will always be grateful, GusDoermann (bacteriophage), whose lab also includedFranklin Stahl, Charlie Steinberg, and David Krieg.This diverse group of geneticists made noteworthycontributions while at the Division, then (with the ex-ception of the Russells who stayed on at Oak Ridge)eventually accepted professorial positions at majoruniversities and research positions elsewhere.

Charlie “Nick” Carter, who had been associatedwith Hollaender at the NIH, was asked to form a bio-chemistry unit with its research keyed toward con-nections with genetics (i.e. nucleic acids). AlthoughCarter had some laboratory experience in the area, heput together a small, rather hodgepodge group whohad very little prior knowledge of the nucleic acids.Waldo Cohn and Joe Khym were invited from the an-alytical division at ORNL. Although Cohn’s graduateand post-graduate training at the University of Califor-nia, Berkeley, and Harvard had been biological, he andKhym had spent the past five WWII years immersedin the heroic job of separating the isotopic elements byion-exchange chromatography, a method developed bythat group at ORNL. Dave Doherty, at Wisconsin, wasa whiz in the organic synthesis of novel biochemicals(e.g. warfarin with Professor Link). I had come froma lab at Duke University directed by Hans Neuraththat was noteworthy for its basic research on proteins.Early on, John Totter, who did have some backgroundwith the nucleic acids, joined us on leave from the Uni-versity of Arkansas. How quickly we all changed ourinterests! Within months we became one unit, concen-trating on the biochemistry of RNA and DNA, whichat that time, was in its primitive stages of development.

2. The internucleotide structure of RNA

To ascertain the basic structure of the nucleic acids,most investigators in the field, including Carter in ourgroup, were separating nucleic acid-related productsby paper chromatographic techniques. Cohn, on the

other hand, was determined to apply his knowledgeof ion-exchange chromatography for the separationof these materials. Very soon thereafter Cohn wasso successful at this effort that his name became thebyword in the field. He was able to separate virtuallyall of the bases, nucleosides, and nucleotides withsuch pristine purity that his assignments of the opti-cal densities of most of these compounds became theinternational standards. Our work was directed moretoward RNA than DNA, the structure of RNA inmany ways being more complicated. I am remindedthat George Brown’s chapter in the 1953 issue ofAnnual Review of Biochemistry referred only to PNA,the pentose of RNA not yet being firmly establishedas ribose [1]. This was also the same year that Watsonand Crick announced the helical structure of DNA. Igot into the area of nucleic acids in a rather indirectway. Totter et al. [2] were studying the uptake of 14Cformate into chicks and rats under the influence offolic acid (more later about this experiment). In addi-tion to analyzing the incorporation of this isotope intoDNA thymidine and purines, they also were curiousabout the uptake into RNA. At this point, I volun-teered to try my experience with protein separationsfor the separation of an RNA product. I was able toget a reasonable preparation for the time; a single, butrather broad peak, in the ultracentrifuge. It may havebeen the first application of guanidinium salts for thispurpose [3]. In any case, I became convinced thatnucleic acids would be my lifetime research direction.

Cohn had found that alkaline hydrolysis of RNAproduced two mononucleotide isomers of the baseshe called a and b. They had the phosphoryl groupslinked to the 2′ and 3′ positions of ribose. This raisedthe question of whether, in RNA, 2′- to 5′-linkagesexisted (DNA, by necessity, has only the 3′- to5′-attachments). We even had the clever notion thatthese 2′- to 5′-linkages would lead to branchedstructures for RNA. Clever, maybe, but wrong. Tomy knowledge, no branched structure of RNA hasbeen reported. Our subsequent hydrolysis of RNAwith pancreatic ribonuclease led to products —pyrimidine mononucleotides and a variety of small,pyrimidine-ending oligonucleotides — whose endgroup phosphorus was found only at the 3′-positionof ribose. These experiments were followed laterwith digests by intestinal and snake venom phos-phodiesterases that produced only the 5′-end groups.


E. Volkin / Mutation Research 488 (2001) 87–91 91

itself very quickly. Exactly the properties required forwhat we called X, the unstable intermediate we hadpostulated. . . .” Jacob then goes on to fault himselffor not recognizing this final piece to the puzzle. TheRNA, then, became not only an integral part of thelactose story but became universally accepted as theintermediary in carrying the genetic code from DNAto the ribosomes for the synthesis of specific proteins.

And so it goes. A nucleic acid derived from ourearlier work on the primary structure of RNA that wecalled DNA-like RNA, and a metabolic intermediarycalled X, deduced from many years of investigationof the lactose operon, became the same thing. Thisnucleic acid was given the name messenger RNA(mRNA).

Acknowledgements

I would like to thank E.S. “Liz” Von Halle for help-ing me recollect some of the Biology Division’s earlygeneticists and Lois Thurston for her invaluable aid inthe preparation of this manuscript. Another article onthe discovery of mRNA was published in TIBS at therequest of Jan Witkowski (E. Volkin, What was themessage? TIBS 20 (1995) 206–209).

References

[1] G.B. Brown, Nucleic acids, purines, and pyrimidines, Ann.Rev. Biochem. 22 (1953) 141–178.

[2] J.C. Totter, E. Volkin, C.E. Carter, Incorporation of formateinto the nucleotides of ribo- and desoxyribonucleic acids, J.Am. Chem. Soc. 73 (1951) 1521–1522.

[3] E. Volkin, C.E. Carter, The preparation and properties ofmammalian ribonucleic acids, J. Am. Chem. Soc. 73 (1951)1516–1520.

[4] A.D. Hershey, Nucleic acid economy in bacteria infected withbacteriophage T2. II. Phage precursor nucleic acid, J. Gen.Physiol. 37 (1953) 1–23.

[5] W.E. Cohn, The anion exchange of ribonucleotides, J. Am.Chem. Soc. 72 (1950) 1471–1478.

[6] E. Volkin, L. Astrachan, Phosphorus incorporation inEscherichia coli ribonucleic acid after infection withbacteriophage T2, Virology 2 (1956) 149–161.

[7] E. Volkin, L. Astrachan, The absence of ribonucleic acid inbacteriophage T2r+, Virology 2 (1956) 594–598.

[8] E. Volkin, L. Astrachan, RNA Metabolism in T2-InfectedEscherichia coli. The Chemical Basis of Heredity, JohnsHopkins University Press, Baltimore, 1957, pp. 686–694.

[9] E. Volkin, Ribonucleic acid turnover in phage infection,in: Proceedings of the Fourth International Congress onBiochemistry, Vol. 7, Pergamon Press, 1958, pp. 212–224.

[10] E.P. Geiduschek, Regulation of expression of the late genesof bacteriophage T4, Ann. Rev. Genet. 25 (1991) 437–460.

[11] E. Volkin, J. Astrachan, J.L. Countryman, Metabolism of RNAPhosphorus in Escherichia coli infected with bacteriophageT7, Virology 6 (1958) 545–555.

[12] J.L. Countryman, E. Volkin, Nucleic acid metabolismand ribonucleic acid heterogeneity in Escherichia coli, J.Bacteriol. 78 (1) (1959) 41–48.

[13] E. Volkin, L. Astrachan, Intracellular distribution of labeledribonucleic acid after phage infection of Escherichia coli,Virology 2 (1956) 433–437.

[14] L. Astrachan, E. Volkin, Effects of chloramphenicol onribonucleic acid metabolism in T2-infected Escherichia coli,Biochem. Biophys. Acta 32 (1959) 449–456.

[15] F. Jacob, J. Monod, On the regulation of gene activity, ColdSpring Harbor Symp. Quant. Biol. 26 (1961) 193–201.

[16] F. Crick, What Mad Pursuit: A Personal View of ScientificDiscovery, Basic Books, 1988.

[17] F. Jacob, The Statue Within: An Autobiography, Basic Books,1988.

90 E. Volkin / Mutation Research 488 (2001) 87–91

in these systems as well. Separation of the subcellularconstituents of phage-infected bacteria revealed thatthe component of highest RNA specific activity wasa particulate fraction that resembled membranes [13].We called this unique RNA, DNA-like RNA.

Labeling experiments using 32P orthophosphateconvinced us that the isotope was being incorporatedinto species of RNA hitherto absent in the host E. coli.But since RNA is synthesized utilizing 5′-nucleotideprecursors, and alkaline hydrolysis yields only the2′- and 3′-nucleotide products, the possibility existedthat we were only measuring the uptake into thenearest neighbor of a preformed RNA and not thecomposition of a separate species. To check for thispossibility, we carried out T2 pulse-chase experimentsusing 14C formate as tracer. Formate was known tobe a precursor in the in vivo synthesis of nucleic acidpurines, becoming the 2 and 8 carbon atoms in thepurine ring. These experiments elegantly confirmedour data using 32PO4, whereby the RNA A/G 14Cratio was 2:1 at the early stages of infection, this ratioundergoing changes at later times after infection [9].An unusual by-product of these experiments typifiesthe genetic control by DNA. Here we observed thatin addition to the expected uptake of formate intoRNA purines, this precursor somehow found its wayinto the RNA pyrimidines as well [9]. To my knowl-edge, formate incorporation into pyrimidines has nototherwise been shown. This was not the case withT7-infected cells or uninfected E. coli in experimentscarried out at the same time, nor was it found in testscarried out years earlier with Carter and Totter, in theRNA pyrimidines of chick and rat tissues. But mostgratifying to us was the observation that the 14C ratioin U and C was 2:1 in T2-infected cells, mimickingthe T/hmC ratio of T2 DNA [9].

In the midst of these experimentations, Gus Doer-mann urged me to expand my knowledge of this fieldby taking the phage course at Cold Spring Harbor.I suspect that part of his reasoning was to free hislaboratory from my constant visitations. In any case,the wonderful atmosphere there — Mark Adams’phage course itself, the regular interaction with topmolecular biologists from this country and abroad,the total immersion in all aspects of bacteriophage —suggested to me that if there is such a place as Heavenfor molecular biologists, it must be just like that 1957summer at Cold Spring Harbor. It was during my

stay there that I had the opportunity to discuss ourfindings at length with Al Hershey. Hershey, who hadbeen doing experiments with the protein synthesis in-hibitor, chloramphenicol, suggested that we see howour system responded with this compound. A numberof these experiments [14] under a variety of condi-tions made it clear that the synthesis of this uniqueRNA was directly associated with the synthesis ofprotein, but we were not able to determine the mech-anism of this association. The exact manner of thisRNA-protein connection would be revealed by Jacoband Monod some 3 years later [15].

4. DNA-like RNA is mRNA

The response to our work by the scientific com-munity was mixed. It was received as being of somemajor significance by most biochemists and molecularbiologists. There followed invitations to present ourwork at FASEB symposia and Gordon Conferences, ata number of universities, and as a major contributor tothe International Congress of Biochemistry in Viennain 1958. As part of the tour of European laboratoriesthat year, I was invited to present our data to a smallselect group of phage workers at an Abbey outside ofParis. Most of those in attendance were phage geneti-cists, and I recall them being totally unresponsive tomy talk.

It was during this time that Francois Jacob andJacques Monod at the Pasteur Institute had beendeveloping the complex mechanism of the lactoseoperon [15]. An integral part of their postulate wasthe necessity of the existence of an unstable in-termediate that carried the information from DNAto produce the enzyme galactosidase. It was at anow-famous informal meeting in Cambridge, St.Patrick’s Day, 1960, that Jacob, Francis Crick, andSidney Brenner suddenly — with great excitement— realized the importance of our findings. To quotefrom Crick’s What Mad Pursuit [16]: “Brenner hadseen the answer. . . It is difficult to convey two things.One is the flash of enlightenment when the idea wasfirst glimpsed. . . The other is the way it cleared upso many of our difficulties”. From Jacob’s book, TheStatue Within [17], “[This Volkin-Astrachan RNA]had two remarkable properties: it had the same basecomposition as DNA; on the other hand, it renewed

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