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

DNAVolume 2, Number 2, 1983Mary Ann lieben, Inc., Publishers

Remaining Speakers' Abstracts from the Third AnnualCongress for Recombinant DNA Research

TRANSFER AND EXPRESSION OF FOREIGN GENES IN PLANTS

J. Schell, M. Van Montagu, J.P. Hernalsteens, L. Willmitzer,J. Leemans, H. Joos, L. Otten, H. De Grève, M. Holsters, P. Zam-bryski, L. Herrera and A. Depicker

Laboratorium voor Genetika, Rijksuniversiteit Gent, Belgium

Laboratorium voor Genetische Virologie, Vrije UniversiteitBrussels, Belgium

Max-Planck-Institut für Züchtungsforschung, Köln-

FRG

Large plasmids in Agrobacterium tumefaciens (Ti) and A.rhizogenes (Ri) enable these bacteria to transfer a defined DNAfragment (T-DNA) into the plant cell nucleus and to covalentlyintegrate this T-DNA segment in chromosomal DNA, thus creatinga new locus at a number of possible sites. The mechanism under-lying DNA transfer is still poorly understood. Genetic evidenceindicates that a relatively large segment including the so-called vir region and the T-DNA region of Ti-plasmids are trans-ferred. In established tumour lines only the T-region is foundto be inserted in the chromosomal DNA and thus stably maintain-ed. A 25 bp direct repeat located at both extremities of the T-region on the Ti-plasmid seems to be important for the integra-tion of the T-DNA. No functions located within the T-region are

required for either transfer or integration. Large foreign DNAsequences of 50 kb or more, experimentally inserted within theT-region, are efficiently transferred and integrated in theplant chromosomal DNA. The plasmid derived T-DNA was shown toconsist of a number of well defined transcriptional units tran-scribed by the host RNA polymerase II and coding for a numberof different functions i.e. enzymes involved in opine synthesisand functions involved in the inhibition of plant differentia-tion. Separate functions suppressing root respectively shootformation have been identified. Removal of these tumor control-ling genes does not affect DNA transfer or integration. Thus itwas possible to design modified Ti-plasmids that can insertforeign genes in plant cells from which normal plants can be re-generated that express the foreign genes and transmit them sexu-ally with normal mendelian seggregation ratios. In order to ex-press foreign genes in plants they have to be inserted behindplant promotor sequences. Several such constructed genes havebeen made and introduced in Tobacco plants and this expressionhas been studied.

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DNAVolume 2, Number 2, 1983Mary Ann Liebert, Inc., Publishers

Remaining Poster Session Abstracts from theThird Annual Congress for Recombinant DNA Research

FORMATION OF LEFT-HANDED Z-DNA IN THE SV40 NUCLEOSOME-FREETRANSCRIPTIONAL ENHANCER REGION by Alfred Nordheim and AlexanderRich, Massachusetts Institute of Technology, Cambridge, Mass.02139.

Left-handed Z-DNA is an alternate form of the DNA doublehelix which is favored in sequences containing alternation ofpurine and pyrimidine residues. Z-DNA can be stabilized underthe torsional constraint of negative supercoiling. An assaysystem was developed that allows the identification of Z-DNAsegments in supercoiled circular DNA by the use of Z-DNA speci-fic antibodies (1). An investigation of supercoiled SV40 DNArevealed the existence of three major anti-Z-DNA antibody bind-ing sites. Two sites are located at the Sph I sequence in the72 bp repeated segments that are known to enhance early viraltranscription. A third Z-DNA site is located just outside these72 bp repeats between positions 258 and 265. A possible in-volvement of Z-DNA formation on activation of eukaryotic pro-moter regions is postulated. The potential effects of Z-DNA onthe structure of chromatin is discussed.

(1) Nordheim et al., Cell 31, 1982, 309-318.

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THIRD ANNUAL CONGRESSFOR

RECOMBINANT DNA RESEARCH

HOW DOES BACTERIOPHAGE T4 SHUT OFF E. COLI PROTEIN SYNTHESIS?Nancy J. Casna and David A. Shub, Department of BiologicalSciences, SUNY at Albany, Albany, N.Y. 12222.

We have inserted a fragment of E_. coli DNA (spanning the endof lacl and the beginning of lacZ) into the dispensable regionof the rllB gene of bacteriophage T4. In this chimeric gene,rllB peptides terminate at nonsense codons as they enter thelac insert. Any ribosome beginning translation at the normallacZ initiator codon will produce a fusion protein containingthe distal 90% of rllB, in the correct translational readingframe. Such a protein should result in an rllB phenotype(growth on a lambda lysogen). However, since these phage arerllB , the host regulatory signals do not function in T4. Itis well known that phage T4-infected cells discriminate againstE. coli-specific transcription and translation. We have gen-erated pseudorevertant phage, which have regained rllB function.We hope some of these will have suffered mutations in the phagegenes that block host-cell gene expression. We have begun tocharacterize these mutants, both genetically and biochemically.

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TRANSCRIPTION OF THE 5S GENE IN PLASMID DNA AND GENOMIC BOVINEAND XENOPUS DNAS BY HELA CELL RNA POLYMERASE III. Chen-Yeh Su,John J. Furth and Gary H. Lee. University of Pennsylvania,Philadelphia, PA 19104

RNA was transcribed by RNA Polymerase III in a cytosolextract of HeLa cells using as templates plasmid DNA containingthe Xenopus laevis oocyte 5S gene-pseudogene cluster, andgenomic DNAs. RNA was evaluated by polyacrylamide gel electro-phoresis, hybridization to 5S DNA, nearest-neighbor analysisand fingerprinting. The 5S gene was efficiently transcribedfrom plasmid DNA. While RNA synthesis initiated from thepromotor of the Xenopus 5S pseudogene in plasmid DNA thetranscript terminated in the pBR322 vector. Little 5S RNA was

synthesized from genomic Xenopus DNA. A similar pattern ofRNA synthesis was observed with Xenopus erythrocyte and Xenopusliver and kidney DNAs as templates. Under similar reactionconditions, RNA 5S in size was transcribed from bovine DNA.At low UTP concentration, RNA a few nucleotides shorter than5S RNA was transcribed from the Xenopus gene-pseudogenecluster in plasmid DNA and genomic bovine DNA. Similarpatterns of RNA were observed with RNA transcribed from bovinethymus, bovine liver and bovine lymphosarcoma DNAs; thispattern was very different from that of RNA transcribed fromXenopus DNA. A third pattern was observed with RNA transcribedfrom mouse DNA. These results suggest (a) the distribution ofRNAs synthesized from genomic DNA by RNA Polymerase III isdetermined by the species from which the DNA is obtained andnot the cell type, and (b) a species barrier exists when HeLacell RNA Polymerase III transcribes the 5S gene in genomicXenopus DNA although the enzyme efficiently transcribes theXenopus oocyte 5S gene when in plasmid DNA. (Supportedby NIH Grant GM 10390).

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THE EXPRESSION IN YEAST OF THE E. COLI GALK. GENE ON A CYC1/GALK FUSION PLASMID. B.C. Rymond, R.S. Zitomer, D. Schumperli*and M.J. Rosenberg*, Department of Biological Sciences, StateUniversity of New York at Albany, Albany, N.Y. 12222, *Nation-al Cancer Institute, National Institutes of Health, Bethesda,MD 20205 and Smith Kline and French, Philadelphia, PA 19101.

A series of plasmid shuttle vectors have been constructedjoining the transcriptional and translational initiationsignals of the yeast CYC1 gene encoding iso-1-cytochrome c,and the E. coli galK gene encoding galactokinase. Theseplasmids contain origins of replication and selectable geneticmarkers for both yeast (ars-TRpl) and E_. coli (ori and the Tcrgene of pBR322) and can therefore be selected for and maintainedin either organism. Galactokinase deficient yeast (gall~) weretransformed with these plasmids and successful fusion eventsselected by the ability of these transformants to utilizegalactose. The degree of galactokinase expression was depen-dent upon the point of fusion between the CYC1 promoter and thegalK gene. The level of galactokinase in each transformant was

subject to carbon catabolite repression, characteristic of theCYC1 gene. YRpRl, the plasmid which directs the greatestlevel of galactokinase expression is a result of an in-framefusion between the CYC1 and galK coding sequences. Northernblot analysis of RNA isolated from yeast transformed withYCpRl, a derivative of YRpRl in which a 2 kb DNA fragment con-

taining the centromere of yeast chromosome III has been intro-duced, demonstrates a single band of galK-specific hybridiza-tion, 1850 nucleotides in length. The intensity of this bandvaries with the carbon source used in the growth media. Thelength of this transcript, its pattern of regulation and thefact that it cross hybridizes with a CYC1 specific probe de-rived from DNA sequences 3' to the point of fusion, suggeststhat transcription starts at the normal CYC1 initiation siteproceeds through the galK and CYC1 coding sequences and stopsat the CYC1 termination site. During anaerobic growth the CYC1gene is transcriptionally inactive. Consequently the CYCl/galKfusion gene is unable to support the anaerobic growth of yeasttransformants on galactose containing media. EMS and UV muta-genesis of these transformants has allowed the isolation ofseveral mutants permissive for such anaerobic growth. Thenature of these mutations is currently being investigated.This galK fusion system offers the advantage of both selectionfor (by growth on galactose) or against (selection against gal-1-P accumulation in ga!7~ and/or gal10^ cells) galK expression.Additionally, the sensitive enzyme assay from crude cell ex-tracts and the ability to estimate the galactokinase levels intransformants based on their staining properties make thissystem attractive for general use in studying gene regulation.

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SEPARATION OF DNA FRAGMENTS DIFFERING BY SINGLE BASE SUBSTITU-TION: APPLICATION TO ß °-THALASSEMIA IDENTIFICATION.Stuart G. Fischer, Nadya Lumelsky and Leonard S. Lerman, Centerfor Biological Macromolecules, State University of New Yorkat Albany, 1400 Washington Avenue, Albany, NY 12222

Lambdaphage DNA fragments 536 base pairs in length differingby single base pair substitution were clearly separated indenaturing gel electrophoresis for mutations within the firstcooperatively melting sequence. The correspondence betweengradient displacement of the mutants and calculated change inhelix stability permits substantial inference as to the type ofsubstitution. We have applied this system to recombinant plas-mids containing human & globin genes and have detected a 272base pair fragment containing a single base pair substitutionwhich confers __. °-Thalassemia.

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GENOME FUSION MEDIATED BY A SITE-SPECIFIC DNA INVERSION SYSTEM.K.E. Kennedy, S. Iida, J. Meyer, and W. Arber Dept. of Micro-biology, Biozentrum, CH-4506 Basel, Switzerland

The genome of the general transducing phage PI contains a3 kb invertible segment called the C loop flanked by 0.6 kbinverted repeats. The C loop is hybridizable with the inverti-ble G loop of bacteriophage Mu (1). Both inversion systems are

related to that in Salmonella, which controls phase variation(2,3). Our studies suggest that in PI, as in Mu (4), inversioncontrols host range. We have analyzed a collection of inser-tion and deletion mutations in the C loop region and constructedmulticopy vectors carrying portions of it. We have thus locatedthe gene ein (£ inversion) in a 600 bp region immediately adja-cent to the outer end of the left inverted repeat. The sitesat which the Cin recombinase acts to invert the C loop, cix(Conversion crossover), have similarly been mapped at the outerends of the inverted repeats (5). Using pairs of analogous ein"1"and Acin plasmids carrying cix sites, we have shown that theein gene product can catalyze intermolecular recombination re-

quired for inversion (6). The ein plasmids can form dimers ina recA host at 10-fold greater frequency than theirZ-cJLncounterparts. Additionally, cin+ plasmids can be transduced bya Acin PI strain at frequencies up to 1000-fold freater thanthose observed for plasmids lacking ein. High frequency trans-ducing phage were readily isolated. Physical characterizationof their genomes showed them to be phage-plasmid cointegrateswith the structures expected to result from recombination be-tween cix sites.

1. Chow L.T., Bukhari A.I. (1976) Virology 74: 242-2482. Kamp D., Kahmann R. (1981) Mol. Gen. Genet. 184: 564-5663. lino T., Kutsukake K. (1980) CSH Symp Quant. Biol. 45: 11-164. Giphart-Gassler M. et al (1982) Nature 297: 339-3425. Iida S. et al (1982) EMBO Journal 1: 1445-14536. Kennedy K.E. etal (1983) Mol. Gen. Genet, (in press)

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RECOMBINANT DNA RESEARCHTMGenBank - The Genetic Sequence Data Bank

* * // *Wayne P. Rindone , Harold M. Perry , Walter B. Goad , Howard S. Bilofsky

and Christine K. Carrico %

Bolt Beranek and Newman Inc.10 Moulton St.Cambridge, MA 02238

fT-10, Mail Stop 465Los Alamos National LaboratoryLos Alamos, NM 87545

%National Institute of General Medical SciencesNational Institutes of HealthWestwood Building, Room 9195333 Westbard Ave.Bethesda, MD 20016

TMGenBank , the Genetic Sequence Data Bank, is a U.S. Government-sponsored internationally available repository of all published or

deposited nucleic acid sequences, catalogued and annotated for sitesof biological interest. The GenBank database, which is available inthe form of computer-readable magnetic tape or an annual printedcompendium, serves investigators around the globe in all scientificresearch environments. Online access to the central GenBank computeris also available to anyone, but only a limited number of users canbe accomodated at any one time. The Data Bank was created in 1982by the National Institute of General Medical Sciences of the NationalInstitutes of Health(NIH) in response to a critical scientific needfor a timely, centralized, accessible genetic sequence data bank.Cosponsors include the National Cancer Institute, the National Insituteof Allergy and Infectious Diseases, and the Division of ResearchResources of the NIH, as well as the National Science Foundation,the Department of Energy and the Department of Defense. The currentmonthly update of the GenBank database contains entries for 902published sequences as well as 6 additional deposited sequences,comprising a total of approximately 800,000 bases.

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

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