Copyright 0 1992 by the Genetics Society of America

Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

Neurospora: The Organism Behind the Molecular Revolution

David D. Perkins

Department of Biological Sciences, Stanford University, Stanford, Calgornia 94305-5020

U NDER the title “Fifty Years Ago: The Neuro- spora Revolution,” HOROWITZ (1 99 1) has cele-

brated an anniversary of the epochal 1941 paper of BEADLE and TATUM, which reported the first mutants with biochemically defined nutritional requirements. HOROWITZ’S account and others (HOROWITZ 1973, 1985, 1990; LEDERBERC 1990) have focused on the people who were involved, the genesis of their ideas, and the role of the 1941 results in transforming biology. The present essay will be concerned mainly with the research organism that was so important to the success of the initial experiments. Neurospora possesses a combination of features that made it an ideal choice not only for accomplishing the original objectives set by BEADLE and TATUM but also for a continuing succession of contributions, including many in areas that transgress the bounds of biochem- ical genetics and molecular biology. 1 shall begin by outlining the story of Neurospora prior to BEADLE and TATUM and then go on to sketch its subsequent history. The previous accounts have stressed biochem- ical genetics and molecular biology. I shall consider other aspects as well, focusing first on genetics, con- tinuing with a summary of research accomplishments of all sorts, and concluding with a consideration of the potential usefulness of Neurospora for population studies.

The vegetative phase of Neurospora was described and used for experiments by French microbiologists nearly 100 years before BEADLE and TATUM (PAYEN 1843; MONTAGNE 1843). In the warm, humid summer of 1842, bread from bakeries in Paris was spoiled by massive growth of an orange mold. A commission was set up by the minister of war to investigate the cause of the infestation and to make recommendations. The commission’s report (PAYEN 1843) includes a colored plate which shows colonies, mycelia, conidiophores and conidia of the “champignons rouges du pain.” An experiment in photobiology is described. Colonies

Genetics 130 687-701 (April, 1992)

grown in the light quickly became bright orange. Colonies grown in the dark, however, remained white for more than 8 days, but the white colonies developed orange pigment within 2 hr when they were brought into the light. Thermal tolerance was also studied (see PAYEN 1848, 1859). These results were cited by PAS- TEUR (1 862) in reporting his own experiments on the survival of mold spores, which helped to refute theo- ries of spontaneous generation.

The next experimental study of Neurospora began in Indonesia during Dutch colonial times. In market- places of East Java, bright orange cakes are displayed. These consist of Neurospora grown on soybean or peanut solids from which oil and protein for curd have been pressed. The Javanese inoculate the solids with conidia to create an appetizing and highly nutri- tious food called oncham, which has a mushroom-like taste (WENT 1901a; SHURTLEFF and AOYACI 1979; Ho 1986). Producing oncham is a cottage industry which has probably gone on for centuries and which continues today.

A Dutch plant physiologist, F. A. F. C. WENT, was stationed at the famous Buitzenjorg (now Bogor) Bo- tanic Gardens in Java at the turn of the century. WENT was attracted by the orange oncham fungus and started experimenting with it. He was frustrated be- cause humidity in Java is sa great that the organism grew through the cotton plugs of his culture tubes. WENT (1904) also found Neurospora in Surinam, where he noted that the fungus was used to process cassava meal in preparation of an indigenous alcoholic beverage. Back home in Utrecht, he described the oncham fungus and its culture (WENT 1901a) and used it for a series of studies on the effects of various substrates on enzymes such as trehalase, invertase and tyrosinase (WENT 1901 b). WENT (1 904) also studied the effect of light on carotenoid production. With knowledge of WENT’S work, PRINGSHEIM (1 909) in- cluded Neurospora in a study of oxidases, and KUNKEL

688 D. D. Perkins

(1 9 13, 19 14) used it in studies of chemical toxicity. All these observations were made using the vegetative phase of the organism and the asexually produced powdery conidia (vegetative spores).

The association of Neurospora with heat and fire must have been known from the earliest times. We now know that the sexually produced heat-tolerant ascospores remain dormant until exposed to heat. Heat activation of ascospores explains the occurrence of Neurospora both in bakery infestations and on burned vegetation. Numerous records going back over a century describe large orange areas following volcanic eruptions in tropical areas. In New Guinea, tribesmen traditionally set hillsides on fire to flush game, and Neurospora bloomed fobowing the burns. In Brazil, MOLLER (1 90 1) described an orange fungus growing on burned vegetation (and on maize bread). A typically ascomycete sexual phase appeared in his cultures, and the sexual fruiting bodies (perithecia) and ascospores were later identified as Neurospora. In Japan, Neurospora made a dramatic appearance following the great Tokyo earthquake and fire of 1923. Within a few days, burned and scorched trees became festooned with orange. Mycologists in two laboratories cultured the organism. KITAZIMA (1 925) observed perithecia in his cultures, and going back to the source, discovered that perithecia were present under the bark of trees in the Temple of Shiba. Orange progeny were obtained from single asco- spores. TOKUGAWA and EMOTO (1924) studied sur- vival of the fungus following exposure to moist and dry heat, and identified the orange pigment as a carotenoid.

Neurospora is commonly seen following agricul- tural burning in warm, moist climates. Sugar cane appears to be an ideal substrate. Ascospores are no doubt activated by burning in the fields and by heating in the mill. Bales of bagasse (fiber from which the sap has been pressed) become orange. In Australia, solids from refinery filters are spread on fields as fertilizer. Large orange colonies develop on this filter mud. Honey bees can be seen visiting the colonies and filling their pollen baskets with the brightly colored conidia (SHAW and ROBERTSON 1980).

The modern history of Neurospora begins in the mid-1920s with material from sugar cane bagasse. The key person was BERNARD DODGE. Like BEADLE, DODGE had grown up on a farm. He worked for years as a school teacher and managed to complete his bachelor’s degree only at age 39. He published his first paper at the age of 40 and was already past 50 when he began to work with Neurospora (ROBBINS 1962). Prior to the Neurospora work, DODGE was the first to discover heat activation of ascospores (1912) and to describe mating types in ascomycetes (1 920), both in Ascobolus.

About the time KITAZIMA was examining his orange fungus in Japan, CHARLES THOM, a colleague of DODGE’S at the Department of Agriculture mycology and pathology laboratory in Arlington, Virginia, was studying cultures of orange mold from sugar cane bagasse in Louisiana. THOM was of the opinion that the orange fungus, then called Monilia sitophila, lacked a sexual stage. However, C. L. SHEAR, the head of the laboratory, found perithecia in one of THOM’S plates. The material was given to DODGE for analysis. The success of DODGE’S experimental crosses kindled his enthusiasm, and Neurospora became his main lifelong interest.

DODGE’S first Neurospora paper, with SHEAR in 1927, goes far beyond the conventional taxonomic descriptions of genus and species. Cultures of the orange fungus had been obtained from many sources. Isolates were assigned to the new genus Neurospora on the basis of their grooved ascospores. (Prior to 1927, the vegetative stage had successively been called Oidium aurantiacum, Penicillium sitophilum and Mo- nilia sitophila.) DODGE showed that the cultures in- cluded three species which were set off from one another by their crossing behavior. Hybrid perithecia from crosses between different species developed slowly and were unproductive or poorly fertile. Al- though a conventional morphologically based taxo- nomic species description was provided for each spe- cies, crossing behavior was implicitly taken into con- sideration and used to assign strains to the designated species. This innovation contrasted with the purely morphological criteria then used by mycologists and clearly anticipated the idea of biological species long before the concept was formalized.

Two species with eight-spored asci, Neurospora crassa and Neurospora sitophila, were shown to be heterothallic: individual haploid cultures from single ascospores were unable to enter the sexual cycle. They fell into two mating types, defined because crosses could occur only between strains of opposite mating t Y Pea

DODGE carried out the first tetrad analysis with N . crassa, showing that the mating types segregated 4:4 in individual asci. The asci were obtained as groups of eight ascospores that had been spontaneously ejected from the perithecia. The Neurospora asco- spores were activated by heat, as in Ascobolus. In contrast to the eight-spored species, isolates of Neu- rospora tetrasperma, with four-spored asci, appeared to be homothallic. Cultures from single ascospores were usually self-fertile. A few self-sterile progeny were produced, however, that behaved as though they were heterothallic. DODGE (1927) was shortly to de- scribe the cytological basis of this “pseudohomothallic” behavior of N . tetrasperma, showing that individual ascospores were usually (but not always) heterokar-

Perspectives 689

yons that contained haploid nuclei of opposite mating (:;\HI. (;. 1,lSl>b:GREN

types. DODGE lost no time in communicating his enthusi-

a s m . At Columbia University, he urged T. H. MOR- GAN and the Drosophila group to use Neurospora. He traveled to Cornell for a seminar. Among the gradu- ate students in the audience were GEORGE BEADLE and BARBARA MCCLINTOCK. BEADLE ( 1 966) later re- called how the students, familiar with then-recent results of E. G. ANDERSON using Drosophila attached- X half-tetrads, were able to point out to DODGE how the second-division segregations he described in Neu- rospora could be explained by crossing over between chromatids at the four-strand stage.

DODGE soon moved to a job as plant pathologist at the New York Botanical Garden. In addition to his official duties, he managed to continue experiments with Neurospora. These included pioneering work on interspecies crosses and on mutations affecting ascus development. He was intrigued by heterokaryons and obtained combinations of strains that showed hetero- karyotic vigor (DODGE 1942). In the next 30 years he published nearly 50 papers on the genetics, cytology, morphology and life cycle of Neurospora.

DODGE’S enthusiasm resulted indirectly in the re- cruitment of CARL LINDEGREN, who did the most significant genetic work with Neurospora prior to BEADLE and TATUM. In 1928, LINDEGREN moved to California from Wisconsin, where he had obtained a Master’s degree in Plant Pathology. He visited T . H. MORGAN to inquire about continuing graduate work at the California Institute of Technology, where MOR- GAN had come with his Drosophila group to head the new Biology Division. LINDEGREN found MORGAN using dissecting needles in an attempt to isolate Neu- rospora ascospores from an agar plate (see LINDEGREN 1973). MORGAN suggested that LINDEGREN work with Neurospora. Following a visit to DODGE, LINDEGREN chose the species N. crassa as best suited for genetic work. He developed highly fertile wild-type strains,

identified mutants that could be used as markers and discovered the first linkages. The linked genes pro- vided confirming proof that crossing over occurred at the four-strand stage. Genetic maps were constructed for two linkage groups using mating type, centro- meres, and morphological mutants. But at about the time BEADLE and TATUM were turning from Drosoph- ila to Neurospora, LINDEGREN abandoned Neuro- spora to begin work on Saccharomyces. His last Neu- rospora paper (LINDEGREN and LINDEGREN 1942), written with his life-long collaborator GERTRUDE LIN- DEGREN, was submitted just as the Stanford workers were about to obtain their first biochemical mutant. LINDEGREN’S Neurospora papers are listed in BACH- MANN and STRICKLAND ( 1 965).

Neurospora was used for several other investiga- tions during the decade before 194 1 . I first heard of it in a plant physiology course taught by DAVID GOD- DARD, who used N. tetrasperma in studies of ascospore activation and dormancy (GODDARD 1935, 1939). BUTLER, ROBBINS and DODGE (1941) demonstrated that biotin was the sole growth factor requirement. In England, WHITEHOUSE ( 1 942) subjected LINDEGREN’S tetrad data to a detailed analysis and went on to produce his own five-point map of the mating-type chromosome of N. sitophila. I t was DODGE and LIN- DEGREN, however, who developed the genetics of Neu- rospora during the 1930s and made the organism known to geneticists. The accumulated information enabled DODGE ( 1 939) to assert: “The fungi in their reproduction and inheritance follow exactly the same laws that govern these activities in higher plants and animals.”

Additional information regarding early history can be found in the introductory sections of SHEAR and DODGE (1 927), MOREAU-FROMENT (1956), and PER- KINS and BARRY (1977), and in essays by RYAN and OLIVE ( 1 96 l ) , TATUM ( 1 96 1 ), LEDERBERG (1 990). SRB ( 1 973), CATCHESIDE (1 973) and LINDEGREN ( 1 973).

690 D. D. Perkins

” I hree genetics textbooks published in 1939 (STUR- TEVANT and BEADLE, SINNOTT and DUNN, AND WAD- DINGTON) included accounts of Neurospora in the context of recombination and sex determination, with diagrams showing the relation of crossing over to second division segregation in the linear ascus.

In February 194 1 , BEADLE wrote to DODGE regard- ing stocks. His letter begins “Dr. Tatum and I are interested in doing some work on the nutrition of Neurospora with the eventual aim of determining whether the requirements might be dependent on genetic constitution.” Eight months later, their paper reporting success in obtaining nutritional mutants was submitted to the Proceedings of the National Academy of Science.

Obtaining the first biochemical mutants and pro- posing the one-gene one-enzyme hypothesis were only two of many advances in which Neurospora played a pioneering role. The problems for which it has since been used are extremely diverse, often ranging far afield from the biochemical genetics that first made it famous. For example, Neurospora soon made funda- mental contributions to understanding the mechanism of recombination. It was also used to resolve the great confusion existing at that time about fungal chromo- somes and their behavior in meiosis.

In 1944, BARBARA MCCLINTOCK visited Stanford University at BEADLE’S invitation; KELLER (1 983, pp. 1 13-1 18) describes the visit. MCCLINTOCK’S long ex- perience with maize enabled her to show convincingly that the chromosomes of Neurospora and their be- havior in the ascus were typically eukaryotic. Using simple light microscopy, she went far beyond the original objective of determining the chromosome number. She outlined the details of meiosis and de- scribed the seven chromosomes. The smallest Neu- rospora chromosomes are now known each to have a 1 C DNA content less than that of Escherichia coli. She showed that they were nevertheless individually rec- ognizable by their distinctive morphology at pachy- tene (MCCLINTOCK 1945; for photographs comparing Neurospora and maize pachytene chromosomes see Figure 6 in PERKINS 1979). She went on to describe pachytene pairing in a translocation heterozygote and to record the ascus types that resulted from different modes of segregation when the translocation was het- erozygous. At the end of her two-month stay in Cali- fornia there was no longer any question: it was clear that fungal chromosome cytology, like fungal ge- netics, is basically similar to that of plants and animals.

The 1941 paper of BEADLE and TATUM opened up exciting possibilities just at a time when war was diverting funds from pure to applied research and when young scientists were moving either into applied research or into the military. BEADLE’S success in keeping his group intact and in obtaining support

BARBARA MCCI.IN.I.OCK

testifies to his confidence in the importance of the research and his persuasiveness as to its value. (There was then no National Science Foundation and no program of external research support by the National Institutes of Health. BEADLE turned to the Rockefeller Foundation and the Nutrition Foundation, and to pharmaceutical firms. See BEADLE 1974; KAY 1989.)

Not everyone was persuaded. HOROWITZ (1979) describes how some geneticists continued to resist the idea that individual enzymes were specified by single genes. And BEADLE (1974) recalls a wartime visit by the mycologist, CHARLES THOM. After being shown some of the striking morphological mutants that were known to segregate as single-gene differences, THOM took BEADLE aside and advised him “What you need is a good mycologist. Those cultures you call mutants are not mutants at all. They are contaminants!”

At the war’s end in 1945, the Neurospora work had progressed substantially and was widely known. (When I returned to Columbia University from the army, DOBZHANSKY told me that the two highlights in biology during the war had been HUXLEY’S book Evolution the Modern Synthesis and BEADLE and TA- TUM’S Neurospora mutants.) Students were attracted to Neurospora. So also were established scientists who had previously been working on other organisms: D. G. CATCHESIDE, STERLING EMERSON, NORMAN GILES, HERSCHEL MITCHELL, FRANCIS RYAN and MOGENS WESTERGAARD. During this period, Neurospora also provided the first introduction to research for numer- ous individuals who were later to become known for their work with other organisms. Among those whose careers began in this way were EDWARD ADELBERG, BRUCE AMES, AUGUST DOERMANN, NAOMI FRANKLIN, LEONARD HERZENBERG, DAVID HOGNESS, BRUCE HOLLOWAY, ESTHER LEDERBERG, JOSHUA LEDERBERG, NOREEN MURRAY, NORORU SUEOKA and CHARLES

Perspectives 69 1

YANOFSKY; see, for example, RYAN and LEDERBERG (1 946).

The Neurospora approach was soon extended to other fungi such as Ophiostoma and Ustilago. GUIDO PONTECORVO, who had previously worked on Dro- sophila with H. J. MULLER, began his program with Aspergillus nidulans. Genetic work flourished on Po- dospora, Sordaria, Ascobolus, Coprinus and Schizo- phyllum. Biochemical mutants were obtained in Schi- zosaccharomyces, Chlamydomonas and even in a flow- ering plant, Arabidopsis (LANGRIDGE 1955).

Application of the Neurospora approach to bacteria was not long delayed. Auxotrophic mutants of E. coli were obtained by CHARLES GRAY (a Stanford under- graduate) and TATUM (1 944), and independently by ROEPKE, LIBBY and SMALL ( 1 944). These made pos- sible the 1946 demonstration of recombination in E. coli and opened the way for the explosive development of bacterial genetics.

Saccharomyces was a relatively slow starter. Heter- othallism with two mating types was discovered by CARL and GERTRUDE LINDEGREN only in 1943. The first induced auxotrophic mutations and the first link- ages were reported in 1949. Eleven workers attended the first yeast conference in 196 1 (VON BORSTEL 1963), compared to 92 participants at a Neurospora conference held the same year (DE SERRES 1962). (Attendance at international yeast meetings now ex- ceeds 1 OOO!)

Advantageous features of Neurospora that were recognized as novel and noteworthy in the 1940s are now largely taken for granted because the same fea- tures are shared in various combinations by many other organisms that have since come into common use. Neurospora differed in important ways from the animals and plants used by most geneticists in 194 1 . It was haploid. All four products of individual meioses could be recovered, and in such a way that centro- meres were readily mapped. Heterokaryons could be formed. Nutritional requirements were defined and simple. Stocks could be preserved in suspended ani- mation, effectively conferring immortality on individ- ual strains. In addition, growth was rapid, generation time short and fecundity high. Propagules suitable for plating were produced abundantly. Pure cultures could readily be obtained and tested for auxotrophic mutations.

Neurospora ascospores are large enough to permit manual isolation without a micromanipulator. Work- ers were initially intrigued by the ability to map cen- tromeres, and genetic analysis was mostly done at first by laboriously dissecting the spores from linear asci in serial order. With time, it was realized that ordered ascus analysis is rarely necessary and that for most purposes random ascospores provide the needed in- formation with far less effort (see PERKINS 1953).

When entire asci are needed for such purposes as studying interference, obtaining double mutants, or identifying chromosome rearrangements, it was found that unordered asci, shot from the perithecium as octets, can easily be obtained in large numbers (STRICKLAND 1960). Addition of a centromere marker made these unordered groups essentially as informa- tive as intact asci (e.g., PERKINS et al. 1986).

Neurospora conidia are a boon for transferring, plating, transforming, preserving stocks and sampling wild populations. These powdery vegetative spores are potentially hazardous as airborne contaminants, however. Laboratory practices were quickly devel- oped that minimized the risk. It was found that if simple precautions are taken, there is no reason why Neurospora cannot coexist in the same laboratory with bacteria, yeast or slowly growing microorga- nisms.

The rapid linear growth of Neurospora (which can exceed 4 mm/hr) is a great advantage for many pur- poses, but for platings it was necessary to develop appropriate media containing colonializing agents such as sorbose (TATUM, BARRATT and CUTTER 1949) or to use genetic variants with restricted growth, such as the conditional colonial mutant cot-1.

Reliable and economic methods were developed for maintaining permanent stocks in suspended animation in silica gel, by lyophilization, or by freezing. These methods (WILSON 1986) enable the Fungal Genetics Stock Center ( 1 990) to carry over 7000 Neurospora strains, with no need for periodic serial transfers.

Along with successes, Neurospora workers inevita- bly experienced frustrations and disappointments. It was initially hoped that new mutations might reveal previously undiscovered essential metabolites, but none were found. (A prospective new amino acid, tentatively named neurosporin, proved to be a crys- talline mixture of isoleucine, valine and leucine; see KAY 1989). An elegant scheme to use heterokaryons for quantitative studies of dominance (BEADLE and COONRADT 1944) proved impractical because many laboratory stocks were heterokaryon incompatible, but this finding opened up the study of vegetative incompatibility and led to the finding that genes re- sponsible for this incompatibility are numerous and are highly polymorphic in natural populations.

Recombinant DNA research with Neurospora was initially impeded by regulatory guidelines that first denied permission to proceed, then required that a disabling mutant be built into recipient strains. After permission was granted, it was found that genes intro- duced by transformation were poorly recovered from crosses, although they remained stably integrated in the chromosomes during vegetative growth. The poor sexual transmission proved to be due to RIP (repeat- induced point mutation), a process that mutates du-

692 D. D. Perkins

plicate genes during the sexual phase (see below and SELKER 1990). RIP was then shown to provide an effective means of achieving targeted gene mutation, an asset which more than compensated for the incon- venience of poor transmission.

Inevitably, other organisms sometimes proved to be superior to Neurospora for particular purposes. For example, bioassays using induced auxotrophs were first developed in Neurospora during the war years, bu t bacteria proved to be so much faster for bioassay that Neurospora was not used to any extent.

Beginning in the 195Os, Neurospora played a cen- tral role in studies of recombination, providing the first proof of gene conversion (MITCHELL 1955) and revealing its main features (see below). Targeted reg- ulation of local recombination frequencies by rec genes that are unlinked or nonadjacent was discovered in Neurospora (CATCHESIDE,~ESSUP and SMITH 1964). Random ascospores of Neurospora continue to be the main source of information on recombination control of this type. Other fungi proved to be superior for recombination studies that required tetrads, however. Conversion frequencies were found to be much higher in yeast and Ascobolus than in Neurospora, while Sordaria and Ascobolus both had the advantage of numerous viable, readily scorable, autonomously ex- pressed ascospore mutants. Neurospora was still a major source of information on gene conversion in the early 1960s when molecular models for eukaryotic conversion and crossing over were proposed by HOL- LIDAY and by WHITEHOUSE and HASTINGS. However, by the mid-I970s, when the more detailed Aviemore model was put forward by MESELSON and RADDING, the most extensive and most critical data came from asci of Saccharomyces, Sordaria and Ascobolus. As a result of this trend, one geneticist whose interests focused almost exclusively on recombination models asked me bluntly in 1984 what I had been doing since the demise of Neurospora!

In fact, the change of emphasis away from recom- bination may have been a blessing in disguise for Neurospora genetics. In my own laboratory, it re- sulted in attention being given to other problems which might otherwise have been neglected. Chief among these was the study of chromosome re- arrangements (see PERKINS 1979). Because deficiency ascospores remain unpigmented while nondeficiency spores are black, Neurospora proved ideal for detect- ing and diagnosing rearrangements. Meiotic mutants were examined cytologically and genetically, together with other mutants affected in development of the sexual phase. I also began to collect and analyze Neu- rospora from natural populations. This led, among other things, to the discovery of Spore killer elements, which bear a striking formal resemblance in their behavior to Segregation distorter in Drosophila, the

t-complex of mice, and gamete eliminator in tomato. Far from being defunct, Neurospora continues to

be a superb research organism. At the present time, it is used as the primary research object in about 70 laboratories in North America and 25 laboratories in 16 countries abroad. It remains the microorganism of choice for numerous specific problems. The knowl- edge and the genetic resources that have been ac- quired during 65 years are invaluable assets. But the most important factor responsible for its wide use is probably an exceptionally happy combination of traits that makes it suitable for research on problems span- ning the entire range from molecules to populations.

The versatility of the organism is illustrated by the examples gathered below. Many of the contributions that will be cited were pioneered using Neurospora. Some of the advances were the first for filamentous fungi, others for the fungal kingdom, and others for all eukaryotes. However, the object in citing them is not to stress priority but to illustrate the variety of research areas to which Neurospora has contributed significantly. The list is far from complete. For ex- ample, no attempt has been made to cover the exten- sive work on specific enzymes or pathways, or on novel biochemical mutants; for documentation of many of these see PERKINS et al. ( 1 982).

Nutritional mutants were used for many purposes. Inter- mediate steps in biosynthetic pathways were determined. By 1944, at least seven different genes had been identified that were involved in the synthesis of arginine (SRB and HOROW- ITZ 1944). [For early work on biosynthesis, see the reviews by HOROWITZ (1950) and by VOCEL and BONNER (1959).] In contrast to what is often the situation in bacteria, genes concerned with successive steps of the same biosynthetic pathway were shown not to be clustered but to be scattered through the genome (for review see HOROWITZ 1950). An apparent exception, the aro cluster-gene (GROSS and FEIN 1960), proved to make a single protein product with seg- ments that specify five separate enzymatic activities (GAERT- NER and COLE 1977).

The first conditional biochemical mutants were identified (STOKES, FOSTER and WOODWARD 1943; MITCHELL and HOULAHAN 1946). Temperature-sensitive mutants were used for testing the one-gene one-enzyme hypothesis (Ho- ROWITZ and LEUPOLD 1951). Different alleles at a locus were shown to produce forms of an enzyme with qualita- tively different properties (HOROWITZ and FLING 1953). Some mutant strains that lacked a specific enzymatic activity were shown to produce a protein that cross-reacted dith antibody against the enzyme (SUSKIND, YANOFSKY and BON- NER 1955).

Complementation of allelic mutations was demonstrated, first between nuclei in heterokaryons (FINCHAM and PATE- MAN 1957; GILES, PARTRIDGE and NELSON 1957), then between the protein products in vitro (WOODWARD 1959; for review see FINCHAM 1966).

Translational suppression was analyzed for the first time at the molecular level (YANOFSKY 1956). Perhaps the best understood mechanism of metabolic suppression was de-

Perspectives 693

scribed, involving suppression of arg-2 by pyr-3d and pyr-3a by ~ r g - 1 2 ~ (DAVIS 1967; REISSIG, ISSALY and ISSALY 1967). These studies entailed the discovery of two genes for one enzyme, one gene for two enzyme activities, and duplicate enzyme activities for two pathways.

Proof was obtained for the channeling of pathway-specific enzymes in separate pools (WILLIAMS, BERNHARDT and DAVIS 1971). Compartmentation of metabolic pools and pathways within vacuoles, cytosol and mitochondria was established and studied in detail (WEISS 1973) (for reviews see DAVIS 1986; DAVIS and WEISS 1988).

Unlinked genes concerned with the same pathway were shown to be coordinately controlled (GROSS 1965). Cros- spathway (general) control of amino acid biosynthetic en- zymes was discovered (CARSIOTIS and LACY 1965; CARSI- OTIS, JONES and WESSELINC 1974). Convincing evidence for positive control was provided for the first time in eukaryotes in a pioneering study of the regulation of sulfur metabolism (MARZLUF and METZENBERG 1968); sulfur regulation in eukaryotes has since been analyzed most fully in Neurospora (for review see FU et al. 1990). A hierarchy of regulatory elements involved in phosphate metabolism was identified and, on the basis of genetic evidence, the novel concept was proposed that regulation is not limited to interactions be- tween regulatory complexes and the DNA sequences of succeeding elements in the regulatory cascade, but that it also involves direct interaction between the protein products of regulatory genes (METZENBERG and CHIA 1979) (for review see METZENBERG 1979).

Mutants with a wide spectrum of altered vegetative mor- phologies were obtained and analyzed (e.g., GARNJOBST and TATUM 1967) and biochemical defects were identified in some of them (for review see MISHRA 1977). The morpho- logical mutant crisp-1 (one of LINDEGREN'S first markers) was shown to lack adenylate cyclase activity (TERENZI, FLA- WIA and TORRES 1974). (The important regulatory signal cyclic AMP is therefore absent and must be dispensable in Neurospora, unlike Saccharomyces.) The process of coni- diation was studied in wild type and in mutants (SPRINGER and YANOFSKY 1989). Genes with greatly elevated expres- sion during conidial differentiation, identified by BERLIN and YANOFSKY (1985), were used in studying regulation (e.g. , ROBERTS and YANOFSKY 1989).

Mutants affected in development of the sexual cycle were examined (for review see RAJU 1992b). One of these (RAJU 1986) appears to be the Neurospora counterpart of the po lp i to t i c mutant in maize, which BEADLE described and studied early in his career.

Electrodes were successfully inserted into Neurospora hyphal cells (SLAYMAN and SLAYMAN 1962). Electrophysio- logical studies showed that glucose transport is driven by a transmembrane proton gradient (SLAYMAN 1970; for review see SLAYMAN 1987); unlike animal cells, the plasma mem- brane potential is maintained primarily by proton flux rather than by potassium and sodium fluxes (SLAYMAN 1965). Other transport systems were shown to be driven by a proton-cotransport mechanism (SLAYMAN and SLAYMAN 1974). Mutant strains were obtained that can be grown indefinitely as protoplasts, without a cell wall (EMERSON 1963; SELITRENNIKOFF, LILLEY and ZUCKER 1981). These were used to isolate and characterize plasma membranes, to

prepare plasma membrane vesicles and to demonstrate that membrane ATPase is a proton pump (SCARBOROUGH 1975; for review see SCARBOROUGH 1978).

Kinetic and genetic studies of amino acid transport (by

DEBUSK, and WILLIAM THWAITES and LAKSHMI PENDYALA) identified several transport systems with broad substrate specificities, unlike the highly specific "permeases" of bac- teria but resembling the broad-specificity systems of mam- malian cells, which had been based on kinetic evidence alone. Four major amino acid uptake systems were charac- terized, distinct from those in yeast (for review see PALL 1970).

Maternal transmission was demonstrated for a class of non-Mendelian respiratory defects (MITCHELL, MITCHELL and TISSI~RES 1953). A non-Mendelian cytochrome defect was transferred between vegetative strains by injecting mi- tochondria (DIACUMAKOS, GARNJOBST and TATUM 1965). Mitochondria were shown to increase in number by division of preexisting mitochondria rather than being formed de novo (LUCK 1963). DNA was isolated from mitochondria for the first time (LUCK and REICH 1964). Progeny were shown to receive mitochondrial DNA only from the maternal par- ent, in both interspecific and intraspecific crosses (REICH and LUCK 1966; MANNELLA, PITTENCER and LAMBOWITZ 1979). A cyanide-insensitive alternative oxidase was identi- fied for the first time in fungi (LAMBOWITZ and SLAYMAN 197 1).

The first sequencing of a nucleic acid from mitochondria (HECKMAN et al. 1978) revealed unique features of initiator tRNA that foreshadowed the discovery of numerous unex- pected features of mitochondrial genomes (for review see BREITENBERGER and RAJBHANDARY 1985). A protein-cod- ing gene was shown to be located within an intron of another mitochondrial gene (BURKE and RAJBHANDARY 1982), ex- tending a discovery in yeast to the filamentous fungi. Self- splicing of a mitochondrial intron was first demonstrated (GARRIGA and LAMBOWITZ 1984) and the first mutants were found that are affected in the splicing of mitochondrial RNA (MANNELLA et al. 1979). Reverse transcriptase was first shown to be present in mitochondria (AKINS, KELLEY and LAMBOWITZ 1986; KUIPER and LAMBOWITZ 1988). Ty- rosyl-tRNA synthetase was shown to play an essential role in splicing (AKINS and LAMBOWITZ 1987).

Several key discoveries concern the mechanisms respon- sible for the import into mitochondria of polypeptides that are synthesized on cytoplasmic ribosomes. Pools of com- pleted polypeptides were shown to be present in the cyto- plasm (HALLERMAYER, ZIMMERMAN and NEUPERT 1977). Different mitochondrial receptors were indicated to be re- sponsible for the import of different precursor polypeptides (ZIMMERMAN, HENNIG and NEUPERT 1981). The first se- quence was obtained for the precursor of a nuclear-coded protein of the mitochondrial inner membrane or matrix (VIEBROCK, PERZ and SEBALD 1982). Contact sites function- ing in import were demonstrated between inner and outer mitochondrial membranes (SCHLEYER and NEUPERT 1985). A processing protease responsible for cleaving targeting sequences in the mitochondrial matrix was purified (HAW- LITSCHEK et al. 1988).

Mitochondrial plasmids were found, with sequences un-

GABRIEL LESTER, DAVID STADLER, MARTIN PALL, GIB

694 D. D. Perkins

related to those of mitochondrial DNA (COLLINS et al. 198 1). Strains were discovered that became senescent following integration of plasmids into the mitochondrial DNA (RIECK, GRIFFITHS and BERTRAND 1982; for review see BERTRAND and GRIFFIrHS 1989). Horizontal transfer of mitochondrial plasmids was shown to occur, independently of mitochon- drial DNA (MAY and TAYLOR 1989; GRIFFITHS et al. 1990; COLLINS and SAVILLE 1990).

The first circadian rhythm in fungi was discovered, man- ifested as periodic conidiation that provided a permanent record as bands were formed along a growth continuum (PITTENDRIGH et al. 1959). Mutations were obtained that affect the free-running period length of the circadian clock (FELDMAN and HOYLE 1973). Clock-controlled genes were identified that are transcribed only at specific times in the circadian day (LOROS, DENOME and DUNLAP 1989). For reviews see FELDMAN and DUNLAP (1983), LAKIN-THOMAS, COT$ and BRODY (1 990) and DUNLAP (1 990).

Resetting the circadian clock was shown to be mediated by a blue-light photoreceptor (SARGENT and BRICCS 1967). Other effects of blue light were studied, including the induction of carotenogenesis, formation of protoperithecia, and phototropism of perithecial beaks (for review see DEGLI- INNWENTI and RUSSO 1984). Mutants were identified that are blind to photoinduction (HARDING and TURNER 1981).

Heterokaryons (for review see DAVIS 1966) were used, first to study dominance and complementation, then to transfer mitochondria, plasmids and transposable elements, to rescue and maintain lethal or deleterious mutations, to map deficiencies, and to determine mutation frequencies.

Use of Neurospora heterokaryons led to the discovery of vegetative (heterokaryon) incompatibility (for review see PERKINS 1988). It was found that the mating type locus functions vegetatively as a heterokaryon incompatibility lo- cus (BEADLE and COONRADT 1944; SANSOME 1945). Other genes controlling the formation of stable heterokaryons (het genes) were identified and mapped (GARNJOBST 1953). Mi- croinjection of incompatible cytoplasm or extracts was shown to be lethal to recipient cells (WILSON, GARNJOBST and TATUM 1961). Partial diploids heterozygous for a het locus were obtained and shown to be highly abnormal (NEWMEYER and TAYLOR 1967; PERKINS 1975). An un- linked suppressor was discovered that neutralizes the vege- tative incompatibility function of the mating type genes but not their mating function (NEWMEYER 1970). Polymorphic het genes were found to be so numerous that they effectively preclude formation of heterokaryons in natural populations of N. crassa (MYLYK 1976).

The mating type genes A and a were cloned, sequenced, and shown to be present in a single copy per genome, with characteristics quite unlike those of the mating type genes of yeast. Although A and a occupy precisely the same locus, their DNA sequences were found to contain no recognizable homology (GLASS et al. 1988) (for reviews see METZENBERC and GLASS 1990; GLASS and STABEN 1990). Mutations had earlier been obtained that inactivate the mating type genes (GRIFFITHS and DELANGE 1978). Genes were identified that are transcribed preferentially during sexual development, and cloned sequences of these mating-specific genes were used to obtain, by RIP and gene disruption, mutant strains in which sexual development is impaired (M. A. NELSON

and R. L. METZENBERG, unpublished results). Attraction of trichogynes to cells of opposite mating type was shown to be mediated by a diffusible mating-type specific pheromone (BISTIS 1983).

Cytological techniques were perfected and details of meiosis and ascus development and of chromosome mor- phology and behavior were examined by light microscopy, both in wild type and in mutants (for reviews see RAJU 1980, 1992b). Synaptonemal-complex karyotypes were obtained by reconstructing meiotic prophase nuclei from thin sections (GILLIES 1972). Recombination nodules were shown to be correlated with reciprocal crossing over events at pachytene and to exhibit positive interference (GILLIES 1972, 1979; BOJKO 1989). Synaptic adjustment of the synaptonemal com- plex was shown to occur in inversion heterozygotes (BOJKO 1990). Neurospora was the first filamentous fungus for which intact DNA molecules from entire individual chro- mosomes were separated electrophoretically, extending the maximum chromosome length that was then physically re- solvable (ORBACH et al. 1988).

Tetrad analysis using a long multiply marked chromo- some arm showed that meiotic crossing over and interfer- ence closely resemble those in Drosophila and Zea mays (PERKINS 1962).

The first definitive proof of gene conversion was accom- plished in Neurospora (MITCHELL 1955). Important char- acteristics of conversion were delineated, especially by MARY MITCHELL, MARY CASE, NOREEN MURRAY and DAVID STAD- LER; see FINCHAM, DAY and RADFORD (1 979).

Genes were discovered that dramatically control the fre- quency of recombination at unlinked or nonadjacent target sites (CATCHESIDE, JESSUP and SMITH 1964), with recombi- nation reduced by dominant alleles at the controllinp loci (for review see CATCHESIDE 1975).

N . crassa was the first fungus to have all linkage groups mapped genetically (BARRATT et al. 1954) and assigned to cytologically distinguished chromosomes (for review see PERKINS and BARRY 1977). Tester strains that incorporate translocations were devised and greatly speeded linkage detection and mapping (PERKINS et al. 1969). Genes at nearly 700 loci and breakpoints of more than 300 rearrangements have been mapped (PERKINS et al. 1982; PERKINS 1990; PERKINS and BARRY 1977; and D. D. PERKINS, unpublished results). The conventional maps have been complemented using restriction fragment length polymorphisms (METZEN- BERG et al. 1984, 1985; METZENBERG and GROTELUESCHEN 1990) and random amplified polymorphic DNA markers (RAPD mapping) (WILLIAMS et al. 1991).

Genes specifying 5s RNA were shown to be dispersed through the genome in single copies (FREE, RICE and METZ- ENBERG 1979; SELKER et al. 1981). Telomeres were cloned and shown to have a DNA sequence identical to that in Homo sapiens (SCHECHTMAN 1987, 1990). Random breaks in ribosomal DNA sequences of the nucleolus organizer region were shown to acquire telomere sequences de novo (BUTLER 1991).

Following MCCLINTOCK (1 945), chromosome re- arrangements of various types were identified and put to many uses (for review see PERKINS and BARRY 1977). Be- cause ascospores that contain deficiencies fail to darken, frequencies of ejected asci with different numbers of black

Perspectives 695

and nonblack spores could be used to distinguish different rearrangement types (PERKINS 1974). Genetic analysis of insertional translocations has been more thorough than in other organisms (see, for example, PERKINS 1972). Numer- ous quasiterminal rearrangements with chromosome seg- ments translocated to telomeres or subtelomere regions were also studied. Insertional and terminal rearrangements were shown to generate partial-diploid progeny (DE SERRES 1957; ST. LAWRENCE 1959; NEWMEYER and TAYLOR 1967). The duplications obtained as segmental aneuploids from insertional and terminal rearrangements proved useful for mapping (PERKINS 1975) and for studying vegetative incom- patibility (NEWMEYER 1970; PERKINS 1975), instability (NEWMEYER and GALEAZZI 1977), and dominance and dos- age of regulatory genes (e.g., METZENBERG and CHIA 1979). Partial-diploid progeny from crosses heterozygous for ter- minal rearrangements were found to revert frequently to euploid condition, usually by loss of the translocated seg- ment.

Heterokaryons were used to recover and characterize recessive lethal mutations (ATWOOD and MUKAI 1953; DE SERRES and OSTERBIND 1962), to determine the frequency of recessive mutation for loci throughout the genome (DE

SERRES and MALLING 1971; STADLER and CRANE 1979), and to study mutagenesis, DNA repair, and dose-rate effects (STADLER and MOYER 1981; STADLER 1983; STADLER and MACLEOD 1984). The spectra of mutational lesions were examined for different mutagens and genotypes (e.g., DE SERRES and BROCKMAN 199 1 ; KINSEY and HUNG 198 1).

An excision-repair mutant was obtained that shows in- creased sensitivity solely to UV, the first example of its type in eukaryotes (ISHII, NAKAMURA and INOUE 1991). A subset of mutagen-sensitive mutants was shown to be abnormally sensitive to histidine and hydroxyurea, and to cause chro- mosome instability (SCHROEDER 1986); this includes mem- bers of two epistasis groups (KAFER 1983). Many mutants of this subset were found to have abnormal deoxyribonucleo- tide triphosphate pools (SRIVASTAVA and SCHROEDER 1989). Histidine and hydroxyurea were shown to cause chromo- some instability in the absence of any mutation causing mutagen sensitivity (NEWMEYER, SCHROEDER and GALEAZZI 1978; SCHROEDER 1986), and histidine was found to cause breaks or nicks in DNA (HOWARD and BAKER 1988).

An endo-exonuclease of Neurospora was characterized and shown to be immunochemically related both to the RecC polypeptide of E. coli and to an endo-exonuclease that is deficient in the rad52 mutant of Saccharomyces (FRASER, KOA and CHOW 1990).

The first DNA-mediated transformation in a sexual fun- gus was achieved in Neurospora (N. C. MISHRA, SZABO and TATUM 1973). [Aspergillus niger had been transformed ear- lier by SEN, NANDI and A. K. MISHRA (1969).] The proto- trophic character, putatively due to transformation, was poorly transmitted through crosses (N. C. MISHRA and TA- T U M 1973), behavior now attributable to RIP but then a cause for skepticism. With the advent of DNA technology and efficient transformation methods (CASE et al. 1979), integration of transforming DNA was found to be primarily nonhomologous. [For a review see FINCHAM (1989).]

Inactivation of duplicated DNA sequences was found to occur premeiotically during the period of proliferation be-

tween fertilization and fusion of nuclei (SELKER et al. 1987; for review see SELKER 1990). This phenomenon, termed repeat-induced point mutation (RIP), was shown to involve methylation and C to T mutation in both copies of duplica- ted sequences. RIP can be used to achieve targeted gene inactivation following transformation. [Premeiotic inactiva- tion of duplicated segments has since been shown to occur in other fungi; for review see SELKER (1 990).] Independ- ently, BUTLER and METZENBERG (1989) found that the number of ribosomal DNA repeats in the nucleolus organ- izer region undergoes change during the same premeiotic period that is subject to RIP.

An active transposable element was identified, the first to be characterized molecularly in filamentous fungi (KINSEY and HELBER 1989). This LINE-like element was shown to be transmitted from one nucleus to another in heterokar- yons (KINSEY 1990).

Methods were devised for sampling natural populations, and wild-collected strains were analyzed (PERKINS, TURNER and BARRY 1976; for review see PERKINS and TURNER 1988). Discrete orange colonies found on burned vegetation in warm, moist climates were shown usually to represent pure haploid clones of Neurospora from different asco- spores. Fertility in crosses to standard reference strains was shown to be a convenient and reliable criterion for deter- mining the species of wild-collected isolates. New hetero- thallic species were described. Homothallic Neurospora spe- cies, devoid of conidia, were also discovered (see FREDERICK, UECKER and BENJAMIN 1969).

Genetic polymorphisms at the protein level were shown to be abundant in natural populations of heterothallic spe- cies (SPIETH 1975), not a foregone conclusion for a haploid organism. Numerous vegetative incompatibility loci were identified and shown to be polymorphic (MYLYK 1975, 1976). Wild populations were shown to carry a load of phase-specific recessive mutations that adversely affect meiosis and the sexual diplophase (LESLIE and RAJU 1985). The nonselective abortion of asci in crosses between inbred strains of a normally outbreeding species provided an ex- ample of inbreeding depression in fungi (RAJU, PERKINS and NEWMEYER 1987).

Chromosomal elements (“Spore killers”) were discovered that show meiotic drive, resulting in the death of meiotic products that do not contain the element. Recombination was shown to be blocked in the chromosomal region that contains the killer element, reminiscent of SD in Drosophila and the t-complex in mice (TURNER and PERKINS 1979; for review see TURNER and PERKINS 199 1).

Length mutations in mitochondrial DNA were studied in different N . crassa populations (TAYLOR, SMOLICH and MAY 1986) and were used to construct a phylogenetic tree for four different species (TAYLOR and NATVIC 1989). Mito- chondrial plasmid DNAs were also compared in different populations and species (NATVIG, MAY and TAYLOR 1984).

Clearly, Neurospora research has till now been con- cerned mostly with genetic, cellular and molecular mechanisms. Relatively little attention has been paid to evolutionary biology, or to population genetics, which has been based since its beginnings almost ex- clusively on plants and animals while the fungal king-

696 D. D. Perkins

dom has been largely ignored. Yet the fungi offer certain advantages for studying populations, not least of which is haploidy during the vegetative stage. Imag- ine what could be done if it were possible to sample animal or plant populations by obtaining individual sperm or pollen grains and growing them up into immortal haploid or homozygous individuals. The equivalent of this is accomplished routinely in Neu- rospora, where orange haploid colonies that origi- nated from single ascospores are readily sampled in the wild, propagated in the laboratory, and main- tained as permanent viable stocks in suspended ani- mation. Some 4000 strains are already available that have been obtained in this way from populations in many parts of the world. What has been learned from them so far suggests that Neurospora can perhaps become for the population genetics of haploid orga- nisms what Drosophila has been for diploids (for re- view see PERKINS and TURNER 1988).

As with Drosophila, attention in the laboratory has been focussed primarily on one Neurospora species, N . crassa, but other species have also come into use. The known Neurospora species range from highly outbred to highly inbred. Some are heterothallic and cross-fertilizing, others are homothallic and self-fertil- izing. One species, N . tetrasperma, does not fall into either category and has been termed pseudohomo- thallic. Like its counterparts in other genera, it rep- resents a breeding system that is based on heterokar- yosis and is therefore unique to the fungi. N . tetra- sperma normally perpetuates itself as a self-fertile heterokaryon containing haploid nuclei of both mat- ing types. Most conidia and ascospores are heterokar- yotic, producing self-fertile cultures that behave as though they were homothallic. A minority of the spores are homokaryotic, however, resulting in self- sterile, functionally heterothallic cultures. The species is therefore predominantly inbred, but it retains the capacity for outbreeding as a ready option (RAJU 1992a). This diversity of life styles in the various Neurospora species shows promise for comparative studies.

After many years of asking “how” questions about the way that Neurospora functions, we should now be in a strong position to ask “why” questions about adaptations, populations and evolutionary origins. Re- search on molecular, cellular and genetic mechanisms is certain to continue. It remains to be seen whether the promise of Neurospora for population genetics will be fulfilled.

Sixty-five years have passed since SHEAR and DODGE named and described Neurospora, and 50 years since BEADLE and TATUM thrust it into prominence. In 1952, DODGE felt that he would soon be able to assert, “The old red bread-mold has at last come into its own.” Developments since then have taken his favorite

organism far beyond what he could have imagined. Neurospora continues to be a source of innovations and surprises.

This essay is dedicated to the memory of B. 0. DODGE on the 120th anniversary of his birth. The summary of research contri- butions benefited from discussion with numerous colleagues. Their comments are much appreciated. I am indebted to the Library of the New York Botanical Garden, Bronx, New York, for the pho- tograph of DODGE, to HERSCHEL ROMAN for that of LINDEGREN, and to MARJORIE M. BHAVNANI for the 1947 photograph of MC- CLINTOCK. Work on Neurospora in my laboratory has been sup- ported since 1956 by grant AI 01462 from the National Institutes of Health.

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