remaining abstracts from the second annual congress for recombinant dna research

DNAVolume 1, Number 3, 1982Mary Ann Lieber!, Inc., Publication

Remaining Abstracts From the Second Annual Congressfor Recombinant DNA Research

THE CLONING AND IDENTIFICATION OF THE PRODUCT OF THE dnaEGENE OF ESCHERICHIA COLI, Mary M. Welch and Charles S.McHenry, Department of Biochemistry and Molecular Biology,University of Texas Medical School, Houston, Texas 77025

It has been previously established that dnaE mutationslack the enzymatic activity associated with DNA polymeraseIII of Escherichia col i (Gefter et al. (1971) PNAS 68,3150). Polymerase III contains three subunits, a, e, and0, of molecular weight 140,000, 25,000, and 10,000, respec-tively (McHenry and Crow (1979) JBC 254, 1748). Toidentify which of these polypeptides is encoded by dnaEwe have subcloned the dnaE gene from the Carbon collectionplasmid pLC 26-43. Three successively smaller plasmidshave been constructed. The smallest of these, pMWE-303,contains only 4.5 kilobases of the Escherichia col i genomeand has the ability to rescue dnaE temperature sensitivemutations. By using an adaptation of the "Maxicell"system of Sanear, Hack, and Rupp (J. Bacteriol. (1979) 137,692) we have identified the dnaE gene product as the a

subunit of DNA polymerase III. The polypeptide encoded bythe cloned dnaE gene co-migrates with the a subunit ofpartially purified DNA polymerase III. Insertions offoreign DNA into the unique Hind III site in this con-structed plasmid simultaneously disrupt the ability of theplasmid to rescue dnaE temperature sensitive mutations andto direct synthesis of the a subunit in the Maxicell .

system. Removal of this foreign DNA leads to the regen-eration of a plasmid which will again rescue dnaE mutations.Thus, the dnaE gene codes for the 140,000 dal ton a subunitwhich is a component of both polymerase III and polymeraseIII holoenzyme. (This work is supported by a researchgrant from NIH and an NIH postdoctoral fellowship to M.W.

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SECOND ANNUAL CONGRESSFOR

RECOMBINANT DNA RESEARCH

CONSTRUCTION AND ENZYMATIC SELECTION OF BACTERIOPHAGE G4GENOMES WITH MODIFIED J-F INTERCISTRONIC REGIONS. Uwe R.Müller, Department of Microbiology, School of Medicine,East Carolina University, Greenville, North Carolina.

The potential to form perfect hairpins in the intercist-ronic regions of viral genomes has been postulated to bearsignificantly on the regulation of gene expression. Tostudy this structure-function relationship I have designeda novel technique which may be generally applicable to cir-cular, double-stranded DNAs, to construct and enzymaticallyselect viable bacteriophage G4 mutants with modified J-Fintercistronic regions. The unique SsT II site within theJ-F intercistronic region was used to linearize G4 RF DNA.After enzymatic removal of the 3' protruding ends, genomeswere recircularized with T, DNA ligase. Thus the SsT IIsite was regenerated in wt molecules, but was lost in thedesired mutant genomes. Subsequent treatment with SsT IIlinearized only wt DNAs, which retained <1% of their trans-fection activity. Second cleavage at another unique site(PsT I) linearized all mutant molecules, but inactiviatedthe wt genomes by cleavage into two fragments. Approx-imately 80% of the plaques obtained by transfection weremutants. At least two types were isolated with deletions oftwo or more nucleotides. The deletions occur in the loopregion of a potential hairpin structure which is thought toact as a terminator of transcription.

A SIMPLE, EFFICIENT ROUTE TO N-ACYL DERIVATIVES OF DEOXY-RIBONUCLEOSIDES.Thomas Horn and Charles F. HoyngDepartment of Organic Chemistry, Genentech, Inc.South San Francisco, California

A general procedure for the preparation of N-acyl deoxy-ribonucleosides has been investigated. The di-(trimethyl-silyl)- derivative is generated and, without isolation, istreated with an acyl chloride. Following hydrolyticwork-up the N-acyl derivatives of adenosine, guanosine,and cytidine are obtained in high yields.

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Chinese Hamster Ovary Cell line mutants resis-stant to DNA Polymerase inhibitor, Aphidicolin.JJ<. Vishwanatha and U.C. Mishra*, Department of BiologyUniversity of South Carolina, Columbia, SC

Aphidicolin, a tetracyclic diterpenoid, is an inhibitorof eucaryotic DNA synthesis through inhibition of DNA Poly-merase a. The parental cell line (CH0-K1) used is sensitivea low concentration (0.2 ug/ml) of aphidicolin. Mutants re-sistant to a high concentration (2 yg/ml) of aphidicolinhave been isolated by a stepwise selection procedure. Themutants have an increased doubling time (22 hrs) compared tothe parent (16 hrs). Synthesis of DNA studied by 3h Thymi-dine incorporation showed that the mutants continue DNA syn-thesis at a diminished rate while it is totally shut down inthe parental cell line. A comparison of DNA polymerase acti-vities indicated that the mutant had elevated levels (2.4fold) of polymerase a. DNA polymerase from the wild typeand mutant cell lines were found equally sensitive to thedrug in U\_ vitro assay. An increased dTTP pool observed inthe mutant, when grown in the drug media, was not associatedwith any change in Thymidilate synthetase and Thymidine ki-nase activities or in sensitivity to Hydroxyurea. By intro-duction of markers like TK-, APR]"-, and HGPRT-into the pa-rental and mutant cell lines, cell hybridizations were per-formed which showed that the aphidicolin resistance is adominant trait. The dominance of the mutation was also evi-dent in hybrids between the CHO cell line and Chinese ham-ster lung (V-79) cell line. The availability of markersamong the different aphidicolin resistant mutants should aidus in determining the complementation among the mutants andto further decipher the nature of drug resistance.(Supported by a NIH grant # GM 29216-01).

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THE POLY(A) SEGMENT OF mRNA: INVOLVEMENT IN MATURATIONAND NECESSITY FOR FUNCTION.

M. Zeevi* and J.E. Darnell Jr. Molecular and Cell BiologyDepartment, The Rockefeller University New York, N.Y.10021 and * Biotechnology General(Israel) Ltd, KiryatWeizmann Rehovot 76326, Israel.

Splicing of newly formed nuclear RNA transcripts hasbeen demonstrated during adenovirus type 2 (Ad-2) mRNAformation in HeLa cells in the presence of 3'-deoxyade-nosine ("cordycepin", 3'dA), a drug that stops poly(A)addition to nuclear RNA. The correctly spliced - poly(A)lacking mRNA molecules have been found in the cytoplasmin association with the polyribosomes. The accumulationof labeled nuclear adenovirus specific RNA complementaryto early regions la, lb and 2 of the adenovirus genomewas approximately equal in 3'dA treated and control cells.At the initial appearance of newly labeled Ad-2 RNA (10min.) in the cytoplasm there was one-half as much labeledRNA in 3'dA treated cells as in the control. However,control cells accumulate additional mRNA in the cytoplasmvery rapidly in the first 40 min. of labeling while the3'dA treated cells do not.Therefore, we conclude that: a)Although poly(A) additionusually precedes splicing during mRNA formation, poly(A)is not required for splicing and b): It appears that thecorrectly spliced poly(A) mRNA molecules that are labeledin the presence of 3'dA can be transported with the same

exit time from the nucleus and can be translated in thecytoplasm but have a much shorter half-life than the poly(A) mRNA molecules from control infected cells.

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NEW CHROMATOGRAPHIC METHODS FOR THE ISOLATION OF VIRUSES,PLASMIDS AND RESTRICTION ENDONUCLEASE FRAGMENTS.Lawrence A. Haff, Pharmacia Fine Chemicals, Piscataway, N.J.

• 08854.

New Chromatographie procedures were devised for pre-paring viruses, viral DNA, plasmids and restriction frag-ments. These procedures were all based upon gel filtrationusing new composite polyacrylamide/dextran gels. These gelsare characterized by their extreme porosity and biologicalinertness, permitting separations based upon size alone.Viruses, such as F-l bacteriophage, were isolated chromato-graphically in good yield with Sephacryl. Plasmid DNA(pBR 322) was isolated by a single step of Sephacryl S-1000chromatography requiring no CsCl centrifugation nor ribo-nuclease treatment. Sephacryl S-1000 was also used tofractionate large amounts of restriction fragments, fromvarious digests, up to 20 kilo base pairs in size. Theisolated DNA was biologically active and free of inhibitorsof modifying enzymes such as restriction endonucleases,kinases, and ligases. Several factors such as columnlength, flow rate, ionic strength and eluents used wereimportant parameters effecting resolution. The greatestadvantages of preparing nucleic acids by gel filtration,rather than by alternative methods, are its low cost,rapidity, simplicity, high capacity, and lack of exposureto interfering contaminants and reagents.

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CLONING AND CHARACTERIZATION OF DNA FROM FLOW-SORTEDMAMMALIAN CHROMOSOMES. Jeffrey K. Griffith, James H. Jett,Ronald A. Walters, Brian D. Crawford, Paul J. Jackson,Mark A. Wilder, John L. Hanners, Judith M. Buckingham, andL. Scott Cram. Life Sciences Division, Los Alamos NationalLaboratory, Los Alamos, New Mexico 87545.

Mitotic chromosomes from Chinese hamster M-31 cells werestained with propidium iodide and purified by fluorescentactivated chromosome sorting. Approximately 2.1 and 2.5 ugof DNA purified from chromosomes one and two, respectively,was partially restricted with EcoRl and cloned into lambda-phage Charon 4A. Plaque forming units sufficient to stat-

istically represent 2.5 and 4.0 equivalents of chromosomeone and two were obtained, respectively. Parallel aliquotsof the sorted chromosomal DNAs were nick translated andcharacterized by hydridization with total Chinese hamstergenomic DNA. The hybridization kinetics of the two tracerswere identical with approximately 35% of the chromosomalDNA reacting with repetitive genomic DNA and 65% withunique DNA. These two tracers were used to determine thepurity and completeness of the two recombinant chromosomeDNA libraries. A maximum of 60-65% of each of the chromo-some-specific tracers was hybridized by DNA purified fromthe homologous DNA library. In contrast, reactions involv-ing either the heterologous chromosomal tracer or a

similiarly prepared total genomic DNA tracer plateaued12-22% lower than the paired homologous chromosomal DNAreactions. These results indicate that both common (i.e.,repetitive) and chromosome-specific (i.e., unique) DNAsequences are represented within the two libraries andthereby demonstrate the discreteness of the two flow-sorted chromosomal DNA populations. Although the homologoushybridization reactions indicate that the representationof the unique chromosomal DNA sequences is incomplete,the heterologous reactions suggest that most, and likelyall of the repetitive DNA sequences within the genome are

also represented in both chromosomes. A more detailedcharacterization of these recombinant libraries, inparticular the representation of the unique chromosomalDNA sequences, is in progress. (This work was performedunder the auspices of the US Department of Energy.)

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EXPRESSION OF BACTERIAL 3-GALACTOSIDASE IN MAMMALIAN CELLSGynheung An, Katsuhiko Hidaka and Louis Siminovitch, Depart-ment of Medical Genetics, University of Toronto and TheHospital for Sick Children, 555 University Avenue, Toronto,Ontario, Canada, M5G 1X8.

We have made a recombinant plasmid containing the genefor bacterial 3-galactosidase close to the SV40 early pro-moter. In this hybrid, pGAK293, the structural gene oftufB-lacZ fusion which produces a functional fusion3-galactosidase in Escherichia coli was directly placeddownstream from the SV40 early promoter with no ATG startcodon in the leader region. CHO and L cells contain verylow levels of endogeneous 3-galactosidase under our condi-tions. Transfection with the pGAK293 plasmid of eitherCHO or L cells led to the expression and appearance of theenzyme. In CHO cells, the amount of 3-galactosidase percell increased at least 50 fold 24 hours after removal ofthe DNA used for transfection. Activity per cell was maxi-mum at 24 hours but total activity continued to increasein the culture for three days. The assay is simple, rapid,sensitive, and economic. This recombinant should thereforeprovide a convenient system to study control mechanism ofeukaryotic genes.

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remaining abstracts from the second annual congress for recombinant dna research

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