Remaining Abstracts from the Fifth Annual Congress for Recombinant DNA Research

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<ul><li><p>DNAVolume 4, Number 2, 1985Mary Ann Lieben, Inc., Publishers</p><p>Remaining Abstracts from the FifthAnnual Congress for Recombinant</p><p>DNA Research</p><p>EPSTEIN-BARR VIRUS GENES IN LATENT GROWTH TRANSFORMING INFECTION</p><p>Elliott Kieff, Timothy Dambaugh, Kevin Hennessy, Susan Fennewald,David Whang, Mary Hummel, Minas Arsenakis and Bernard Roizman</p><p>The Epstein-Barr virus (EBV) genome is a 170 bp, linear doublestrand DNA molecule. The virus causes infectious mononucleosis,is believed to be an etiologic agent in human Burkitt lymphoma,and is remarkably efficient in acute transformation of B lympho-cytes into continuously proliferating lymphoblasts. The entirevirus genome persists in the proliferating cells as a multicopyepisome or less frequently as integrated DNA. The infection isnon-permissive for virus replication . Only a few virus genesare characteristically expressed in the growth transformed Blymphocytes. These genes are likely to be involved in mainte-nance of cell proliferation, in episome maintenance and inregulation of expression of other virus genes.</p><p>The four virus genes expressed in latent infection are tran-scribed from three non contiguous regions of the virus genome.Two genes; one encoding a pair of small non polyadenylated RNAssimilar to adeno VA RNAs and the other encoding a intranuclearprotein are clustered near the left end of the genome. Thisnuclear protein is probably required for initiation of growthtransformation. Another nuclear protein gene is encoded by thecentral region of the genome. Data of Yates and Sugden indicatethat this nuclear protein is required for episome maintenance. Amembrane protein is encoded by the right end of the genome. Themembrane protein is likely to be involved in ion transport. Italso probably generates the new antigenic reactivity in theplasma membrane which makes the growth transformed cell subjectto killing by immune T cells in latently infected humans. Thecurrent status of studies of expression of these genes inheterologous systems will be discussed.</p><p>183</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>TRANSLATIONAL STRATEGIES IN ADENOVIRUS-INFECTEDCELLS. Robert Schneider, Jan Kitajewski, John Logan &amp;Thomas Shenk Department of Molecular Biology,Princeton University, Princeton, N.J. 08544</p><p>Two viral elements have been shown to influencethe efficiency with which mRNAs are translated withinadenovirus-infected cells: the VA RNAs and thetripartite leader sequence. The VA RNAs are twosmall polymerase Ill-coded transcripts. Viralmutants have been constructed which fail to producethese RNAs. Mutant-infected cells contain normallevels of mRNAs late after infection, but reducedquantities of their encoded polypeptides. The VARNAs function by preventing inactivation of elF-2subsequent to infection. The RNAs prevent activationof the PI kinase and subsequent phosphorylation ofthe elP-2 alpha subunit. They have likely evolved toantagonize the effects of interferon anddouble-stranded RNA which may be generated withininfected cells. The second viral element whichinfluences translation is the tripartite leadersequence, a 200 nucleotide, 5' noncoding segmentattached to all mRNAs which are encoded by the majorlate transcription unit. This sequence enhances theefficiency with which mRNAs are translated late butnot early after infection.</p><p>184</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>HOMEOTIC GENES AND THE CONTROL OF DEVELOPMENT.</p><p>Walter J. Gehring, Mozentrum, University of Basel, 4056 Basel, Switzerland</p><p>The genetic information which is transmitted from generation togeneration contains a precise developmental program which controlsontogeny, and it also includes historical information reflectingevolution. Development is based on the differential expression ofthe genetic information in a precise spatial and temporal pattern.The nature of the genetic events that orchestrate early embryonicdevelopment and direct the cells to organize themselves accordingto a distinct body plan is one of the central problems of biology.In Drosphila mutations have identified a group of genes, thehomeotic genes, which are involved in the control of the bodyplan. Recombinant DNA technology has allowed the isolation ofseveral homeotic genes. In the course of our studies on theAntennapedia gene, we discovered a low level repeat sequenceshared by several homeotic genes which had been cloned previously.Since this repeat sequence can be used to isolate other homeoticgenes from random populations of DNA segments in gene libraries,we named it homeobox. The term box refers to the fact that thecrosshomology is limited to a short and highly conserved DNAsegment of approximately 180 basepairs. The homeobox has beenused to isolate more than a dozen additional homeotic genes fromDrosophila. An evolutionary study shows that homeobox containinggenes are not confined to insects, but they also occur in verte-brates including man. DNA sequence analysis indicates that thehomeobox codes for a highly conserved protein domain, which formspart of the homeotic proteins. By computer search, a small butsignificant degree of crosshomology between the homeobox and theMAT genes of yeast has been found. The 4MAT genes code forproteins which control the expression of a set of unlinked genesinvolved in mating and sporulation. There is accumulating evidencethat they may accomplish this by binding of their protein productsto regulatory sites near the 5' ends of those genes which theyregulate. This suggests a similar mode of action for the homeoticproteins. In situ hybridization of cloned DNA to RNA in frozensections of Drosophila embryos indicate that the homeotic trans-cripts accumulate in specific body segments according to the bodyplan and that the cleavage nuclei express these transcripts in aposition-dependent fashion. The mechanism of action of thehomeotic genes is under investigation.</p><p>185</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>Induction of pColE3-CA38 hic-proinsulin gene fusion causes E.colicell lysis.T. Verriet, T.J. Beardall, P.C.K. Lau, and L.P. Visentin.National Research Council of Canada; Ottawa; Ontario; CanadaK1A OR6.</p><p>The colicin operon of plasmid pColS3-CA38, which is ccmposed ofthe col, irrm and hie genes, is under the control of the mitomycinC inducible ool gene promoter. The hie gene is involved in lacunafomation, colicin release, cell death and lysis. The N-tenriinusof the Hie protein has a sequence siroilar to known signal peptidesof secreted proteins of bacteria (1).In previous experiments we have shown that a colicin E3-humanproinsulin fused protein was produced upon induction of E. coliwith iritomycin C but this product was not secreted (2). In thisstudy we attempt to induce the synthesis and secretion of humanproinsulin by fusing the signal sequence of the hie gene withthe proinsulin gene. The fusion was made in pVT22 (2) at thePvuII site of the hie gene using EcoRl linkers. Induction with0.5, ug/ml of mitomycin C of an E. coli culture carrying therecornbinant plasmid was rapidly followed by the production ofhuman proinsulin as monitored by radioimmunoassay. Cell lysiswas observed after 60 to 90 mins of induction. These results areconsistent with the previous observations (1) that the C-terminusof the Hie protein is not essential for its lytic function.Attempts to characterize the fused Hic-proinsulin protein byin vivo pulse labelling of E. coli proteins with 0-^C] aminoacidswere unsuccessful. Different conditions known to increase foreignprotein stability in E. coli were tested. These included the useof various E. coli strains (including a Ion mutant) and varioustemperature-growth conditions. The hie signal sequence was alsofused to a tetramer of human proinsulin genes. None of theseconditions were found to increase the stability of the fusedproduct.The results presented here demonstrate the utility of fusion withthe hie gene signal sequence for induction and relea.se of foreigngene products from E. coli without the usual requirement ofenzymatic, chemical or mechanical processing.References.(1) R.J. Watson et al., (1984) Gene 29 175-184(2) T. Vernet et al., (1985) Gene (In press)</p><p>186</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>THE MAJOR IMMEDIATE EARLY PROMOTER OF CYTOMEGALOVIRUS(TOWNE STRAIN) MEDIATES EFFICIENT EXPRESSION OF THEGENOMIC BOVINE GROWTH HORMONE GENE AND TISSUEPLASMINOGEN ACTIVATOR cDNA IN CHINESE HAMSTER OVARYAND HELA CELLS.D.P. Palermo, D.R. Thomsen, R.J. Brideau, and L.E. Post. The UpjohnCompany, Kalamazoo, Michigan.Mammalian expression vectors have been constructed which employ the</p><p>major immediate early (IE) promoter of cytomegalovirus (CMV Townestrain) and include selectable markers derived from SV2neo or SV2dhfr.The promoter was used to direct the expression of the genomic form ofbovine growth hormone (bGH) in dhfr deficient Chinese hamster ovary(CHO) as well as HeLa cells. Selection for dhfr positive or G418 resistantphenotypes respectively, resulted in the isolation of similarly expressingcell lines capable of accumulating approximately 2 yg/lO^ cells by 48hours post seeding as assayed via standard radioimmunoassay. Subsequentselection of a gene amplified derivative of the CHO line by adaptation togrowth in 30 uM methotrexate resulted in a 29 fold increase in bGHoutput, bringing the amplified expression level to 58 yg/lO^ cells at48 hours post seeding. The promoter was also tested for its ability toefficiently express the cDNA for tissue plasminogen activator (tPA).3' regulatory elements derived from bGH genomic sequences were usedas a polyadenylation signal for the tPA transcripts. CHO transfectantswere tested for tPA activity by standard fibrin plate assay. Supernatantsfrom an unamplified clone were shown to have accumulated approximately0.108 yg tPA/10^ cells by 72 hours post seeding. Initial amplificationaccomplished by adapting this line to growth in 100 nM methotrexateresulted in a mixed cell population synthesizing 4.2 yg tPA/10^ cells 72hours after seeding. These experiments serve to demonstrate the utilitynot only of the CMV IE promoter, but of the bGH 3' regulatory elementsas well in expression in two mammalian systems of both genomic andcDNA sequences.</p><p>187</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>SELECTIVE SYSTEMS FOR THE ISOLATION OF HUMAN DNA REPAIRGENESJames C. Fuscoe, Larry H. Thompson, and Anthony V. CarranoBiomdical Sciences Division, Lawrence Livermore National Laboratory,Livermore, CA</p><p>The Chinese hamster ovary cell-line mutants EM9 and UV20 aredefective in repairing damaged DNA. EM9 has a reduced ability torepair DNA strand breaks and is noted for its highly elevated frequencyof sister-chromatid exchange, a property shared with cells fromindividuals with Bloom's syndrome. UV20 is extremely sensitive toboth mitomycin C and ultraviolet radiation and has a deficiency in theincision step of nucleotide-excision repair, which are properties associatedwith cells from individuals with xeroderma pigmentosum. Selectivesystems have been developed to learn whether normal human cells possessgenes capable of correcting the biochemical defects in these mutants.We have demonstrated genetic complementation of both mutants byfusion-hybridization with human fibroblasts and lymphocytes, and bytransfection with DNA from hybrid cells. By Southern blot hybridizationwith a human-specific probe, the hybrids and transformants were foundto contain human DNA. Further, independent transformants sharedcommon human restriction fragments. These restriction fragmentsmust be part of or closely linked to the repair genes and, thus, providea means of isolating the genes. Work performed under the auspicesof the U.S. Department of Energy by the Lawrence Livermore NationalLaboratory under contract No. W-7405-ENG-48.</p><p>188</p></li><li><p>FIFTH ANNUAL CONGRESSFOR</p><p>RECOMBINANT DNA RESEARCH</p><p>Comparison of Pooled DNA Samples Reveals Polymorphic RestrictionFragment Frequency Differences Between Disease and ControlPopulations. Carolyn Strange, Norman Arnheim, Michle Manos, and HenryErlich, Cetus Corporation, Emeryville, CA 94608.</p><p>A new rapid method has been developed and used to search forrestriction fragment length polymorphisms (RFLP) that are in linkagedisequilibrium with disease-associated loci. We examined DNApolymorphisms within the HLA class II loci associated with susceptibility toIDDM (insulin-dependent diabetes mellitus) by genomic blot analysis withDC-beta and DR-beta cDNA probes. To facilitate the search forinformative RFLP, we compared pooled DNA samples from IDDM patientswith DNA pools from random control individuals instead of theconventional approach of examining individual DNA samples from the twogroups. Several specific polymorphic restriction fragments associated withIDDM were revealed using this economical and rapid approach. Therestriction enzymes and probes identified as informative in this screeningwere then used to analyze HLA-DR typed IDDM families, homozygoustyping cells, and unrelated individuals to determine the association of thespecific restriction fragments with HLA-DR type and the frequency incontrol and IDDM populations. Some individual polymorphic fragmentsassociated with IDDM correlated strongly with HLA-DR3 (e.g., DC, Rsal2.7 kb; DR, TaqI 7.3 kb), while others correlated strongly with HLA-DR 4(DC, Rsal 1.5 kb; DR, TaqI, 2.6 kb). Some IDDM-associated fragments(e.g., DR, TaqI 10 kb) subdivided the serologically defined HLA-DR typeand represent new and highly informative markers for IDDM susceptibility.</p><p>189</p></li></ul>

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