speakers' abstracts from the fourth annual congress for recombinant dna research

DNAVolume 3, Number 1, 1984Mary Ann Lieber!, Inc., Publishers

Speakers' Abstracts from the Fourth AnnualCongress for Recombinant DNA Research

RETROVIRUSES AS PROBES FOR MAMMALIAN DEVELOPMENTRudolf Jaenisch, Klaus Harbers, Michael Kuehn, Ange-lika Schnieke, Jürgen Löhler, and Klaus Kratochwil*,Heinrich-Pette-Institut für Experimentelle Virologieund Immunologie an der Universität Hamburg, Martini-strasse 52, 2000 Hamburg 20, Federal Republic ofGermany; *österreichische Akademie der Wissenschaften,Institut für Molekularbiologie, Billrothstrasse 11,A-5020 Salzburg, Austria

Experimental insertion of the Moloney leukemia vi-rus into the germ line has resulted in an embryonicrecessive lethal mutation in Mov-13 mice. Integrationof the proviral genome occurred at the 5' end of theal(I) collagen gene blocking formation of stable mRNA.Sequence and SI mapping analyses were performed tocharacterize the position of the proviral genome inrelation to the transcriptional map of the mutatedgene. The results indicated that the virus has in-serted into the first intron 19 bp 3' of the intron-exon boundary. Insertion of retrovirus-like or trans-posable elements into introns has led to mutations ofgenes in a number of other systems including mice,myeloma cells, plants, yeast and Drosophila.

Sequence comparison showed a striking homology ofexon sequences and sequences up to 215 bp 5' of themRNA start between the mouse and the human al(I) col-lagen gene. This indicates that the sequences 5' ofthe mRNA start are highly conserved during evolution,suggesting that this region has an important role inthe control of tissue specific collagen expression.

Embryos homozygous at the Mov-13 locus are arrestedin development between days 11 and 12 of gestation.This is the time when abundant transcription of theal(I) collagen gene starts, suggesting an essentialrole of type I collagen for midgestation development.Histological examination of day-12 embryos revealeda general cell necrosis without obvious malformation.In vitro organ explantations are being performed forfunctionally testing the role of collagen in organo-génesis.

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FOURTH ANNUAL CONGRESSFOR

RECOMBINANT DNA RESEARCH

EXPRESSION OF FOREIGN GENES IN HIGHER PLANTS, lohn D. Kemp,Ph.D., Agrigenetics Corporation, Advanced Research Division, 5649 E.Buckeye Rd., Madison, WI 53716

The insertion of genes encoding beneficial traits (such as diseaseresistance, yield improvement, and stress tolerance) into well-adaptedcrop plants is potentially of great agricultural importance. The demon-stration that the soil bacterium Agrobacterium tumefaciens can insertspecific regions of its tumor-inducing plasmid into plant nuclear DNA hasstimulated extensive research into the use of these plasmids as vectorsfor gene transfer. Furthermore, A. tumefaciens can infect a wide rangeof plants making it suitable for genetic engineering in numerous plantspecies. Sequences coding for the bean seed protein phaseolin have beeninserted into the transferred region of the tumor-inducing plasmid. Thesesequences are expressed after transfer to a wide range of plant species.Experiments such as these have demonstrated that eukaryotic genes canbe expressed to yield detectable levels of proteins in alien plant cells.

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THE MOLECULAR GENETICS OF TRANSPOSABLE ELEMENTS IN PLANTS

Jeffrey L. BennetzenDepartment of Biological Sciences

Purdue UniversityWest Lafayette, Indiana 47907

Transposable element systems were first described in maizethrough the pioneering work of McClintock. In the ensuing twentyyears, the ability of these "controlling" elements to transpose,to cause chromosomal breakage and rearrangement, and to inducemutations was heavily documented in corn. Since then,transposable elements have been identified genetically or

biochemically in many bacteria, fungi, animals, and plants. Inbacteria, these elements are apparently important in mobilizinggenetic traits (e.g. antibiotic resistance) for transfer throughthe bacterial ecosphere. The role(s) of these ubiquitoustransposable elements and their mechanisms of action are onlyvaguely understood in eukaryotes. Several uniquely useful aspectsof corn biology and the many years of sophisticated controllingelement genetic research have allowed maize to retain it'sprominence as the genetic system for study of transposableelements. Regulation of the transposition and chromosome breakagereactions induced by transposable elements, alterations in thatregulation, reversible and irreversible changes in the state of an

element, and secondary changes inherent to transposable elementsand the loci they are associated with have all been well-dissectedgenetically in maize. We are just now beginning the task ofanalyzing these phenomena at the molecular level. Elementsrepresenting four families in maize, one in soybean, and one inAntirrhinum majus have been cloned. These elements have many ofthe structural features seen in transposable elements from othersystems, including inverted terminal repeats and short directduplications of DNA at the site of insertion. The form andfunction of these elements; the timing and mode of their variousactivities, are rapidly coming to light. Discussion of theseobservations will demonstrate how well the molecular data were

predicted by the genetics and will also indicate the numerous new

questions that have been raised.

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STRUCTURE AND EXPRESSION OF MAIZE ZEIN GENES. Brian Larkins,Department of Botany and Plant Pathology, Purdue University,West Lafayette, Indiana 47907.

The storage protein in maize seed is composed of a group ofalcohol-soluble polypeptides known as zeins. These proteinsare synthesized by membrane-bound polyribosomes in the develop-ing endosperm, and are secreted into the lumen of the roughendoplasmic reticulum (RER). Within the lumen of the RER zeinsassociate to form dense masses called protein bodies. Zeinproteins can be separated into several groups of polypeptideswith apparent mol wts of 27,000, 22,000, 19,000, 15,000, and10,000 daltons. The Mr 22,000 and Mr 19,000 polypeptides are

the most abundant, and account for approximately 80% of thetotal protein based on coomassie blue staining. The poornutritional quality of maize seed protein results from the lowlevels of lysine and tryptophan in the zeins as well as theirhigh isoleucine/leucine ratio. In most normal genotypes lysineconstitutes only about 17» of the total amino acids. In mutantssuch as opaque-2, the lysine content is increased to 3% due toa 507» or greater reduction in zein proteins. However, seedsize is smaller in the mutants and the yield is reduced.Furthermore, the mutant kernals are brittle, and food productsmade from them retain this brittle characteristic. Since zeingenes that encode proteins with more lysine do not exist inthe maize genome, one way to improve the lysine content ismodify the genes through in vitro mutagenesis and geneticallyengineer them into the maize genome. This requires that weunderstand the structure of the proteins and the regulation ofthe genes encoding them. Our studies have shown that the Mr22,000 and Mr 19,000 zeins have a highly ordered structure,which appears to be different from that of the Mr 27,000, Mr15,000 and Mr 10,000 proteins. The Mr 22,000 and Mr 19,000proteins are encoded by a large multigene family, whereas theother proteins appear to be encoded by only a few genes. Inspite of the difference in the numbers of genes encoding theseproteins, the levels of mRNAs may be very similar. The genesencoding zeins have a structure typical of most eukaryoticgenes, although they do not contain introns. Experiments arein progress to transfer these genes to other cells where wecan study their transcription and the effect of in vitromutagenesis on protein synthesis and processing.

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PLANT MITOCHONDRIA!. GENOMEC. S. Levings, III, Genetics Department

North Carolina State UniversityOur current understanding of the maize mitochondria! genome is

presented with special emphasis on those features which areunique to higher plants. The mitochondrial genomes of higherplants are larger and more variable than those of otherorganisms. Only a few mitochondrial genes have been identifiedin maize, however, some of these are not encounterred in themitochondria of fungi and mammals. Maize and wheat mitochondrialgenomes code for three rRNAs, 5S, 18S and 26S. A 5S rRNA is notfound in the mitoribosomes of fungi and animals. Sequence andsecondary structure analyses of the maize 18S rRNA indicate thatit is parkaryotic in nature even though it does not contain atypical Shine-Daigarno sequence at its 3' end. Plantmitochondria code for their own tRNA species. Maize mtDNAcarries the protein-encoding gene for cytochrome oxidase, subunitII. It has been suggested that the cytochrome oxidase, subunitI; cytochrome b; ATPase, alpha and 9 subunits; and a ribosomalprotein are encoded by maize mitochondrial genes. Severalunassigned open-reading frames have also been identified.Preliminary studies of transcription and RNA processing indicatethat there are multiple transcriptional units, the 5' ends ofprimary transcripts are not capped, and the 31 ends are notpolyadenylated. Finally, although the molecular basis is not yetclear, there is abundant evidence suggesting that maize mtDNAcodes for factor(s) responsible for the trait, cytoplasmic malesterility. Maize mitochondria sometimes contains plasmid-likeDNAs that range in size from 7.5 to 2.0 kb and are in addition tothe usual mtDNA. In the male-sterile cytoplasm S (cms-S), twoplasmid-like DNA, designated S-l (6.4 kb) and S-2 (5.4 kb) arefound. Sequence analysis shows that these molecules areterminated by 208 bp inverted repeats and that they containextensive open reading frames. Transcript mapping studiesindicate that these open- reading frames are transcribed.Studies of cms-S cytoplasms which have spontaneously changed fromthe male-sterile to the male-fertile phenotype reveal that the S-1 and S-2 molecules have disappeared. As expected, transcriptsassociated with plasmid-like DNA are no longer found in therevertants. The plasmid-like DNAs have proteins covalentlylinked to their 5' ends similar to adenovirus and Bacillus phageDNAs. Electron microscopy (EM) observations indicate that theseDNAs exist in the native state as circular molecules presumablydue to intra-molecular attractions between their terminalproteins. EM investigations have revealed molecules occurringwith a frequency of 1% that are thought to be replicativeintermediates. Studies of these intermediates suggest that themolecules replicate by a mechanism known as strand-displacement.The terminal protein of the plasmid-like DNAs probably primes forDNA replication and is encoded by these DNAs.

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GENETIC ANALYSIS OF RHIZOBIUM INFECTION. S. R. Long, T.W.Jacobs, T.T. Egélhoff, E.L. deHostos, J.T. Mulligan, J.K. Tu,R. Sanga, R.F. Fisher. Department of Biological Sciences,Stanford University, Stanford CA 94305.

The symbiotic nitrogen-fixing bacterium, Rhizobium meliloti,invades the root cells of its host plant, alfalfa. Thisinvasion is highly host-specific, and involves bacterialredirection of host cell wall production. Rhizobium alsostimulates cell division in otherwise non-dividing host cells, a

property it shares with its close relative, Agrobacteriumtumefaciens. We have identified several bacterial gene regionsrequired for nodulation. Mutations in these regions result ina Nod" (non-nodulating) phenotype. The cloned genes fornodulation were obtained by the following procedure: a

pLAFRl-based clone bank was conjugated into each nodulationmutant strain. Groups of transconjugants were inoculated ontoalfalfa plants; the subpopulation of cells in which a clone withthe normal nodulation gene was in residence, and which thereforehad a Nod phenotype, were selected by isolating nodules fromthese plants and obtaining bacteria from inside them. Thebacteria resident in the nodules contained the cloned nodulationgenes. Carrying out such experiments, we have determined thatthere are three "nod" gene regions which occur within 45kilobases of the R. meliloti plasmid-linked nif genes. We havecarried out physical and genetic analysis of one nodulation generegion. Cloned fragments from this region were subjected totransposon Tn5 mutagenesis, and the mutations were moved intothe R. meliloti genome by marker exchange. This produced a

series of mapped Tn5 mutations, whose nodulation properties were

examined by inoculating them onto plants. These tests revealeda 3.6 kb DNA region in which transposon insertions completelyprevent nodule development. We have carried out pairwisecomplementation tests of these mutants, which indicate theorganisation of this region into four to six complementationunits, ranging in size from 300 bp to approximately 1100 bp. Theproducts of two of the complementation groups have beenexpressed in vitro, and the sequence of one of them has beendetermined. To understand the function of these genes, we are

carrying out assays in other bacterial backgrounds. Theseclones confer upon Agrobacterium tumefaciens the ability toinduce nodules on alfalfa, but the frequency is low and thestructure incomplete. The genes do not appear to control hostrange as simple dominant loci, since they do not extend the hostrange of other Rhizobium species. To assay the effect of the nodgene products at the molecular level, we are constructingstrains in which one or more of the gene regions isoverexpressed. We will examine these strains in associationwith intact plant cells.

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TRANSFER AND REGULATION OF MAMMALIAN AND FUNGAL METALLOTHIONEINGENES, D. Hamer, A. Carter, B. Felber, A. Leone, G. Pavlakis,C. Schmidt, and M. J. Walling, Laboratory of Biochemistry,National Cancer Institute, NIH, Bethesda, MD 20205

The metallothioneins are small, cysteine rich proteins thatchelate heavy metals such as zinc, copper, cadmium, and mercury.The synthesis of these abiquitous proteins is homeostaticallyregulated by heavy metal ions. We have cloned métallothioneingenes from mice, humans, and (in collaboration with T. Butt atSmith Kline French) from the yeast Saccharomyces cerevisiae.These genes, and mutants derived from them, have been transferedinto homologous and heterologous host cells using a variety ofvectors. Expression has also been examined in cells with trans-acting mutations that influence metallothionein gene expression;these include cells from patients with Menke's disease, an X-linked disorder that results in deficient circulating copper,and in selected copper-sensitive and copper-resistent yeaststrains. These experiments show that: 1) metallothioneinexpression is transcriptionally regulated by upstream sequences;2) these sequences interact with positively acting cellularfactors; and 3) there are probably multiple steps in theinduction pathway.

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EXPRESSION OF FOREIGN GENES IN TRANSGENIC MICEG. Stanley McKnight, Robert E. Hammer and Ralph L.Brinster, Department of Pharmacology, university ofWashington, Seattle, WA. 98195 and Laboratory ofReproductive Physiology, University of Pennsylvania,Philadelphia, PA. 19103.

Fertilized mouse eggs were injected with eitherthe chicken transferrin or ovalbumin genes and theoffspring and their progeny were examined for (a) thepresence of chicken gene sequences, (b) expression ofmRNA and protein from the injected genes and (c)regulation of mRNA levels by estrogen. The resultsdemonstrate that 15-30% of the offspring frominjected eggs carry the foreign DNA and can transmitthese sequences to progeny. The transferrin gene wasexpressed actively in the liver of 5 of the 6 miceexamined and in those 5 animals the levels oftransferrin mRNA in liver were approximately 10 foldhigher than the levels in brain. This apparent tissuespecificity was also seen in several generations ofoffspring. Chicken transferrin was secreted into theserum of transgenic mice and reached levels as highas 250ug/ml. The transferrin gene is normally inducedin chicken liver by both estrogen administration andnutritional iron deficiency. We examined 3independent lines of transgenic mice to determinewhether the chicken transferrin gene would maintainits responsiveness to estrogen. In all 3 lineschicken transferrin mRNA was induced 2 fold after 10days of stimulation with diethystilbestrol. The mousetransferrin gene was also induced by estrogentreatment as judged by a 2-3 fold increase in serumtransferrin. Our results indicate that both the mouseand chicken transferrin genes are responding toestrogen in transgenic mice and that the induction issimilar to the 2-3 fold induction we have previouslyreported in chickens. The ovalbumin gene was notexpressed actively in mouse liver although detectablelevels of mRNA were seen in other tissues. Inconclusion, we suggest that tissue-specific signalson the transferrin gene are sufficiently conservedsuch that this gene is partially recognized andpreferentially transcribed in mouse liver. Theestrogen response indicates that the mouse estradiolreceptor can recognize and regulate the expression ofa chicken gene. Supported by grants from the NIH andNSF.

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white LOCUS DNA TRANSFORMATION IN DROSOPHILA: DOSAGE COMPENSATION,zeste INTERACTION AND POSITION EFFECTS. Gerald M. Rubin, TulleHazelrigg and Robert Levis, Department of Biochemistry, Universityof California, Berkeley, CA 94720

P-element mediated DNA transformation was used to generatetransformants carrying segments of DNA from the white locus ofDrosophila melanogaster. The vast majority of transduced copiesof an 11.7 or 14.3 kb segment of DNA from white successfully res-cued the white- eye-color phenotype when inserted in many differ-ent chromosomal locations. However, two transformants with abno-mal eye pigmentation

-

apparently a consequence of the genomicpositions of the transduced white gene - were also recovered. Inall seven cases tested, autosomal insertions of white, which isdosage compensated in its normal location on the X chromosome, re-tained the property of being dosage-compensated. In contrast tothe relative insensitivity of eye color pigmentation and dosagecompensation to genomic position, the transduced white DNA seg-ments differed widely in their interactions with the zeste1 muta-tion, ranging from greater than normal repression by zeste1 toinsensitivity to the presence of zeste1.

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ÏEAST TELOMERES AND ARTIFICIAL CHROMOSOMES, J.W.Szostak,I.E.Claus and A.W.Murray, Dana-Farber Cancer Institute and Dept.of Biological Chemistry, Harvard Medical School, Boston, MA 02115

Telomeres are special DNA structures that provide stable,fully replicatable DNA termini. We have used the ends of thelinear extrachromosomal rDNA of Tetrahymena to study the structureand function of telomeres in yeast. The Tetrahymena ends work as

stable ends on linear plasmids in yeast. Deletion mappingexperiments have shown that the only sequences required for thisfunction are the cluster of C-C-C-C-A-A repeats found next to theterminus of the rDNA. We have cloned yeast telomeres by removingone Tetrahymena end from a linear plasmid by restriction enzymedigestion; such DNA tranforms yeast very poorly, and thus providesa powerful selection for the presence of a functional telomere.Yeast telomeres are similar in many ways to the Tetrahymena end,but have a different sequence composed of irregular repeats:C1 _-A. Tetrahymena ends become larger during replication in yeastby about 200 bp. This increase in size is due to the addition ofsequences similar to those found in yeast telomeres. Possiblemechanisms for this reaction will be described. The stability oftelomeres may reflect an equilibrium between reactions such as

exonucleolytic degradation and incomplete replication, which tendto shorten the telomeric DNA, and the C1 _A addition reaction. Wehave also studied another set of DNA processing reactionsinvolving telomeres. An inverted repeat of C^A clusters isprocessed within the cell into two new functional telomeres. Thisreaction can proceed with as much as 1.7kb of DNA at the center ofthe inverted repeat, and provides a simple method for theconstruction of new linear plasmids. We suggest that this processmay be involved in the separation of telomeres which accidentallybecome ligated together.

We have constructed artificial chromosomes by combiningcloned origins of replication, centromeres and telomeres.Centromeres do not function properly on short linear plasmids (<15 kb ), but do appear to work well on long linear plasmids ( >50kb ). Such artificial chromosomes pair with each other and disjoinproperly in meiosis. The fidelity of both mitotic and meioticsegregation is lower than that of natural chromosomes. Possiblereasons for this, and for the effect of size, will be discussed.The telomeres and adjacent sequences of yeast chromosome III havebeen removed and substituted with Tetrahymena telomeres; theresulting chromosome appears to function normally in mitosis andmeiosis.

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A SIGNAL WITHIN THE LAMB PROTEIN THAT SPECIFIES AN OUTER MEM-BRANE LOCATION. S. A. Benson, E. Bremer, and T. J. Silhavy,NCI-Frederick Cancer Research Facility, P.O. Box B, Frederick,Maryland 21701.

Escherichia col i strains containing two new lamB-lacZfusions have been isolated and characterized. One strain,BRE103, produces a hybrid protein that contains the completeLamB signal sequence plus 39 ami no acids of the mature LamBprotein fused to a large functional COOH-terminal fragmentof ß-galactosidase. The other strain, BRE120, produces ahybrid protein that is slightly larger and contains thesignal sequence plus 49 ami no acids of mature LamB. Bothfusions contain sufficient information to allow export ofthe hybrid protein from the cytoplasm; however, only thelarger hybrid protein is found in the outer membrane. Thesefusions, together with previously described lamB-lacZ fusionshave enabled us to define more precisely the minimum amountof lamB required to initiate protein export. In addition,they reveal a new export signal, termed outer membrane signal,that directs LamB to the outer membrane.

Research sponsored by the National Cancer Institute, DHHS,under Contract No. N01-C0-23909 with Litton Bionetics, Inc.

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GENETIC ANALYSIS OF PROTEIN STABILITY AND FUNCTION

Robert T. Sauer, Dept. of Biology, M.I.T., Cambridge MA 02139

We are studying the effects of single and double ami no acidsubstitutions on the thermal stability and the DNA bindingproperties of repressor proteins from bacteriophages lambdaand P22. In many cases, these studies establish the roles ofspecific amino acid side chains and functional groups in theprocesses of protein folding and DNA recognition.

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P53, A CELLULAR TUMOR ANTIGEN, Arnold J. Levine, Nancy Reich,Diane Pennica and David Goeddel, State University of New Yorkat Stony Brook, Department of Microbiology, School of Medicine,Stony Brook, New York 11794 and Genentecn, Inc. 460 Point SanBruno Blvd., So. San Francisco, California 94080

P53 is a cellular protein of 53,000 MW whose levels are some

10-2,000 fold elevated In a wide variety of transformed cellswhen compared with normal cells. Higher levels of p53 in trans-formed cells are Independent of the transforming agent (chemi-cals, viruses, Irradiation, genetic predisposition, etc.) and an

homologous protein has been found in primate and rodent cells. InSV40 and adenovirus transformed cells p53 is found in an oligo-merlc protein complex with the viral encoded tumor antigens knownto be required for the transformation event.

In nontransformed cells p53 protein has a short half life (20-30 minutes) and its rate of synthesis varies as a function of thecell cycle. In G arrested cells stimulated by the addition offresh serum into 5-phase, the steady state levels of p53 m-RNAand protein Increase In the late G. period. Transformed cellsand nontransformed cells have similar levels of p53 m-RNA. TheIncreased levels of p53 protein in transformed cells result froman enhanced post-trans I atlona I stability of p53 protein resultingin a longer half life for this protein. The alterations In thelevels of p53 In transformed cells might then have some causaleffect upon the regulation of cellular replication.

A 1.6 Kb c-DNA clone derived from p53 m-RNA has been obtainedand the nucleotide sequence determined. Several Interestingaspects of the protein predicted from this sequence will bediscussed.

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ACTIVATION OF THE MYC ONCOGENE BY CHROMOSOMAL TRANSLOCATION.Michael D. Cole, Department of Biochemistry, St. LouisUniversity School of Medicine, St. Louis, MO 63104.

Chromosomal translocations are a consistent feature of manytumor cells, most notably in mouse plasmacytomas and Burkittlymphomas. We have shown that the chromosomal translocationbreakpoint occurs at the c-myc oncogene in plasmacytomas andthat the oncogene becomes linked in a 5' to 5' orientation tothe immunoglobulin C gene through an abortive switching eventin the majority of tumors. In Balb/c tumors, the breakpointalmost always occurs within 400 bp of the boundary between the1st c-myc exon (which is untranslated) and the 1st intron. Wehave been particularly interested in the relationship betweenthe sequences within the c-myc gene and the normal Ig switchsignals and in the transcriptional control of the translocatedgene. We have sequenced the translocation breakpoint in twotumor lines and compared them to other sequenced breakpoints.A tetranucleotide sequence (GAGG) was found 11-12 bp from thebreakpoint in 5 out of the 5 tumor lines in which c-myc has re-combined with S . No other conserved sequences were found andno homology with the S region was apparent. These results sug-gest that if switch recombinases are involved in the transloca-tion, they recognize a sequence within c-myc that is unlike anyof the signals previously proposed for productive switches.This raises the possibility that the initial break within c-mycwas made by some other DNA endonuclease activity. Data willalso be presented which maps the transcription initiation siteswithin the translocated gene.

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HUMAN TRANSFORMING GENES. Michael Wigler, OttavioFasano, Elizabeth Taparowsky, Daniel Birnbaum,Mitchell Goldfarb and Jörgen Fogh*. Cold SpringHarbor, NY and *Sloan Kettering Institute, Rye, NY.

Use of the NIH3T3 focus assay has revealed thewidespread presence of activated ras oncogenes inhuman tumor cell DNA. The three ras genes found sofar encode proteins with remarkably conserved aminoacid sequence. Systematic in vitro mutagenesisindicate that amino acid substitution at multiplesites lead to the activation of the transformingpotential of the normal ras gene product. We havebegun to utilize cotransformation of NIH3T3 cells and|tumorigenicity in nude mice as an assay for new human1tumor genes. Our initial experience indicates thisassay to be far more sensitive than the focus assay,and reveals a set of new "tumor" genes which are notdetectable by previous bioassays.

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INTERPLAY BETWEEN STRUCTURE AND TOPOLOGY IN ACTIVECHROMATIN Charles R. Cantor, Department of HumanGenetics and Development, College of Physicians andSurgeons, Columbia University, New York, NY 10032

Recent studies from many laboratories provide anumber of tantalizing hints that nucleosome structureand placement may be different in actively transcribedgenes. Other results indicate that torsional stress mayplay a role in eukaryotic gene expression in spite ofthe fact that most of this stress appears to besequestered within nucleosomes. Here, I will describefour studies that we have recently carried out incollaboration with other laboratories to examine thepossible relationship between structure and torsionalproperties of chromatin. Actively transcribed ribosomalchromatin appears to contain nucleosomes with anunusual structure that potentially has alteredtopology. Globin chromatin shows evidence of structuralchanges correlated with gene activity. DNA inminichromosomes behaves with more torsional rigiditythan one would expect from simple models. This impliesthat chromatin structure may well provide effectivebarriers to the diffusion of torsional stress so thatperturbed local structures will behave as smallsupercoiled domains. Finally, calculations show thatsmall supercoiled domains may promote different DNAconformational changes than large ones. Thesecalculations may shed some light on the possiblestructures of SI nuclease hypersensitive sites.

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TRANSCRIPTION FACTORS THAT CONFER PROMOTER SPECIFICITY TO RNAPOLYMERASES I AND II. William S. Dynan, R. Marc Learned andRobert Tjian, Dept. of Biochemistry, University of California,Berkeley, CA 94720

Fractionation of whole cell extracts capable of carrying outselective and accurate transcription in vitro has revealed sever-al factors that allow RNA polymerases I and II to discriminatebetween promoters. One of these factors, SP1, acts together withRNA polymerase II and at least one other cellular factor to faci-litate transcription of simian virus 40 early and late promoters.Analysis of deletion mutants shows that SP1 dependent transcrip-tion of the SV40 early promoter requires sequences within the 21base pair repeats located at 60-100 bp upstream from the initia-tion sites for early transcription. In a DNase footprintingassay, incubation of SP1 with promoter DNA leads to protection ofsequences in the same 21 bp repeat region. This result indicatesthat SP1 is able to recognize and bind to sequences in the up-stream promoter element independently of other components of thetranscriptional machinery. Recently we have found that SP1 alsorecognizes and binds specifically to the upstream control se-quences of a cellular promoter isolated from CV1 monkey cells(McCutchan and Singer (1981) PNAS 28:95). In addition, SP1 ap-pears to activate transcription of the monkey promoter. Thesefindings suggest that SP1 may be a promoter-specific factor thatcontrols the transcription of a number of different viral andcellular promoters.

A different example of promoter-specific transcription factorsis seen when template containing a rRNA promoter is analyzed byin vitro transcription. Extracts from mouse and human cells bothcontain a factor that is required for transcription and that isreadily separated from the endogenous RNA polymerase I. We havepurified the factor from human cells approximately 10,000 foldby column chromatography and identified a polypeptide of 42,000daltons that copurifies with the transcriptional activity. Thisfactor, called SL1, is responsible for the species specificityof ribosomal transcription. For example, the human factor, inconjunction with the mouse or human RNA polymerase I, allowstranscription of the human ribosomal promoter but does not allowtranscription of the mouse ribosomal promoter. Antibodies raisedagainst the 42,000 dalton protein binds preferentially to thenucleolus of primate cells but not mouse cells, and specificallyinhibits transcription initiation from the human rRNA promoterbut not from the mouse promoter. These findings indicate thatSL1 is a transcription factor that imparts promoter recognitionto RNA polymerase I and that it can discriminate between rRNApromoters from different species.

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CHROMATIN STRUCTURE NEAR TRANSCRIPTIONALLY ACTIVE GENES.G. Felsenfeld, J. Nickol, B. Emerson, D. Jackson and J. McGhee.Laboratory of Molecular Biology, National Institute of Arthritis,Diabetes, and Digestive and Kidney Diseases, National Institutesof Health, Bethesda, Maryland 20205.

The structure of the 30 nm thick chromatin fiber and thenucleosomes of which it is composed are now reasonably wellunderstood. Are these structural elements also present in theneighborhood of transcriptionally active genes? We presentevidence in the case of the chicken adult ß globin (ß ) gene thata large proportion of the coding region in chromatin isolatedfrom erythrocytes is packaged in nucleosome-like particles withphysical properties quite similar to those of bulk nucleosomes.In contrast, the 5' flanking region includes a DNA segment about200 base pairs long, hypersensitive to nuclease digestion, thatappears to be nucleosome-free in cells in which the ß gene isexpressed. We have examined some of the physical and chemicalproperties of the DNA in this region. We have also identified a

factor, present in erythrocyte nuclei, which is able to bindspecifically to this region. When histones are assembled in thepresence of the factor onto plasmids containing this DNAsequence, the nucleoprotein complex shows hypersensitive behaviorlike that seen in vivo.

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DEVELOPMENTAL CONTROL OF GLOBIN GENE EXPRESSION, HaroldWeintraub and Jonathan G. Izant, Hutchinson Cancer ResearchCenter, 1124 Columbia Street, Seattle, WA 98104

We have found a supranucleosomal particle that is responsiblefor packaging inactive genes. This structure may beresponsible, in part, for the massive suppression (10 -10 orgreater) of tissue-specific gene expression in non-expressingtissues (e.g., refs. 1 & 2). In order to try to understand thegenetic circuits that regulate whether a given gene is expressedor not and whether it is or is not packaged into thesesupranucleosomes, we have begun an analysis that attempts toturn-off the expression of a given gene — in the ideal case, a

"regulatory" gene — given the possibility that such a gene willbe included within a pool of recombinant DNA clones derived by"plus-minus" screens of cDNA libraries from different tissues.The method depends on microinjection of a recombinant DNAplasmid where the anti-sense RNA is transcribed by a suitablyplaced promoter. As a model system, we have shown that in theappropriate conditions an anti-sense Herpes T.K. constructionprevents the expression of normal Herpes T.K. activity, possiblydue to the formation of RNA-RNA hybrids.1. Groudine, M. and Weintraub, H. (1975) Rous Sarcoma Virus

Activates Embryonic Globin Genes in Chicken Fibroblasts.Proc. Nati. Acad. Sei. USA 72, 4464-4468.

2. Ivarie, R.D., Schacter, B.S., and O'Farrell, P.H. (1983) Thelevel of expression of the rat growth hormone gene in livertumor cells is at least eight orders of magnitude less thanthat in anterior pituitary cells. Molec. Cell. Biol. 3_,1460-1476.

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ENHANCERS AS TRANSCRIPTIONAL CONTROL ELEMENTS

Peter Gruss and Hans SchölerZentrum für Molekulare Biologie und Institut für Mikro-biologie der Universität Heidelberg, Im NeuenheimerFeld 364, 6900 Heidelberg, Federal Republic of Germany

Enhancers are eis essential transcriptional controlelements, which can increase the transcriptional acti-vity of native and foreign genes. This activity can beexerted relatively independent of distance and orienta-tion with respect to their coding region. The mechanismby which enhancers/activators potentiate transcriptionis unknown. We suggested previously on the basis ofhost-specific activation the possibility of an involve-ment of host-specific molecules. It was also proposedthat activators are bidirectionally active "super"entry sites for components of the transcription machin-ery. In order to test an involvement of cellular trans-cription factors we established transfection conditionsallowing for efficient competition in vivo. In aneffort to identify the competition capacities of de-fined genetic elements in this assay, we found that itwas the enhancer element which actively competed forenhancer-mediated transcriptional activities. Thissuggests the presence of a limited number of cellularfactors involved in enhancer function. Some enhancers(e.g., MSV) show a striking host-range effect. Usingthe transfection-competition assay we demonstrated thatall enhancers/activators assayed so far utilize thesame set of cellular factors, although in a host-specific manner. On this basis, our data are compatiblewith the hypothesis of enhancers acting as "super"entry sites for cellular transcription factors. Theyare also in good agreement with host-specific acti-vation due to different binding affinities of cellularmolecules to various enhancers.This work was supported by BMFT (Bundesministerium fürForschung und Technologie, Bonn) grant BCT 0364/1.

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STUDIES ON THE MOLECULAR MECHANISMS OF HEAT SHOCK GENEACTIVATION IN DROSOPHILA. Carl S. Parker and Joanne Topol,California Institute of Technology, Division of Chemistry, Pasadena,California 91125.

An extensively purified RNA polymerase II transcription factor fromDrosophila binds specifically to the regulatory site of several heat shockgenes. There are two tandem binding sites for the heat shock genetranscription factor on the Hsp 70 gene. Binding to these sites isapparently ordered with the site closest to the TATA box binding thefactor first. The first site must be present for binding to occur on thesecond site. A second transcription factor has also been isolated fromDrosophila cultured cells. This factor binds to the TATA homology aswell as the start point of transcription and a portion of the non-translated leader region of several genes. This factor is required for invitro transcription of the actin and histone genes of Drosophila. Thisfactor, however, is not required for in vitro transcription of the Hsp 70gene when the heat shock gene specific transcription factor is present.

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THE ORGANIZATION, FUNCTION AND EVOLUTION OF GENES UP 1HE MAJORHISTOCOMPATIBILITY COMPLEX. Leroy Hood, Division of Biology,California Institute of Technology.

The major histocompatibility complex (MHC) of mammals encodestwo families of genes, class I and class II, whose cell-surfaceproducts participate in various aspects of the immune response.The class I genes fall into two categories, those encodingtransplantation antigens (K, D, L) and those encodinghematopoietic differentiation antigens (Qa and TL); the former are

present on all somatic cells whereas the latter are present onlyon subpopulations of bone marrow-derived cells. The class II orla (immune response-associated) genes are expressed on B cells,macrophages, and some T cells. Transplantation antigens serve asrestriction elements permitting cytotoxic T cells to recognizeforeign antigen in the context of self and the la antigens servethe same function for regulatory T cells. The functions of thehematopoietic differentiation antigens are unknown.

We have isolated most of the class I genes from the inbredBALB/c mouse and most of the class II genes from several strainsof mice. There are 31 class I genes falling into eight cosmidclusters, each of which can be mapped to one of four recombinationregions in the MHC (K, D, Qa, and Tla). We have identified sevenclass II genes encoded within 200 kb of DNA in the I region of theMHC.

Fibroblasts (mouse L cells) have been transformed by class Iand II genes and B cells have been transformed with class IIgenes. These transplantation antigens and la antigens function inboth cell types. I_n_ vitro mutagenesis (e.g., exon shuffling)experiments have begun to delineate the various functional regionsof these cell-surface recognition molecules.

The class I and II genes exhibit homology to one another and tothe antibody genes. Recent data suggest that the T-cell receptorgenes also are homologous to these gene families. Thus the threeantibody gene families and at least one T-cell receptor genefamily as well as the class I and II gene families constitute a

supergene family, presumably derived from a common ancestralmultigene family. Thus the multigene family itself appears to bea unit of evolutionary diversification. Moreover, these multigenefamilies may evolve such that their gene products may interactwith one another to form sophisticated recognition molecules(e.g., light and heavy immunoglobulin genes); they may regulatethe functioning of another family (e.g., la genes and T-cellreceptor genes); or they may evolve to assume new and distinctfunctions (e.g., T-cell and B-cell receptor genes). These genefamilies may have evolved special mechanisms for theirevolutionary diversification.

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GENETIC APPROACHES TO CHROMOSOME STRUCTUREJohn R. Roth, Department of Biology, University of Utah, SaltLake City, Utah 84112

The chromosomes of the bacteria _E_. col i and Salmonella havebeen accurately mapped. A remarkable finding is that, althoughthese bacteria have diverged widely in their microsequencehomology, their chromosome maps have been conserved almostcompletely. What selective forces act to preserve genetic maporder?

To approach this question we've developed methods forselecting and mapping chromosome rearrangements. The long-termgoal is to determine what rearrangements are possible and whateffect rearrangements have on cell growth. Thus far we'vestudied duplications, transpositions and inversions. Severalgeneral points have emerged.

Duplications are frequent in bacteria. They're generated byrecombination between homologous sequences located at distinctpositions in the chromosome. All are unstable and return to thenormal haploid structure following recombination/ segregation ofthe extra copy. Almost all regions of the chromosome can beduplicated including the origin of replication. Largeduplications put a burden on the cells protein syntheticmachinery and can be deleterious. The only region we've beenunable to duplicate includes the terminus of chromosomereplication. Duplications may play an important role inallowing cells to adapt to novel growth conditions. Theserearrangements can increase gene dosage so as to increase thelevel of growth limiting gene products. In addition,duplications can serve to fuse genes to foreign promoters. Asmall repetitive sequence, present within many opérons mayprovide the homology which permits formation of these novelduplicative operon fusions.

Inversions are extremely rare in bacteria. It's not yetclear whether this is due to mechanistic problems in formingduplication or whether inversions occur and lead to a non-viablecell.

We entertain seriously the notion that the chromosome isstructured to favor adaptively valuable, reversibleduplications. Maintaining this capability may put selectiveconstraints on evolution of the genetic map.

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speakers' abstracts from the fourth annual congress for recombinant dna research

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