DNAVolume 1, Number 1, 1981Mary Ann Liebert, Inc., Publishers

Speakers' Abstracts from the First Annual Congresson Recombinant DNA Research

Actin and Tubulin Gene Families in Drosophilia. J. Natzle,S. Tobin, F'Sanchez, E. Zulauf, U. Rolest, andB. McCar-thy. Dept. of Biochemistry and Biophysics, University ofCalifornia, San Francisco, CA94143 and Dept. of Genetics,University of California, Berkeley, CA. 94720.

The existence of several similar, but not identical,members of gene families coding for structural proteins,such as actin and tubulin, raises several issues. Suchfamilies are typified by those for Drosophila actin, of whichthere are six copies at dispersed, unlinked loci, andDrosophila tubulin where a-tubulin and 0-tubulin are eachencoded by four genes, again at unlinked loci. All of thesegenes have been isolated and are being characterized. Suffi-cient differences occur in the primary sequences of in-dividual gene copies to preclude the possibility that they are

simply duplicates coding for the same protein. Even greaterdifferences occur in 5' and 3' flanking sequences, making itpossible to quantitate individual messengers by hybridiza-tion with specific probes. Such experiments have revealed a

great deal of specificity in the expression of actin andtubulin genes during development. For example, a probespecific for the actin gene located at 79B hybridizes withlarval RNA, but not with RNA of embryos, pupae, or Kccells. Similarly, a probe specific for one of the foura-tubulin genes reacts with RNA of early embryos, but notRNA isolated from other stages. These data imply that themultiple genes exist to code for different forms of thesestructural proteins, and that their expression is individuallycontrolled.

Mechanisms for Expression of Cloned Genes in Hetero-specific Environments. S.N. Cohen. Dept. of Biochemistry,Stanford University, Palo Alto, CA. 94305.

The construction of E. coli plasmids that enable theisolation and characterization of transcriptional andtranslational control signals from heterospecific DNAsources will be described. A novel two-planed cloningsystem for obtaining effectively regulated high-level expres-sion of foreign genes will be discussed, along with recent in-vestigations involving the construction and characterizationof DNA cloning systems for antibiotic-producing Strep-tomyces.

Cloning of Mouse H2 Genes. P. Kourilsky. Unité deBiologie Moléculaire du Gène, ER CNRS 201 and SCN IN-SERM 20, Institut Pasteur, Paris, France.

Messenger RNA coding for the major transplantationantigens in the mouse was partially purified and cloned inE. coli. H2 specific cDNA probes were isolated. DNA se-

quencing indicated that the clones characterized so far cor-respond to at least two alíeles and provided information on

the primary structure of the third domain, the trans-membrane part and the cytoplasmic portion of H2 heavychains in the d haplotype. The number of genes sharing se-

quence homology with these probes in the mouse genomehas been estimated and several genes have been cloned. In-formation on their structure will be reported and the datawill be discussed with regard to the genetic origin or thepolymorphism of H2 antigens.

Factors Involved in the Transcription of PurifiedEukaryotic Genes In Vitro. R.G. Roeder, N. Heintz,B. Honda, D. Lee, D. Luse, T. Matsui, J. Segall, andB. Shastry. Washington University School of Medicine,St. Louis, MO 63110.

Eukaryotic genes can be accurately transcribed in solublesystems containing purified DNA templates and cellular ex-

tracts with either endogenous or exogenous (purified) RNApolymerases. Both initiation and termination have beendemonstrated for class III genes, including 5S and tRNAand adenovirus VA RNA genes. Accurate initiation(mimicing exactly that found in vitro) has beendemonstrated for several class II genes, including (i)adenovirus (early, intermediate, late) transcription units,(ii) mammalian globin genes, and (iii) amphibian andhuman histone genes (in the latter case specific terminationevents are also apparent). Various studies indicate that thetranscription events observed in these systems are mediatedby factors which are neither tissue nor species specific and,consequently, that other factors/mechanisms are necessaryto explain the regulation observed in vivo. Our furtherstudies have emphasized an analysis of the various factorspresent in the soluble extracts in order to understand theirmechanism of action (and ultimately, their modulation byother factors). There appear to be at least four such factorsfor class II genes (as assayed with the major late promotorof adenovirus) and one has been purified to homogeneity.These factors are chromatographically separable fromthose required for transcription of class III genes. The lat-ter include one component (TFIIIA) specific for 5S RNAgenes and at least two other factors (fractions) which couldbe common for the class III genes analyzed. The 5S genespecific factor (a protein of about 40 000 daltons) has beenpurified from Xenopus oocytes. This protein binds both tothe 5S RNA gene control region and forms a stable 7S RNPcomplex with oocyte 5S RNA (suggesting a potential feed-back initiation mechanism). The level of this protein variesgreatly during development, and cultured somatic cells ap-pear to contain an altered form (slightly larger) of this pro-tein. The implications of these findings for 5S RNA genecontrol will be discussed, as well as the further purificationand analysis of the other class II and class III genetranscription factors.

71

72 SPEAKERS' ABSTRACTS

Definition of the SV40 Early Transcriptional ControlRegion and Its Relation to the T-Antigen Binding Sites.R. Tjian and D. C. Rio. Dept. of Biochemistry, Universityof California, Berkeley, CA 94720.

The transcription of simian virus 40 (SV40) in produc-tively infected monkey cells is a temporally regulated pro-cess which is in part modulated by the viral A gene product(T-antigen). We have recently shown that SV40 T-antigen iscapable of .repressing SV40 early transcription in vitro.Under conditions of the transcription assay, T-antigen isable to bind specifically to the T-antigen binding sites,which are located at +50 to +25 bp (site I), +5 to -20 bp(site II), —45 to —70 bp (site III) from the major in vivoearly RNA cap site ( + 1). Here we report the use of in vitromutagenesis and recombinant DNA techniques to definethe limits of the SV40 early transcriptional control region.

A cell-free RNA synthesizing system was used to assaythe ability of cloned mutant viral DNA templates to initiateRNA polymerase II transcription. Run-off RNA productsof discrete length are efficiently synthesized when transcrip-tion is directed by DNA restriction fragments containingwild-type SV40 early promoter sequences. Deletionmutants DL 11 and DL 27, generated in vitro by Bal 31nuclease, remove the TATAA (Hogness) box at —25 to

—

30, yet exhibit an unaltered level of in vitro RNA syn-thesis, suggesting that this sequence is not essential for earlytranscription initiation. However, deletion mutants DL 24,26, 105, and 108 show markedly reduced ability to directthe synthesis of discrete run-off RNA products. These dele-tion mutants serve to define the 5' and 3' borders of theSV40 early transcriptional control region at sequenceslocated from -60 to

—

160 bp. The region at —60 to -80bp, defined by mutants DL 24 and 26, lies within or directlyadjacent to the third SV40 T-antigen binding site (

—

45 to-70 bp). Thus it is probable that the T-antigen mediatedrepression of early SV40 transcription is due to a directcompetition between the RNA polymerase II transcriptioncomplex and T-antigen for adjacent overlapping bindingsites on SV40 DNA. To test further this idea, we have con-structed a hybrid plasmid containing the SV40 T-antigenbinding sites placed 200 bp distal to the major adenovirus 2(Ad2) late promoter. Transcription from this Ad2 late pro-moter was not inhibited by saturating levels of T-antigen inthe in vitro assay. Thus, these findings are consistent withthe idea that repression of transcription by the SV40T-antigen involves a direct steric blockage at the RNApolymerase binding site due to overlapping regulatory, se-

quences.

Chromosome Structure of Globin Genes. H. Weintraub.Dept. of Genetics, Fred Hutchinson Cancer ResearchCenter, Seattle, WA 98104.

TATAA+ 50 +25 +5 -20-i ' I-1 II I—*—

We have analyzed the chromosome structure of both thea and ß globin gene cluster in three cell types present atvarious stages of erythroid differentiation in developingchick embryos. These three cell types are (1) embryonicerythroblasts synthesizing embryonic a- and /3-globinchains, (2) adult erythroblasts synthesizing adult a- andß-globin chains, and (3) precursor red cells not synthesizingglobin mRNA. We show, using three assays ofchromosome structure (DNase I sensitivity of active genes;in vitro run-off transcription; and DNA undermethylation),that the globin genes are inactive in precursor cells. Duringdifferentiation to the embryonic lineage, the embryonic,but not the adult genes become active, while during dif-ferentiation to the adult erythroid lineage, the adult genes,but not the embryonic genes become activated. Theseresults are discussed in terms of a model where domains ofchromosome structure determine which globin gene is ac-

tive. Finally, we have also focused on the biochemical basisfor the DNase I sensitivity of active genes. We have shownthat two proteins, HMG 14 and 17, are responsible for in-ducing a DNase I sensitivity upon active nucleosomes.Since these proteins have a preferential affinity for activenucleosomes we have coupled them to an affinity columnand show that active nucleosomes can then be purified byvirtue of their binding to the HMG 14/17 column.

Promoter Sequences of Eukaryotic Genes Transcribed byRNA Polymerase B. P. Chambón. Laboratoire deGénétique Moléculaire des Eucaryotes du CNRS, Unité 184de Biologie Moléculaire et de Génie Génétique de PIN-SERM, Institut de Chimie Biologique, Faculté deMédecine, Strasbourg, France

In vitro genetic techniques were used to study the se-

quence requirements for initiation of specific transcriptionin vitrojínd in vivo.

Deletion mutants were constructed around the putativepromoters of the Adenovirus-2 major late, simian virus 40(SV40) early, and chicken conalbumin genes. Specifictranscription in vitro by RNA polymerase B together with a

HeLA cell extract was used as a test for promoter function.With this approach it has been shown that the TATA box(or Goldberg-Hogness box) region of homology (centeredat about 28 base pairs upstream from the 5'-end of thesegenes) is essential for directing specific initiation oftranscription in vitro within a narrow area. The essentialrole of the TATA box was unequivocally demonstrated bythe strong "down-effect" of single base mutations in theTATA box. It was also observed that the overall promoterefficiency is influenced by the particular sequence sur-

rounding the RNA start site (cap site). It appears thereforethat the promoter region for accurate initiation ún vitro islocated within a segment of about 40 bp which includes the

RNA Pol II _uControl region

-45 -70 -160-j III _L- -1

SPEAKERS' ABSTRACTS 73

mRNA start site and some sequences which are situated im-mediately upstream and contain the TATA box.

The promoter role of these sequences for specific initia-tion of transcription in vivo was demonstrated with SV40recombinants bearing various deletions around the 5'-endof th SV40 early genes. However, SV40 early genes are notexpressed in deletion mutants lacking sequences locatedabout 200 bp upstream from the mRNA start site. The re-

quirement for these far upstream sequences which are

dispensable in vitro and have no counterpart in prokaryotesmay reflect the importance of chromatin structure in thecontrol of gene expression in eukaryotic cells.

Studies on the Mechanism of RNA Splicing in Yeast./. Abelson, C. Peebles, G. Knapp, R. Ogden, P. Johnson,and R. Ng. Dept. of Chemistry, University of California,San Diego, LaJolla, CA 92093.

1. Intervening Sequences in tRNA Genes. The tRNAprecursors that accumulate in the yeast temperature-sensitive mutant, ts-136, contain intervening sequences. Asoluble extract from yeast splices these precursors. The in-tervening sequence is removed intact and the ends of thehalf-tRNA sized molecules are joined to form a maturetRNA molecule. This splicing reaction can be operationallyseparated into two distinct steps: the endonucleolyticcleavage of the pre-tRNA and the ligation of the halves. Inthe absence of ATP only the cleavage reaction occurs pro-ducing the intervening sequence and half-tRNA molecules.Similar half-tRNA molecules appear as kinetic in-termediates or accumulate if splicing is inhibited with pure,mature tRNA. These half-tRNA molecules have beenpurified and can be efficiently ligated in an ATP-dependentreaction that is also inhibited by added mature tRNA.

This separation of activities has allowed us tocharacterize the products and intermediates of the splicingreaction and thereby to gain an insight into the mechanismof splicing. The cleavage of the precursor occurs by a sim-ple scission of two phosphodiester bonds leaving5'-hydroxyl and 3'-phosphate termini on the intervening se-

quence and the halves. The 3'-phosphate is required for thesubsequent ligation of the 5'-half to its cognate 3'-half.

We have made progress in the characterization of thetRNA splicing enzymes. The activités can be physicallyseparated. The endonuclease appears to be a membraneprotein. It fractionates with the yeast membranes and isreleased from membranes by treatment with non-ionicdetergent. In preliminary experiments it appears that theendonuclease is associated with the nuclear membrane. Theligase is not strongly associated with membranes but ratheris a soluble protein. Both activities have been partiallypurified.

2. The Actin Gene in Yeast Contains an Intervening Se-quence. The yeast, Saccharomyces cerevisiae, is known tocontain the highly conserved and ubiquitous protein, actin.We have used cloned actin sequences from Dictyosteliumdiscoideum to identify and clone the actin gene in yeast.Hybridization to genomic fragments of yeast DNA suggestthat there is a single actin gene in yeast. We have determinedthe nucleotide sequence of that gene and its flankingregions. The sequence of the gene reveals an intervening se-

quence of 309 bp in the coding sequences at the 5' end of the

gene. The existence and location of the intervening se-

quence was verified by sequencing the 5' terminus of the ac-tin mRNA using the dideoxy chain termination technique.This similarity of the splice junction sequences in this geneto those found in higher eucaryotes suggests that yeast mustpossess a similar splicing enzyme.

Gene Control Definitely Occurs at Several Levels in AnimalCells. J. E. Darnell, Jr. Molecular Cell Biology Dept.,Rockefeller University, New York, NY 10021.

A brief review of transcription unit design, including as anew example, an exploration of the boundaries of themouse 0-globin transcription unit, will be given. Knowingthe boundaries of transcription units and something aboutprimary nuclear transcripts then allows an assessment oftranscriptional control. Several types of transcriptionalcontrol are apparent during adenovirus infection andtissue-specific mRNAs also can be readily demonstrated tobe largely under transcriptional control.

With proper assays several changes in the levels of severaldifferent adenovirus mRNAs can be demonstrated not tobe due to transcriptional controls but in one case to dif-ferential processing and in other cases to differentialcytoplasmic stability.

Studies on Gene Structure, Expression, and Regulation.B. W. O'Malley. Dept. of Cell Biology, Baylor College ofMedicine, Houston, TX 77025.

Our goal is to understand the regulation of gene expres-sion in eucaryotic cells. We have worked with the chickenoviduct system in which synthesis of egg white proteins(ovalbumin, ovomucoid, etc.) are induced by steroid hor-mones (estrogen and progesterone). We have isolated theovalbumin and ovomucoid genes and determined theircomplete structure. They are each complex genes whosestructural sequences are interrupted by seven interveningsequences. Evolutionary studies on the ovomucoid geneshow that the structural sequences (exons) correspond tofunctional domains of this protein. Studies on thechromosomal structure of these genes reveal that they are

present in large DNA-sensitive "loops" which are tran-scribed only at the boundaries of the genes. Our studies in-dicate that the steroid hormonal regulation of the expres-sion of these genes is at the transcriptional level. The syn-thesis of a premessenger RNA is induced and a splicing en-

zyme system then removes all intervening sequences. This"processing" of the primary transcript appears to be carriedout in a definable compartment of the nucleus and mayutilize the assistance of a Ul RNA template. The inductiveeffect of steroid hormones on the expression of these genesappears to be mediated by a specific cytoplasmic feceptor-hormone complex which translocates to the nucleus andbinds onto chromosomal acceptor sites. Subsequent to thisnuclear event, a hormone-mediated event occurs at the ef-fector sites of inducible genes to stimulate their rate oftranscription.

Origin and Function of Avian Retrovirus TransformingGenes. J.M. Bishop. University of California, San Fran-cisco, CA 94143.

74 SPEAKERS' ABSTRACTS

Avian retroviruses induce neoplastic transformation in a

variety of cells. Prototypes include Rous sarcoma virus(RSV)/fibroblasts; MC29 virus (MCV)/fibroblasts,epithelial, and myeloid cells; avian erythroblastosis virus(AEV)/fibroblasts and erythroblasts; and avian myeloblas-tosis virus (AMV)/myeloblasts. The genetic loci (or onco-

genes) responsible for oncogenesis by each of these virusesderive from separate loci (or "proto-oncogenes") in normalvertebrate DNA. The proto-oncogenes for RSV, MCV,AEV, and AMV, isolated from DNA libraries of chickenand mammalian DNAs, display properties of conservedand essential cellular genes: they are present throughoutvertebrates; they have complex topography, yet reside atconstant positions within the genome of particular species;they are not linked to identified replicative genes of en-

dogenous retroviruses; and they give rise to distinctiveRNA transcripts in both growing and resting cells.

The oncogene of RSV (src) encodes a tyrosine proteinkinase (pp605rc). A closely related protein (pp60"ro'o-irr)and enzymatic activity are detectable in uninfected avian,rodent, and human cells. Both pp60wand pp60"TO'°-îrc are

tightly associated with the plasma membrane; one domainof pp605rr is bound to the membrane, another isexposed —apparently at the cytoplasmic surface. It appearsthat pp60OT and pp60pro'0~src may carry out similar func-tions in the cell, and it is therefore conceivable that srctransforms cells by introducing excessive amounts of an

otherwise normal cellular protein.The oncogenes of MCV and AMV give rise to single pro-

teins whose pleiotropic effects apparently account for allforms of tumorigenesis by these viruses. By contrast, theoncogene of AEV contains two separately expressed do-mains that may individually mediate transformation oferythroid cells and fibroblasts. The topography of the AEVoncogene is mirrored in the structure and expression of itscellular progenitor, which is also divided into two separate-ly expressed domains. The oncogenes of RSV, MCV, AEV,and AMV may each abort the developmental program of atleast one cellular lineage. We suggest that the cellular pro-gentors of these oncogenes are tissue-specific regulators ofgrowth and/or differentiation, and that the same specificitypersists in the function of the viral gene products.

The preceding findings conjoin to suggest thatretroviruses may have excerpted vital regulatory elementsfrom the vast pool of cellular genes; as a consequence, theseelements would be readily accessible for study. We furtherspeculate that the cellular progenitors of retrovirus on-

cogenes may be equivalent to "cancer genes" —either thegenetic loci at fault in human pedigrees afflicted with a highrisk of cancer, or their alleged counterparts, the geneswhose mutation in somatic cells lead to spontaneous or

chemically induced neoplasia.

Acquisition and Retention of Amplified DihydrofolateReducíase Genes in Cultured Animal Cells. R. T. Schimke.Dept. of Biology, Stanford University, Palo Alto, CA94305.

Methotrexate (MTX) resistance is obtained in mouse andhamster cells by step-wise selection in increasing MTX con-centrations. Resistance is a result of selective amplification

of DNA sequences containing the dihydrofolate reductase(DHFR) gene. In stably resistant cell lines, the DHFR genesare associated with expanded regions of a singlechromosome (homogeneously staining region-HSR). Inunstably resistant cells the DHFR genes are present on extra-chromosomal, acentromeric elements called double minutechromosomes (DMs). We estimate the repeat length of theamplified DNA to be 500-1000 kb. Preliminary results sug-gest that the DNA sequences present in the HSR and on DMsare similar, and thus raise the question of whether DMs are

precursors or products of HSRs, and whether DMs mediateexchange of DNA between chromosomes or between cells.

Molecular Cloning of Bacterial Genes Involved in Sym-biotic Nitrogen Fixation. J. Shine. Dept. of Genetics,Research School of Biological Sciences, Australian Nation-al University, Canberra, A.C.T. 2601 Australia.

Biological nitrogen fixation resulting from the symbioticinteraction between different species of Rhizobium andleguminous plants requires the coordinated expression ofspecific genes from both partners. In order to obtain an

understanding of the symbiotic process at the molecularlevel, we have made use of transposon mutagenesis andrecombinant DNA techniques in an approach to theanalysis of the genes controlling symbiosis in differentspecies of Rhizobium.

The transposon Tn5 (coding for kanamycin resistance)has been introduced into the genome of several Rhizobiumstrains. Selection is made for kanamycin resistance and dif-ferent transposon-induced mutants need only be screenedonce on plants to determine which colonies carry insertionsgiving rise to a symbiotic defect. Thereafter, the mappingof the symbiotically important gene is carried out by map-ping the drug-resistant gene and a limited number of planttests can be done to confirm the association between drugresistance and symbiotic defect. Total DNA isolated fromindividual colonies of such mutants has been digested withEco RI endonuclease and cloned into the Eco RI site of theplasmid pBR322. Recombinants carrying the Tn5transposon and flanking Rhizobium sequences have beenselected by kanamycin resistance. Such cloned DNAfragments, when radioactively labeled in vitro, provide ex-

cellent hybridization probes to examine the organization ofcorresponding sequences in other Rhizobium species, andhave also been used to isolate the corresponding wild-typegenes from clone banks of Rhizobium DNA.

In order to analyze the structure and function of cloned"symbiotic" genes, it is necessary to be able to reintroducethe gene into the original mutant Rhizobium and observe a

restoration of symbiotic capacity. This has been carried outby subcloning the wild-type symbiotic genes into the con-

jugative plasmid RP4. E. coli cells harboring such a recom-

binant plasmid have then been mated with the originalTn5-induced mutant strain to confirm the presence of sym-biotic gene sequences by the ability of the cloned fragmentto repair the symbiotic defect. Restriction endonucleaseanalysis of DNA isolated from such cells demonstrates thepresence of both mutant and wild-type gene sequences.This suggests that the observed restoration of symbioticcapacity has occurred by complementation and hence that

SPEAKERS' ABSTRACTS 75

the entire symbiotic gene is contained within the clonedfragment.

The ability to clone symbiotic genes by this approach willallow us to investigate in detail the molecular genetics ofsymbiotic nitrogen fixation. Furthermore, analysis of theeffects of introduction of these cloned genes into otherRhizobium species should provide some insight into themechanisms involved in host specificity.

Bacterial Expression of Multiple Human Leukocyte Inter-feron Genes. D. V. Goeddel, P. W. Gray, D. Leung,P. Week, and E. Yelverton. Genentech, Inc., South SanFrancisco, CA 94080.

Eight distinct human leukocyte interferon cDNA cloneshave been isolated from a cDNA library. Six of these are

full-length clones coding for pre-interferon molecules of188 or 189 amino acids. These six interferon genes havebeen directly expressed in E. coli as mature IFs of 165 or

166 amino acids (preceded by a methionine). In addition,several hybrid interferon genes have been constructed andexpressed in E. coli. The interferons produced displaydistinct target-cell specificities in tissue culture antiviralassays.

The Rearrangements of Antibody Genes. L. E. Hood. Div.of Biology, California Institute of Technology, Pasadena,CA 91125.

Antibody molecules are comprised of light (L) and heavy(H) chains that fold into discrete molecular domains whichcarry out two categories of functions —pattern recognitionor antigen binding [the variable (V) domain] and effectorfunctions such as complement fixation [the constant (C)domains]. Light chains are encoded by several discretecoding segments or exons that are separated by interveningsequences—leader (L), variable (V), joining (J), and cons-

tant (C). Heavy chains are encoded by these exons as well as

several additional exons —diversity (D) and membrane (M).Two distinct types of DNA rearrangements occur duringthe differentiation of antibody-producing or B cells. (1) V-J(or V-D-J) joining juxtaposes the various gene segmentsthat encode the variable region in association with the Qigene. (2) Then CH switching may displace the V regioncoding elements to any other class or subclass of CH gene.The DNA recombination mechanisms that mediate each ofthese types of DNA rearrangements are quite distinct andclearly are developmentally controlled. In addition, severalaspects of antibody expression require an RNA splicingmechanism. I will discuss our latest data on the organizationand rearrangement of heavy-chain gene segments and uponRNA splicing with special emphasis on the various combin-atorial mechanisms the vertebrate immune system has em-

ployed to amplify information. Some of the mechanismswhereby DNA rearrangements and somatic mutation leadto antibody diversification will also be discussed.

Structure and Expression of the Growth Hormone Set ofGenes. J.D. Baxter, A. Barta, G. Cathala, S.R. Spindler,N. Eberhardt, G. Bell, S. Mellon-Nussbaum, J. Shine,R. Richards, J. A. Martial, M. Wegnez, andN.E. Cooke.

Howard Hughes Medical Institute Laboratories, Dept. ofMedicine and Biochemistry and Biophysics, and the Meta-bolic Research Unit, University of California, San Fran-cisco, CA 94143.

Growth hormone (GH), prolactin (Prl), and lacental lac-togen (PL) form a set of probable evolution-related genes.Their expression is controlled by several factors includingthyroid and glucocorticoid hormones. cDNAs for thesegenes have been cloned, sequenced, and in some cases usedto synthesize the respective hormones in bacteria. Thesegenes display substantial nucleic acid sequence homology,although the strong preference for G and C in the thirdposition of the codons for GH and PL is not present in Prl.Genomic DNAs to human(h) GH, rat(r) GH, hPL, hPrl,and rPrl have also been analyzed and in some cases com-

pletely sequenced. There are several hGH and hPL genes;these are all located on chromosome 17 (in some cases closelinkage has been established), whereas Prl is on

chromosome 6. Each gene contains five exons and four in-trons located in equivalent positions. The common

ancesteral gene may have evolved by duplication of a

smaller gene, since there are four regions of internalhomology in the coding structure. Two of these are in thesame exon —even in the Prl gene which probably divergedfrom the GH-PL ancesteral gene over 350 million yearsago. One exon does not contain any of these regions of in-ternal homology and may correspond to a different func-tional domain of the protein. One intron of the rGH gene isof particular interest in that the presence of repeating struc-tures and AT-rich regions suggest how rearrangementsthrough evolution may result in the expansion and contrac-tion of introns. A comparison of gene structure and proteinand RNA products suggests that in at least three cases therecan be alternative splicing of the pre-mRNAs to yield multi-ple hormone forms from the same gene. Studies in culturedrat pituitary (GH3, GC, GH«) cells indicate that glucocor-ticoid and thyroid hormones regulate GH gene transcrip-tion. In addition, hormones and serum factors also affectRNA processing, including the relative abundance of pre-mRNA and mature mRNA as well as the relative ratios ofthe different mature mRNAs generated by alternative splic-ing. Thus, a study of these genes reveals interesting featuresof gene structure and evolution, RNA processing, and hor-monal regulation.

Synthesis, Processing, and Secretion of Eukaryotic Pro-teins in the SV40 Monkey Cell System. D. Hamer.Laboratory of Biochemistry, National Cancer InstituteNIH, Bethesda, MD 20205.

We have constructed SV40 recombinants carrying thehepatitis B virus surface antigen gene, the human growthhormone gene, and a variant human growth hormone gene.These molecules have been propagated in cultured monkeykidney cells, the permissive host for SV40, as virus par-ticles. Cells infected with the SV40-hepatitis recombinantsynthesize 3 million molecules/cell/day of hepatitis B sur-

face antigen. The antigen is glycosylated, secreted into themedia, and assembled into 22-nm particles in-distinguishable from those in the serum of chronic hepatitis

76 SPEAKERS' ABSTRACTS

carriers. Similarly, cells infected with the growth hormonerecombinants produce growth hormones that are ap-propriately processed and secreted. The level of expression,40 million molecules/cell/day, is similar for both genes.The variant protein binds to growth hormone antibody,though less efficiently than the normal protein, and also togrowth hormone receptors. These results indicate that theSV40 monkey cell system will be useful for producing andcharacterizing eukaryotic proteins that must be post-translationally modified or assembled to obtain biologicalactivity.

The Control of Expression of Transformed Genes. R. Axel.Institute of Cancer Research, Columbia University, NewYork, NY 10032.

Transformation results in the integration of heterologousDNA into the chromosome of the transformed host. Wehave used hybridization in situ to demonstrate the linkageand insertion of most, if not all, cotransformed sequencesinto the recipient cell chromosome. Insertion is notrestricted to a unique chromosome or chromosomal region,but frequently occurs at a site of gross chromosomal rear-

rangements. Genomic blot hybridization along withhybridizations in situ now permit us to correlate chromo-somal and molecular events associated with insertion andexcision of DNA in the chromosome. Expression of trans-formed genes provides an assay for the functional role ofDNA sequence organization about specific genes. We are

currently employing in vitro mutagenesis in concert withtransformation to define a block of nucleotides essentialfor the expression of the viral tk gene, the hamster aprtgene, and the major Drosphila heat-shock gene integratedin the chromosome of mouse fibroblasts.

High-Efficiency Tranformation by Direct Microinjectionof DNA into Cultured Mammalian Cells. M.R. Capecchi.Dept. of Biology, University of Utah, Salt Lake City, UT84112.

Direct microinjection of DNA by glass micropipettes intonuclei of cultured mammalian cells can be used (1) to studythe immediate expression of a gene, much as one would usethe Xenopus oocyte injection system and (2) to study DNA-mediated transformation of mammalian cells.

When the herpes simplex virus thymidine kinase gene(HSVtk) was delivered into nuclei of LMtk" cells, a mousecell line deficient in thymidine kinese activity, essentially100% of the cells expressed tk enzymatic activity. As a testsystem for studying gene expression in injected cells, theHSVtk transcription of signals were removed and substitutedwith presumed retro-virus promoter sequences. These ex-

periments were done in collaboration with P. Luciw.The number of injected LMtk" cells capable of indefinite

growth in a tk* selective medium (that is, transformants)depends on the nature of the plasmid DNA into which theHSVtk gene was inserted. One cell in 100-200 cells that re-ceived nuclear injections with pBR322/tk DNA gave rise toa viable colony when grown in HAT medium (i.e., a tk*selective medium). The transformation frequency increasedto one in five injected cells when specific SV40 DNA se-

quences were also introduced into the HSVtk plasmid.

Simlarly high transformation frequencies are routinely ob-served when ASV-LTR sequences are introduced intopBR322/tk.

With the microinjection procedure, transformation fre-quency is relatively insensitive to DNA concentration anddoes not depend on the presence of a carrier DNA. Most ofthe transformants were stable in nonselective medium as

soon as they could be tested.When multiple copies of the SV40 or LTR vectors were

injected into LMtk" cells, either as linear or circularmolecules, most of these sequences are found in thetransformants integrated into host sequences as head to tailconcatamers. Transformants containing single copies of thevector sequences can be obtained by injecting only a fewplasmid molecules per cell (less than five).

Yeast Vectors for Functional Analysis of Yeast Genes andChromosomes. R. W. Davis, M. Thomas, K. Struhl, D. T.Stinchcomb, S. Scherer, M. Fasullo, and C. Mann. Dept.of Biochemistry, Stanford University, Palo Alto, CA94305.

Saccharomyces cerevisiae (baker's yeast) can betransformed with DNA using the procedure of Hinnen,Hicks, and Fink. The cell wall is enzymatically removedand DNA is taken up by the cell upon addition of calciumand polyethylene glycol. No carrier DNA is used. Fourgeneral types of yeast vectors have been developed. Theyare double vectors containing sequences that allow selectionand maintenance of DNA sequences in both E. coli andyeast. Selection and maintenance in E. coli is achieved byuse of the E. coli vector pBR322 which contains a Col Elreplicator, and ampicillin- and tetracycline-resistant genes.Selection in yeast is generally achieved by incorporating in-to the vector one or more yeast genes in one of the aminoacid, purine, or pyrimidine biosynthetic pathways: Themajor variable in these vectors is the manner in which theyare maintained in a yeast cell. Yip vectors are maintained inyeast cells by integration into a yeast chromosome byhomologous recombination. The recombination can occurin the selectable marker or in the inserted yeast sequences.YEp vectors contain sections of the yeast plasmid Scplwhich allow autonomous replication in a yeast cell. Wehave transformable yeast strains which are devoid of theendogenous yeast plasmid and upon transformation with a

YEp vector, the yeast contains about 30 copies of the vectorsequences per haploid genome. YRp vectors are maintainedin a yeast cell as an autonomously replicating sequence byincorporating chromosomal DNA sequences called ars

(autonomously replicating sequences). These vectors can

also integrate into a yeast chromosome which is notdeleterious and does not result in rearrangements in theDNA sequence. YCp vectors contain sections of DNAwhich have centromere function. We have isolated a DNAsegment on chromosome IV near the TRP1 gene, whichbehaves, during meiosis and mitosis, as a yeast centromere.These vectors are apparently maintained stably in the yeastcell in a single copy state. Transforrriation of yeast cells nor-

mally results in the addition of genetic information. Arecently developed technique, however, allows the replace-ment of sequences following transformation. This process

SPEAKERS' ABSTRACTS 77

has been termed transplacement. Two vectors have beendeveloped for transplacements, termed TRpl4 and TRpl5.Integration by homologous recombination can bestimulated, thus targeted by cleaving the targeting DNA se-

quence on the vector. These linear molecules are found to

integrate at a much higher frequency by homologous

recombination. The integration results in complete repairof the cleaved DNA sequence. These four vector systemsare now being used to study gene structure, chromosomestructure and the regulation of gene expression. They can

be used to manipulate DNA sequences on an autonomouslyreplicating vector or on a yeast chromosome.