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

DNAVolume 2, Number 1, 1983Mary Ann Lieber!, Inc., Publishers

Speakers' Abstracts from the Third AnnualCongress for Recombinant DNA Research

Genes Modulating Behavior in Aplysia, Richard H. Scheller,Linda B. McAllister, James Jackson, James Schwartz, Eric R.Kandel and Richard Axel, Center for Neurobiology and Behaviorand Institute of Cancer Research, 701 West 168th Street, NewYork, N.Y. 10032

Invertebrates provide sinple model systems for the studyof the cellular and molecular basis of behavior. Egg-layingin the mollusc, Aplysia, is characterized by a stereotypedbehavioral array which results from the activation of neuralcircuits dependent upon the coordinate release of severalsmall neuroactive peptides. The behavioral array is thoughtto be initiated by the A and B peptides which ultimatelycause the release of a set of peptides including egg-layinghormone, ELH, which directly mediate the egg-laying process.We have sequenced the genes encoding the A and B peptides, aswell as ELH. Each of the three genes encodes a polyproteinprecursor in which the active peptides are flanked byinternal cleavage sites providing the potential to generatemultiple small peptides. Surprisingly, each of the genesconsists of sequences homologous to A or B peptide, as wellas ELH. Although these genes share significant nucleotidehorology, they have diverged such that different member genesexpress functionally related but non-overlapping sets ofneuroactive peptides in different tissues. These datasuggest that a single genetic unit may encode the informationdictating a complex pattern of behaviors.

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THIRD ANNUAL CONGRESSFOR

RECOMBINANT DNA RESEARCH

An Unusual Family ofMobile Genetic Elements in Higher EukaryotesLarry Kcdes, Dan Liebermann, Rob Maxson, Joel Weinthal, and Geoffrey Childs ,

Department ofMedicine, and Barbara Hoffman-Liebcrmann and Stanley Cohen,Department ofGenetics, Stanford Medical School, Palo Alto, CA, 94305

We have discovered in deuterostomes a family of transposable elements, the TUfamily, which is structurally heterogeneous and whose members have long terminalinverted repeat sequences (IVRs).

Tu Elements Are Heterogeneous But Have Homogeneous Termini. The first elementwe found (TU1) was a 3.6 kb insert in a histone H2B gene of the sea urchinStrongylocentrotus purpuralus. Structural analysis of different TU elements isolatedfrom genomic S. purpuralus DNA, has revealed a general conservation of the overallorganization of the elements but with important structural and nucleic acid sequenceheterogeneity. The TU inserts are all flanked by an 8 base pair direct repeat of thegenomic DNA. The termini are exactly complementary IVRs often several hundredbase pairs long. The outer domains (OD) of the IVRs are themselves made up of adegenerate 15 base pair direct repeating sequence with no homology to the innerdomains (ID) of the terminal IVRs. The very outermost 100 base pairs of the ODs ofthree TU elements are identical. F.lcctron microscopy of other TU elementsdemonstrates, however, that the lengths of the both the IVRs and the middle (M)segments is highly variable. Restriction enzyme and nucleotide sequence analysisdemonstrate differences in IVR and M sequences as well. Hybridization experimentsrevealed that different TU elements carry different M segments that exhibit no

homology to each other.There Are Hundreds Of IVRs Per Genome. We have used the OD, ID and M

segments of cloned TU elements to identify, isolate and examine related sequences in S.purpuralus and other sea urchin species. Southern blots of genomic DNA from a seriesof sea urchin species demonstrated that there are hundreds of copies of the TU IVRs pergenome but that there arc far fewer copies of the M segment associated with TU1.

Astro Blots Determine The Linkage Of TU Domains And Their Copy Numbers. Wedeveloped a novel double plaque lift method (Astro Blots) for simultaneouslyquantitating the number of OD, ID, and M segments in various sea urchin genomes anddetermining their physical linkage. The results show that many copies of the ODsequence are present in all sea urchin species but that the M fragment from TU1 ispresent in high copy number only in species closely related to S. purpuralus. In thosecases, all the M sequences arc physically linked to an IVR sequence. In distantly relatedspecies the M segment is present cither in low copy numbers and not linked to the IVR,or not present at all. We conclude that different M segments predominate in TUelements of different species. These studies have allowed us to construct an evolutionaryscheme for the assembly of these elements and suggest a role for these elements in thedispersion and amplification of repeating sequences.

TU Family IVR Sequences arc Present in All Vertebrates including Man. Strong,discrete and multiple bands of hybridization were detected with the IVR sequences ingenomic Southern transfer experiments with DNA from all vertebrates examinedincluding Xenopus levis, chicken, mouse, monkey and man. No hybridization wasdetected with the TU1 M segment probe. There arc no homologous sequences in E. colior Drosophila mclanogaslcrD~NA. 'ITic number of homologous segments in transformedhuman cells (I IcLa) is 3-4 fold more than in normal human DNA.

*

Curren! address: Department of Genetics. Albert liinslcin College of Medicine48



REGULATION OF YEAST HISTONE mRNA SYNTHESIS. LynnaHereford and Mary Ann Osley, Sidney Färber CancerInstitute and the Department of Microbiology andMolecular Genetics, Harvard Medical School, 44Binney St., Boston, MA 02115.

Yeast histone mRNA levels are strictly controlledduring cell division such that they are maximallyaccumulated during the S phase of the cell cycle.This periodic accumulation is the result ofdistinct, yet complementary, transcriptional andpost-transeriptional regulatory mechanisms.Transcriptional regulation, which is evidenced bythe restriction of histome mRNA synthesis to aninterval between late G-^ and early S, appears to bemediated by the replicative state of the histonegenes themselves. This conclusion derives from theobservation that periodic transcription isabsolutely dependent upon the presence of functionalorigins of replication (ARS elements) which arelocated at the 3' ends of the H2B genes within eachof the two genetically unlinked, divergentlytranscribed H2A-H2B gene pairs. This potentiallynovel regulatory mechanism will be discussed indetail.

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DEFINITION OF CRITICAL NUCLEOTIDES WITHIN THE ENHAN-CER/ACTIVATOR REGION OF SV40Peter Gruss, Microbiology, University of Heidelberg,Im Neuenheimer Feld 230, 6900 Heidelberg, WestGermany, and Hans Weiher, Dept. of Molecular Biology,Wendell M Stanley Hall, University of California,Berkeley, Cal. 94720, USA

Activators or enhancers are control elements,which drastically increase the transcriptional acti-vity of certain genes. They differ from promoterelements in their ability to excert this functionrelatively independent of distance and orientationwith respect to the coding region. The overall acti-vity of the enhancer/activator is host-dependent,suggesting a possible involvement of host specificfactors. Although activator elements often seem toreside within repeated sequences, little informationis available concerning the nucleotides involved inthe functional activity. In an effort to definecritical nucleotides we have constructed and analyzedmutants containing multiple nucleotide exchangeswithin the SV40 72 bp repeat. Our results define nu-cleotides essential for the enhancer function. Compa-rison of this sequence with nucleotide sequences inother known activators allows the identification ofpotential related activator core elements.

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THE RELATION OF CHROMATIN STRUCTURE TO GENE EXPRESSION FOR ADROSOPHILA GLUE PROTEIN GENE. Steven K. Beckendorf, WilliamMcGinnis, Antony W. Shermoen, and Jill Heemskerk. Departmentof Molecular Biology, University of California, Berkeley, CA94720

The expression of Drosophila glue protein genes is inducedin larval salivary glands by the molting hormone ecdysone.The chromatin structure near one of these genes, Sgs-4,changes drastically when the gene is active. In the nuclei oflate third instar salivary glands, Sgs-4 is activelytranscribed and a complex of five DNase I hypersensitive sitesappears on the 5' side of the gene. Two of these are locatednear the site of transcription initiation while the otherthree lie between -300 and -500. Analysis of Sgs-4 under-producers suggests that the upstream hypersensitive region isinvolved in regulating gene expression. In several strains offlies, base substitutions or small deletions in this regionare correlated with changes in the pattern of DNase hyper-sensitivity and with reduction or elimination of RNA produc-tion. In one case, dosage compensation of this X-linked geneis eliminated by a small deletion in this region. One under-producer has, instead of changes in the -300 to -500 region, a1.3 kb transposable element inserted just upstream from itsTATAA sequence. This insertion results in four size classesof Sgs-4 transcripts, two of which begin within the element.Despite this large sequence rearrangement, each transcriptretains the normal Sgs-4 temporal and tissue specificity. Themechanism determining this specificity must, therefore, beable to act in a relatively distance-independent manner.

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REGULATION OF CLONED METALLOTHIONEIN GENES, Dean H. Hamer,Anthony D. Carter, Barbara Felber, Marie-France Jubier, GeorgeN. Pavlakls, Carl J. Schmidt and Maryjane Walling, NationalCancer Institute, National Institutes of Health, Bethesda, MD20205

The metallothioneins are ubiquitous heavy metal bindingproteins whose synthesis is inducible by both heavy metals andglucocorticoids. We have cloned and initiated a structuraland functional analysis of metallothionein genes from mouse,monkey and man. The mouse metallothionein-I gene is trans-criptionally regulated by cadmium when introduced intocultured cells on SV40 and BPV vectors. Analysis of deletionand linker-scanning mutants, using a convenient E. coligalactokinase read-through assay, shows that the regulatorysequences lie within a small region close to the 5' end of thegene. The cloned mouse gene does not respond to dexamethasonetreatment, even though the chromosomal genes in the same cellsretain their inducibility, indicating that heavy metals andglucocorticoids regulate this gene by independent mechanisms.Metallothionein genes can also be used as dominant selectivemarkers, by conferring resistance to cadmium, and to obtainthe efficient and regulatable expression of other gene productssuch as human growth hormone.

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SYMBIOTIC NITROGEN FIXATION; ANALYSIS OF THE REGION ENCODINGHOST-SPECIFIC NODULATION IN RHIZOBIUM sp.

J. Shine , P.R. Schofield, K.F. Scott, B.G. Rolfe,J.M. Watson

Centre for Recombinant DNA Research,Australian National University, Canberra, Australia

The symbiotic association between plants and bacteria ofthe genus Rhizobium is the result of a complex interactionbetween the bacterium and its host, requiring the expressionof both bacterial and plant genes in a tightly co-ordinatedmanner. Bacteria bind to the emerging plant root hairs andinvade the root tissue through the formation of an infectionthread. The plant responds to this infection by the develop-ment of a highly differentiated root nodule. These nodulesare the site of synthesis of the bacterial enzyme complexnitrogenase, which reduces atmospheric nitrogen to ammonia.This fixed nitrogen is then exported into the plant tissue andassimilated by plant-derived enzymes.

Genes encoding both nodulation (nod) and nitrogenfixation (nif), two of the major requirements for effectivesymbiotic nitrogen fixation, have been isolated from severalRhizobium strains by molecular cloning. In many cases these'symbiotic' genes are closely linked on a large plasmid. Invitro mutagenesis and introduction of these cloned genes intoother bacterial species has permitted a detailed analysis ofthe structure and function of the genes involved in the nodand nif phenotypes.

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MOLECULAR CLONING AND ORGANIZATION OF NODULIN GENES: A GROUPOF HOST GENES INVOLVED IN ROOT NODULE SYMBIOSIS, D.P.S. Verma,F. Fuller, and J. Lee, Plant Molecular Biology Laboratory,Department of Biology, McGill University, 1205 DocteurPenfield Avenue, Montreal, CANADA H3A 1B1

The development of symbiotic association between a legumeplant and the soil bacteria, Rhizobium sp. requires theexpression of several specific host genes. Using immuno-logical and hybridization techniques, we have identified insoybean, several nodule-specific host gene products. Towardsisolation of the genes encoding for nodule-specific proteins(nodulins), a cDNA library was constructed from poly A(+) RNAof nodules and screened for abundant nodule-specificsequences. Five unique species which represent more than 50%of the nodule-specific clones, were identified by cross-

hybridization experiments. The most abundant of these encodethe well characterized nodule protein, leghemoglobin. Theother four species were designated as Nod A, Nbd B, Nod C andNod D and were found to be present in the nuclear genome atlow copy number. On the other hand, leghemoglobin isrepresented by a closely related family of sequences. Thesegenes are induced only following infection of the plant byRhizobium and their transcripts represent 15% (leghemoglobin),6.5% (Nod A) and 0.6-1.11 (Nod B, C and D) mole fraction ofnodule polysomal RNA. Hybrid-selection of nodule RNA,followed by in vitro translation, indicated that Nod A-Dsequences encode polypeptides of 44, 27, 24 and 100-120thousand molecular weights respectively. Since thesepeptides reacted specifically with antisera to noduleproteins, the data suggest that they represent nodulinsequences. Genomic fragments, representing some of thesesequences, have been isolated and their organization iscompared with those of leghemoglobin genes. Using Alu-HaeIIIand EcoRI libraries of soybean, we have isolated most of thesequences representing leghemoglobins including some pseudoand truncated genes. Several of these genes are linked onthe chromosome and are flanked by sequences that aretranscribed more abundantly in root and leaf tissues. Thereare also some specific repeat sequences present in betweenthese genes. A detailed analysis of this region of thechromosome might provide some insight into the mode ofregulation of these genes following infection of the plant byRhizobium.

* Supported by grants from Natural Sciences and EngineeringCouncil of Canada

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SEED PROTEIN GENE STRUCTURE AND EXPRESSION IN NORMAL ANDMUTANT SOYBEAN LINES. Robert B. Goldberg and Diane Jofuku,Department of Biology, UCLA, Los Angeles, CA 90024, Lila 0.Vodkin, Seed Research Laboratory, USDA, Beltsville, MD 20705

A number of soybean inbred lines have been identifiedwhich are defective in the expression of various seed proteingenes. In an attempt to uncover DNA sequences which might beimportant for the control of seed protein gene expression, wehave investigated seed protein gene structure and function istwo mutant lines, one which lacks seed lectin (Le" line) andone which is devoid of Kunitz trypsin inhibitor activity(Kti- line).

In normal, Le+Kti+ lines, lectin and Kunitz trypsininhibitor gene expression is under strict developmental control(1,2). The primary control level appears to be transcriptional(2). Recent studies using a runoff transcription system withisolated nuclei have indicated, however, that lectin and Kunitztrypsin inhibitor genes are not regulated coordinate!y in theformal sense (L. Walling and R.B. Goldberg, unpublisheddata). Both liquid (1,2) and filter hybridization studiesshowed that lectin and Kunitz trypsin inhibitor proteins areencoded by small gene families containing approximately twoand five genes each, respectively. R-loop studies failed toreveal detectable introns in any lectin or Kunitz trypsininhibitor gene investigated to date.

Mutant Le- and Kti" lines were shown to contain thesame number of lectin and Kunitz trypsin inhibitor genes astheir normal, Le+ and Kti+ counterparts. In contrast,Le" and Kti" embryos were found to possess reduced amountsof lectin and Kunitz trypsin inhibitor mRNAs respectively.These reduced mRNA levels correlate well with the extent ofseed protein reduction in each line. For example, the Le"line which has no detectable seed lectin, has <0.01% thenormal level of lectin mRNA. On the other hand, the Kti"line, which has significant Kunitz trypsin inhibitor cross-reacting material, has 10% of the Kti* Kunitz trypsin inhibitormRNA level. These data suggest that each mutation is due toeither a transcriptional or a post-transcriptional lesion.

A detailed study of lectin gene structure in Le+ plantsshowed that one gene encodes seed lectin, while the other isa diverged gene of unknown function. In Le" and Le+ plantsthe latter gene region is identical; however, the Le" seedlectin gene was shown to contain a 3.4 kb insertion elementwhich resembles a transposable element. Preliminary studiesfailed to reveal any obvious differences in Kunitz trypsininhibitor gene structure or organization in Kti+ and Kti"plants. Together, these data suggest that the Le" phenotypeis a result of an insertional inactivation of the seed lectingene, and that a point mutation and/or a small chromosomalalteration may be responsible for the Kti- mutation.1. Goldberg et al. (1981), Dev. Biol. 83, 201. 2. Goldberg etal. (1981), Dev. Biol. 83, 218.

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STRUCTURAL DETERMINANTS FOR INDUCTION OF GENE EXPRESSION. BertW. O'Malley, Department of Cell Biology, Baylor College ofMedicine, Houston, Texas, 77030

Studies have been carried out to determine the chromosomalstructure of inducible genes and the interactions of inducers(eg., steroid hormone receptors) with regulatory regions ofthese genes. When oviduct nuclei are digested with DNase I,the sex steroid inducible ovalbumin and X and Y genes werepreferentially digested as compared to total genomic DNA. Infact, it was noted that these genes are included within a100,000 base DNA domain which is sensitive to DNase I; withinthis domain, the non-transcribed flanking sequences exhibiteda DNase I sensitivity which was indistinguishable from thatof the transcribed regions. However, this entire region wasresistant to DNase I digestion in spleen, liver and erythrocytenuclei, while a globin gene sequence was resistant in oviduct,spleen and liver nuclei but sensitive in erythrocyte nuclei.These giant DNase I sensitive domains provide the capabilityfor gene expression and appear to be a result of the moleculardifferentiation process since they are cell-specific and con-tain potentially expressible genes of that cell type. Repeti-tive sequences within this domain have been mapped and a parti-cular subfamily termed CR1 has been shown to exist only at theborders of this large DNase I-sensitive domain. The regionof homology between these CR1 sequences extends over a regionof approximately 160 base pairs. When the CR1 sequences arecompared with mammalian ubiquitous interspersed repetitive DNAsequences (human Alu and mouse Bl families), several regionsof homology are evident. Also, tissue specific methylationhas been detected at the 3'-border of these DNase I-sensitivedomains. Restriction enzyme digestion of nuclear matrix and/or scaphoid preparations has permitted the isolation of DNA se-qeunces attached to these nuclear support structures. Theseexperiments permit us to design a theoretical model for thearrangement of genomic DNA into expressible and nonexpressibleregions via its attachment to the nucleoskeleton. Finally,studies have been carried out to determine the regions ofinteraction of steroid hormone receptors with inducible genes.These results suggest a preferential binding of receptor pro-tein in the 5'-region of these genes.

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HEMOGLOBIN SWITCHING IN GOATS, J.B. Lingrel, S.G. Shapiro, T.M.Townes, S.M. Wernke, S.E. Spence and P.I. Liberator, University ofCincinnati Medical Center, Dept. of Microbiology and MolecularGenetics, Cincinnati, OH 45267.Many animals, including man, synthesize different hemoglobins (Hb)

at different times of development depending on the globin genesexpressed at that stage. For example, in man e and ç globin genes are

expressed during embryonic development, and their globins combine toform embryonic Hb. Later a and y genes are expressed to produce HbFor fetal Hb. During adult lilfe, a, 6 and 8 chains are active, resultingin the formation of adult hemoglobins HbA and HbA2- Goats and sheepgo through a series of similar switches, e and ç globin genes are

expressed during early embryonic development, giving rise to embryonicHb while a and y genes become active as erythropoiesis moves to thefetal liver and bone marrow. The globins coded for by these genes giverise to HbF. However, rather than switching directly from fetal toadult Hb, as do humans, goats exhibit an extra switch. Just beforebirth, y globin gene expression is repressed and ßC globin genes becomeactive resulting in the production of preadult Hb, HbC. During thefirst year of life, ßC globin gene expression is switched off and ßAglobin genes become active. This results in the formation of adult Hb,HbA, which is composed of a and ßA subunits. Therefore, the goatprovides an interesting model system for the study of hemoglobinswitching. To understand the basis for these developmental switches,we have cloned the globin genes of the goat and studied their sequenceorganization. Several interesting features have emerged. The geneorder is very different from humans and is as follows: e'-e"-\|>ßX-ßC_eIILeIV_ljjßZ_ßA_eV# The first four genes include two embryonic genes,a pseudogene and the preadult gene. This cluster is followed by asecond set of four genes which resembles that of the first set, namelye", e'V, ipßZ anA gA# The ßA ¡s followed by a fifth embryonic gene,suggesting that another set of four genes may follow the ßA gene.Thus, it appears that there has been a triplication of a four-gene set inthe goat and it is likely that the y gene, which has not yet been linkedto this cluster, is the final member of the third set. This arrangementraises some interesting questions concerning the regulation of theglobin genes in the goat. The three genes which are expressed atdifferent times, namely ßA, ßC and y, are not arranged in develop-mental order and sequencing studies reveal that they are very homolo-gous. The only major difference in these three genes is the presence ofa insertion-like element in the second intervening sequence. It isinteresting to speculate that this movable element may be involved inthe developmental expression of these genes. The a locus contains a Cglobin gene followed by duplicated a genes. The a globin genes are

virtually identical with their homology breaking off just proximal to theCCAAT box at the 5' end and after the polyadenylation site at the 3'end. It is possible that the sequence involved in the regulation of thesegenes is located within this conserved region.

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SYNTHETIC OLIGONUCLEOTIDES AS HIGHLY SPECIFICHYBRIDIZATION PROBES. R. Bruce Wallace, Ph.D. Departmentof Molecular Genetics, City of Hope Research Institute,Duarte, CA 91010

We have examined the effects of base-pair mismatch onthe stability of duplexes formed between synthetic oligo-nucleotides and several natural occurring DNA sequences.Several types of mismatches (A-C, G-T, A-A, and T-T) havebeen examined using oligonucleotides from 11 to 20 basesin length. In each case, the duplexes containing themismatch were less stable than the corresponding duplexeswithout mismatches. Hybridization conditions could bedetermined where the mismatched duplexes would not form,while the perfectly matched ones would. Under these con-ditions the oligonucleotides are highly specific probescapable of distinguishing DNA sequences differing by aslittle as a single nucleotide. The specificity of oligo-nucleotide hybridization has been used in several ways (1)to identify mutants generated by oligonucleotide directedmutagenesis, (2) to identify, from a recombinant phagelibrary, the cloned gene for a specific member of a multi-gene family using one or more oligonucleotide probes spe-cific for that member, (3) to detect the single basechange responsible for several human genetic diseases bydirect hybridization of oligonucleotides to genomic DNA.

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EXPRESSION OF THE a2 TYPE I COLLAGEN GENE Benoit deCrombrugghe, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland 20205

Examination of the methylation pattern of the chick a2 typeI collagen gene in different tissues suggests that the 5' por-tion of this gene is never methylated even in tissues which donot express the gene. However, the body of the gene is alwaysmethylated whether or not the gene is expressed. It is likelythat a mechanism prevents the methylation of a DNA segment atthe 5' end of this gene. In contrast to this invariantmethylation pattern, a site that is hypersensitive to DNase I,located in the chromatin containing the promoter of this gene,is only detected in cells which express this gene, not incells which do not express it. However, the same DNase Ihypersensitive site is found in chick embryo fibroblasts (CEF)and in Rous sarcoma virus transformed fibroblasts (RSV-CEF)although the rate of transcription of type I collagen RNA is8 to 10 times lower in RSV-CEF than in CEF. We conclude that(1) the absence of methylation at the 5' end of this gene,(2) the DNase I hypersensitivity, and (3) the p60src mediatedinhibition of transcription are caused by three different andindependent mechanisms. These probably reflect three dif-ferent levels of control for this gene.

We have also examined potential regulatory signals in thea2(I) collagen gene by cloning these sites in recombinantplasmids and by studying their role in chick cells by DNA-mediated transfection. In these plasmids the bacterial genefor chloramphenicol acetyl transferase (CAT) was placed underthe control of the a2(I) collagen promoter. The levels of CATenzyme are determined in cell extracts 20 to 50 hr after DNA-mediated transfection and reflect the activity of the promoter.Deletions in the a2(I) collagen promoter clearly alter theselevels and indicate which parts of the a2(I) collagen promotersequence are important for expression. We have also replacedan SV40 polyA addition site present in the expression vectorsused by tandem polyA sites present at the 31 end of the typeIII collagen gene. This substitution increases the levels ofCAT 8- to 10-fold after DNA transfection. This stimulation isprobably due to an increased stability of the correspondingmRNA. Therefore, sequences at the 31 end of genes can alsoinfluence their level of expression.

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EXPRESSION OF A METALLOTHIONEIN-GROWTH HORMONEFUSION GENE IN MICE: R. L. Brinster1, R. D.Palmiter2 and R. M. Evans3. l-Laboratory of Repro-ductive Physiology, School of Veterinary Medicine,University of Pennsylvania, Philadelphia, Pa. 19104,¿Department of Biochemistry, Howard Hughes MedicalInstitute, University of Washington, Seattle, Wa.98195,and 3Tumor Virology Laboratory, The SalkInstitute, San Diego, California 92138.

Studies have demonstrated that a fusion genecomposed of the mouse metallothionein I promoter/regulator fused to the herpes simplex viral thymi-dine kinase (HSV-TK) structural gene is expressed inseveral tissues of mice resulting from eggs micro-injected with the gene (Brinster et al., Cell 27,223, 1981). The fusion gene is transmitted toprogeny and expression of the gene is induced byheavy metals which normally regulate metallothionein(Palmiter et al., Cell 2_9, 701, 1982). A cloned ratgrowth hormone structural gene that is expressed inmouse fibroblasts (Doehmer et al., PNAS 79, 2268,1982) was substituted for the HSV-TK structural genein the fusion construction, and the new gene (MGH)injected into fertilized mouse eggs. From 21offspring, 7 were shown to contain the MGH gene, and6 of these grew 2-3 times more rapidly than litter-mates (Palmiter et al., Nature 300, 615, 1982).Some of the animals were almost twice the size ofcontrols. The gene is transmitted to progeny andthe progeny that carry the gene are larger thansiblings that do not have the fusion gene.

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METALLOTHIONEIN AND RELATED HEAVY METAL BINDINGPROTEINS: GENE STRUCTURE AND REGULATION OFEXPRESSION.

Michael Karin, Department of Microbiology, U5C School of Medicine,2011 Zonal Avenue, Los Angeles, CA 90033.

Metallothioneins (MTs) are low molecular weight, cystein rich, heavymetal binding proteins. MTs and related heavy metal binding proteinsare ubiquitously distributed among the animal and plant kingdoms. Inall species which have been examined, their expression is induced bythe same heavy metal ions that bind to the proteins.The human genome contains about twelve different MT genes. Wehave been studying the regulation of expression of the MT-II. gene.The expression of this gene is regulated by both heavy metal ions andglucocorticoid hormones. The cloned human MT-II. gene retains itsresponsiveness to both classes of inducers, after introduction into ratfibroblasts. By fusion of the 51 flanking region of human MT-II» geneto the structural portion of the HSV-TK gene, we have renderea theTK gene inducible by both heavy metal ions and glucocorticoidhormones. Therefore this DNA fragment containing 770 b.p of 5'flanking sequence contains all the necessary information fortranscriptional activation by both inducers. Introduction of this DNAfragment on an SV40 derived vector into CV1 cells leads to activationof the endogenous MT-II gene, after the vector has replicated andreached a high copy number. These results suggest that MT geneexpression is under repressor control and that the repressor binds tothe y flanking region of the genes, inhibiting their transcription. Webelieve this repressor to be the apoprotein (apothionein). Heavy metalions binding to the apoprotein lead to transcriptional activation due toderepression.To gain more information about the mechanisms controlling geneexpression by heavy metal ions we have been studying an organismamenable to modern genetic analysis: Saccharomyces cerevisiae. Thiswork is done in collaboration with Dr. Seymour Fogel at UC Berkeley.The complete nucleotide sequence of the CUP1 locus of yeast, whichcontrols resistance to copper ions has been determined. We havemapped two transcription units within this locus which is amplified asan intact unit in copper resistant strains. One of the transcriptionunits is copper inducible and contains an open reading frame for a lowmolecular weight cystein rich protein: copper chelatin. The chelaingene can be placed on a free replicating plasmid and introduced intocopper sensitive cells. Due to expression of the chelatin gene thosecells become copper resistant. The expression of the chelatin gene isstill controlled by copper, after placing it on a plasmid with only 1^0b.p. of 5' flanking sequences. The same 5' flanking sequence whenfused to other structural genes renders them copper inducible. Themechanisms controlling expression of the yeast gene seem quitesimilar to the ones controlling expression of the human genes.

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ADENOVIRUS VAI RNA IS REQUIRED FOR EFFICIENT TRANSLATION OFVIRAL mRNA AT LATE TIMES AFTER INFECTION. Cary Weinberger,Robert Schneider and Thomas Shenk, Department ofMicrobiology, Health Sciences Center, State university of NewYork, Stony Brook, NY 11794.

Adenovirus type 2 (Ad2) encodes two small polymeraseIll-transcribed RNAs which are produced in large amountssubsequent to infection. These transcripts are termed VAI(major species) and VAII (minor species) RNAs. Mutationsconstructed within the Ad2 VAI or VAII intragenic controlregions prevent cell-free transcription of the altered genesusing HeLa cell extacts (Fowlkes and Shenk Cell 22, 405-413,1980). Now we have "rebuilt" VA RNA genes carrying deletionswithin their intragenic control regions back into completevirus. dl330 lacks 29 bp within its VAI gene; it producesVAII RNA, but no detectable VAI RNA. d!328 lacks 17 bpwithin its VAII gene; it synthesizes VAI RNA but nodetectable VAII RNA. dl328 grows as well as its wild-typeparent, but ¿1330 grows more slowly and produces a 20-foldlower yield than the parent. The defect in dl330 is clearlydue to a lack of VAI RNA since the mutant grows to wild-typelevels in the presence of an SV40 recombinant which containsand expresses the VAI RNA gene.

The VAI"" defect has been localized to late viraltranslation. Late viral mRNAs are capped, spliced,polyadenylated and present in the cytoplasmic compartment ofinfected cells at normal levels, but they are translatedinefficiently. Several lines of evidence suggest thetranslational defect occurs at the level of initiation. Meanribosome transit times are identical in mutant and wild-typeinfected cells. dl330-infected cells contain large amountsof inactive, 74S ribosomes. There are fewer active polysomesin dl.330-infected cells than in wild-type infections, andthey are considerably smaller. Experiments are in progressto identify more precisely the step during initiation oftranslation at which VAI RNA functions.

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ACTIVATION OF THE TRANSFORMING POTENTIAL OF CELLULAR ONC GENES.T. G. Wood, D. G. Blair, M. L. McGeady and G. F. VañrJe" Woude.

The molecular elements essential for enhancment or activationof the transforming potential of the Moloney sarcoma virus(MSV) one qene (mos) have been identified. The long terminalrepeat (LTR) of the cloned MSV proviral DNA has been shown toenhance the biological transforming activity of the viral mossequence (v-mos). In these assays DNA recombinants containinga single LTR in either a 51 or 31 position relative to v-mosresulted in equivalent transforming efficiencies. Analysis ofRNA transcripts expressed in cells transformed by these recom-binant DNAs suggested that the 51 LTR provided transcriptionalcontrol signals for promotion and initiation of the mos_ contain-ing RNA, while an LTR introduced 31 to v-mos contributed signalsfor polyadenylation of mos containing RNA transcripts. Theseresults suggested that sequences responsible for initiation andpolyadenylation of v-mos containing RNA transcripts were ac-quired from either vector, host or carrier DNA sequences. Theabsence of sequences insuring either initiation or polyadenyla-tion of the mos containing RNA did not preclude enhancement ofthe transforming efficiency of v-mos suggesting that othersequences within the LTR are responsible for enhancement.This was directly demonstrated when a 300 base pair (bp) segmentderived from the 5' portion of the LTR was shown to providethe same level of enhancement as the entire 588 bp LTR sequence.This segment lacked signals for initiation and polyadenylationbut contained the 73 bp tandem duplication.

We have demonstrated that the transforming potential ofthe normal cellular mos sequence (c-mos) was activated byintroducing an LTR 51 to c-mos. These analyses suqgested thatnormal cellular one qenes can be activated by sequences withtranscriptional enhancing properties such as are present in theLTR. While c-mos was activated by an LTR 31 to it, the effi-ciency was several thousand fold lower than the level obtainedwith constructs containing an LTR 51 to c-mos. By removingvarious portions of the normal mouse sequences 5' to c-mos, wehave identified a 500 bp region of the normal mouse sequenceswhich intereferes with activation of the transforming potentialof c-mos. Since c-mos expression has not been observed innormal cells, this region may be involved in the control ofexpression of this gene.

These analyses have been useful to define the molecularelements (one genes and enhancers) and their arrangementswhich will result in transformation.

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HUMAN TRANSFORMING GENES: M. Wigler, K. Shimizu,M. Goldfarb, E. Taparowsky, Y. Suard and 0.Fasano, Cold Spring harbor Laboratory, ColdSpring Harbor, New York 11724

Three distinct genes have been isolated fromDNA of human tumor cell lines which are capable oftransforming NIH3T3 cells following DNA mediatedgene transfer. All three are members of a genefamily related to the viral ras oncogenes. Themechanism of activation of these genes from normalgenes will be discussed as will a new assay forhuman tumor genes.

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THE REGULATION OF CELLULAR TRANSCRIPTS BY SV40 INDUCED CELLGROWTH AND TRANSFORMATION by Arnold J. Lev I ne, Department ofMicrobiology, State University of New York at Stony Brook,Stony Brook, New York 11794.

A c-DNA library was prepared from the m-RNA fraction ofSV40 transformed Balb/3T3 mouse cells. A number of distinctc-DNA clones were Identified, using colony In situ hybridiza-tion, that detected higher levels of m-RNA In an SV40 trans-formed cell line than nontransformed cells. Similarly thesec-DNA clones detected greater than 100 fold higher levels ofm-RNA In SV40 Induced tumor tissue when compared with RNA*sderived from normal mouse tissues. Infection ofnontransformed cells with SV40 stimulated the levels of RNAcomplementary to the cloned c-DNA Inserts. The kinetics ofstimulation of these RNA's and the levels obtained afterInfection varied with different cloned c-DNA's tested. Thesecellular transcripts could be regulated by either the trans-formed state and transforming genes or the actively growingstate of transformed cells compared to their nontransformedcell counterparts. In SV40tsA transformed cell lines (SV40tsAmutation In the large T-antlgen gene) the growth potential ofthe cells appears to be an Important variable in regulatingthe levels of some of these transcripts.

Some of the mur I ne c-DNA clones hybridize under stringentconditions with human DNA Indicating strong nucleotidesequence conservation. c-DNA clone 85 Is derived from a

family of genes (at least five distinct genomic clones) whosetranscripts appear to be regulated by cell growth or replica-tion. Studies are in progress to characterize the propertiesof these genes and gene products.

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REPLICATION OF THE GENOME OF HEPATITIS B-LIKE VIRUSES,Jesse Summers and William S. Mason, Institute for CancerResearch, Fox Chase Cancer Center, Philadelphia, PA 19111

Replication of the genome of hepatitis B-like viruses hasbeen studied in an animal virus model, the duck hepatitis Bvirus (DHBV) (1). DHBV-infected hepatocytes persistently pro-duce DNA-containing virions but incur no apparent cytopathicconsequence. We have found that the DNA in these virions isnot synthesized as a consequence of semi-conservative repli-cation, but is reverse-transcribed de_ novo from an RNA tran-script of the genome (2) (3). The process resembles thatoccuring in retroviral infections during synthesis of the DNAprovirus. Two major differences however are (i) that reversetranscription occurs as a step in virus assembly in HBV-likeviruses, while in retroviruses it is an early step in initia-tion of infection, and (ii) synthesis of the minus strand ofDHBV DNA is apparently initiated on a protein primer (4), whilein retroviruses a cellular t-RNA is utilized.

Certain aspects of the overall strategy of genome replica-tion by HBV-like viruses will be discussed; i.e. the formationand maintenance of a transcriptionally active form of DHBV DNAin the cell as an early step in viral infection, and theassembly of an RNA transcript of the genome ("pre-genome") intonucleocapsid cores, and the subsequence reverse transcriptionof this RNA transcript into DNA during maturation of thevirion.

1. Mason, W. S., Seal, G. and J. Summers. A virus of Pekinducks with structural and biological relatedness to human hepa-titis B virus. J. Virol J36: 829-836 (1980).2. Mason, W. S., Aldrich, C., Summers, J. and J. M. Taylor.Asymmetric replication of duck hepatitis B virus DNA in livercells. Proc. Nati. Acad. Sei. (USA) _79: 3997-4001 (1982).3. Summers, J. and W. S. Mason. Replication of the genome ofa hepatitis B-like virus by reverse transcription of an RNAintermediate. Cell ^9: 403-415 (1982).4. Molnar-Kimber, K., Summers, J., Taylor, J. M. and W. S.Mason. A protein is covalently bound to minus strand DNA inter-mediates of Duck Hepatitis B virus. J. Virol, in press (1983).

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EXPRESSION OF P21 RAS PROTEIN IN TRANSFORMED MAMMALIAN CELLSAND IN E. COLI. Barbara Fleurdelys#, Robert Bassin#, GordonHager#, Edward M. Scolnick*, and Mark E. Furth0. laboratoryof Tumor Virus Genetics, Nati. Cancer Inst., Bethesda, MD 20205*Merck Sharp and Dohme Research Lab., West Point, PA "MemorialSloan-Kettering Cancer Center, New York, N.Y. 10021

The ras oncogene of the Harvey murine sarcoma virus en-codes a 21,000 dal ton protein (p21) which efficiently trans-forms susceptible cells to malignancy. A kinetic analysis oftransformation was performed by placing p21 expression underthe control of the hormonally inducible promoter from the longterminal repeat (LTR) sequence of the murine mammary tumor virus(MMTV). Transformed clones of murine NIH 3T3 fibroblasts wereselected after uptake of plasmid DNA containing the viral rasgene inserted downstream from the MMTV LTR. When these cellswere cultured in hormonally defined medium, the maintenance ofthe transformed phenotype was strongly dependent on the presenceof dexamethasone, and was temporally coupled to the synthesisof p21.

In order to facilitate biochemical studies of p21, theHarvey viral ras gene was expressed in E_. coli using a plasmidvector from M. Inouye, SUNY. The construction introduced asubstitution of four amino acids at the amino-terminus of theprotein. The p21 polypeptide was identified in cells carryingthe ras plasmid by immunoprecipitation with sequence andconformation specific monoclonal antibodies against epitopeson Harvey viral p21. Phosphorylation of p21 was detected invivo by metabolic labeling with 32Pi. The protein synthesizedin bacterial cells was partially purified and displayed theguanine nucleotide binding activity and the GTP-specificautophosphorylation activity associated with p21 purified fromvirally transformed mammalian cells. Thus, these activitiesare intrinsic to the protein. Tryptic peptide and phospho-amino acid analysis revealed that phosphorylation was restric-ted to the characteristic unique threonine residue (position59 in Harvey viral p21).

The p21 encoded by the oncogene of BALB sarcoma virus,bas, which is nearly identical to ras of Harvey sarcoma virus,also was expressed in E_. coli. Its properties were similarto those of p21 ras. However, like the product of the normalcellular Harvey-type ras (bas) gene, the bacterially producedp21 bas was not phosphorylated, apparently because it lacksthe specific threonine target site.

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

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