poster session abstracts from the third annual congress for recombinant dna research

DNAVolume 2, Number 1, 1983Mary Ann Lieber!, Inc., Publishers

Poster Session Abstracts from the ThirdAnnual Congress for Recombinant

DNA Research

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PURIFICATION of NUCLEICACIDS.John A. Thompson and Robert W. Blakesley. Bethesda ResearchLabs, Inc., Gaithersburg, MD 20877.

A nucleic acid chromatography system (NACS) has been de-veloped using a weak anion exchange resin(l). Studies withsingle-stranded oligodeoxyribonucleotides (enzymatic/synthetic), double-stranded DNA restriction fragments, andhigh m.w. RNA molecules (viral, messenger, ribosomal) haveshown that once bound to NACS, nucleic acids are eluted byapplying a salt solution of specific ionic strength which ishigher than that used for binding. Fractionations of thesecomplex mixtures are executed by applying a linear salt gra-dient of increasing ionic strength and separations are usuallyaccording to the length (m.w.) of the polynucleotide. Hence,lower m.w. nucleic acids elute before higher m.w. due to thepresence of fewer total negative phosphate groups. However,studies with different isoacceptors of tRNA molecules andseparated strands of DNA restriction fragments have shown thatthe available charge density for a given molecule may bedifferent from that calculated due to either base sequence,secondary/tertiary structure, pH or presence of organic modi-fiers. Fractionations according to secondary/tertiarystructure allows for routine purification of supercoiled DNAmolecules (plasmid, viral, mitrochondrial, chloroplast).Numerous post-column functionality tests have shown thatnucleic acids purified (greater than 90% recovery regardlessof molecular weight) by NACS maintain a biological activitythe same as or more, several different, defined particlesizes have been successfully utilized as a basis for opera-ting NACS in different chromatography systems (HPLC,peristaltic pump, gravity flow).

J. A. Thompson, R. W. Blakesley, K. Doran, C. J. Hough andR. D. Wells, "Methods in Enzymology" 100, Part B(1983).

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THIRD ANNUAL CONGRESSFOR

RECOMBINANT DNA RESEARCH

AN AUTOMATED AND RAPID SYNTHESIS OF THE DOUBLESTRANDED LAC-OPERON DNA

G. Alvarado-Urbina and M. MichalakBIO LOGICALS - DNA Research Chemistry Section7 Hinton Avenue North, Ottawa, OntarioK1Y irPl Canada

The double stranded lac-operon DNA of the following sequence:

y AATT CTT GTG AGC GGA TAA CAA G 3'3' GAA CAC TCG CCT ATT GTT CCT AG 5'

has been synthesized by an automated stepwise addition procedure usingthe polymer support approach and appropiately fully protected bases(T,C,A and G) as previously described. (G. Alvarado-Urbina et al,Science, 2ik 270 (1980)). The deoxyribonucleosides were transformed toeither phosphomonochloridites or piperidino or morpholinophosphoramidites.

DMTO

R-P-OCH3

R:CI; ^N. ; JH^ OC^AAA/-©

The cycle time for each step is 18 min. A comparative study of thedifferent synthesis procedures used in terms of the overall yield andquality of the synthetic DNA was assessed. The highest yield wasobtained using the phosphomonochloridite as an intermediate. The yieldfor a single strand lac operon DNA varied from 1.2-1.6 mg per gram ofpolymer support.

The isolation and characterization of these two 23-unit long pieceswas accomplished using TLC, HPLC and polyacrylamide gelelectrophoresis and will be presented. Sequence of the isolated DNAwas confirmed by a modification of Maxam-Gilbert sequencingprocedure. (Banaszuk A.M., et al - Analytical Biochem (1982). In press).

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NONRADIOACTIVE DETECTION OF CLONED VIRAL SEQUENCES

Robert G. Pergolizzi, Steven Vogel, Barbara E.Thalenfeld, K. Steven LaForge, Huey Yang, KennethH. Johnston, Stanley Kline, Dollie Kirtikar, AshokPurohit, and Dean Engelhardt. Enzo Biochem, Inc.,New York, N.Y.

Cloned sequences derived from Epstein-Barrvirus, Herpes virus (types I and II), andHepatitis B virus were used as models for thedevelopment of a nonradioactive detection systemon Southern blots. These clones were nicktranslated with 2'-deoxy-UTP-5-allylamine coupledto either biotin (1) or maltotriose. These probeswere then hybridized to Southern blots (2) ofrestriction digests of the above viral DNAs in thepresence or absence of masking cellular DNA. Cotanalysis revealed no difference in hybridizationkinetics between substituted and native probes.Detection of hybridized probe DNA was accomplishedby incubating the blots with a complex composed ofhorseradish peroxidase coupled to eitherstreptavidin (for biotinylated hybrids) or ConA(for glycosylated hybrids). Addition of theappropriate substrate results in the rapiddevelopment of an identifiable coloredprecipitate. There was no detectable binding ofDNA to non-homologous sequences and generalbackground was extremely low.

Blots in which successive lanes containeddecreasing amounts of DNA showed that a DNA bandof 10-20 pg. can be detected. This sensitivity iswithin the range for single copy genes on Southernblots of eukaryotic genomic DNA.

(1) Langer, P.R., Waldrop, A.A., and Ward,P.C.Proc.Nat.Acad.Se i.,78;6633-66 37 (1981)

(2) Southern, E. Methods Enzymol.,68 ; 152-176(1979)

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IN SITU HYBRIDIZATION/DETECTION OF VIRAL SEQUENCESUSING NONRADIOACTIVE PROBES

B.E.Thalenfeld, S.Vedbrat, R.G.Pergolizzi,P.O'Hern, H.-L.Yang, K.H.Johnston, A.Purohit,S.Kline, S.Vogel, D.Engelhardt, and W.Prensky.Enzo Biochem, Inc., New York, N.Y.

We have developed a system for thenon-radioactive detection of specific viralpolynucleotide sequences using biotinylated DNAprobes. Adenovirus (Ad 2) DNA and both genomicand cloned Herpes Simplex virus DNA sequences(HSV-1) were labeled by nick translation with2'-deoxyuridine triphosphate 5-allylamine-biotin(1). Probe DNA was hybridized to Carnoy-fixedcells infected with the above viruses at varyingconcentrations and for varying lengths of time.Within fifteen minutes, sufficient biotinylatedDNA was hybridized to permit the visualization ofintracellular viral sequences. Detection wasbased on the interactions between biotin andstreptavidin or biotin and anti-biotin antibody.By linking either fluorescein or a

color-developing enzyme to these complexes, thecellular localization of the hybridizedbiotinylated probe could be visualized. Byvarying prehybridization treatments, both DNA andRNA viral-specific sequences could be identified.This assay system provides a simple, rapid,non-radioactive and safe means of detecting viralinfections.

(1) Langer,P.R., Waldrop, A.A., and Ward,P.C.Proc.Nat.Acad.Sei.,78; 6633-6637

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GLYCOSYLATED DNA PROBES FORHYBRIDIZATION/DETECTION OF HOMOLOGOUS SEQUENCES

Jannis Stavrianopoulos, Dollie Kirtikar, StanleyKline, Robert Pergolizzi, Christine Brakel,Kenneth Johnston, Callie Stathopoulos, DivinaGatica, Peter Pulkrabek, Elazar Rabbani and NormanKelker. Enzo Biochem, Inc., New York, N.Y.

In an effort to obtain safe, rapid,nonradioactive methods for labeling DNA we havedeveloped procedures for the production anddetection of glycosylated DNA. This work wasinitiated by the observation that glucosylated DNA(from phage T4) is precipitated by ConA. Aglucose substituted thymidine-triphosphateanalogue (2'-deoxyuridine-5-allylamine-maltotriose-5'-triphosphate) was synthesized andused to label DNA in a nick translation reaction.The rate and extent of incorporation were similarto thymidine triphosphate which it replaced.Reassociation kinetics showed that double helixformation was unperturbed by the presence of thesugar residues. Using standard procedures, theglucosylated probes were hybridized to specificDNA sequences on nitrocellulose filters.Hybridized probe was detected enzymatically by thesequential addition of ConA, a naturallyglycosylated enzyme and a color producingsubstrate, e.g., horseradish peroxidase (HRP) andH^Op plus diaminobenzidine. Enzyme activity islocalized at the hybridized glucosylated probe bythe tetravalent Con A which binds bothDNA-attached glucose and a sugar moiety on HRP.Signal amplification was achieved by premixing ConA with enzyme and adding the complex to thehybridized DNA. In this way, less than 5picograms of nitrocellulose-bound DNA could bedetected. The procedure has been usedsuccessfully in Southern blotting techniques.Examples will be presented.

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CLONING AND EXPRESSION OF CHICKEN GROWTH HORMONEIN £. COLIBoone, T., Murdock, D., Tallen, M., Martin, F.,Hockman, H., Altrock, B., DeOgny, L., Lai, P.,Wypych, J., Langley, K., Rudman, C., Stebbing, N.,and Souza, L., Applied Molecular Genetics Inc.,1900 Oak Terrace Lane, Thousand Oaks, CA 91320

Complementary DNA made from chicken pituitarymRNA was cloned into pBR322-SV40 vectors.Recombinants were screened for chicken growthhormone (cGH) sequences using a bovine growthhormone cDNA derived probe. The clone containingthe largest insert (cGH5) of the eight found tohybridize to bGH was sequenced by the dideoxymethod using M13-cGH5 clones as templates. Thesequence of cGH5 revealed the following featuresof the gene: 1) at least 50 bases of 5'untranslated sequence, 2) codons for a 26 aminoacid leader, 3) codons for a 190 amino acid matureprotein and 4) more than 100 bases of 3'untranslated sequence. Construction of a vectorfor the direct expression of cGH was carried outby priming a single stranded copy of the gene inphage M13 with a synthetic oligomer complementaryto the first 13 nucleotides coding for the first 5amino acids of the mature form of cGH. The finalform of the reconstructed cGH gene contains an ATGcodon adjacent to the first codon and uniquerestriction sites at either end of the geneallowing easy cloning of the gene into variousexpression vectors. Expression in _E_^ coli of cGHusing a trp promotor pBR322 vector was monitoredby polyacrylamide gel electrophoresis ofradiolabeled maxicells, whole cell extracts, andby RIA of crude bacterial lysates. The proteinmade in maxicells and whole cell extracts has a

molecular weight similar to natural cGH(Mr24,000). bGH isolated from bovine pituitarieswas labeled with 125j an¿j used in conjunction witha-cGH to generate a heterologous RIA to determinethe levels of cGH expression in E^ coli.Recombinant cGH purified from _E^ coli to near

homogeneity contains an N-terminal methionine asdetermined by protein sequence analysis. Thismaterial shows biological activity in variousanimal systems.

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RECOMBINANT DNA RESEARCHABSTRACT FORM

EXPRESSION OF pBR322 CODED AMPICILLIN RESISTANCE AS A SECRETED3-LACTAMASE PRODUCT IN BACILLUS SUBTILIS.Gordon L. Williams and Lucie K. Fritz, Battelle, Pacific North-west Laboratories, Richland, WA 99352

Several chimeric plasmids were produced by introducing a1.7 Kb fragment of the Staph aureus plasmid pC194 into pBR322.These plasmids replicate and express Chloramphenicol resistancein E^_ coli and B^ subtilis, as well as Staph. In the course ofisolating bacteriophage promoter regions with these plasmids, aclone was isolated which spontaneously expresses a high levelof Ampicillin resistance in the host B. subtilis. Assay ofscraped plates containing polyvinyl alcohol indicates that theprotein product is secreted by the cells in large quantities.This fact is confirmed by cell-free assay and isoelectricfocusing results, which also demonstrate the product to be thepBR322 coded AMP resistance protein, rather than another native3-lactamase produced in minor quantities by B. subtilis. Re-striction analysis of the plasmid indicates an unexpected.65 Kb insertion, upstream from the AMP coding region. DNAfrom this region hybridizes by Southern blotting with DNA fromthe bacteriophage 0105, but not with pC194. No homology isdetected by blotting the .65 Kb fragment against chromosomalDNA from b\_ subtilis or E_^ col i. Preliminary sequence dataindicates lack of homology between the insert and the siteof integration, except for a 29 base pair repeated sequenceat either end of the insert. Insertion occurs at the upstreamend of the Tn3 transposible element, which contains the AMPresistance gene of pBR322. The inserted element and thechimera are stably maintained in B. subtilis and E. coliunder CAM or AMP pressure.

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INTRODUCTION AND EXPRESSION OF THE HUMAN a1-ANTITRYPSINGENE IN CULTURED RAT LIVER CELLS. Xiao-zhou Shen*,Vincent J. Kidd, Eugene C. Lai, and Savio L. C. Woo.Howard Hughes Medical Institute and Department of CellBiology, Baylor College of Medicine, Houston, Texas 77030and *Institute of Zoology, Academia Sinica, Beijing, China.al-Antitrypsin is an important plasma proteinase in-

hibitor synthesized in the liver. It inhibits a number ofserine proteases including polymorphonuclear leukocyteelastase which is the principle enzyme capable of degrad-ing polymerized elastin. There is a genetic deficiency inman which is characterized by an abnormal phenotype of theprotein, and the deficiency is associated with a number ofdiseases including chronic obstructive pulmonary emphy-sema, infantile liver cirrhosis, renal disease, arthritisand some malignancies. The study of molecular pathologyhas shown the deficiency of al-antitrypsin is caused bya single amino acid substitution in the peptide chain. Inorder to study the deficiency at the molecular level, wehave isolated the human chromosomal al-antitrypsingene. Mapping and sequencing analyses have shown that thegene contains 4 intervening sequences and is approximatelylOkb in length to code of a mature mRNA of about 1400 nu-

cleotides. The availability of the cloned human al-antitrypsin gene has made it possible to study its expres-sion and regulation after transferred into cultured eu-

karyotic cells. In this study, a 15.2-kilobases DNA frag-ment of cloned human al-antitrypsin gene containing allof its structural segments, and intervening sequences aswell as 5' and 3'-flanking regions was subcloned into theEcoRI site of pSV2neo vector DNA. The chimeric moleculewere introduced into cultured normal rat liver cells(Clone-9 and BRL-3A) by the calcium phosphate precipita-tion procedure. Transformants were selected by an amino-glycoside antibiotic, G418, at a dosage of 0.2mg/ml.Transformed colonies were expanded and the presence of hu-man al-antitrypsin gene in these cell lines was estab-lished by Southern hybridization. The molecular organiza-tion of the integrated human al-antitrypsin gene was ob-served to be identical to the original gene clone in mostof the transformed lines, and the copy number of the generanged from 1 to 10. The expression of the human al-antitrypsin gene in the heterologous liver cells was mea-sured by Northern blot analysis. At least some of the 6transformants produced an al-antitrypsin RNA speciesthat comigrated with authentic al-antitrypsin mRNA on

denaturing agarose gels. The gene transfer system wouldthus permit the analysis of expression of the normal anddeficient al-antitrypsin genes in order to gain furtherinsight into the deficiency syndrome. (X.-z. S. is a fel-low supported by Rockefeller Foundation).

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A 7.5KB DNA INSERTION IN THE APOLIPOPROTEIN A-I (APO A-I)GENE IS RELATED TO THE DEVELOPMENT OF PREMATUREATHEROSCLEROSIS IN HUMANS. Sotirios K. Karathanasis,Vassilis I. Zannis, and Jan L. Breslow. Harvard MedicalSchool and Children's Hospital Medical Center, Boston, MA02115.

Two sisters with skin and tendon xanthomas, cornealopacities, and severe premature coronary atherosclerosiswith low HDL levels and deficiencies of two apolipoproteins,apo A-I and apo C-III, have recently been described. Thelesion in these individuals has been studied in thefollowing manner: Synthetic oligonucleotide probes for apoA-I were constructed based on it's amino acid sequence.These were used to select cDNA clones from a recombinant DNAlibrary made from human liver mRNA. A cDNA clone, pAI-113,corresponding to apo A-I amino acids 94-243 was used toprobe a human genomic library and select the normal apo A-Igene. This gene was sequenced and shown to have 3 exons and2 introns. Southern blot analysis utilizing pAI-113 as a

probe revealed differences between normal and apo A-Ideficient individuals after digestion with several differentrestriction enzymes. Therefore, the lesion in thesepatients could not be explained by a single basesubstitution but required either a deletion or insertion ofDNA which upon mapping had to involve the region upstream ofthe probe. A portion of this region of the normal apo A-Igene was cloned and when used to probe the patients' DNArevealed the presence of a 7.5kb insertion in the region ofthe 3' end of the second intervening sequence. Thisinsertion is presumed to affect apo A-I synthesis and resultin low HDL levels and severe premature atherosclerosis.Genomic blot analysis of chromosomal DNA prepared from thefirst degree relatives of these patients showed that this7.5kb DNA insertion segregates as a typical autosomalrecessive Mendelian trait.

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A NEW DELETION OF THE hGH-VARIANT AND hCS GENES.John S. Parks, Jan E. Herd and Peter V. Nielsen,Department of Pediatrics, Emory University, Atlanta,Georgia and Department of Gynecology, University ofCopenhagen, Herlev, Denmark.

The hGH and hCS gene cluster on human chromosome17 consists of at least 5 members. Genes containedin 2.8 kb Eco RI fragments of cellular DNA code forthe fetal placental hormone hCS. Fragments of 2.6kb contain the gene for normal pituitary hGH and asecond, non-allelic hGH-V gene which codes for ahormone with similar biologic potency but little RIAcross-reactivity. Large Eco RI fragments of 9.5 kbcontain an nhGH-liken gene with unknown sequence andfunction. Extensive base sequence homology amongthe genes has provided opportunity for deletionswithin the hGH/hCS gene cluster. The phenotypesdemonstrated by individuals with deletions of hGH orhCS genes provide insight into the physiologicalsignificance of these genes and their products.Maternal serum obtained during the first pregnancyof a 23 year old Danish woman was found to containno detectable hCS by RIA. As in other reportedinstances of hCS deficiency, the infant was ofnormal length and weight at birth. Genomicrestriction analysis of white blood cell DNA usingan hCS cDNA probe disclosed a new variety of hGH-Vand hCS gene deletion. Eco RI digests of theinfant's DNA showed hybridizing fragments of 10.0,8.0 and 2.6 kb with no detectable hybridization at9.5 or 2.8 kb. Bgl II and Mspl digests showedabsence of the fragments normally associated withhCS and hGH-V genes. Comparison of the child'srestriction patterns with those of her parentsshowed that she was a compound hétérozygote. Shehad inherited an hGH-V and hCS gene deletionidentical to the previously described deletion (DNA1:251-257) from her mother and a different hGH-V andhCS deletion, characterized by an 8.0 kb Eco RIfragment containing the "hGH-like" gene, from herfather. These findings demonstrate heterogeneity ofextended gene deletions responsible for thebiochemical phenotype of hCS deficiency. They alsoprovide confirmatory evidence that the products ofthe hGH-V and hCS genes are not required formaintenance of pregnancy or for normal intrauterinegrowth.

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CLONING OF cDNA ENCODING THE PRE-ß-SUBUNIT OF MOUSE THYRO-TROPIN. J.A. Gurr, J.F. Catterall* and I.A. Kourides, MemorialSloan-Kettering Cancer Center and *The Population Council andThe Rockefeller University, New York, N.Y. 10021.

Thyrotropin (TSH) is a glycoprotein hormone consisting oftwo dissimilar, non-convalently bound glycosylated subunits, aand 3. The TSH-a subunit is probably identical with that forFSH, LH, and CG within a species, whereas the ß subunit isunique and confers biologic specificity to the complete hormone.The a and ß subunits are synthesized from separate mRNAs. Wereport here the cloning and nucleotide sequencing of mouse TSH-ß cDNA. Double-stranded cDNA was synthesized from sucrosegradient-purified poly(A)+ mRNA from a mouse thyrotropic tumorusing reverse transcriptase, inserted into the Pst I site ofthe plasmid pBR322 using poly(dC)-poly(dG) tailing and clonedin E. coli RRI. Plasmids containing cDNA encoding the TSH-ßsubunit were identified by cell-free translation of hybrid-selected mRNA and immunoprecipitation with TSH-ß-specificantibody. The nucleotide sequence of a cDNA, 595 nucleotidesin length, was determined by the chemical degradation methodof Maxam and Gilbert and the entire amino acid sequence of themouse TSH-ß subunit was deduced. The pre-TSH-ß subunitcontains a 20 amino-acid amino terminal signal sequencefollowed by a 118 amino acid mature TSH-ß protein. There is85-90% homology in amino acid sequence between mouse TSH-ßsubunit and human, pig, and cow TSH-ß with complete con-servation of all 12 cysteine residues. The mouse subunit isunique in that it contains an additional 5 or 6 amino acidsat its carboxyl terminus compared to the bovine or human andpig subunits, respectively. Computer-assisted metric analysishas been used to detect homologies between the nucleotidesequences of mouse TSH-ß, mouse a, and human CG-ß. TSH-ß mRNAfrom mouse thyrotropic tumor was estimated to be 750nucleotides in length by hybridization with labeled TSH-ß cDNA.

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THE MOUSE GLANDULAR KALLIKREIN GENE FAMILY

*A.J. Mason, B.A. Evans, D. Cox , J. Shine and R.I. Richards,8entre for Recorfoinant DNA Research.and Department of,enetics, Research School of Biological Sciences, Canberra.*Department of Biochem. and Biophys, University of California,San Francisco, California 94143, U.S.A.

The glandular kallikreins represent a class of trypsin-like enzymes which form a distinct sub-group of the mammalianserine proteases. They are characterized by their ability torelease vasoactive peptides (kinins) from kininogen. Howeverthe observed kininogenase activity of different kallikreins ishighly variable. This finding, along with the demonstrationthat certain kallikreins are involved in the specific matura-tion of growth factors, indicates that these enzymes perform a

range of physiological functions. In an attempt to define allthese different activities, we have decided to determine thenumber and structure of genes encoding members of the kalli-krein family.

A kallikrein cDNA clone was used to screen a mousegenomic library giving a total of 29 unique clones. Res-triction mapping of these clones reveals that kallikrein genesare frequently closely linked, with intergenic spacer regionsin the order of 4 kb. Mapping of a series of overlappinggenomic clones has shown that three of the kallikrein genesare arranged in a cluster, separated from genes on either sideby at least 10 kb. In other cases, genes appear to bepresent in pairs. A comparison of our genomic DNA cloneswith fragments seen in genomic blots indicates that there are25-30 separate kallikrein genes within the mouse genome.

We have demonstrated that all of these genes are localized onchromosome 7 by Southern blot analysis of genomic DNA isolatedfrom a series of mouse-Chinese hamster somatic cell hybridsknown to contain different combinations of mouse chromosomes.

Differences in the coding potential of several genesappear to be related to the substrate specificity of thedifferent enzymes. This observation, as well as the appar-ently large number of kallikrein genes, is consistent with theproposal that a range of different kallikreins perform a widevariety of physiological functions.

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ISOLATION OF A BACTERIAL cDNA CLONE CODING FOR THE BETASUBUNIT OF RAT LUTEINIZING HORMONE. Mark A. Tepper andJames L. Roberts, Department of Biochemistry and Center forReproductive Sciences, Columbia College of Physicians andSurgeons, N.Y., N.Y. 10032Luteinizing hormone (LH) and follicle stimulating hormone

(FSH) are part of a family of hormones called theglycoprotein hormones. Each member of this family containstwo noncovalently linked subunits designated alpha and beta.Within a given species, the a-subunits of each hormone areall identical and the ß-subunits differ greatly. Thus, theß-subunit appears to specify the biological activity of thehormone. As an initial step in understanding the molecularmechanism by which LH and FSH are regulated, we have cloneda cDNA for the ß-subunit of rat LH. This cDNA was isolatedfrom a library made by the reverse transcription of mRNAextracted from the anterior pituitary of ovariectomizedrats. Two thousand recombinants were screened byhybridization to a P-labeled human LH gene clone [giftfrom John Fiddes and Karen Talmadge, Cold Spring HarborLabs, described in Boorstein et. al., Nature 300, 419-422(1982)]. Three bacterial clones were isolated thathybridized strongly to human ß-LH. Sequence analysis ofplasmid DNA from these clones shows that they code for ratß-LH. The largest cDNA insert of 400 base pairs contains theentire coding sequence of rat ß-LH, but is missing thehydrophobic amino terminal signal sequence. Translation ofthe rat ß-LH cDNA sequence into amino acid sequence revealsan 85% and 73% homology with those of porcine and humanß-LH, respectively. This cDNA clone is also being used tostudy the regulation of ß-LH gene expression by gonadalsteroids. Pituitary RNA from ovariectomized and castratedrats as well as ovariectomized with estadiol replacementtherapy is being analyzed by dot blot hybridization. Thecellular distribution of the ß-LH mRNA sequences in ratpituitary is being studied by i_n situ hybridizationhistochemistry.

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AN EVOLUTIONARILY CONSERVED LARGE REPETITIVE ELEMENT LIES 3KILOBASES 3* TO AN ADULT MOUSE BETA GLOBIN GENE. Frank H.Burton, Charles F. Voliva, Marshall Hall Edgell, and Clyde A.Hutchison III, Department of Microbiology and Immunology,University of North Carolina, Chapel Hill NC 27514.

A large repetitive element at least 6 kb long occursroughly 3 kb 3' to the adult mouse beta globin gene, Hbb-BetaOne. This element is a member of a large interspersed repeatfamily which we call MDR1 (Mus domesticus Repeat One). MDR1has been partially characterized by a number of otherlaboratories as well as our own. We have extended theestablished map of this family by 500 base pairs. This mapclosely corresponds to the map of the large repetitiveelement in the globin locus.

The structure of this repetitive element differs betweenthe globin haplotypes Hbb-s and Hbb-d (Weaver, et al., Cell24, 403-411, 1981). Sequence rearrangements cause some ofthese structural differences. In one case a sequence near orwithin the 5' end of the element in one haplotype isrelocated to the opposite end in the other haplotype. In asecond case a sequence interrupts the 3' region of theelement in one haplotype and is deleted in the otherhaplotype. In both cases these sequences are single-copy DNA.Consequently, the sequence deleted from the repetitiveelement in the second example is not found in the genome ofthis mouse.

This repetitive element is similar in its size andlocation to the 6.4 kb repeat which occurs 3 kb 3' to thehuman adult beta globin gene. Both human genomic DNA andcloned DNA containing the 6.4 kb human repeat hybridize tofragments derived from the mouse globin region repeat. Theseresults suggest that these repeats, and consequently themouse MER1 family and human Kpn I family, are evolutionarilyrelated.

We have developed a two step hybridization procedure foridentifying sequences in a gene cluster which are eitherdispersed or contiguous subdomains of a large repeat family.This procedure, independent of restriction-map comparisons,allows us to diagnose the repetitive sequence in the mouseadult globin region as an organized structure rather than afortuitous aggregation of smaller unrelated repeats. In thismethod filter-bound DNA is first hybridized to unlabeledgenomic DNA (vrtiich contains large repetitive sequences) andthen, after washing, to a labeled probe which is nothomologous to the filter-bound DNA but is homologous to adifferent portion of the large repetitive unit. This "DNAsandwich technique" is analogous to the RNA sandwichprocedure of Dunn and Hassell (Cell JL2, 23-36, 1977), and hasother potential applications related to establishing thelinkage of non-homologous DNA sequences.

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THE LONG TERMINAL REPEATS AND ASSOCIATED FLANKING SEQUENCES OFA MOUSE VIRUS-LIKE GENETIC ELEMENT. Clague P. Hodgson, PaulaK. Elder and Michael J. Getz, Department of Cell Biology, MayoClinic/Foundation, Rochester, MN 55905.

Mouse VL30 elements are a class of retrovirus ortransposon-like 5Kb DNA sequences. Multiple copies arearranged in a generally polymorphic fashion in all testedrepresentatives of the genus Mus, and at least some of theseare expressed as a 30S RNA which can be pseudotyped in murinetype C retroviruses and transmitted to heterologous cell types(1,2). In contrast to endogenous mouse retroviruses, however,the accumulation of VL30 RNA appears to be highly responsiveto epidermal growth factor (EGF) stimulation of quiescentcells in culture (3). One VL30 element examined here has beenfound to contain a set of 601bp terminal redundancies whichbear many similarities to retroviral long terminal repeats(LTRs); they are direct repeats with short inverted repeattermini, they appear to duplicate 4bp of cellular DNA uponintegration, and they contain sequences analogous to retro-viral transcriptional control signals. Unlike most murineretroviruses, an open reading frame of 57 amino acid residuesfollows the potential transcription start site, and is encodedentirely within the LTR. Although little sequence homologywas found to the LTRs of murine retroviruses, there arenevertheless several short, but distinct homologies to regionsof murine retroviruses which are believed to facilitatetranscription, replication, or integration. First, a set of36bp tandem repeats in the VL30 LTR is partially homologous toa set of 72bp tandem repeats in the Moloney murine sarcomavirus which may function as an enhancer of transcription.Second, a lObp sequence at one terminus, including theinverted terminal repeat, is part of a homology with murineleukemia viruses (MuLV) which includes the (-) strand tRNAprimer binding site. Lastly, a strong homology to the (+)strand origin of Moloney murine leukemia virus replication isfound adjacent to the opposite LTR. Together, these dataestablish that VL30 elements are structurally analogous tointegrated retrovirus proviruses and certain classes oftransposable genetic elements. The existance of sequence ho-mology with regions of MuLV proviruses believed to function inreplication and integration provides a molecular basis for thenotion that VL30 elements replicate and disperse themselves byMuLV-assisted mechanisms. This work was supported by NIHgrant GM25510 and by the Mayo Foundation.1. Courtney, M.H., et al., J. Virol. 43:511-518, 1982.2. Sherwin, S.A., et al., J. Virol. 26:257-264, 1978.3. Foster, D.N., et al., Proc. Nati. Acad. Sei. USA, in

press, 1982.

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INHIBITION BY PROTAMINE GENE OF TRANSFORMATION OF MOUSE Ltk" CELLS BY A HSV-1 tk GENE.J.C. States, J. Jankowski, J. Eames & G.H. Dixon, Dept. ofMedical Biochemistry, University of Calgary, Calgary,Alberta Canada, T2N 4N1.

We have utilized the calcium_phosphate precipitationmethod to co-transfer mouse L tk" cells with a herpessimplex virus-1 thymidine kinase (HSV-1 tk) gene and anintact rainbow trout protamine gene. In addition, we haveused several modifications of the protamine geneencompassing inclusion of an SV-40 origin of replication ora HSV-1 tk gene promoter region, a fusion gene of the tkpromoter and protamine mRNA coding region, and a plasmidcontaining both the native protamine and HSV-1 tk genes.The transformation frequency with the HSV-1 tk gene asmeasured by colony growth in selective HAT medium wasdecreased by the presence of the native protamine gene on aseparate plasmid in the co-transformation. The presence ofHSV-1 tk gene elements in the plasmid containing theprotamine gene counteracted the protamine gene inhibition oftransformation by the HSV-1 tk gene. The fusion gene showedless ability to inhibit the establishment of colonies by thetransformation with the HSV-1 tk gene, as did the protamineplasmid containing the SV-40 origin of replication. Thenative protamine gene does not appear to be expressed withinthe first two days of culture in non-selective media, but,after 4 weeks, RNA hybridizable to a nick-translatedprotamine DNA probe is present. In contrast, such RNA isproduced continually in cells transformed with an intactprotamine gene with a HSV-1 tk promoter located in theregion 5' to it. These results suggest that expression ofan unaltered protamine gene in heterologous cells causescell death under conditions which limit their growth.(Supported by a grant to G.H.D. from the Alberta ProvincialCancer Hospitals Board and Alberta Heritage Foundation forMedical Research Post-doctoral Fellowships to J.J. andJ.C.S.)

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USE OF VARIOUS RECOMBINANT PLASMID TO STUDY PROTAMINE GENEEXPRESSION IN VITRO. Jacek, M. Jankowski and Gordon H.Dixon, University of Calgary, Calgary, Alberta, Canada,T2N 4N1.

An in vitro approach has been used to study troutprotamine gene expression. Different recombinant plasmidscontaining trout protamine genes were used as a template.Initially pTPlOl, a Bam HI-Eco RI subclone of the originallambda charon 4A protamine clone was used as a templatebut since 1.5 kb of this plasmid was uncharacterized, new

plasmids pJBRP and pJP22 were constructed in which a 920b.p. Bgl II

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Bam HI protamine containing fragment wasinserted into the Bam Hi site of pBR 322 (pJBRP) orbetween the Bam HI and Mbol 1666 sites of pBR 322 (pJP22) .

Using the HSV tk gene, recombinant plasmids wereconstructed in which the promoter fragment of tk(PvuII-Bglll) was inserted tandemly upstream from the 5'region of the intact protamine gene (pJPltk) or this tkpromoter was inserted between Pvu II

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Bam HI sites in pBR322 (pB3). A plasmid with a new fusion gene in which theprotamine gene is under the control of the tk promoter(Pvu II - Mlu I fragment) was also constructed (pM7). Forin vitro transcription, a HeLa cell lysate system was

prepared and the RNA transcription products, afterglyoxalation, were electrophoretically analysed on 5%polyacrylamide gels. After linearization, plasmids pJBRPand pJP22 after cutting with Bgl II, Hpa II, Rsa I, Fole Iand Bam HI showed good correlation of the sizes of thetranscripts with those expected from the known protaminegene sequence. Transcription is a-amanitin (1 ug/ml)sensitive. When pJPltk, pB3 or pM7 plasmids were comparedas a templates, we have found that the natural protaminepromoter is much stronger than the HSV-1 promoter tk. Thetk promoter in the presence of protamine promoter (pJPltk)enhances transcription of protamine gene. (Supported byM.R.C. of Canada and the Alberta Heritage Foundation forMedical Research).

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REGULATED EXPRESSION OF HUMAN INTERFERON GENES INCHINESE HAMSTER OVARY CELLS. F. McCormick, M. Trahey, G.Ringold*, M. Innis, Cetus Corp., 600 Bancroft Way, Berkeley, CA.,*Dept. of Pharmacology, Stanford University, Stanford CA.

We have studied the expression and regulation of human IFN genesin CHO cells. IFN genes (beta-1, alpha-61 and alpha-76) were engi-neered for expression directed by their own promoters or by the SV40early promoter. Plasmids containing these genes and mouse dihydro-folate reducíase (dhfr) cDNA sequences were transfected into dhfr-CHO cells and dhfr+ transformants tested for IFN expression.Genomic sequences 5' to IFN coding regions were required forinducible expression of beta-IFN and alpha-IFN. In both cases, IFNactivity could be induced by double-stranded RNA or infection withNewcastle Disease Virus. The levels of expression per cell wereextremely sensitive to cell density. IFN genes utilizing SV40promoters were not inducible and expression was insensitive to celldensity. Cells containing many copies of the cloned dhfr gene wereselected by exposing populations to increasing concentrations ofmethotrexate, and IFN expression was measured in surviving^ cells.One cell line, resistant to 30 nM methotrexate secreted 10° U ofglycosylated human fibroblast IFN per 106 cells per day after super-induction. This level is 100 times higher than that of the cell linefrom which it was selected and appears to be a result of co-amplification of the transfected beta-IFN gene. The biological andbiochemical properties of the IFN secreted by these cells will bepresented.

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HAMSTER GENE SEGMENT CODING FOR ASPARTATE TRANSCARBAMYLASECOMPLEMENTS E. COLI PYR B MUTANT. Jeffrey N. Davidson and Lee A.Niswander. ETeanor Roosevelt Institute for Cancer Research andDepartment of Medicine, University of Colorado Health SciencesCenter, Denver, CO 80262

The multifunctional CAD protein in mammalian cells carriesthe first three enzymes of pyrimidine biosynthesis (carbamylphosphate synthetase II, aspartate transcarbamylase, dihydro-orotase). Although all three activities are covalently linkedon a single polypeptide, each enzyme is organized as anindependent, functional domain. A cDNA sequence correspondingto a portion of the 3' end of the Syrian hamster CAD gene andinserted in place of the Tet region of pBR322 was kindly pro-vided by Dr. George Stark. This plasmid was transformed into¡E_. coli Pyr B (aspartate transcarbamylase") and Pyr C (dihydro-orotase") mutants in order to test for complementation. Trans-formants were selected for growth on ampicillin (the plasmidmarker) or on medium free of uracil. None of the Pyr C trans-formants could grow in the absence of uracil, while all thePyr B transformants were both drug resistant and prototrophicfor uracil. Plasmid DNA was isolated from a few Pyr B trans-formants. Their patterns of DNA fragments after digestion withPst I were identical to that of the original cDNA plasmid. Inaddition, plasmid DNA from primary transformants could yieldprototrophs after secondary transformation of Pyr B mutants.Unlike the £. coli enzyme, the aspartate transcarbamylase ofthe Pyr B transformants failed to be inhibited by CTP orstimulated by ATP. The activity of the transformants could beimmunoprecipitated by antiserum which specifically binds ham-ster CAD protein. These results demonstrate that a cDNAsequence coding for a domain of hamster CAD protein cancomplement an E. coli mutant defective in pyrimidine biosyn-thesis. This data is essential in establishing the order ofthe DNA sequences encoding the enzyme activities of the CADprotein as: 5'-dihydroorotase-carbamyl phosphate synthetase II-aspartate transcarbamylase-3'. Finally, this may be a generalmeans for the isolation and genetic analysis of other mammaliangenes. Supported by grants from the American Cancer Society(FRA242) and the National Institutes of Health (HD02080).ERICR contribution #420.

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A SEQUENCING PROTOCOL WHICH EMPLOYS IODINATED-SINGLESTRANDED M13 PROBES TO "WALK" ALONG A DNA MOLECULE--SEQUENCESOF A MOUSE LYSINE tRNA GENE AND A PUTATIVE tRNA PSEUDOGENE.Jang H. Han and John D. Harding. Dpmt. of Biological Sciences,Columbia Univ. N.Y. 10027

We have employed the following protocol to sequence mouse

tRNA genes cloned in lambda phage. M13 subclone banks are

prepared, screened with tRNA and the hybridizing M13 clones are

sequenced. As a novel feature, iodinated, single-stranded DNAfrom the sequenced M13 clone is used to detect additional M13clones which contain sequences partially overlapping and alsocontiguous to that determined initially. The iodinated probeswill also identify M13 clones containing the same sequencecloned in the opposite orientation in M13. Thus it is possibleto rapidly "walk" along a DNA molecule and determine itssequence on both strands.

A 1.8 kb EcoRI-Xbal mouse DNA fragment contains a single lystRNA (anticodon UUU) gene. The putative lys tRNA differs fromsequenced lys tRNA at 5 positions, all variable in eucaryotictRNAs. The flanking regions are not generally homologous topublished human and Drosophila lys tRNA gene sequences. How-ever, the mouse gene contains a 14 bp region comprising 13A-T bp, located 30-44 bp from the 5' end of the coding region.Cognate A-T rich regions are present in the human andDrosophila genes. The coding region is flanked by 2 11 bpdirect repeats, similar to those associated with Alu sequences.

A 395 bp sequence derived from a different lambda clonehybridizes weakly with tRNA but contains no identifiabletRNA coding regions. A putative pseudogene structure ispresent which contains analogs of the D, Anti-codon and T-pseudoU stems and loops. Several, but not all, of thesemi- and invariant residues characteristic of eucaryotictRNAs are present. Mutations include the loss of the amino-acyl stem and insertions and deletions in the loops.

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"QUICK-BLOT" TECHNIQUE FOR mRNA IMMOBILIZATION DIRECTLY FROMWHOLE CELLS. David Gillespie and Joel Bresser, Barry AshbeeLeukemia Research Laboratories, H.L. Orlowitz Institute for theStudy of Cancer and Blood Diseases, Hahnemann University, Phil a.PA 19102

Message RNA (mRNA) dissolved in Nal will adsorb to nitro-cellulose membranes. At low temperatures, binding of ribosomalRNA, native DNA and denatured DNA is poor. Immobilized mRNAis available for molecular hybridization and can be translatedinto proteins or reverse transcribed into DNA. When intactcells are disrupted, dissolved in Nal and filtered throughnitrocellulose membranes, mRNA selectively binds to the filtermaterial. This immobilized RNA can also be used for molecularhybridization, translation or reverse transcription. Detailsof the method will be presented.

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ANALYSIS OF THE env GENE OF MOLECULARLY CLONED MCF/MULVRECOMBINANTS ISOLATED IN VITRO WHICH ARE CAPABLE OF TRANS-FORMING CELLS IN CULTURE. G.E. Mark and U.R. Rapp,Laboratory of Viral Carginogenesis, National CancerInstitute, Frederick, MD 21701

Several MCF class recombinant MuLVs have been isolatedfrom IdUrd induced C3H/MCA 5 cells in culture and molecularlycloned. Two of these viruses are capable of transformingmink lung epithelial cells to anchorage independent growth,as well as producing tumors when infected into newborn NFS/Nmice. Restriction maps and heteroduplex analysis revealthese genomes to be closely related to an AKV parent; one

genome (CI-3) was full length while the other (CI-4) con-tained a deletion identical to that found in spleen focusforming virus (SFFV). 2408 nucleotides of CI-3 MA,including the MCF envelope gene, has been sequenced andcompared to ecotropic AKV and dualtropic Mo-MCF sequences.The recombination junctions are within the polymerase geneand 15 nucleotides 5' of the gp70/p15e cleavage site. The597 nucleotide Prp15e sequence contains 5 base changesrelative to AKV, occurring close to the recombinationaljunction, the U3 sequences are identical to those of AKV(T1 #101 is absent). The MCF glycoprotein region is approxi-mately 99$ homologous to that of Mo-MCF. The SFFV-likedeletion of CI-4 has removed 696 nucleotides which code for 3of the 6 glycosylation sites of gp70 and the extensivehydrophobic regions surrounding the gp70/p15e junction.Interestingly, this delection is flanked by a direct repeat(TGGTANCGGGA). These data will be presented in detail aswell as mRNA and protein characterizations of the transformedcells in an attempt to uncover the mechanism oftransformation.

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CHANGES IN CHROMATIN OF THE BRAIN OF RATS DURING AGINGM.M. Chaturvedi and M.S. Kanungo, Biochemistry Laboratory,Dept. of Zoology, Bañaras Hindu Univ., Varanasi 221005, India

Earlier studies from this laboratory using nuclei of thebrain of rats of various ages have shown that acetylation andbutyrate-mediated hyperacetylation of histones and subsequenttranscription decrease as a function of age. The possibilityof such changes occurring in the chromatin due to conformâtionalchanges in the chromatin of the brain were probed by two endo-nucleases, DNase I and micrococcal nuclease (MCN), using 20,45 and 90 week old rats. The kinetics of digestion by DNase Ishow that the digestibility of chromatin decreases significantlywith age. Furthermore, the production of 10 and 20 bp fragmentsis far less in older rats as seen by denaturing polyacrylamideslab gel electrophoresis. However, neither the digestibilityof DNA nor the production of monomer DNA (200 bp) or its multi-ples by MCN change with age. These data show that conforma-tional changes occur in the higher order structure of chromatinwith progressive age. Such changes may limit the access of a

large molecule like DNase I to the closely spaced (10.4 bp)cutting sites on DNA. MCN being a smaller molecule, may haveaccess to its cutting sites which are spaced far apart (200 bp).Hence, no differences in the digestion by MCN are observed withprogressive age.

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CODING STRUCTURE ANALYSIS AND CLASSIFICATION OF POLYNUCLEOTIDE SEQUENCES: SOME APPLICATIONS AND IM-PLICATIONS. A. 0. Nazarea, Cancar for Studies la Scat. Kech, & Thernodynaalca, University of Taxa«ac Austin, Austin, Taxaa 78712.

A voluminous corpus of raw DNA sequence data haa now bacom« accasaibla owing to enormous advan-ces in the direction of automated sequencing. And the catalogue of raw data can only grow at anever increasing rate. This fact, as well aa the discovery of highly repeated sequences, conservedsequences, and genomic activation by inaertlon and transpoaable elements and by singlet mutation,underscore the need for a sufficiently encompassing analytical method on the characterization andand interpretation of Information encoded is polynucleotide sequences. There now exists a repre-sentation theory for the analysis and claaslfication of such sequences based on the use of pseudo-scalar gauge encoding (A.D.Nazarea, Proc.Nati.Acad.Sei.,to appear). In this representation, allpossible sequences of a given length form a commutative (or Abelisn) group, and it la in Che con-text of thia exhauatlve group of aaquencea that the analysis of Information encoding is attemptedusing autocorrelations! and Fourier spectral methods. The talk will briefly explain this theory,together with some of its possible applications and implications. In parallel with the above,a simple conceptual model of a nonequilibrlum parametric sequence generator (with a well charac-terized structural parameter y) has been developed, based on interferometric principles (A.D.Naza-rea, Adv.Chem.Phys.,to appear). The model Is capable of generating both palindromic (dyadicallysymmetric) and non-palindromic fundamental (block) repeating sequence units (FRO's). Informationretrieval from such artificially generated sequences (in terms of the so-called Y-peaka) will bediscussed in Che talk. The sequence autocorrelation for a single-stranded chain produced by thesequence generator with r-0.9 and a 256—mer length is shown In Fig.A. From the sequence autocor-relation function can be directly derived the Fourier spectral signature of the sequence, shown inFig.B for the same sequence length and i-O.tt. The prominent peak is called the Y-peak and is themain (diagnostic) feature of the spectral signature of the 256-mer sequence. It can be shown that

Fig. Ae

¡oh

_J_J

Fig.B

the characteristic FRU corresponding to the spectral signature displayed above (Fig.B) Is a palin-dromic subsequence P -(PyPuPuPy), together with a Pu spacer. Here, Pu and Py stand for purlne andpyrimidine beses, respectively. Thus, the entire 256-mer sequence can be written as a compound tan-dem repeat: poti/((PyPuPuPy)Pu). Note that any sequence generated as above is of 'first order1 inthe following specialized sense: that the sequence does not necessarily take into account the givenfact chat some additional periodicity constraint (involving dlnucleoclde periodicity, for example)must be satisfied (E.N.Trifonov & J.L.Sussman, Proc.Nati.Acad.Sei.,77(1980),3816) if, to follow theexample, the strand (and Its complement) were required, chromatin-like, to fold smoothly Into a

nucleosomal configuration— implying (as a 'second order' constraint) that the sequence as a wholepossess a well defined flexural anisotropy related to the pitch of the nucleosomal fold. The appli-cations which will be discussed in Che rest of the talk will include the conservation of informa-tion In chimaerlc (spliced) sequences, and the changes in the spectral signature of a sequence as

a result of a singlet base change (point mutation). This latter will be Illustrated using the pointmutation data on the T24 oncogene (C.J.Tabln, S.M.Bradley, C.I.Bargmann, R.A.Weinberg, A.G.Papageor-ge, E.M.Scolnick, R.Dhar, D.R.Lowry s C.H.Chang, Nature,300(1982),143; E.P.Ready, R.K.Reynolds, E.Santos & M.Barbacid, Nature,300(1982),149).

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EFFICIENT RECOVERY OF FUNCTIONALLY INTACT MESSENGER RNAFROM AGAROSE GELS, VIA TRANSFER TO A DEAE-MEMBRANE

Lene' J. Holland and Lawrence J. WanghDepartment of Biology, Brandeis University, Waltham, MA 02254

PolyA+ RNA can be fractionated with maximum resolution byelectrophoresis in agarose gels containing the reversibledenaturing agent methymercuric hydroxide. We have developed asimple procedure for the efficient recovery of functionallyintact mRNA molecules separated in these gels. RNA iselectrophoretically transferred from the gel to a commerciallyavailable diethylaminoethyl (DEAE) membrane. The pattern ofRNA bound to the membrane is a faithful replica of the RNAresolved in the gel. The efficiency of transfer is nearly 100%for RNAs up to 4000 nucleotides in length, the largest sizetested. Methylmercuric denaturation of the RNA is reversed bysoaking the DEAE-membrane with bound RNA in 20 mM ammoniumacetate. The membrane is then sliced into sections such thateach section contains a sharply limited set of mRNA moleculeshighly enriched for particular sequences. RNA molecules whichdiffer by as little as 200 nucleotides can readily be separatedby this method. Elution of RNA from the membrane isaccomplished at 65° using a high ionic strength buffercontaining 6M guanidine-HCl, 17 mM EDTA, 17 mM sodium acetate,50 ug tRNA, pH 6.5. Under these conditions the efficiency ofelution is at least 95% for RNAs of all sizes up to 4000nucleotide, the largest molecules tested. Use of NaCl ratherthan guanidine-HCl in the elution buffer results in recovery ofonly a small fraction of large RNA molecules. Messenger RNAisolated by this procedure is suitable for subsequent enzymaticreactions. We have used the method for analysis of mRNA fromXenopus liver. The recovered RNA could be translated intofull-length polypeptides and could be reverse transcribed intocomplementary DNA. On the basis of the translation assay weestimate an overall yield of 10% for a particular messenger RNAof 2300 bases. Thus, specific mRNA species can be recoveredfrom yg quantities of polyA+ RNA in an amount sufficient forsubsequent molecular manipulation.

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poster session abstracts from the third annual congress for recombinant dna research

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