poster session abstracts from the first annual congress on recombinant dna research

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

Poster Session Abstracts from the First Annual Congresson Recombinant DNA Research

Isolation and Expression of Genes Encoding InducibleHepatic Proteins. J.F. Morrow, R.S. Stearman, W. R.Pearson, J.J. Windle, CG. Peltzman, D.A. Potter, A.M.Benson, and P. Talalay. Dept. of Microbiology and Phar-macology, Johns Hopkins University, Baltimore, MD21205.

We have constructed recombinant plasmids encodingtwo inducible proteins synthesized by murine hepatocytes.We have used these recombinants to demonstrate large in-creases in the concentrations of the mRNAs for these pro-teins upon induction.

One of these polypeptides, SAAL (serum amyloid A), isone of the acute phase serum proteins. Their concentra-tions are elevated naturally by infection, inflammation, or

malignant neoplasia, and experimentally by a factor frommacrophages or by bacterial lipopolysaccharide. SAALmRNA concentration is elevated at least 500-fold, probably1000-fold or more. SAAL is 2.5% of the total protein syn-thesized by liver 22 hr after lipopolysaccharide administra-tion. Actin biosynthesis also increases, fivefold, and serum

albumin synthesis and its mRNA are diminished to one-

third of normal. The synthesis of SAAL is tissue-specificsince its mRNA was not found in kidney.

The other recombinant plasmid encodes the majorglutathione transferase of murine liver. Dietary antiox-idants, including BHA, which protect against car-

cinogenesis elevate transferase activity up to tenfold. Theenzyme catalyzes the inactivation of some mutagens. Theconcentration of transferase mRNA is elevated about25-fold by BHA. It becomes the major mRNA of murineliver.

Cloning of Rabies Virus Specific Glycoprotein andNucleocapsid Protein mRNA. A. Anilionis, W. Wunner,and P.J. Curtis. The Wistar Institute, 36th St. at Spruce,Philadelphia, PA 19104.

Double-stranded complementary DNA was synthesizedfrom rabies virus infected cell poly(A) RNA and insertedinto the Pst I site of pBR322. Clones specific for theglycoprotein and nucleocapsid protein mRNA were iden-tified by their ability to hybridize to a specific mRNA whichhad been detected by microinjection into Xenopus laevisoocytes. The glycoprotein inserted sequence contains 1.75kbp and lacks only 35 nucleotides from the 5' terminus ofthe glycoprotein mRNA.

Structure and Function of the Collagen Gene. G. Vogeli,H. Ohkubo, Y. Yamada, M. Sobel, M. Mudryj, I. Pastan,and B. de Crombrugghe. NIH, Bethesda, Md. 20205.

We have isolated the chick a-2 (type 1) collagen gene in aseries of overlapping clones. The gene is 38 kb long. Itscoding information is subdivided into more than 50 exons

which are interrupted by introns of various sizes. Many of

the exons that encode the helical portion of collagen havean identical length of 54 bp, implying that the ancestralgene for collagen arose by amplification of a single geneticunit. As a first step to study the expression and the regula-tion of the a-2 collagen gene in appropriate in vivo and invitro systems, we have examined the structure of its pro-moter. We have determined the location of the transcrip-tion initiation site within this gene by a primer extensionexperiment. A small DNA fragment labeled at its 5' end,containing sequences located in the 5' proximal exon of thegene, was elongated by reverse transcriptase using as

template poly(A) containing RNA isolated from chick em-

bryo calvaría. A single extension product was obtained.The precise location of the 5' end of the a-2 collagen RNAwas determined by SI protection. The result of this experi-ment is in excellent agreement with the size of the extensionproduct. The DNA sequence around the RNA initiationsite was determined. A canonical Goldberg-Hogness se-

quence TATAAATA is found 23 bp and a CAT box 75 bppreceding the start site. This CAT box is located within an

area where several large inverted symmetrical sequences are

found which are overlapping and hence mutually exclusive.These symmetries could play a role in the regulation of thisgene.

Cloning and Characterization of a Virulence Gene from a

Phytopothogenic Pseudomonas. L. Comai and T. Kosuge.Dept. of Plant Pathology, University of California, Davis,CA 95616.

Pseudomonas savastanoi, a pathogen of olive andoleander plants, causes a disease called "knot," character-ized by the development of tumors or galls on infectedplants. Indoleacetic acid (IAA), produced by thebacterium, is a determinant of tumor formation sincemutants lacking in IAA production (Iaa" mutants) are

avirulent. A fragment of the indigenous plasmid pIAAlwas cloned in E. coli and was shown to carry the first gene(iaaM) of the pathway synthesizing IAA from tryptophan.Physical characterization of this gene led to the discoverythat certain spontaneously occurring Iaa" mutants

originated from insertion mutations in the IAA region. Themodalities of expression of the cloned gene in E. coli and P.savastanoi are also taken into account.

Agrobacterium Mini-Ti Plasmid Coded Proteins are Syn-thesized in E. Coli Mini-Cells. M. Hagiya, S.-T.ïLiu, andC.I. Kado. University of California, Davis, CA 95616.

Agrobacterium tumefaciens ID 1422 harbors a Ti plasmidof 28 Mdal. This mini-Ti plasmid confers the usual on-

cogenic and phenotypic properties similar to those of thelarger 120-Mdal Ti plasmid counterparts, namely, on-

cogenicity on a number of plant hosts and opine synthesis.Mini-Ti plasmid DNA fragments, generated by restrictionendonuclease Sal I and Pst I, were cloned in E. coli mini-

79

80 POSTER SESSION ABSTRACTS

cells producing strain P67-54 using pBR325 as the vector.All cloned fragments, representing the entire pTil422plasmid, directed the synthesis of distinct proteins in themini-cells. The plasmid sequence directing the synthesis ofeach of these proteins was located on a physical map of themini-Ti plasmid. Proteins with molecular weight of 45 000,24 500 and 16 000 from Pst I fragment pTL2015 cor-

responded to those reported in the crown gall tumor cells.

Prolactin-Deficient Variants of Rat GH3 Pituitary TumorCells. R.D. Ivarie and J.A. Morris. Dept. of Molecular andPopulation Genetics, University of Georgia, Athens, GA30602.

GH3 cells normally synthesize and secrete the two pitui-tary proteins, prolactin (rPRL) and growth hormone(rGH). From a heavily ethyl methane sulfonate mutagen-ized population, low-producing rPRL variants have beenisolated at a frequency of 10-20%. The rate of intracellularprolactin synthesis in the variant cells was reduced 40- to100-fold compared to wild-type cells, while rGH synthesisvaried less than twofold. This decrease was also paralleledby a decrease in the level of translatable pre-rPRLmessenger RNA. Although rPRL expression was greatlydecreased in the variants, its repressibility by glucocor-ticoids was unimpaired. Furthermore, there was a coor-dinate loss in the expression of another protein, termedp21, whose synthesis is glucocorticoid-repressible andthyroid hormone inducible in GH3 cells. However, the ex-

pression and regulation of other glucocorticoid and thyroidhormone sensitive gene products was unaltered in thevariants. Protein p21 expression was coordinately regainedin revertant cells expressing normal levels of prolactin.

The stability of the low-producing phenotype differed intwo variants. One (B2) gave rise to wild-type revertants at10-20% frequency even after three rounds of serialsubcloning for the low-producing phenotype, while another(B3) was more stable in that only one revertant has beenfound among 47 subclones. By contrast, no low-producingrPRL variant has yet to be found among 57 subclones ofunmutagenized GH3 cell populations.

Repeated Sequences in the Human Growth Hormone andPlacental Lactogen Gene Region. V.J. Kidd and G.F.Sounders. University of Texas System Cancer Center,Houston, TX 77030.

The human polypeptide hormones placental lactogen(hPL) and growth hormone (hGH) provide an interestingmodel to study the tissue-specific expression of two closelyrelated genes and the possible regulatory role of their sur-

rounding DNA sequences. It is known that hGH and hPLare located on band q22-24 of chromosome 17. Morerecently, we have demonstrated that these genes are linkedin the arrangement 5'-hGH-hPL-3' with 15 kb of intragenicDNA separating them.

Subsequent analysis of 38 kb of DNA in and around thisgene cluster has shown an interesting and complex arrange-ment of repetitive sequences. Hybridization with a clonedrepresentative of the Alu family repeat, BLUR 2, has iden-tified at least ten areas of this reiterated sequence in thecluster. Preliminary evidence indicates Alu repeats both 5'

and 3' to the chromosomal gene as well as in the structuralgene. Since the coding regions of these genes contain noAlu family sequence, it follows that the sequences must bepresent in the intervening sequences of both the hGH andhPL genes. In addition, heteroduplex analysis and two-dimensional electrophoresis blot hybridization experimentsindicate a complex arrangement of inverted and tandemrepeats which are unrelated to the Alu family sequence.

Transcription of the DNA Genome of an AutonomousParvovirus. R.C Bates, J.T. Patton, P.R. Burd, andE.R.Stout. Dept. of Biology, Virginia Polytechnic Institute andState University, Blacksburg, VA 24061.

Parvoviruses are small icosahedral viruses that contain a

predominantly single-stranded DNA genome of about 1.5x 106 d (5.5 kb). These viruses are highly dependent on

host-supplied functions for transcription and replication ofthe viral genome, thus providing a useful probe ofeukaryotic gene expression. We have examined thetranscription process of an autonomous parvovirus, bovineparvovirus (BPV). Viral-specific RNA purified from BPV-infected cells was shown by hybridization to be complemen-tary to the virion DNA strand and by poly(U)-Sepharosechromatography to be polyadenylated. To determine thetime of onset of viral mRNA synthesis, RNA isolated fromthe nucleus and cytoplasm of infected cells at differenttimes p.i. was hybridized to BPV virion DNA or to plasmidDNA containing BPV Eco RI A-fragment immobilized on

nitrocellulose filters and also by the Northern RNA blottingtechnique. BPV RNA was initially detected in both nucleusand cytoplasm at 10-12 hr p.i. By 16 hr p.i. five predomi-nant viral RNAs (3.8, 3.4, 3.6, 2.6, 1.8 kb) were detected.All of these RNA species appeared simultaneously between12 and 16 hr p.i. Further analysis of these RNA species byelectrophoresis of Sl-nuclease-treated BPV DNA-RNAhybrids showed an additional RNA (5.2 kb) in the nucleusbut not the cytoplasm of infected cells. The 2.6- and 1.8-kbviral RNAs were found to be associated with polysomes ofinfected cells. To determine if the RNAs were spliced, BPVDNA-RNA hybrids were digested with SI and analyzed on

alkaline gels. The 3.8-, 3.4-, and 2.6-kb viral RNAs were

not spliced, while the 1.8-kb viral "RNA was determined tohave a single splice producing two exons of 1.1 and 0.7 kb.A transcription map of the BPV RNAs is currently beingprepared.

Cloning of the Silent 5 Globin Gene from Old World Mon-keys. S.L. Martin and A.C. Wilson. University of Califor-nia, Berkeley, CA 94720.

A gene-duplication event in the common ancestor of allhigher primates led to the genes encoding the 5 and ßpolypeptides of hemoglobin. With the exception of OldWorld monkeys, 5 and ß globin genes are coexpressed inprimate adults, where 6 globin is minor in amount (1-5%)but appears to be functionally equivalent to ß. In OldWorld monkeys, however, è globin is not found in theblood. By using restriction endonuclease mapping of totalgenomic DNA, we have demonstrated the ô gene is presentin six Old World monkey species: rhesus, baboon, talapoin,African green monkey, colobus, and langur. Furthermore,

POSTER SESSION ABSTRACTS 81

no major alterations are apparent in the DNA structure ofthe region containing the ô and ß globin genes of OldWorld monkeys relative to other primates who do expresstheir ô gene. In order to understand the molecular basis forthe Old World monkeys' lack of ô globin, and to measure

the rate of sequence divergence in this ostensibly silentgene, we have cloned the 5 and ß globin genes from two

distantly related species—

colobus and rhesus. GenomicDNA was digested to completion with Bgl II and theresulting fragments were separated by preparative discon-tinuous electrophoresis. We obtained three fractions,enriched with respect to the 7.7-kb fragment bearing therhesus 6 gene, the 5-kb fragment bearing the rhesus ß gene,and the 13-kb fragment bearing both the ô and ß genes ofthe colobus monkey. Each was ligated into the Bam HI siteof Charon 30 and packaged in vitro. The resulting recombi-nant phage were screened with a nick-translated fragmentcontaining the human 5 gene from a genomic clone.

> Plaques hybridizing to the probe have been purified and are

being characterized and compared to their human counter-parts by restriction analysis and annealing methods prior to

sequencing. Preliminary results indicate that the monkey ôregion has been evolving more slowly than would be ex-

pected for a silent gene.

New Mechanism of the Control of Gene Expression and itsLoss in Carcinogenesis. M. Hillar, Z. Stolzmann, andS. Allen. Dept. of Biology, Texas Southern University,Houston, TX 77004.

Low-molecular-weight peptides (deprimerones) are ex-

tractable from deproteinized polysomal poly(A)-mRNA,DNA, and nuclear RNA at alkaline pH with ethanolic solu-tions. These three groups of peptides are further frac-tionated on Sephadex G-25 column, thin-layer chroma-tography on cellulose plates, and by HPLC on /iBondapakC,8. Isolated peptides control transcription and initiation oftranslation in reconstituted cell-free systems and stabilizedouble-stranded structure of DNA. Peptide levels are de-creased in neoplastic tissue preparations (Novikoffhepatoma, mouse lymphosarcoma, mouse fibrosarcoma)by about 40-70%. Extraction of deprimerones from depro-teinized DNA of normal cells amplifies several times itstemplate activity for transcription. Such an effect is not ob-served in tumor cells. Injected daily to tumor-bearing mice(with fibrosarcoma) they dramatically stop the growth oftumor.

Deprimerones represent natural, endogenous regulatorsof gene expression, loss of which during the carcinogenesisis responsible for cancer expression (deprimerone theory ofcarcinogenesis).

Molecular Cloning of a Human Adenylate Kinase Gene.N.C. Mishra. Dept. of Biology, University of SouthCarolina, Columbia, S.C. 29208. U. Schmeidl, S. Rahr,R.H. Schirmer, and H.P. Vosberg. Max Planck Institutefür Medizinische Forschung, Heidelberg, W. Germany.

Muscle development has been studied as a model systemfor the understanding of the control of gene expression dur-ing differentiation. Several of the muscle proteins havebeen purified to homogeneity and their amino acid sequences

are known. The availability of homogeneous DNA probesspecific to the sequence of mRNA coding for human mus-

cle proteins would be extremely helpful for the analysis ofthe control of gene expression at the molecular level. Herewe report the isolation and characterization of mRNA spe-cific for human adenylate kinase. The mRNA prepared byimmunoprecipitation was used as a template for makingcDNA and finally dsDNA; the latter was ligated into theplasmid DNApBR322. The hybrid plasmid was used totransform E. coli cells.

Transcriptional Efficiency of the ampC /3-Lactamase Geneof E. Coli K12 in a Mutant with a Defective ampC At-tenuator and in Up-Promoter Mutants. T. Grundström,B. Jaurin, T. Edlund, and S. Normark. Dept. ofMicrobiology, University of Umeâ, S-901 87 Umeâ,Sweden.

We have sequenced the eiitire ampC /3-lactamase gene ofE. coli K12, including the regulatory region for transcrip-tion preceding the structural gene. In vivo expression fromthe wild-type ampC gene of the E. coli chromosome is low(about 10"" of total cell protein) due to both a moderatestrength of the promoter and a transcriptional terminatoridentified in the 59-bp long ampC leader. About 93% of in-itiated transcripts terminated at position 41 in vitro. Muta-tions leading to increased transcription of ampC have beenDNA sequenced and characterized by in vitro transcription.They fall into two groups: up-promoter mutations andmutations in the attenuator. One of the mutations is the in-sertion of a G-C basepair between the —10 and —35regions of the /3-lactamase promoter. This mutation in-creased initiation of transcription 15-fold.'

A second mutation is a C-G to A-T transversion occur-

ring within a 9-bp long entirely G-C containing dyad sym-metry that is the terminator stem of the ampC attenuator.The mutation weakened the thermodynamic stability of theterminator stem and abolished termination of transcriptioncompletely. The relative synthesis of the ampC /3-lactamaseincreases with the growth rate in wild-type E. coli K12. Thisgrowth-rate-dependent response was unaffected by the up-promoter mutant presented above, but was absent in theattenuator mutant. We therefore suggest that the ampC at-tenuator mediates the growth-rate-dependent regulation ofampC

Based on the nucleotide sequence of the leader, we

hypothesize that the degree of interaction with ribosomeswhose concentration in the cell is proportional to thegrowth rate, is the regulating entity. Translation in thebeginning of the leader directly protects the first half of thedyad symmetry from interaction to form the terminatorstem of the attenuator. The correlation between sequencechanges and promoter efficiency in up-promoter mutationsof the ampC gene is under study. ¡iv

Regulation in the Str Operon of E. Coli. E. Lifson, Dept. ofMicrobiology, Box 672, J. Zengel, andL. Lindahl. Dept. ofBiology, University of Rochester, Rochester, NY 14642.

The Str operon codes for four proteins: two structuralcomponents of the 30s ribosomal subunit and two auxiliaryproteins involved in the elongation step of protein transía-


tion in E. coli. We have evidence to suggest there exists a

second promoter in the Str operon. We are examining thephysiological significance of this secondary promoter bycloning fragments of the Str operon into the plasmidpKO-1. This plasmid vector system allows the fusing ofpromoters to the gal K gene. We are able to measure pro-moter strength under different growth conditions bymeasuring the expression of the galactokinase gene. We are

also quantifying the RNA(s) from this operon to supportthe promoter studies.

High-Efficiency DNA Transfection and Rapid Detection ofTransfected DNA Expression. G. Milman. Johns HopkinsUniversity, 615 N. Wolfe St., Baltimore, MD 21205.

DEAE dextran mediated DNA transfection in suspen-sion routinely gives gene expression in 0.1-1% of thetransfected cells. Transfection occurs with almost equal ef-ficiency in human diploid skin fibroblasts, monkey BSCcells, mouse L or 3T6 cells, and rat cells. Unlike calciumphosphate mediated transfection which yields stablytransformed cell lines, DEAE dextran mediated transfec-tion appears to produce cells which only transiently expressDNA analogous to the abortive transformation describedfor polyoma and SV40 viruses. However, the high efficien-cy of DEAE dextran mediated transfection allows one toscreen individual cells during the 1-7 days of transient geneexpression. Transfection with a bacterial plasmid contain-ing the genes for herpes thymidine kinase and polyomaT-antigen was detected by an in situ autoradiographic assayfor thymidine kinase and/or an in situ immunofiuorescenceassay for T-antigen. In principle, it should be possible todetect the expression of any transfected gene which can beassayed by these or other in situ assays. This may be par-ticularly useful when there is no selective method to obtainstable transformants with a gene. It should be emphasizedthat one obtains analytical data, and that the transfectedcells once fixed and stained are not available for furtherstudy. Nevertheless, DEAE dextran mediated transfectionand rapid in situ analysis of transient gene expression mayin many cases provide an alternative to calcium phosphatemediated stable transformation.

Dexamethasone Inhibits a-Fetoprotein Gene Expression./. Chiu, P. Commers, and R. Massari. Dept. of Biochem-istry, University of Vermont, College of Medicine, Burling-ton, VT 05405.

a-Fetoprotein (AFP) is synthesized at a high level in theliver during fetal life, with the level in serum decreasingafter birth. There is very little AFP synthesis in the liver ofadult animals. The decrease of AFP synthesis in adult liveris not due to an alteration in translation. The low level ofAFP in the liver of an adult rat results from a lack of AFPmRNA.

We found that AFP serum level is prematurely reducedin newborn rats by the administration of glucocorticoids.The magnitude of the response is so great that within 3-4days the AFP level in serum dropped to 2.5% of the controllevel. Similar changes in liver AFP concentration were alsodetected. We have also examined ornithine decarboxylase

which is known to be induced by glucocorticoids. Our dataconfirmed the results from other laboratories that gluco-corticoid administration enhances ornithine decarboxylaseactivity in liver. These results indicate that glucocorticoidsare selectively inhibiting liver AFP concentration ratherthan causing general inhibition of protein synthesis in thisexperimental system.

Several other known chemical and hormonal inducers ofliver enzyme activities were also studied. None of them af-fected the AFP levels in serum and liver cytosol. To deter-mine if the inhibitory effect of steroids on AFP productionis glucocorticoid specific, we treated newborn rats withseveral different steroids. It is evident from our data thatglucocorticoids have the specific inhibitory effect.

The reduction in AFP levels is not due to a change ofdistribution of AFP molecular variants, an inhibition ofsecretion of synthesized AFP by the liver, or a disruptionof liver polysomes. Glucocorticoids decrease the AFPlevels in hormone-treated rats by suppressing synthesis ofAFP. AFP polysomes from normal rats were as large as

those isolated from the livers of dexamethasone-treatedrats. However, the amount of AFP-producing polysomes inhormone-treated rat liver is only 14% of the controls. Byhybridization assays, it was found that dexamethasone-treated livers contained decreasing amounts of AFP mRNAsequences in liver cytoplasmic RNAs.

The effect of glucocorticoids on AFP mRNA transcrip-tion was studied in hormone-treated isolated newborn ratliver nuclei. 3H-labeled RNA was synthesized in vitro andisolated from nuclei of control and hormone-treated livers.The AFP mRNA sequence in newly synthesized RNA wasdetermined by hybridization to immobilized recombinantDNA containing AFP gene sequences. Administration ofdexamethasone to newborn rats strongly suppressed AFPmRNA transcription. Dexamethasone effects on the rate ofAFP mRNA turnover are also considered.

Abelson Murine Leukemia Virus: Molecular Cloning of In-fectious Integrated Proviral DNA. A. Srinivasan, E.Premkumar Reddy, and S.A. Aaronson. Laboratory ofCellular and Molecular Biology, National Cancer Institute,Bethesda, MD 20205.

The integrated proviral genome of Abelson murineleukemia- virus (A-MuLV) was cloned in Xgt WES. XBbacteriophage following Eco RI digestion and enrichmentof proviral sequences by sequential RPC-5 columnchromatography and agarose gel electrophoresis. Recombi-nant DNA clones containing a 7.8-kbp Eco RI insert were

shown to have the entire integrated Abelson-MuLVgenome with both 5' and 3' ends flanked by mink cellularDNA sequences. This DNA fragment was shown tö inducefocus transformation upon transfection of NIH/3T3 cells.Moreover, focus-forming virus could be rescued fromtransformed nonproducer cells upon superinfection with a

type-C helper virus. A polyprotein of MW 120 000 (pl20)containing MuLV gag gene determinants was invariablydetected by immunoprecipitation analysis of individualtransformants induced by the 7.8-kbp DNA. Molecularlycloned integrated A-MuLV in its infectious form should be


of use in elucidating the mechanisms involved in transfor-mation by this virus.

Expression of the Polyoma Small T-Antigen Gene in E.Coli. A. Horwich, A.H. Koop, and W. Eckhart. Salk In-stitute, San Diego CA 92138.

We have fused a cloned segment of the polyoma virusgenome, including the coding region for the smallT-antigen, to either a 203 base pair or a 95 base pairregulatory s'egment of the E. coli lactose operon. Thehybrid genes, cloned in E. coli, express proteins of 26 000or 24 000 daltons molecular weight, which are immuno-precipitable with polyoma antitumor serum. The structureof the proteins, their resemblance to the small T-antigen,and the level of expression are related to the regulatory seg-ment used.

Expression of Pseudomonas Fluorescens /3-Galactose-De-hydrogenase in E. Coli K12. P. Buckel and E. Zehelein.Boehringer Mannheim GmbH, Postfach 120, D-8132 Tutz-ing, Germany.

In order to investigate the heterologous expression ofPseudomonas genes in E. coli we have clonedPseudomonasfluorescens DNA in a cosmid/£. coli system.Genomic DNA was cleaved by partial digestion with Sau3A and introduced into the Bam HI site of the cosmid pJC75-58. Recombinant molecules were transduced into E. coliafter in vitro packaging. Conditions did not permit detec-tion of /3-galactose dehydrogenase (EC 1.1.1.48) from Ps.fluorescens, so we used a modification of the immuno-screening method described by Broome and Gilbert.Radioactive labeling of the antibodies was replaced by an

enzyme-linked immunosorbent assay (ELISA)./3-Galactose dehydrogenase synthesis was found once per

530 E. coli transductans examined. Expression was lowerthan in the repressed Pseudomonas strain. Subcloning ofthe /3-galactose dehydrogenase gene and linkage to a strongE. coli promotor yielded an E. coli strain that produces 100times more of the enzyme than the induced Ps. fluorescens.

Measurement of Pro-opiomelanocortin mRNA Levels inthe Tissues of Rats and Mice. N.C Birnberg, O. Civelli,M. Comb, and E. Herbert. University of Oregon, Eugene,OR 97403.

We have been studying the tissue distribution andregulation of pro-opiomelanocortin (POMC) gene expres-sion in rodents. Using the 150-bp cDNA probe to mouse

endorphin cloned by Roberts et al., we have developed a

quantitative solution hybridization assay and a Northernblot assay for POMC mRNA. POMC mRNA has beenmeasured in the mouse anterior pituitary tumor cell lineAtT20-D16v, the anterior pituitary, intermediate lobe,hypothalamus, and amygdala. In addition, we demonstrateregulation by the synthetic glucocorticoid dexamethasoneof POMC mRNA levels in the AtT20 cell line.

We have fractionated RNAs isolated from the above-mentioned tissues on agarose gels, transferred them tonitrocellulose sheets and hybridized them with 32P-labeled

cDNA. POMC mRNA behaves as a 1.2-kb RNA whenglyoxal denatured. The intermediate lobe expresses thePOMC gene(s) at 2000 ppm compared with 70 ppm in theanterior lobe and 2 ppm in the hypothalamus. The AtT20cell line, behaving as a pure population of anterior lobe cor-

ticotrophs, expresses POMC at about half the level of theintermediate lobe.

Primary Transcription Unit of the Mouse /3-Major GlobinGene. E. Hofer and J.E. Darnell, Jr. The Rockefeller Uni-versity, New York, NY 10021.

Using labeled RNA produced by in vitro transcription inisolated nuclei from DMSO-induced Friend cells, it wasshown that at least 80% of the transcripts from the /3-majorglobin gene locus originated near (most likely at) the cap siteof the /3-globin mRNA, whereas transcription proceededpast the poly(A) addition site for at least 1000 nucleotides.The existence of continuous transcription beyond thepoly(A) site was demonstrated by (1) equimolar amounts oflabeled nascent RNA bound to cloned DNA fragmentsrepresenting the 5' and 3' portions of the mRNA sequence,the large intervening sequence, as well as regions beyondthe poly(A) site of the mRNA; (2) specific TIoligonucleotides diagnostic of the region immediatelyfollowing the poly(A) site of the nuclear RNA identified inhnRNA labeled in isolated nuclei; (3) size fractionation ofthe labeled nascent chains. These results suggest the necessi-ty for an endonucleolytic cleavage of a primary transcriptfollowed by polyadenylation in the process of creating the3' end of the /3-major globin mRNA.

A comparison of uninduced and DMSO-induced Friendcells further demonstrated that the same transcriptionalunit is functional in uninduced cells, although at a muchreduced level. Addition of DMSO to the cell culture in-creased the transcriptional activity of the /3-major globingene 10- to 20-fold as measured by in vitro transcription inisolated nuclei, therefore accounting for most, if not all, ofthe increase observed in /3-globin mRNA levels.

Isolation and Characterization of Genomic Clones En-coding Chicken Cell Heat-Shock Proteins. G. Aliperti,P.M. Kelley, CD. Clark, and M.J. Schlesinger.Washington University School of Medicine, St. Louis, MO63110.

The pattern of proteins synthesized by chicken embryofibroblasts (CEF) changes dramatically after these cells are

incubated at 45°C for a few hours (heat shock). Threeproteins —P22, P?6, and P,5 —can account for up to 50% ofthe cell's protein synthetic capacity immediately after heatshock. We have isolated recombinant DNA clones thatcontain chromosomal DNA sequences coding for the mostabundant of these proteins, P22. A library of randomchicken DNA fragments inserted into the vector X Charon4A was screened with cDNA made from heat-shocked CEFtotal mRNA which had been hybridized previously to totalmRNA from CEF incubated at 37°C. Four different cloneshave been isolated; two of these contain chromosomalDNA sequences coding for P22, as demonstrated by in vitrotranslation of hybrid-selected mRNA. Restriction endonu-


clease analysis is being performed to identify further DNAfragments containing heat-shock sequences.

Regulation of Leucine Transport Genes in E. Coli. C Landickand D.L. Oxender. University of Michigan, Dept. ofBiological Chemistry, Ann Arbor, MI 48109.

Leucine is transported into E. coli by at least two high-affinity systems (LIV-I and leucine-specific system). Allfour known genes for high-affinity leucine transport (UvK,UvJ, UvH, and UvG) are closely linked and have been clonedon a plasmid vector, pOXl. The level of high-affinityleucine transport (which in turn controls the concentrationof internal leucine) is controlled primarily by the expressionof the genes for the periplasmic LIV- and leucine-specificbinding proteins, UvJ and UvK, respectively. These genesare separated by approximately 1 kb and each is preceded bya 300-500 base pair regulatory region. LivJ and UvK andtheir regulatory regions are the result of a gene duplication,having diverged approximately 20% in amino acid se-

quence. While bearing little similarity to the transcriptionattenuation regions found in biosynthetic opérons, eachbinding protein gene regulatory region appears to producea terminated transcript in an in vitro transcription system.LivJ and UvK are regulated separately but in an interdepen-dent fashion: UvK is expressed maximally when UvJ isinactivated by mutation or deletion. Two promoterlikesequences precede UvK, while only one recognizable pro-moter has been found preceding UvJ. Expression of eachgene is affected by mutations in rho, the gene for transcrip-tion termination, and in UvR, a negatively acting regulatorygene mapping separate from the leucine transport generegion. These effects are additive on one another, sug-gesting independent regulatory mechanisms. We are cur-

rently studying the expression of these genes both in vivoand in vitro as a model for the evolution of regulatorymechanisms and also to determine how the expression ofgenes, whose products are compartmentalized away fromtheir sites of synthesis, is regulated.

Molecular Cloning of a Family of Coordinately RegulatedGenes: The a, ß and y Chains of Fibrinogen. G.R. Crabtreeand J.A. Kant. NIH, Bethesda, MD 20205.

Recently we have found that defibrination of Sprague-Dawley rats with Malayan pit viper venom produces a 15 to38-fold increase in the levels of translatable fibrinogenmRNA in the liver. This probably occurs through afeedback-like mechanism in which a plasmin degradationproduct of fibrinogen, fibrinogen fragment E, stimulatesincreased synthesis of fibrinogen. We have used thisphenomenon to obtain cDNA clones for the a, ß, and 7chains of fibrinogen. A cDNA library of approximately9000 clones was created in pBR322 from total induced ratliver poly(A) RNA by the poly dG-dC tailing method. Apart of this library was screened using plaque hybridizationto 32P-labeled cDNA prepared from induced and nonin-duced mRNA. Clones giving more intense signals with theinduced 32P cDNA were used for hybrid selection ofmRNA. Of eight cloned cDNAs which selected mRNA,two contained inserts coding for the a chain, one for the ß

chain, and one for the 7 chain. Rescreening the cDNAlibrary with these probes has allowed identification ofclones containing sequences for the great majority of theamino acid coding regions of each chain. This general ap-proach should be useful to obtain recombinant plasmidsfor families of coordinately regulated genes.

tRNA Gene from the Syrian Hamster Genome. R.P. Hartand W.R. Folk. Dept. of Biological Chemistry, The Uni-versity of Michigan Medical School, Ann Arbor, MI 48109.

We have cloned from the Syrian hamster genome a

2.1-kb Bam HI fragment that hybridizes specifically to la-beled tRNA. Analysis by Southern hybridization localizesthe tRNA-specific sequences to a 115-bp region of the frag-ment. The DNA sequence of this region reveals a tRNA-like structure followed by two stretches of thymineresidues. The RNA that hybridizes to this clone is currentlybeing purified and sequenced. Studies of the evolutionarydiversity of this gene in various mammals have been in-itiated.

Nucleotide Sequence of a Cloned Structural Gene Codingfor the Polypeptide Hormone Calcitonin. J. W. Jacobs,J.T. Potts, Jr., N.H. Bell, and J.F. Habener. HarvardMedical School, Massachusetts General Hospital, Boston,MA 02114.

Calcitonin (M, = 3500), a calcium-regulating hormone,is synthesized by way of proteolytic cleavages from a pre-cursor glycoprotein (Mr = 17 000). To determine the struc-ture of the precursor we have cloned and sequenced a

cDNA encoding a substantial portion of the precursor.Double-stranded DNA was prepared from poly(A) RNAobtained from a calcitonin-producing rat medullary car-

cinoma of the thyroid (MTC). cDNAs were inserted intoplasmid pBR322 and cloned in E. coli x!776. Clones con-

taining coding sequences for calcitonin were selected byhybridization arrest translations and confirmed by transla-tions of hybrid selected mRNAs. A cloned cDNA of 376 bpwas isolated and sequenced by chemical methods. The nu-

cleotides encoding the amino acid sequence of calcitoninwere located near the C-terminus of the precursor flankedat both ends by nucleotides coding for basic amino acidsand cryptic protein sequences. At the C-terminus the calci-tonin sequence is extended by gly-lys-lys-arg followed by a

cryptic 16 amino acid sequence before a stop codon ap-pears. Amino-terminal to the calcitonin sequence is a lys-arg sequence preceded by a cryptic 25 amino acid sequence.The basic residues are characteristic of prohormone cleav-age sites and the glycine immediately following theC-terminal prolinamide is typical of cleavages .involvingtransamidation. Analyses by agarose gel electrophoresis ofMTC RNA revealed a predominant mRNA coding for cal-citonin of 1000 bases, as well as less prominent mRNAs of800 and 650 bases. These studies indicate that calcitonin issynthesized by way of both amino and carboxy terminalcleavages from a large prohormone. Calcitonin cDNA willbe used to determine the structure of the genome and tostudy the regulation of gene expression.


Association of RNA Precursors with the Chick OviductNuclear Matrix. J.L. Nordstrom, Dept. of Biochemistryand Biophysics, Texas A&M University, College Station,TX 77843. E. Ciejek, M. Tsai, and B. W. O'Malley. Dept.of Cell Biology, Baylor College of Medicine, Houston, TX77030.

Nuclear matrix was prepared by sequential treatment ofoviduct nuclei with Triton-X 100, DNase, and 2-MNaCl.To minimize endogenous ribonuclease activity, publishedprocedures, were modified such that all steps were performedat

—

20°C (except the DNase treatment which was per-formed briefly at 0°Q. Examination of SDS-PAGE pro-tein patterns and electron micrographs confirmed the isola-tion of intact nuclear matrix structures. RNA was isolatedfrom the nuclear matrix preparations and subjected todenaturing gel electrophoresis. Gels were analyzed byethidium bromide staining and by hybridization of Nor-thern blots to cloned DNA probes for ovalbumin,ovomucoid, 5.8S ribosomal, and Ul RNA. All of theprecursors to ovalbumin and ovomucoid mRNAs and the45S and 32S precursors to ribosomal RNA were associatedalmost exclusively with the nuclear matrix fraction. By con-

trast, mature ovalbumin and ovomucoid mRNAs were

distributed approximately equally between matrix and non-

matrix fractions. Finally, mature ribosomal RNAs and allof the small nuclear RNAs (including Ul RNA) were foundalmost exclusively in the nonmatrix fractions.

These results indicate that RNA precursors are integratedinto the structure of the nuclear matrix and are consistentwith the hypothesis that the nuclear matrix is the site ofRNA processing. snRNAs, however, appear to reside inseparable ribonucleoprotein particles. If involved in RNAprocessing, snRNPs may represent a mobile element thatacts on a more rigidly bound RNA precursor-protein com-

plex.

Use of Chloroplast DNA Clone Banks for the Study of theEvolution and Organization of the Chloroplast Genome.J.D. Palmer. Stanford University and Carnegie Inst. ofWashington, Dept. of Plant Biology, 290 Panama St., Stan-ford, CA 94305.

Fragments encompassing the entire chloroplast genomesfrom mung bean, pea, and spinach have been cloned intopBR322. Detailed restriction maps reveal that the 148-kb,circular mung bean genome contains the large (25-kb) in-verted repeat sequence found in all other higher-plantchloroplast genomes while the 121-kb pea genome does notcontain any detectable repeated sequences. Thus deletionof one copy of the highly conserved inverted repeat has oc-curred during legume evolution since the divergence of thepea and mung bean lineages. Solution hybridizations in-dicate extensive base sequence homologies between thesetwo genomes, yet filter hybridizations and comparativerestriction mapping indicate that a large number of rear-

rangement events have occurred that have scrambled theorder of these conserved sequences. One striking example isthe presence in mung bean of a 0.5-kb insertion within theribosomal RNA operon which is absent in pea. On theother hand, mung bean and spinach, which both contain

the inverted repeat structure, have much less sequencehomology than pea and mung bean, yet have a remarkablyconserved order and arrangement of genes. The implicationof these studies is that the presence of the inverted repeatstructure serves in some unknown manner as a stabilizingfactor such that rearrangement events are quite infrequentin those chloroplast genomes which contain the invertedrepeat, and that once the inverted repeat structure is lostrearrangements occur at a much higher frequency.

Patterns of transcription of these chloroplast genomeshave been studied in great detail and evidence has been ob-tained for developmental regulation of gene expression dur-ing light-induced greening.

Cauliflower Mosaic Virus as a Recombinant DNA Vectorfor Higher Plants. R.C Gardner, S.D. Daubert, A.J.Howarth, and R.J. Shepherd. Dept. of Plant Pathology,University of California, Davis, CA 95616.

A small, closed circular DNA molecule (8 kb) constitutesthe genome of cauliflower mosaic virus (CaMV). The viralgenome has been cloned and shown to retain infectivity.The putative protein-encoding regions of the virus genomeare currently being characterized as to their tolerance forvarious genetic manipulations. We are using deletions andinsertions to categorize each region as essential or

nonessential to viral propagation. A method for the in-troduction of 12-bp inserts at specific sites in the clonedDNA has been developed. The procedure entails the inser-tion of a selective resistance marker flanked by syntheticDNA sequences. Excision of the resistance marker resultsin a net insertion of 12 by containing a novel Sal I restric-tion site. Among the 6 coding regions of the genome we

have identified some in which the insertion of 12 bp is lethaland others in which the altered DNA retains biological ac-

tivity. Most of these latter mutations modify symptoms. Alarger insert (e.g., a 67-bp fragment containing a func-tionally active lac operator) is lethal when inserted at thenew Sal I site in some of the viable mutants.

We are studying a similar method for the production of20-bp insertions that results in the introduction of a novelPvu I restriction site at the insertion point. Insertions are

made in CaMV DNA fragments that have been subclonedin M13. These will be introduced into full-length CaMV byrecombination. Excision of the resistance marker leaves a

net insertion of 20 bp in this case. This method will test theeffect of relatively small but out-of-phase insertions.

Deletion mutations have also been examined. A 222-bpdeletion in region 3 is lethal. However, we have characterizeda spontaneously occurring 421-bp deletion in region 2that has no effect on viability. A search for complementa-tion between altered regions is underway.

Multiple Transcripts From Nomadic Sequences ofDrosophila Melanogaster. H.E. Schwartz, T. Lockett, andM. Young. The Rockefeller University, 1230 York Ave.,New York, NY 10021.

Transcripts from 17 repetitive, mobile DNA sequencefamilies of Drosophila melanogaster have been studiedboth in tissue culture and developing flies. All of the


nomadic DNA sequences examined produce multiplemolecular weight classes of RNA in cultured cells. Specialattention was given to one such sequence family, copia,which is complementary to the most abundant poly(A)*RNAs from Drosophila. Copia RNAs are also found in em-

bryo, larvae, and adult flies. The multiple molecular weightclasses of copia RNAs apparent in tissue-culture cells are

present at all developmental stages.Copia RNA was examined qualitatively and quantitative-

ly in another species of fly, Drosophila simulons, whichcontains far fewer copia DNA copies. Both species produceabundant copia RNAs, but of different molecular weights.

Regulation of HeLa RNA Metabolism in Adenovirus-5 In-fected Cells. L.J. Krueger, M. Zeevi, and J.E. Darnell, Jr.,Molecular Cell Biology Dept., The Rockefeller University,New York, NY 10021.

Selective translation of mRNA into protein modulatescellular function in many diverse biological systems. Weshow that adenovirus-5 (ade-5) infection inhibits in vivotranslation of cellular mRNA. Quantification of cellularmRNA sequences in uninfected and infected cells was

determined by filter hybridization. The amount ofuninfected cell poly(A)-containing probe hybridizing to themRNA of uninfected and ade-5 infected cells was similar.This suggests that rapid turnover of the mRNA duringade-5 infection does not account for the in vivo shut-off ofHeLa protein synthesis. Analysis of the 5'- and 3'-temini in-dicates that the cellular RNA is structurally intact. Althoughin vivo HeLa cellular protein synthesis ceases at 16 hr post-infection, purified total cytoplasmic mRNA directed thesynthesis of viral and cellular protein in an in vitroheterologous system. Some of the effects of ade-5 infectionon host-cell protein synthesis are modulated through dif-ferential translation. This translational control may act in-dependently or in concert with the proposed post-transcriptional control.

Recombinant DNA molecules are currently being used toelucidate other cellular events which regulate and define thecell-virus interaction. The abundance of approximately 150ds-cDNA-plasmid molecules containing sequences devisedfrom HeLa cellular mRNA has been characterized andranges from >: 1.0 to 0.01% of the uninfected mRNA. Cur-rently, alterations in RNA metabolism during adenovirusinfection are being studied.

Myosin Heavy Chain —How Big Can a Gene Family Get?R.M. Wydro, H. T. Nguyen, R.M. Gubits, and B. Nadal-Ginard. Dept. of Cell Biology, Albert Einstein College ofMedicine, Bronx, NY 10461.

Myosin heavy chain is one of the major contractile pro-teins comprising approximately 15-25% of the protein inadult muscle tissue. From limited biochemical and im-munological data, there appears to be a specific myosinheavy chain in adult smooth muscle, cardiac muscle(possibly a ventricular and atrial specific myosin heavychain), and skeletal muscle (possibly a slow-, and fast-twitch specific myosin heavy chain). In addition there is atleast one type of embryonic myosin heavy chain and a

cytoplasmic myosin heavy chain (of cytoskeletal function).This multiplicity of different myosin heavy chains suggeststhat they may be encoded by a family of related genes. Thepotential for studying similar genes under differentregulatory control has been of major interest in ourlaboratory.

We recently isolated several complementary DNA(cDNA) clones from rat skeletal muscle, cardiac muscle,and a rat myogenic cell line (L6E9) which hybridize to

myosin heavy chain (MHQ messenger RNA (mRNA).These cDNA clones varied in length from 680 to 2300 bprepresenting 9-33% of the 7100 nucleotides MHC mRNA.One of these cDNA clones, pMHC-25 (isolated from theembryonic myogenic cell line L6E9), cross hybridizes toMHC mRNA isolated from adult and embryonic skeletalmuscle and adult cardiac muscle. In a Southern blot ofEcoRI restricted rat genomic DNA, pMHC-25 hybridizedto at least eight bands ranging in size from 2000 to 23000bp. Some of these bands contain more than one hybridizingfragment. Hybridization of genomic Southern's withrestriction fragments of pMHC-25 shows that most of thesebands represent separate genes. This cDNA clone was

therefore used to screen an EcoRI rat genomic library clonedin X phage Charon 4A. We obtained seven clones fromthis screening with inserts ranging from 11 to 160 kb. Fourof these clones have unique restriction maps. Althoughthese four genomic clones cross hybridize to MHC mRNAisolated from different tissues, by increasing hybridizationstringency and by using EcoRI fragments subcloned fromthe genomic clones, we have been able to assign tissue spe-cificity to each clone. One corresponds to adult cardiacmuscle, one adult skeletal muscle, and two to embryonicskeletal muscle MHC genes. Despite the fact that thegenomic clones have relatively long inserts we haveevidence indicating that most of the DNA in the genomicclones do not contain flanking DNA sequences. Surprising-ly, despite the cross hybridization of these clones to MHCmRNA from various tissue, the absence of flanking DNAsequences and the fact that these clones were isolated with a

simple probe, heteroduplex analysis shows that regions ofhomology between the different clones are restricted tothree to four regions of a few hundred base pairs inter-spersed within regions of nonhomology. This homologycluster is surrounded by two large regions of nonhomology.

Preliminary analysis with some of the other MHC cDNAclones indicates that the genes detected by pMHC-25 are

only a subset of the MHC genes in the genome. These datasuggest that myosin heavy chain is coded for by a verylarge, complex and interrelated multigene family.

Globin Gene Expression During Chicken Development.D.A. Hansen, E.A. Seftor, J.B. McCabe, andA'.J. Tobin. /

Dept. of Biology and Molecular Biology Institute, UCLA,Los Angeles, CA 90024.

In chickens, as in all other vertebrates that have beenstudied, both the a- and /3-globin gene families are

developmentally regulated. The accessibility of the chickembryo makes this an excellent model system for studyingglobin gene switching. In order to analyze gene regulation


at the molecular level, we have prepared recombinant DNAprobes for individual globin mRNAs, as well as for other(nonglobin) mRNAs present in chicken erythroid cells.These probes were constructed by cloning cDNA preparedfrom total cytoplasmic poly(A)* mRNA of erythroid cellsfrom 5-day embryos and anemic hens. We have classifiedthe recombinant DNAs by repeated colony hybridizations.

We have utilized a clone containing the embryo-specifica-like ir-globin mRNA sequence to investigate gene switch-ing within the a family. By RNA blot hybridizations, we

have determined that ir-globin mRNA is present in 5-dayerythroid cells but undetectable in adult erythroid cells atthe level of one mRNA molecule per 20 cells. Nor can we

detect ir mRNA precursor in the hnRNA of adult erythroidcells. The ratio of the rate of appearance of ir-globinmRNA to that of /3-globin mRNA in adult erythroid cells isless than 1:50 that in the 5-day embryo. These data supporta transcriptional, rather than a posttranscriptional, mecha-nism for globin gene switching.

Structural and Functional Analysis of Two Genes Codingfor the Major Androgen-Dependent Secretory Proteins inRat Seminal Vesicle. P.E. Mansson, B.A. Dickson, D.B.Tully, D.B. Carter, and S.E. Harris. Laboratory ofReproductive and Developmental Toxicology, National In-stitute of Environmental Health Sciences, ResearchTriangle Park, N.C. 27709.

A cDNA library representing the abundant class ofseminal vesicle poly (A*) RNA was constructed in pBR-322using the dG-dC tailing technique. Two cDNA clones were

identified by hybrid arrest translation and DNA sequencingto have inserts containing most of the coding informationfor secretory protein IV and secretory protein V. It was

shown by using the cDNA IV insert as a nick-translatedhybridization probe that cDNA IV does not cross react withthe cDNA V clone indicating that mRNA IV and mRNA Vare fairly nonhomologous. Probes to cDNA IV and cDNAV were used to study the kinetics of induction of mRNA IVand mRNA V by testosterone. Using cDNA IV as probe, a

rat gene library was screened for positive genomic DNAfragments. From 11 positive signals from approximately 3million colonies, four have been plaque purified. Two ofthe genomic clones contain the entire gene and flanking se-

quences on a 3.6- or a 3.3-kb Eco RI fragment, respective-ly. By comparing the other flanking Eco RI fragments ofrat DNA in the two clones and comparing Southern blotsof Eco RI digested total rat DNA from CD rats (outbred,contain 3.6- and 3.3-kb radioactive bands) and Fisher rats(inbred, contain only the 3.6-kb band) using cDNA IV as a

probe, it was tentatively concluded that the two genomicclones contain DNA representing two different allelicforms of the SVS IV gene found in the CD rat population.This is presently being confirmed by heteroduplex map-ping. Seven of the original positive signals obtained from thelibrary using cDNA IV as a probe were rescreened with a

cDNA V probe. One signal was positive with the cDNA Vprobe under conditions where cDNA IV and cDNA V donot hybridize to each other. This indicates that the SVS IVnatural gene and the SVS V natural gene are linked with a

maximum distance of 15 kb between them. The 3.6- and

3.3-kb Eco RI bands containing the SVS IV natural geneare presently being subcloned for extensive restriction map-ping and DNA sequencing. The present level of restrictionmapping of the two clones containing the presumptivealíeles of the SVS IV gene indicates that they contain at leasttwo introns.

Evolution of Mammalian Mitochondrial DNA. W. W.Hauswirth and P.J. Laipis. Dept. of Immunology andMedical Microbiology and Biochemistry and MolecularBiology, J. Hillis Miller Health Center, University ofFlorida, Gainesville, FL 32610.

Comparison of nucleotide sequences and restriction en-

zyme digestion patterns of cloned mitochondrial DNAfrom the livers of pedigreed cows shows that variations inthe nucleotide sequence of mitochondrial DNA existamong individuals of the same breed, as well as betweenbovine breeds. Physical mapping studies to locate these dif-ferences, as well as biochemical studies that examine themolecular details of each sequence change, yield informa-tion about the rates and mechanisms of normal mutationalevents in mammals. In addition, work that comparesrestricton enzyme patterns of a variety of other bovidspecies (water buffalo, yak, American bison) yieldsestimates of mutational rates over long periods of time thatare of evolutionary significance.

A careful analysis of maternally related animals suggeststhat some very rapid sequence variations are occurring. Therapid rate and type of variation we have observed in thislineage suggest that it probably arises from the mechanismof mitochondrial inheritance and may indirectly contributeto evolutionary divergence. Such rapid shifts in mitochon-drial DNA populations between maternally related animalsmay provide a mechanism for selecting and testing thefitness of mitochondrial DNA variants. Additionally itsmechanism may be of importance in understanding geneticvariation in eukaryotic DNA sequences in general.

Complete Sequence of Human Fetal Globin Gene Region.S. Shen, J.L. Slightom, and O. Smithies. Laboratory ofGenetics, University of Wisconsin, Madison, WI 53706.

We have sequenced the entire human fetal globin generegion of over 10 kb and find that the two 7 genes are theresult of a tandem duplication of 5 kb of DNA. A sequenceof 110 bases occurs with about 65% homology three timesin the region: at both ends and at the midpoint. This sug-gests that the ancestral (single 7 gene) chromosome had two

sequences of local homology about 5 kb apart, and that theduplication arose as the result of the occurrence of an une-

qual crossing over between these stretches ¿of localhomology.

We have investigated the differences that have arisensince the original duplication. With the exception of a

region of 1550 bases, the duplicated regions now differ inabout 14% of their nucleotides and have 31 length dif-ferences ranging from 1 to 122 base pairs. The exceptionalregion has only 0.5% base differences and no length dif-ferences and appears to be the consequence of a recent geneconversion. As a result of this conversion, part of the DNA


sequence of the Ay gene has been replaced by the corre-

sponding region of the °7 gene from the same chromosome.The nature of the DNA signals determining the ends of theconverted regions is discussed.

In analyzing the distribution of base replacements alongthe duplicated regions (excluding the converted region) we

have discovered a striking new relationship: the number ofreplacements in a given locality is directly related to the ATcontent of the locality. The probability that the observedrelationship is due to chance is less than 0.0012. For exam-

ple, the AT content of localities with 5% or less basesubstitutions averages 52%; localities with 20% or more

substitutions average 67% AT. The relationship suggeststhat the evolutionary variability of different chromosomalregions can be controlled by selection for or against a highAT content. Thus, the third coding region of length 129nucleotides has an AT content of 45% and differs in onlyone base between the two y genes. We suggest that the lowsubstitution rate is largely a consequence of this low ATcontent.

5S and tRNA Gene Transcription in the Silkworm, BombyxMori. D.G. Morton and K.U. Sprague. University ofOregon, Eugene, OR 97403.

We are interested in identifying the DNA sequencesignals involved in the regulation of transcription of 5S andtRNA genes by RNA polymerase III in the silkworm, Bom-byx mori. We have cloned an Eco RI restriction fragmentof Bombyx mori genomic DNA containing both a singletRNAala gene and one 5S RNA gene, and are studying theactivities of these genes in vitro. Structural analysis revealsthat the two genes are about 4600 bp apart and are orientedin opposite directions. The nucleotide sequences of the two

genes and their flanking DNA have been determined, andsites of transcription initiation and termination in vitroidentified. The two genes share a sequence homology of 6bp, 5'AATTTT3', located at 13-15 nt preceding the sites oftranscription initiation. This sequence has also been foundat the same position for another Bombyx tRNAala genewhich does not share other flanking sequences. This con-

served sequence may play a role in the transcriptionalregulation of these genes, since a requirement for 5' flank-ing DNA has been shown for transcription of a BombyxtRNAala gene by Bombyx RNA polymerase III. Bombyx 5Sand tRNAt'3 genes appear to have some differences in theirtranscriptional regulation; while both genes are active inXenopus laevis oocyte transcription extracts, only thetRNA gene is active in homologous Bombyx mori oocyteand silkgland transcription extracts.

Tools for in vitro and in vivo Genetic Manipulation inPhytopathogenic Pseudomonas and Other NonentericGram Negative Bacteria. N.J. Panopoulos, D.E. Sandlin,B.J. Staskawicz, M. Sato, S. Peters, M. Honma, andC Orser. Dept. of Plant Pathology, University of Califor-nia, Berkeley, CA 94720.

Most plasmid cloning vectors in current use are derivedfrom replicons that have a restricted host range and are not

suitable for the establishment of cloning systems innonenteric bacteria. Plasmid RSF1010 has a broad hostrange among enteric and nonenteric species and is suitablefor cloning of Eco RI and Pst I generated DNA fragments.However, it lacks cleavage sites for several other commonlyused restriction endonucleases. We have constructedderivatives that provide cloning and insertional inactivationcapabilities for Hindlll-, Xhol-, BamHl- and Sailgenerated DNA fragments. These derivatives maintain thebroad host range properties of RSF1010 and have beenused to clone DNA fragments from the plant pathogenPseudomonas phaseolicola. They provide useful alter-natives to RP4- and RK2-derived broad host range vectors,with which they are compatible. pAS8Tcsre/?-/::Tn7 is a

derivative of the hybrid plasmid pAS8 (RP4-ColEl) carry-ing Tn7 insertion in a gene necessary for RP4-specificreplication. It has a broad transmission host range con-

ferred by the RP4 transfer system, but narrow replicativehost range because it is ColEl dependent for its replicationand expresses suicidal properties following transfer to non-

enteric bacteria. The plasmid has been used successfully forthe isolation Tn7 insertion mutants in phytopathogenicPseudomonas and is potentially suitable as a dual purposegenetic vehicle, suicidal Tn carrier and integratively sup-pressible sex factor in gram negative bacteria that are withinthe conjugational host range of RP4 and do not supportstable replication/maintenance of ColEl.

Organization of the Structural Genes in MetaphaseChromosomes. M.T. Kuo and R. Schwartz. Dept. of CellBiology, The University of Texas System Cancer Center,M.D. Anderson Hospital and Tumor Institute, and Dept.of Cell Biology, Baylor College of Medicine, Houston, TX77030.

The ovalbumin (ov) gene is not transcribed in chickenMSB-1 cells in culture, whereas the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene is expressed con-

stitutively. The ov genes in both interphase nuclei andmetaphase chromosomes are not destroyed preferentiallyby the digestion of DNase I. However, the GPD genes are

sensitive to the DNase I digestion in both cases. In addition,the DNase I hypersensitive cleavage sites have been foundaround the GPD gene regions in chromosomes as well as innuclei. These results strongly suggest that (1) thechromosome isolation procedure we used provides a highdegree of fidelity in maintaining the native structure ofchromosomes, and (2) the open configuration of activechromatinin the transcribing stage (interphase) is preservedin the nontranscribing (metaphase) stage. Investigations todetermine if there is any specific DNA segment in these two

gene regions attaching them to the chromosome scaffoldthus far have failed to detect a specific DNA sequenceassociation with the scaffold. These investigations were

performed by dehistonization of the isolated chromosomesby various methods, followed by restriction enzyme diges-tion and the Southern blotting-hybridization technique us-

ing nick-translated recombinant DNA clones as probes.These results suggest that care needs to be taken in inter-

POSTER SESSION ABSTRACTS

preting the chromosome scaffold structure observed underelectron microscopy.

Overproduction of Argininosuccinate Synthetase (ASS) inHuman Cells Due to a Regulatory Mechanism. T.-S. Su,W.E. O'Brien, H.-G. Bock, andA.L. Beaudet. Baylor Col-lege of Medicine, Houston, TX 77030.

The human cell line RPMI-2650 (wild-type) was used tostudy metabolite regulation of ASS and to isolatecanavanine resistant (Can1) variants that overproduce theenzyme. Activity of ASS in nmol/min/mg protein was asfollows: wild-type cells grown in arginine medium, 0.14;wild-type cells grown in citrulline medium, 0.86; and Canrcells grown in either medium, 25.0. Immunological studiesindicated similar relative differences in amounts of enzymeprotein. A marked increase in translatable mRNA for ASSwas demonstrated in Canr cells. A sucrose gradient fractionof mRNA from Canr cells was used to synthesize cDNAthat was cloned in pBR322. A near full-length cDNA clonefor ASS was identified by differential filter hybridizationand the identity confirmed by plasmid-selected mRNAtranslation. Both dot and blot hybridizations revealed thatthe relative amounts of hybridizable mRNA correlated wellwith the varied levels of enzyme activity in all cells and con-ditions tested. The size of the mRNA estimated from theblot hybridization is 1670 ± 50 bp. Using cDNA as theprobe, the restriction pattern of genomic DNA from wild-type and Canr cells showed a complex but identical pattern.Using the 3' end and the 5' end of the cDNA as probe, theblot patterns were suggestive of the presence of family oflinked genes. The data indicated that (1) metabolite regula-tion in wild-type cells and overproduction of ASS in Canrcells are mediated by changes in steady-state levels ofmRNA for ASS; (2) a regulatory difference between Canrand wild-type cells allows a major change in mRNA ac-

cumulation without gene amplification; (3) there may be an

ASS gene cluster in the human genome.

Transformation of Chinese Hamster Ovary (CHO) Cells ByDNA. /. Abraham, J. Sivaswami, and M.M. Gottesman.Laboratory of Molecular Biology, National Cancer In-stitute, NIH, Bethesda, MD 20205.

We have been interested in establishing conditions forDNA transformation of Chinese hamster ovary (CHO)cells, since this cell line has proven of considerable value forgenetic analysis. The introduction of DNA into CHO cellsfrom CHO cells carrying dominant mutations and fromother cell lines would allow linkage analysis, cloning ofgenes by plasmid rescue, and studies on the expression offoreign DNA sequences in well-characterized CHO mutant

backgrounds. Unlike mouse L cells and certain other celllines, CHO cells have until recently proved refractory as

recipients in DNA transfer experiments. We have ap-proached this problem by studying the transfer of thecloned herpes simplex virus gene for thymidine kinase intoCHO cells lacking thymidine kinase by selecting for cellsable to grow in HAT medium. Using a modified protocolbased on the DNA-CaPOi precipitate method, we have

89

been able to transform CHO cells with HSV-TK DNA.Transformation to TK* is linear with increasing amounts ofTK DNA up to a maximum of 64 transformants per 106cells per /¿g of DNA. In our hands, this represents a maxi-mum transformation efficency approximately 2% of thatfound with mouse L TK" cells. Unlike the L cell system, calfthymus DNA as carrier is inhibitory above 20 /¿g per 106cells.

We have used a cloned DNA derived from the 3' portionof ASV 21S mRNA to study cotransformation in the CHOsystem. In analogy to the L cell system we find that thisunselected DNA is efficiently cotransformed with TKDNA, and that this cotransformed DNA exists in high copynumber in the transformed cells. These experimentsdemonstrate that the CHO system will be a useful one forgenetic analysis of the regulation, linkage, and expressionof transformed cloned DNAs and may be usable for selec-tion and rescue of whole cell DNA from strains carryingdominant mutations.

Detection and Cloning of Drosophila melanogaster RepeatedSequences using Two-Dimensional Displays of RestrictionSegments. T.E. Gilroy, S.S. Smith, and CA. Thomas, Jr.Dept. of Cellular Biology, Scripps Clinic and ResearchFoundation, La Jolla, CA 92037.

Drosophila nuclear DNA was cleaved with BamHl andfractionated by electrocution. DNA in the resulting frac-tions was cleaved with Eco RI and electrophoresed in adja-cent lanes of long agarose slab gels. The resulting two-dimensional display contained 2500-4000 DNA bands ofdifferent mobility. Six of the very prominant bands, whichprobably arose from repeated sequences, were recoveredfrom preparative two-dimensional gels. These segmentswere cloned into the BamHl-Eco RI (or BamHl) region ofpBR322. Recombinant plasmids from ApTcs colonieswhich rapidly hybridized to nick-translated Drosophilanuclear DNA were characterized by restriction analysis andSouthern blotting experiments using previously describedDrosophila sequences as probes. Plasmids derived fromband I contain an 830-bp BamHl segment of repeatedribosomal DNA. Plasmids from bands V and VI haveBamHl-EcoRl inserts which are, respectively, the 3.7- and1.3-kb segments from the 5.0-kb repeating element of thehistone gene cluster. Plasmid inserts from bands II, III, andIV do not hybridize to several previously characterizedrepeated sequences of Drosophila DNA (e.g. histone,rDNA, and copia). Bands III and IV each contain restric-tion segments cleaved from two or more families ofrepeated sequences. Southern blotting experiments onnuclear DNA are indicating we have isolated interspersedmiddle repetitive sequences. Laboratory strains are beingcompared to determine if the repeats are mobile. Ex-periments on in situ hybridization to polytene chromosomesare in progress.

Expression of Rous Sarcoma Virus and Murine MammaryTumor Virus Nucleotide Sequences in E. Coli. G. W.Notani. Dept. of Oral and Medical Microbiology, Universi-ty of Minnesota, Minneapolis, MN 55455.


I have used /3-galactosidase fusion vector pMC1403,derivative pGNlOl for the study of expression of viral genesin E. coli. The lac 205 initiation complex in pGNlOl is outof phase with the natural coding sequence of/3-galactosidase. Random fragments from DNase I digestsof cloned RSV and MuMTV genomes were cloned inpGNlOl between the lac205 and lacZ genes (1) to screen

and characterize the viral sequences that provide initiationcomplexes for the expression of viral//3-galactosidase genesand (2) to obtain the expression of inserted viral coding se-

quences using the /ac205 initiation sequence and the lacZindicator gene product. Clone pGNl 17 was found to have a

strong promoter for the expression of the Sre gene. Cor-relation of nucleotide sequences with promoter activity is inprogress. A detailed map of the MuMTV clone PJM7 hasbeen constructed and a complete library of the MuMTVgenome has been cloned in pGNlOl for determining thenucleotide sequence of the viral genome and for thepreparation of viral antigens.

Construction of a Full-Length Double-Stranded cDNAClone of Potato Spindle Tuber Viroid. D.E. Cress andR.A. Owens. U.S. Dept. of Agriculture, Beltsville, MD20705.

A collection of recombinant plasmids containing DNAinserts complimentary to potato spindle tuber viroid(PSTV) RNA was constructed by synthesis of double-stranded cDNA from a polyadenylylated PSTV templateand inserted into the Pst I site of plasmid pBR322 by theoligo (dC)-oligo (dG) tailing procedure. Restriction sitemapping of the largest insert, pDC29 (460 bp), suggestedthat most of the PSTV sequence had been cloned but that a

portion contained an altered restriction pattern. The DNAsequence of this insert was determined, revealing that 94%of the PSTV template had been faithfully copied andcloned and that a portion of this sequence had undergoneduplication and rearrangement, probably during cDNAsynthesis. To construct a complete insert of the PSTVgenome, the largest y4vi7ll-.fifaelll subfragment frompDC29 was ligated at the Avail site to the contiguousy4vcrll-//aelll subfragment from another cDNA clone.Hindlll oligonucleotide linkers were ligated to the Haelllblunt ends of this linear ligation product, and this insertwas cloned into the Hindlll site of pBR322. Restriction siteanalysis of these clones confirmed that full-length cDNAclones of PSTV had been constructed. Digestion of theseDNAs with Haelll releases a 359-bp fragment containingonly PSTV-specific sequences. These recombinant clonesare providing valuable hybridization probes for studyingthe mechanisms of viroid replication and pathogenesis ininfected plant tissue.

NiZ-Transfer into Yeast: A. Stable Chromosomal Integra-tion. A. Zamir, C.V. Maina, G.R. Fink, and A.A. Szalay.B. Extrachromosomal Transfer. D. Morris, J. Noti,F. Osborne, and A.A. Szalay. Boyce Thompson Institutefor Plant Research, Cornell University, Ithaca, NY 14853.

The availability of an amplifiable recombinant plasmidcarrying the entire nif region of K. pneumoniae and thedevelopment of a yeast transformation system make it now

possible to attempt the introduction of «//"genes into yeast.This may expand the number of potential nitrogen-fixingorganisms. These experiments may also serve as model forthe transfer, stability, and expression of a complexmultigenic cluster from bacteria in eukaryotic cells.

A bacterial hybrid plasmid, pWK220, containing the en-tire nitrogen-fixation gene cluster (consisting of at least 15genes) from K. pneumoniae has been used in conjunctionwith an E. coli yeast shuttle plasmid containing the yeastHis 4 gene cluster to cotransform a His 4 minus recipientstrain of Saccharomyces cerevisiae. Of 87 histidine in-dependent clones screened, two were found to contain nifDNA. Restriction and hybridization analyses showed thattwo copies of the nif plasmid (46 kb each) are integrated intandem in the recipient chromosome by recombination be-tween homologous regions in the two transformingplasmids. Chromosomal integration has also been verifiedby tetrad analysis showing that the nif DNA behaved inmeiosis like a true mendelian element. During mitoticgrowth one of the two copies of the «//"region is frequentlylost. The remaining copy of nif is stable even after 40generations in nonselective medium. The total DNAisolated from the stable yeast transformants retains thenitrogen fixing ability when transformed back into E. coliC. This stability makes it now possible to undertake studiesof the expression and regulation of the «//genetic cluster ineukaryotic cells.

The analysis of the transcriptional and translational pro-ducts of the nif opérons in S. cerevisiae may be facilitatedby introducing multiple copies of these genes. We trans-ferred the nif genes from the chromosome of K.pneumoniae to S. cerevisiae on amplifiable plasmidsspecially constructed to be used in in vitro packaging andyeast transformation. These hybrid vectors contain pBR322sequences for replication and selection in E. coli, yeast2-jtm sequences and selectable yeast markers necessary fordivision in yeast, and the cohesive ends of bacteriophage Xwhich allow packaging of recombinant molecules into Xphage heads. The following plasmids have been constructedand are available on request:

Vector

pBTI-1pBTI-5pBTI-8pBTI-9pBTI-10

Markers

Apr Tcr Leu2 XcosApr Leu2 XcosTcr Leu2 XcosTcr Leu2 XcosTcr Trpl Xcos

Size (kb)11.411.312.610.67.6

Cloning Sites

BamHlBamHl Hindlll'BamHlBamHl Hindlll XbalBamHl, Hindlll

Large fragments (about 30-40 kb) of K. pneumoniaeDNA, generated by partial digestion with restriction en-donuclease 5cw3A, were ligated into the BamHl site ofplasmid pBTI 1. The hybrid molecules were introduced intoE. coli by transduction and colonies screened for nif se-


quences by DNA: DNA hybridization. Plasmids containingthe entire «//cluster were used to transform yeast cells.

Studies on the expression of structural genes (K, D, andH) of the nif cluster in S. cerevisiae are now in progress.

Progesterone Induction and Estrogen Repression of theRabbit Uteroglobin Gene. T. Chandra, R. Snead, L.E.Day, D.W. Bullock, and S.L.C. Woo. Howard HughesMedical Institute, Dept. of Cell Biology, Baylor College ofMedicine, Houston, TX 77030.

Uteroglobin is a pregnancy-specific protein in the rabbituterus. The protein accumulates in the uterine lumen andpeaks at 4-5 days of pregnancy. Subsequently the leveldeclines to the basal pre-pregnant level at day 10. An ap-propriate environment, of which uteroglobin is a majorcomponent, in the uterine lumen is imperative for implan-tation of the developing blastocyst which occurs at day 7.Physiological and pharmacological studies have indicatedthat the synthesis of the protein is induced by progesterone.More interestingly, the depletion of the protein in theuterus after 6 days of pregnancy is caused by estrogenrepression which overides the progesterone induction ef-fect. In order to investigate the dual mode of hormonalregulation of mammalian gene expression, we have con-structed a full-length cDNA clone from a partially purifieduteroglobin mRNA and determined its nucleotide se-

quence. The primary sequence analysis of the entire rabbituteroglobin structural gene shows that it is 465 bp in lengthand contains 47 bp of 5'-noncoding sequence, 270 bp ofpeptide coding region following the initiation codon ATGand preceding the termination codon TAG, followed by142 bp of 3'-untranslated sequence. In addition, a polymor-phism within the uteroglobin structural gene has been ob-served between the two strains of rabbits used by us andothers, which involved a G-A transition in the nucleotidesequence resulting in the substitution of an aspartic acidresidue by asparagine at amino acid number 46. Analysis ofsteady-state RNA levels in the uterus at various days ofpregnancy has shown that the induction and repression ofuteroglobin synthesis is the result of accumulation anddepletion of its mRNA, respectively. The chromosomalgene is 2.8 kb in length to code for a mature mRNA of 465nucleotides and contains three intervening sequences,which agrees with the size of the largest precursor RNAdetected by Northern hybridization. The transcription do-main of the gene appears to be coincidental with the 5' and3' termini of the chromosomal gene as revealed by directDNA sequencing.

Enhanced Transformation of Human Fibroblasts by Ori-gin-Defective SV40. M.B. Small, H.L. Ozer, Dept. ofBiological Sciences, Hunter College, New York, NY 10021and Y. Gluzman. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY 11724.

Transformation of normal human diploid fibroblasts bySV40 was examined in a quantitative fashion, with par-ticular attention to an origin-defective SV40 derivative.This molecule was isolated by introduction of a deletion invitro at the Bgll restriction site (map position 0.67) ofwild-type SV40 cloned into a modified E. coli plasmid vec-

tor. The transformation efficiency of origin-defective SV40was compared to that of form I and III wild-type virionDNA, a fragment of wild-type SV40 deleted in the lateregion, and form I and III cloned wild-type SV40 (with andwithout adjoining plasmid sequences). Cells were

transfected by the calcium phosphate precipitation tech-nique. Transformation was assayed both by focus formationand growth in agarose. The following conclusions appearwarranted based on the data to be presented. (1) Origin-defective SV40 is superior in efficiency of transformation toall other SV40 derivatives tested, this enhancement beingindependent of adjoining plasmid sequences. (2) Linearizedmolecules appear to be more efficient than circularmolecules, and plasmid sequences at both ends of the formIII SV40 increase this effect. (3) T-Ag expression and actincable organization are consistant with typical SV40transformants. (4) Preliminary studies varying DNA con-

centration used for transfection suggest that transforma-tion frequencies of greater than 1 in 10 000 can be obtainedin early passage human fibroblasts with this origin-defective SV40 recombinant DNA. Transformants ofhuman fibroblasts generated by this vector may prove par-ticularly useful in the establishment of human cell lines forvarious purposes.

Comparison of Promoters for the T3 and T7 RNAPolymerases. J.N. Bailey and W.T. McAllister. RutgersMedical School, Piscataway, NJ 08854.

Bacteriophages T3 and T7 both encode RNApolymerases that are responsible for transcription of thelate region of the respective phage genomes. Because theseRNA polymerases are structurally among the simplestknown (they consist of a single protein of approximately110 000 daltons), the manner in which they interact withpromoters is of considerable interest. Despite extensivesimilarities between the two phages and their RNApolymerases, neither polymerase will recognize promotersfound in the DNA of the other phage.

To characterize the basis for this template specificity, we

have undertaken a comparative analysis of T3 and T7 pro-moters. We have cloned several T3 promoter containingfragments into pBR322. DNA sequences of representativeT3 promoters have been determined. A comparison ofthese sequences with T7 prompter sequences demonstratesinteresting differences.

Recombinant DNA Stability in the Bacillus subtilisChromosome. S.J. Phillips, G.A. Wilson, andF.E. Young.Dept. of Microbiology, University of Rochester School ofMedicine, Rochester, NY 14642.

We have determined the stability of chromosomal and in-tegrated recombinant thymidylate synthetase genes inBacillus subtilis. We have used resistance to trimethoprimas a selection method to determine the stability of the Thy*phenotype. Bacillus subtilis contains two thymidylate syn-thetase genes, thyA and thyB. These genes can bedistinguished phenotypically by their temperature sensitivi-ty. Mutations in both genes are, required to produce a

thymine requiring cell. Under nonselective conditions, cellsthat are singly ThyA* or ThyB* become Thy" at frequencies


of 6.6 x 10"6 and 2.1 x 10"s, respectively. These frequenciesare statistically indistinguishable using Student's t-test.

'

A set of recombinant plasmids, carrying thymidylate syn-thetase genes from E. coli or B. subtilis phage /322, was con-structed. All of these plasmids transform Thy" B. subtilis toThy* by integrating into the host chromosome. This in-tegration is facilitated by a plasmid-borne homology seg-ment. Integrated plasmid stability varied from a low of8.5 xlO"6 to a high of 1.3 xlO"2. The stability of an in-tegrated plasmid was found to be inversely proportional tothe size of the plasmid-borne homology segment.

In Vitro Transcription of the Cloned Chick a(2)-CoIlagenGene. G. Merlino, G. Vogeli, T. Yamamoto, H. Ohkubo,B. deCrombrugghe, and I. Pastan. NIH, Bethesda, MD20205.

Rous sarcoma virus-transformation of chick embryofibroblasts greatly depresses the synthesis of a(2)-collagenand its corresponding mRNA, suggesting that the activityof the a(2)-collagen gene in RSV-transformed cells ismediated by transcriptional control. To examine this ques-tion, we have isolated the collagen gene and used two 5'fragments as templates for transcription. We have detectedone specific, a-amanitin sensitive transcript from a 3.5 kbgenomic fragment located at the 5' end of the collagen gene.The RNA start site was localized and the orientation of theRNA polymerase Il-dependent transcript was established ina 0.38 kbp subfragment of the same DNA. The startsite of the in vitro made RNA corresponds to the initiationsite of the a(2)-collagen RNA made in vivo. The promoteractivity of this DNA is at least as strong as the RSV pro-moter. A canonical Goldberg-Hogness box (TATAAATA)is found approximately 20 bp upstream from the point ofRNA initiation. We are presently analyzing the sequencesof the in vitro RNA products.

Transcription, Processing, Turnover, and Translation ofCopia RNA. S. Falkenthal, E. Korn, M. Graham, andJ.A.Lengyel. University of California, Los Angeles, CA 90024.

We have investigated the processing of newly transcribedcopia RNA by examining the kinetics of incorporation ofradioactivity into copia-specific sequences in the nucleusand cytoplasm of Drosophila cultured cells. Radioactivityin copia RNA was detected by hybridization to the recom-

binant plasmid cDmll42 which contains the copia se-

quence. The data were analyzed using a compartmentalmodel, which takes into account changing pool specific ac-

tivities (monitored by analysis of the rate of labeling oftRNA) and flows of radioactivity through several process-ing compartments. Rate constants were obtained bysimultaneous computer fit of the data to a system of dif-ferential equations. This method, which has not previouslybeen applied to the processing of specific RNA sequences,allowed various models for copia nuclear RNA processingto be tested. Of the two models which fit the kinetic dataequally well, only one was also consistent with the sizeanalysis of copia nuclear RNA during pulse labeling. Fromthe computer analysis of the data, we conclude that the rateof synthesis of copia RNA is 90 5-kb molecules/cell/min,the equivalent of the transcription of 0.2 molecules/min

from each genomic copia sequence. Most (39-84%) of thecopia nuclear RNA transcripts are adenylated, but only a

small fraction (2-9%) of the adenylated transcripts are ex-

ported to the cytoplasm as mRNA. This analysis of a

specific mRNA shows that intranuclear processing, par-ticularly the step of export of a mature mRNA, can play a

significant role in the control of gene expression.The half-life of copia RNA in the cytoplasm was deter-

mined by pulse-chase experiments to be 9.5 hr; this is 1.6times longer than the half-life of the intermediate decayclass of total poly(A)* cytoplasmic RNA.

The messenger properties of copia cytoplasmic RNAwere investigated by translation of purified copia RNA inan mRNA dependent rabbit reticulocyte lysate. Threepolypeptides of 51 000, 33 000, and 21 000 daltons are

seen.

Blotting of Double-Stranded DNA: Application in RapidGenome Fingerprinting. C. W. Chen, S.S. Smith, and CA.Thomas, Jr. Dept. Cellular Biology, Scripps Clinic andResearch Foundation, La Jolla, CA 92037.

We have developed a versatile technique for transferringdouble-stranded DNA from agarose gel to glass fiber filter.With this technique, the gel was placed on a sheet of What-man GF/A paper, to which a solution of 7-MNaC104 was

fed continuously. The gel was dissolved by the GO; whilethe released DNA was retained on the filter. The retainedDNA can be irreversibly fixed on the filter by uv illumina-tion or exhaustive drying, or extracted with a low-salt buf-fer. The recoverd DNA is suitable for further restrictiondigestion or end-labeling with reverse transcriptase.

We have applied this blotting technique in two-dimensional fingerprinting of genomic DNA: GenomicDNA cleaved with a restriction enzyme was fractionated on

agarose gel, recovered, redigested with a second enzyme,and separated on a gel slab. This resulted in a two-dimensional display of the genome. This method can beapplied to genomes as complex as those of bacteria. Theprocedure is rapid and does not require elaborate equip-ment. Success in the transfer of double-stranded DNA of-fers other possibilities in DNA technology.

Differential Transformation of Haemophilus Influenzae byPlasmid RSF0885 with and without Inserts of Chromoso-mal DNA. N.K. Notani, J.K. Setlow, and D. McCarthy.Brookhaven National Laboratory, Associated Universities,Inc., Upton, NY 11973.

It is known that competent cells of H. influenzae effi-ciently take up only DNA that contains a specific 11-basepair sequence, about 600 copies of which are distributedthrough the Haemophilus genome. The plasmid R.SF0885(MW 3.6 x 106) carrying an ampicillin resistance marker(amp) is taken up poorly by H. influenzae and transformswith a low frequency (up to 0.01%), which could indicatethat the required uptake sequence is absent or altered. Thekinetics of DNA uptake, as measured from transformationby the plasmid, are much slower compared to those bychromosomal DNA, and Haemophilus chromosomal DNAreadily eliminates plasmid transformation by competition.Five plasmid DNA isolates carrying inserts of different


lengths of chromosomal DNA produced in vitro are takenup well by the cells and transform for amp with an efficien-cy at least 100-fold higher than with the plasmid DNAalone. Up to 4% amp transformation was observed withpD7, which contains a chromosomal DNA insert aboutthree times the size of RSF0885 DNA, and l-/*g of plasmidpD7 DNA yields around 2.7 x 107 amp transformants. Thefate of 3H-labeled pD7 used as donor DNA was examinedin competent wild-type cells under conditions of little DNAsynthesis. Following irreversible binding, the intracellularpD7 DNA \vas found to possess substantial transformingactivity in lysates of samples taken up to 60 min. This is incontrast to the intracellular phage HP Ici transfectingDNA, which fragments from very early times following up-take. While transformation of the plasmid without insertsis approximately the same in Rec" strains as in the wild type,pD7 transformation in the highly recombination-deficientreel cells is about two orders of magnitude lower than inthe wild type, indicating that transformation by plasmidscontaining inserts requires the reel gene. However, there isstill no observable fragmentation of the intracellularplasmid DNA.

Expression of the Moloney Murine Sarcoma VirusTransforming Gene. /. Papkoff T. Hunter, and I. Verma.TVL, Salk Institute, P.O. Box 85800, San Diego, CA92138.

In vitro translation of Moloney murine sarcoma virus(Mo-MSV) virion RNA yields a 62K product from the gaggene for viral structural proteins as well as four proteins ofapproximately 37K, 33K, 24K, and 18K. These proteins,which form an overlapping set, can be shown by severalcriteria to be unrelated to viral structural proteins. Hybridarrest translation experiments with cloned recombinantDNA containing sequences from the Mo-MSV transform-ing gene (sre) show that the 37K, 33K, 24K, and 18K pro-teins are synthesized in vitro from the sre gene of Mo-MSVvirion RNA. The specific portion of the Mo-MSV RNAwhich codes for these proteins has been localized by hybridarrest translation using cloned DNA's representing variousregions of the Mo-MSV sre gene. Protein chemistry showsthat the amino acid sequence of these in vitro products isconsistent with the single open translational reading framepredicted from the DNA sequence of the Mo-MSV sre

gene. We are using a similar approach to identify the Mo-MSV sre gene products in virus transformed and MSVDNA transfected cells. In these experiments, MSV sre-derived messenger RNA's, identified by northern analysis,will be purified for in vitro translation.

Events in the Evolution of Pre-Proinsulin. R.J. Douthartand F. Norris. The Lilly Research Laboratories, In-dianapolis, IN 46285.

An analysis of the existing DNA sequences of variouspre-proinsulin genes has been carried out using a VAX11/780 computer. Nine base tandem repeating sequenceswere found in the signal peptide regions of the human, ratII, Syrian hamster, and chicken genes. The human and ratII repeating units are identical. The hamster unit differs byan evolutionary distance (ED) of 1, and the chicken by an

ED of 3 from this sequence. Statistical analysis of randomsequences having the same base composition as these signalpeptide regions establishes that the probability of this ar-

rangement ocurring by chance is very small. We proposethat a small repeating unit reflects the structure of theancestral signal peptide region of insulin.

The signal peptide repeating units were compared to theappropriate pre-proinsulin coding sequences and ED was

plotted versus base position. Significance levels wereestablished using random chains of appropriate base com-

position. The periodicity of the repeating unit appears not

only in the signal peptide region but also in the connectingpeptide region immediately following the known positionof the large intron. This analysis establishes that there isstatistically significant evolutionary relationship betweenthe signal peptide region and the connecting peptide basesequence following the large intron.

New Methods for Liposome-Mediated Delivery and Ex-pression of Nucleic Acids in a Variety of Eukaryotic CellTypes. R. Straubinger, R. Fraley and D. Papahadjopoulos.University of California at San Francisco, San Francisco,CA 94143.

Recent advances in liposome methodology permit the en-

capsulation of large macromolecules, such as nucleic acids,within phospholipid vesicles. Liposome-encapsulated RNAand DNA molecules retain full biological activity and, im-portantly, incubation of the liposome-encapsulated nucleicacids with a variety of cell types has been shown to result intheir intracellular delivery and expression.

Liposome-mediated delivery of SV40 DNA to CV-1monkey cells has been used as a model system for studyingand optimizing liposomal delivery. The efficiency ofdelivery is dependent on incubation conditions, vesicle lipidcomposition, and the number of RNA or DNA moleculesentrapped per vesicle. Under optimal conditions the effi-ciency of liposome-mediated SV40 DNA infectivity (2 X106 pfu//ig DNA) is comparable to that obtained with othertransfection techniques. By fluorescence microscopy, ap-proximately 35% of the cells are found to be T-antigenpositive. Similarly, the incubation of liposome-encapsulated HSV TK DNA with TK" mouse L cells resultsin transformation frequencies comparable to that obtainedwith the carrier-free calcium phosphate procedure.

Liposome-mediated delivery of nucleic acids is notlimited to mammalian cells; the entrapment of plant viralRNA's in vesicles results in higher efficiencies (10-100-fold)of infection of plant cells than other commonly usedmethods. Purified Ti DNA, isolated from A. tumefaciensand encapsulated in liposomes, has been delivered to plantprotoplasts. Transformation is observed at a frequency of10"3, an efficiency approximately 1000-fold higher than freeDNA.

In addition to the many applications of liposome-mediated nucleic acid delivery in vitro, the development ofmethods for targeting liposomes to specific cell types maymake possible the eventual engineering of gene delivery invivo.

Structure of the Human Major Histocompatibility Locus.P.A. Biro, D. Pereira; A. Sood, and S.M. Weissman.


Dept. of Human Genetics, Yale University School ofMedicine, New Haven, CT 06510.

A cDNA clone for the human histocompatibility antigenHLA-B7 was isolated from a cDNA plasmid libraryprepared from RNA from an HLA overproducing humancell line. The library was screened with a synthetic30-nucleotide long DNA probe presumed to be complemen-tary to a portion of the HLA message. The HLA insert was

subsequently excised from the plasmid and used to reprobethis and other cDNA libraries. Further HLA clones havebeen isolated which are different from the original B7clone.

Southern blotting of the clone against human DNAcleaved by a variety of restriction enzymes reveals severaldifferent bands of various intensities which are complemen-tary to the HLA message. About 18 different bands are

seen, some of which may be doublets and this is more thanthe minimum number necessary to account for the HLAgenes (about 4-6). Considerable restriction enzymepolymorphism is observed between the DNA of differentindividuals and the polymorphism occurs between bothstrongly and weakly hybridizing bands. Studies usingprobes specific for the 5' and 3' ends of the message are

under way to demonstrate the presence of intervening se-

quences and subsequently to estimate the total number ofHLA genes in the complex. Further experiments are beingperformed to determine the chromosomal assignment ofeach band to determine which bands cross hybridize withHLA but are not part of the MHC complex.

Ten genomic clones have been isolated from a screeningof 400 000 plaques of a partial EcoRI library of humanDNA in X phage. In contrast, only one /3-like globin clonewas found during the same screening, suggesting the HLAgenes are present at a much higher frequency than theunique globin genes. Each HLA clone was found to be dif-ferent by restriction enzyme analysis and in some casesmore than one HLA gene may be present in the same clone.

Although some clones contain identically sized bandsafter EcoRI digestion, recutting with a second enzymeshowed these bands were not identical and this has madeidentification of possible overlapping clones difficult.However, it suggests there may be a higher order repeatingstructure within the HLA complex. This is supported by theobservation that several clones show small, apparentlyidentical, bands when digested by Xba and by Xba andEcoRI together, even though their Eco RI cleavage patternsare very different. Such small fragments may be part of a

family of sequences which are repeated throughout theHLA complex and possibly elsewhere in the genome andmay be involved in the function of the HLA locus.

Endogenous Viruslike (30S) Gene Organization and Ex-pression in Chemically Transformed Mouse Cells. M.G.Courtney, L.J. Schmidt, and M.J. Getz. Dept. CellBiology, Mayo Clinic/Foundation, Rochester, MN 55905.

Endogenous mouse viruslike (VL) RNA molecules ex-hibit properties that resemble those of replication-defectiveRNA tumor viruses and it has been suggested that they maybe proto-retroviruses. Earlier studies indicated that VLRNA is elevated in various spontaneously transformed cell

lines and that there is a correlation between the level of VLgene expression in various clones of mouse NIH 3T3 cellsand their ability to grow in soft agar.

We have isolated a clone (BVL-1) from a mouse Charon4A gene library which carries one of 20-50 representativesof the VL gene family. Using fragments derived from thisclone as probes we have studied the genomic organizationand expression of VL sequences in two mouse embryo celllines, AKR-2B and C3H/10T'/2, and in two chemicallytransformed derivative cell lines, AKR-MCA andC3H/MCA-58, which were obtained by treatment with thecarcinogen 3-methyl cholanthrene. Southern blots ofEco RI-cut DNA showed no detectable difference in theorganization of VL genes between either cell line and itstransformed derivative, but these and further studies dosuggest a strain specific pattern of VL gene organization.Comparison of the level of VL gene expression in these celllines demonstrates that in both cases carcinogen-inducedtransformation is accompanied by a several fold increase inthe frequency of VL sequences in polysomal poly(A)* RNA.Furthermore, maintenance of nontransformed AKR-2Bcells in the presence of excess epidermal growth factor(EGF) which induces the cells to phenotypically mimic thegrowth characteristics of transformed cells results in a

relatively large enhancement (200-fold) of VL gene expres-sion. It seems, therefore, that VL gene expression may beinvolved in the cells' proliferative response and thatelevated expression in transformed cells may be associatedwith continual mitogenic stimulation provided by the"transforming" growth factors released from these cells.

A 7-kb region of DNA adjacent to the VL gene in cloneBVL-1 contains highly repetitive sequences which are wide-ly dispersed throughout mouse DNA. Sequences homol-ogous to these repetitive elements are expressed in all thecell lines studied and are present at higher concentrations innuclear versus cytoplasmic poly(A)* RNA. These propertiesare reminiscent of the Bl sequence family. This closeassociation of VL genes with repetitive DNA, if shown tobe a general phenomenon, may enhance the mobility ofVL sequences which may be an important feature of theirputative role as protoviruses.

Identification of A cDNA Clone for Bombyx Mori HighCysteine (He) Proteins, and its Use to Study theDevelopmental^ Regulated Accumulation of He RNAs.S.C Bock, D.C. Tiemeier, and M.R. Goldsmith. Universi-ty of California, Irvine, CA 92717.

The chorion (eggshell) of the commercial silkmoth Bom-byx mori is composed of some 70 proteins which are syn-thesized over a 4-day period according to a well-characterized developmental program. A subgroup "ofchorion components, the high cysteine (He) proteins,dominates the synthetic profile in follicles pulse-labeledwith radioactive aminoacids during the very late stages ofchoriogenesis. Furthermore, the translational expression ofhigh cysteine proteins Hc6-Hcll appears to be regulatedcoordinately.

A cDNA clone containing sequences complementary tothose of mRNAs encoding high cysteine proteins Hc6-Hcl 1has been identified. Messenger RNAs were selected byhybridization to plasmid m2410 DNA bound on DBM-


cellulose, and subsequently translated in Xenopus laevisoocytes. The utility of this protocol results from the abilityof frog oocytes to efficiently translate very small amountsof mRNA, and to process the translation products to formswhich comigrate with authentic, secreted chorion stan-dards.

Further studies using cDNA clone m2410 have shownthat transiational expression of high cysteine proteins Hc6-Hcll is closely correlated both in time and in magnitudewith the accumulation of their corresponding RNAs.Future experiments with this plasmid will be directedtowards understanding the molecular basis for coordinateexpression of high cysteine proteins Hc6-Hcll.

Structure and Expression of the Hemagglutinin Gene of theHON1 Strain of Influenza Virus. A.R. Davis, A.L. Hiti,and D.P. Nayak. Dept. of Microbiology and Immunology,UCLA School of Medicine, Los Angeles, CA 90024.

Using a synthetic DNA primer and reverse transcriptionwe have cloned the hemagglutinin (HA) gene of theA/WSN/33 strain (HON1) of human influenza virus. Oneclone contains the entire structural gene for HA, thusfacilitating studies on the expression of this gene in E. coli.We are now studying this expression. The clone has alsobeen used to determine the nucleotide and protein se-

quence. The A/WSN HA gene is 1775 nucleotides in lengthcoding for 565 amino acids. The cRNA contains a 5' non-

coding region of 31 nucleotides, a coding region of 1695nucleotides and a 3' noncoding region of 48 nucleotides.The sequence shows a 17 amino acid "signal" prepeptide atthe amino terminus followed by HA1 (325 amino acids), a

single arginine connecting residue, and HA2 (222 residues)at the carboxy terminus. The comparison with other sub-types reveals many conserved features throughout theprimary sequence in all strains and reinforces the argumentfor a basic architecture required of the hemagglutininmolecule. Comparison of the HO and H2 shows a 67%amino acid sequence homology, which is the highest amongthe subtypes, suggesting a close geneokigic relationship be-tween the two.

DNA Negative Superhelicity of the Ti Plasmid ModulatesAgrobacterium Tumefaciens Induced Tumorigenesis. P.D.Lipetz, R.E. Stephens. Dept. of Radiology, Ohio StateUniversity, Columbus, OH 43210 and A. G. Galsky, Dept.of Biology, Bradley University, Peoria, IL 61625.

The Ti plasmid is a natural vector for introducing TDNA sequences into dicotyledonnus plant DNA; resultingin the formation of crown-gall tumors. Ti plasmid DNAnegative superhelicity, as determined by agarose gel elec-trophoretic mobility, was decreased by incubating A.tumefaciens with novobiocin (Nov) and nalidixic acid (Nal)and increased by incubation in spermidine (Spd) (0.1-10.0mM for all treatments). Increased DNA negativesuperhelical stress is known to favor genetic recombinationby creating single stranded regions which favor nucleationof homologous DNA sequences. At all treatment concen-

trations crown-gall tumorigenic virulency of A. tumefa-ciens on potatoe disks correlates positively with the inducedalterations of DNA negative superhelicity. (10-mM tumor

virulency: 10-mM Spd= 187%, 10-mMNov = 54%, 10-mMNal = 40%). Controls indicate that neither bacterial viabilitynor plant affects were responsible for decreasedtumorigenicity. DNA negative superhelicity may modulateeither the ability of host vector systems to transfer geneticinformation or the expression of such information.

Cloning of Both the Simian Sarcoma Virus Genome and itsHomologous Human one Gene. R.D. Favera, E. Gelmann,R.C. Gallo, and F. Wong-Staal. Laboratory of Tumor CellBiology, National Cancer Institute, NIH, Bethesda, MD20205.

The simian sarcoma virus (SSV) and its helper virus(SSAV) belong to a unique family of horizontally transmit-ted primate retroviruses. SSV is tumorigenic when in-oculated into primates and can transform fibroblasts invitro. This virus is closely related to the gibbon apeleukemia viruses, agents which cause leukemias and lym-phomas in gibbons. Cloned circular SSV and SSAV viralDNA was purified from acutely infected tissue culture cellsand each was cloned in the phage X vector, Charon 21A.Comparison of the SSV and SSAV clones by heteroduplexanalysis and restriction enzyme mapping revealed: (1) SSVcontained a deletion in the region of the polymerase gene.(2) An insertion toward the 3' end of the moleculerepresented the sre gene. (3) This SSV clone contained an

inversion of one terminal repeat unit (LTR) and adjacentsequences.

Since retroviral sre genes have a cellular origin we usedthe cloned DNA as probes to analyze several vertebratespecies, including human, for the presence of SSAV- or

SSV-related squences. In all species tested homologous se-

quences were found with the SSV probe, but not the SSAVprobe. Screening of a phage X human gene library with theSSV probe located the human gene fragment homologousto the SSV sre gene. This fragment is approximately 15 kband contains the region of homology to SSV and flankingsequences. The presence of intervening sequences withinthis human one gene is demonstrated by restriction enzymeand heteroduplex analysis.

Synthesis of Hepatitis B Virus Surface Antigen in E. Coliand in Eukaryotic Cells. P. Charnay, C Pourcel, M.F.Dubois, M. Gervais, A. Louise, C Chany, and P. Tiollais.Unité de Recombinaison et Expression Génétique, IN-SERM U.163, Institut Pasteur, 28, rue du Docteur Roux75724 Paris Cedex 15 and Unité de Recherche sur les Infec-tions Virales, INSERM U.43, Hôpital Saint-Vincent dePaul, 74, avenue Denfert-Rochereau, 75014 Paris, France.

Hepatitis B virus (HBV) is a human DNA virus responsi-ble for acute and chronic hepatitis and which is also in-volved in the etiology of hepatocellular carcinoma. Thevirus has not yet been grown in cell culture. We have clonedthe entire genome in E. coli. This permitted the establish-ment of its nucleotide sequence (3,182 bp). The gene codingfor surface antigen (HBsAg), which is now being used as a

vaccine, was localized. We present the amino acid sequenceof the polypeptide and a model of the viral envelope takingaccount of this sequence and of recent data on the structureof the antigen.


Biosynthesis of hepatitis B surface antigen was achievedin two different systems: (1) The gene for HBsAg was fusedwith the lacZ gene carried by a X phage vector. Infection ofE. coli with the recombinant phage induced synthesis of a

hybrid polypeptide carrying determinants of surface an-

tigen. (2) The HBV genome was introduced in mouse LTK"cells by cotransformation with the cloned herpes simplexvirus thymidine kinase gene. Several copies of HBVgenomes were integrated into high molecular weightcellular DNA. HBsAG was excreted without cell lysis intothe culture medium as particles having the samecharacteristics as those found in human serum.

Characterization of a Mouse Cell Line with Multiple GenesEncoding an Altered Dihydrofolate Reducíase. D.A.Haber, S.M. Beverley, P. Brown, and R. T Schimke. Stan-ford University, Stanford, CA 94305.

We have studied a line of 3T6 mouse fibroblasts which isresistant to 400-nM methotrexate following growth in step-wise increasing concentrations of the inhibitor. This cellline has been used in attempted gene transfer experimentsinto mouse bone marrow cells. Enzymological studies haverevealed that these cells not only contain elevated levels ofdihydrofolate reductase (DHFR, the target enzyme ofmethotrexate), but also express an enzyme that is altered inthat it has a 270-fold reduction in methotrexate binding af-finity and a 20-fold decreased enzymatic turnover number.The altered enzyme is not present in the parental cells whichare resistant to lower methotrexate concentrations andwhich express elevated levels of wild-type DHFR, implyingthat the mutation arose in cells already containingamplified DHFR genes. The normal parental gene is not ex-

pressed in cells showing elevated levels of altered enzyme.Following growth of subcloned cells in the absence ofmethotrexate and the loss of the amplified sequences, thenormal enzyme is reexpressed. These observations, togetherwith the enzymological data, suggest the possibility thatamplified levels of altered genes may be necessary simply tomaintain vital enzymatic activity.

Structures of a Gene and Pseudogene for Rat Cytochromec. R.C Scarpulla, K.M. Agne, J. Barthel, and R. Wu. Sec-tion of Biochemistry, Molecular and Cell Biology, CornellUniversity, Ithaca, NY 14853.

Using as a hybridization probe a coding region subfrag-ment of the cloned iso-1-cytochrome c gene from Sac-charomyces cerevisiae, eight different recombinant phageswere isolated from a Charon 4A-rat genomic library. Restric-tion framents corresponding in size to those found in thephage inserts and homologous to the yeast probe were alsopresent in total rat genomic DNA. The nucleotide sequenceof a 0.96-kb portion of one of these was determined andestablished the existence of a gene encoding a cytochrome cidentical in amino acid sequence to that of mouse. Peptidemapping studies have indirectly shown that the mouse andrat proteins are the same but until now sequence evidencehas been lacking. In contrast to the yeast iso-1 and iso-2cytochrome c genes, neither of which have introns, the ratgene contains a single 105-bp intervening sequence inter-rupting glycine codon 56. Thermal dissociation of the yeast

probe from the homologous rat DNA occurred at 58°C in0.39-M Na* and the areas of greatest nucleotide sequencehomology coincided with four regions of functionally con-strained amino acid sequence. The results establish thatcytochrome c genes may be isolated by interspecieshybridization between widely divergent organisms.

Of the eight different phages which hybridized to theyeast coding sequence, four failed to hybridize to the ratgene coding region. A portion of one of these homologousto the yeast gene was localized on a 1-kb Pst I insert frag-ment. Sequence analysis revealed three short homologousstretches which accounted for hybridization to the yeastcytochrome c gene. Although separated by nonhomologousDNA, the three sequences were in the same order andtranscriptional orientation as they appear in the yeast gene.The most likely explanation for these homologous se-

quences is that they represent an ancient form ofpseudogene that has diverged extensively without selectionand is detectable only by hybridization to the gene of a

lower eucaryote such as yeast. Unlike previously describedpseudogenes for 5S RNA and globin, this one has little or

no significant homology with^the copy of the rat gene thatis currently functional. It therefore appears that remnantsfrom the ancient evolutionary history of a gene can occur inthe genomes of present-day organisms.

Structural Organization of the pyrB Gene Loci in E. Coli.J.R. Wild, W.D. Roof, K.F. Foltermann, and G.A.O'Donovan. Texas A&M University, College Station, TX77843.

The two polypeptide chains that comprise aspartatetranscarbamoylase are encoded by adjacent cistrons ex-

pressed in the order, promoter-operator, catalytic cistron,regulatory cistron (po pyrBc pyrBr). These cistrons havebeen cloned into plasmid pBR322 on a 2.8-kb fragment (theminimal coding requirement is approximately 1500-bp plusregulatory regions). The cloned fragment contained theregulatory regions since repression/derepression could be ac-

complished by manipulating the endogenous uridinenucleotide pools. Deletion of the regulatory cistron did not

impair the expression of the catalytic subunit. As expected,the catalytic trimer possessed no effector response nor did itexhibit homotropic kinetics for aspartate. Insertion of Muinto the structural region of pyrBc had a negativepleiotropic effect on the expression of pyrBr. A colinearmap of the amino acid sequences with restriction sites in thecloned fragment suggests that less than 10-20 basesseparate the carboxyterminus of the catalytic cistron fromthe start of the regulatory cistron. These data support theidea that a single bicistronic operon encodes the catalyticand regulatory polypeptides of aspartate transcarbamylasein E. coli.

A Balb/c Mouse Procollagen Gene: Isolation and Iden-tification of a proal (I) Gene Fragment. J. Monson andB.J. McCarthy. Dept. of Biochemistry & Biophysics,University of California, San Francisco.

We report the isolation and identification by DNA se-

quence analysis of a Balb/c mouse proal (I) gene fragment(MPC1). This represents the first isolation of a proal(I)


genomic fragment from mouse or any other species. The 5'80% of MPC1 cross hybridized to the a domains of thechicken proal(I) and pma2(I) cDNA probes while the 3'20% hybridized almost exclusively to the proal (I) COOH-terminal propeptide domain. The DNA sequence of a

1300-bp fragment from the a domain of MPC1 wasestablished. Five coding sequences were identified; togetherthese specify 126 amino acids which are 95% homologousto the calí proal (I) amino acid sequence from residues 568to 690 of the calf al(I) chain. Three of the coding se-

quences are 54 bp and two are 108 bp in length. In contrastto the findings in chicken, in the mouse proal(I) gene frag-ment U occupies 60% of the wobble positions in the 126codons examined. The 5' and 3' ends of the intervening se-

quences are homologous to the splice junction consensus

sequences. In addition the MPC1 splice junctions have con-siderable sequence complementarity to rat Ula RNA.Finally, an apparently rodent-specific, genomic repetitivesequence has been localized within the a domain of thismouse proal(I) gene.

Temperature-Sensitive Mouse Cells Created by Transfer ofa Mutant Herpes Thymidine Kinase Gene. M.M. Manos,S.D. Munroe, and L.A. McReynolds. Dept. of Biochem-istry, College of Medicine, University of Arizona, Tucson,AZ 85724.

The thymidine kinase (tk) gene of herpes simplex virus-1(HSV-1) has become a useful, easily selectable marker forDNA-mediated gene transfer to; mutant mouse cells (Ltk").

We have utilized a /Ar-gene-containing DNA fragmentfrom HSV-1 mutant tsl 117 in gene transfer experiments.This virus produces a temperature-sensitive tk activity in in-fected cells. The 8.3-kb Hpa I fragment from the viral DNAwas used to transform Ltk" to tk*. The transformants were

characterized by growth properties, cell morphology, flowmicrofluorimetry, incorporation of thymidine and in vitrotk activity.

Transformants exhibit a 70% decrease in thymidine in-corporation into DNA within 4 hr after a shift to thenonpermissive temperature. Transformants also exhibittemperature-dependent growth in modified HAT mediumwhich has a reduced thymidine concentration (1 /¿g/ml).Control cells containing the comparable fragment from thewild-type are not temperature sensitive. The mutant cellpopulations were analyzed by flow microfluorimetry after 3days in modified HAT medium at either 34°C or 39°C. Thefraction of cells in G2 + M was 25 times less at 39°C than at34°C, while the d population was increased at 39CC. Thisdemonstrates that the cells are not randomly arrested in thecell cycle. The transformants can be rescued from lethalgrowth conditions (modified HAT, 39°Q by either a shiftto the permissive temperature, 34°C or by increase in thethymidine concentration.

This system provides temperature-dependent growth as

an additional selective factor for gene transfer, and may of-fer a means of inducing tk gene amplification with in-cremental temperature increase.

The a-1-Antitrypsin Gene and Pulmonary Emphysema.S.L.C Woo, T. Chandra, M. Leicht, Howard Hughes

Medical Institute, Department of Cell Biology, Baylor Col-lege of Medicine, Houston, TX 77030, and K. Kurachi andE. W. Davie. Dept. of Biochemistry, University ofWashington, Seattle, WA 98105.

a-1-Antitrypsin is a plasma protease inhibitor that ac-

counts for 90% of total anti-protease activity in the blood.Reduced levels of this protein in certain individuals con-stitute a genetic disorder known as a-1-antitrypsin deficien-cy which is often associated with chronic obstructivepulmonary emphysema. The deficiency is characterized bythe presence of a mutated a-1-antitrypsin gene which givesrise to a variant Z-type protein instead of the normalM-type protein, and is inherited by an autosomal recessivetrait. The protein is a single polypeptide of 45 000 daltonsin molecular weight and is synthesized in the liver. We haveenriched the a-1-antitrypsin mRNA from a baboon liver byspecific immunoprecipitation of total polysomes. ThemRNA appeared to be greater than 90% pure as analysedby cell-free translation followed by SDS polyacrylamide gelelectrophoresis of the labeled protein products andfluorography. Complementary DNA synthesized from thismRNA preparation was inserted into the Pst I site ofpBR322 DNA by the dC/dG tailing method. The aminoacid sequence of the carboxy terminus of humana-1-antitrypsin is known. In one of the recombinant clonesanalyzed, a stretch of DNA containing these codons hasbeen found to exist immediately preceding a terminationcodon. All codons matched perfectly with the amino acidsequence, except the presence of an isoleucine codon in thebaboon cDNA instead of a methinonine residue in thehuman protein, which could be the result of a single basesubstitution. Another interesting feature of the a-l-anti-trypsin gene is the presence of an ATTAAA hexanucleotidein the 3'-untranslated region instead of the common

AATAAA sequence. This baboon a-1-antitrypsin cDNAclone is being used as a specific hybridization probe toscreen a human chromosomal gene library in order toisolate the corresponding human gene. The availability ofthe human chromosomal a-1-antitrypsin gene will permitfuture attempts to analyze the deficiency syndrome at thegene level.

Analysis of Non-Alu Families of Repeated Sequences fromwithin the Human /3-Globin Gene Complex. P.A. Biro,P. Jagadeeswaran, D. Tuan, B.G. Forget, and S.M. Weiss-man. Dept. of Human Genetics and Internal Medicine,Yale University School of Medicine, New Haven, CT 06510.

The discovery of the Alu family of repeated sequences inthe human genome has facilitated the study of otherfamilies of repeated sequences. We have identified andcharacterized a number from within the human /3-globin likegene cluster.

By Southern blotting small DNA fragments about 1 kb inlength from within this region to human DNA, at least fivedifferent families of repeated sequences have been detected.They form a continuous stretch of over 5 kb and occupymost of the region between the embryonic epsilon and theG 7-globin genes and are repeated between 1 000 and 10 000times in the genome. Restriction enzyme analysis indicatesthat some of them may also occur as part of a longer


repeating unit elsewhere in the DNA. Two of the sequenceshave also been found downstream from the beta globingene and are similarity present in at least one hithertoundescribed /3-like pseudogene clone.

Individual genomic clones complementary to each of thefive families have been isolated from a human partialEco RI library. Hybridization analysis indicates that therepeating elements are not always found in the samenumber or configuration in each clone. One random clonehas been analyzed in detail. It is complementary to about1600 bp of DNA which maps between 4.5 and 6 kbupstream from the G 7-globin gene.

This region of the globin complex has been sequencedand shows a stretch of about 600 bp bounded by an im-perfect inverted terminal repeat of 17 bp bounded by thedinucleotide pair TG-AC. We suggest that this element inthe human genome is the analog of movable elements suchas TY-1 of yeast and copia of Drosophila. The sequencesalso bear partial resemblances to DNA sequences of thelong terminal repeats of the known pro-retroviruses.

The corresponding region from the random clone is be-ing sequenced and shows extensive homology with theglobin clone. However, the terminal sequences of the repeatmay be of a different nature from those described above.

Structural Homology Between a-Fetoprotein and SerumAlbumin. A. Dugaiczyk, S. Law, and J. Hawkins. Dept. ofCell Biology, Baylor College of Medicine, Texas MedicalCenter, Houston, TX 77030.

a-fetoprotein (AFP) is a serum protein produced by allmammals so far studied. It is produced by the yolk sac andfetal liver; after birth, the rate of AFP synthesis in the liverrapidly decreases to an undetectable level. However,reinitiation of AFP synthesis is again observed in in-dividuals with hepatomas or germ cell tumors. Elevation ofserum AFP levels is also observed in a variety ofpathological conditions and in certain human fetal malfor-mations such as neural tube defects. Albumin synthesis is incontrast suppressed in all those pathological conditionswhere AFP production is reinitiated. The exact function ofAFP is not known let alone the molecular mechanismsunderlying the inverse expression of the two proteins.

In order to understand the function of a-fetoprotein, theentire DNA complementary to murine AFP mRNA hasbeen cloned in the plasmid vector pBR322 and its completenucleotide sequence determined. The DNA is 2064nucleotides long, comprised of 41 nucleotides representingthe 5'-untranslated region, 57 nucleotides identifying a

hydrophobic prepeptide of 19 amino acids, 1758nucleotides coding for the 586 amino acids of a-fetoproteinand 153 nucleotides making up the 3'-untranslated regionexcluding 55 nucleotides of poly(A). The deduced amino

acid sequence for AFP reveals extensive homology to serumalbumin. The cysteine residues in AFP are in virtually iden-tical sequence positions to those found in the human serumalbumin polypeptide chain, suggesting that 14 disulfidebridges may be formed throughout the AFP molecule, allexcept one at the same positions as those found in albumin.The AFP molecule can be organized into a three domainstructure almost identical to that of serum albumin, sug-gesting that the two proteins have a common ancestralorigin.

The analysis of structure homology is further extendedinto the chromosomal mouse and human genes for the twoproteins. The human serum albumin gene is localized on

five contiguous Eco RI DNA fragments of chromosomalDNA over 20 kb in length. The detailed structure of thegene will be presented.

Definition of the 5' Flanking Sequences Required forSpecific Initiation of Ovalbumin Gene Transcription inVitro. S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley. Departmentof Cell Biology, Baylor College of Medicine, Houston, TX77030.

An in vitro system has been used to study the initiation oftranscription of the ovalbumin gene. The DNA templatewas a cloned ovalbumin gene fragment that contained 5'flanking sequences, and 393 nucleotides of gene sequence.A HeLa cell crude extract was used as the source of RNApolymerase and initiation factors. Correct initiation was

judged by the size of RNA product, by SI mapping and bythe sizes of transcription products generated fromovalbumin DNA templates truncated at the 3' end. A seriesof deletion mutants were constructed by trimming 5' flank-ing sequences of the ovalbumin DNA template using ex-

onuclease III. The DNAs generated were then cloned inpBR322 and used as templates to determine which se-

quences were necessary for initiation of transcription.Specific initiation of the ovalbumin gene was unaffected bydeletion of all but 61 nucleotides of the 5' flanking se-

quences. However, specific initiation was abolished bydeletion of all but 26 nucleotides of the 5' flanking se-

quences. Thus, a region between 61 and 26 nucleotidesupstream from the cap site, which includes the Hogness box(TATATAT) at position 32-26, is essential for the correctinitiation of the ovalbumin gene. Nevertheless, naturalDNA fragments containing false Hogness boxes not nor-

mally located in the immediate 5' flanking region of an

auhentic gene did not serve as promoters for initiation oftranscription. Recently, we have fractionated the totalHeLa extracts into various components by columnchromatography. Several components from different frac-tions were required for accurate initiation of the ovalbumingene.

poster session abstracts from the first annual congress on recombinant dna research

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