Summary
We isolated the LYS2 gene of S. cerevisiae on an autonomously replicating plasmid in a four-step procedure. First, we identified a recombinant plasmid which expressed a lys2 complementing activity upon yeast transformation of a lys2 mutant. Second, we determined the boundaries of the corresponding transcribed sequence in this plasmid by S1 nuclease mapping of the mRNA. Third, we inactivated the functional chromosomal copy coding for the lys2 complementing activity by directed integration of a plasmid that carried an internal fragment of the transcribed sequence. Fourth, we showed by a complementation test with an authentic lys2 mutant that the integration had inactivated the chromosomal LYS2 gene. This proved that the plasmid isolated in the first step indeed carried the LYS2 gene and not a suppressor of the lys2 mutation. The gene is unusually long (4.0 kb). It was used as hybridization probe in comparing LYS2 regions in various yeast strains and in a first construction of LYS2 based yeast vectors. Such vectors could be very useful because of the easy selection of lys2 mutants from any S. cerevisiae strain.
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References
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 257:104–109
Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196:180–182
birnboim NC, Doly J (1979) A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Bolivar F, Rodriguez RL, Green PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles: a multipurpose cloning system. Gene 2:95–104
Botstein DS, Falko SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW (1979) Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24
Broach JR, Strathern JH, Hicks JB (1979) Transformation in yeast: Development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133
Broquist HP, Stiffey HV (1959) Biosynthesis of lysine from adipic acid precursors by yeast. Fed Proc 18:198
Broquist HP, Trupin JS (1966) Amino acid metabolism. Annu Rev Biochem 35:231–274
Chattoo BB, Sherman F, Atubalis DA, Fiellstedt TA, Mehnert D, Ogur M (1979) Selection of lys2 mutants of the yeast S. cerevisiae by the utilization of α-aminoadipate. Genetics 93:51–65
Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics, a manual for genetic engineering. Cold Spring Harbor Laboratory, New York
Davis RW, Thomas M, Cameron J, St. John TP, Scherer S, Padgett RA (1980) Rapid DNA isolations for enzymatic and hybridization analysis. Methods in Enzymol 65:404–411
Donahue TF, Farabaugh DJ, Fink GR (1980) The nucleotide sequence of the His4 region of yeast. Gene 18:239–249
Fink GR (1966) A cluster of genes controlling three enzymes in histidine biosynthesis in S. cerevisiae. Genetics 53:445–459
Gafner J, De Robertis E, Philippsen P (1983) Delta sequences in the 5′ noncoding regions of yeast tRNA genes. EMBO J, 2:583–591
Hicks JB, Hinnen A, Fink GR (1979) Properties of yeast transformation. Cold Spring Harbor Symp Quant Biol 43:1305–1313
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Hohn B, Hinnen A (1980) Cloning with cosmdis in E. coli an yeast. In: Setlow JK, Hollaender D (eds) Genetic engineering principles and methods, vol 2. Plenum Press, New York London, pp 169–183
Hwang YL, Lindegren G, Lindegren CC (1966) Genetic study of lysine biosynthesis in yeast. Can J Genet Cytol 8:471–480
Karn J, Brenner S, Barnett L, Cesarini G (1980) Novel bacteriophage λ cloning vector. Proc Natl Acad Sci USA 77:5172–5176
Keesey JK Jr, Bigelis R, Fink GR (1979) The product of the his4 gene cluster in S. cerevisiae. J Biol Chem 254:7427–7433
Kingsman AJ, Clarke L, Mortimer RK, Carbon J (1979) Replication in S. cerevisiae of plasmid pBR313 carrying DNA from the yeast TRP1 region. Gene 7:141–153
Maniatis T, Fritsch EF, Sambrock J (1982) Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory, New York
Meyhack B, Bajua W, Rudolph H, Hinnen A (1982) Two yeast acid phosphatase genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences. EMBO J 1:675–680
Mortimer RK, Schild D (1980) Genetic map of S. cerevisiae. Microbiol Rev 44:519–521
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation, a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358
Ratzkin B, Carbon J (1977) Functional genetic expression of eukaryotic DNA in E. coli. Proc Natl Acad Sci USA 74:487–491
Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labeling DNA to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Roeder GS, Fink GR (1980) DNA rearrangements associated with a transposable element in yeast. Cell 21:239–249
Schell MA, Wilson DB (1979) Cloning and expression of the yeast galactokinase gene in an E. coli plasmid. Gene 5:291–303
Sharp PA, Berk AJ, Bergert SM (1980) Transcription map of adenovirus. Methods Enzymol 65:750–768
Shortle D, Haber JE, Botstein D (1982) Lethal disruption of the yeast actin gene by integrative DNA transformation. Science 217:371–373
Sinha AK, Battacharjee JK (1970) Control of a lysine biosynthesis step by two unlinked genes of Saccharomyces. Biochem Biophys Res Commun 39:1205–1210
Smith GE, Summers MD (1980) The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal Biochem 109:123–129
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stinchcomb DT, Struhl K, Davis RW (1979) Isolation and characterization of a yeast chromosomal replicator. Nature 282:39–43
St John TP, Davis RW (1979) Isolation of galactose inducible DNA sequences from S. cerevisiae by differential plaque filter hybridization. Cell 16:443–452
Strathern JN, Newlon CS, Herskowitz J, Hicks JB (1979) Isolation of a circular derivative of yeast chromosome III. Cell 18:309–319
Struhl K, Cameron JR, Davis RW (1976) Functional genetic expression of eukaryotic DNA in E. coli. Proc Natl Acad Sci USA 73:1471–1475
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Sutcliffe JG (1979) Complete nucleotide sequence of the E. coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90
Truffa-Bächi P, Cohen GN (1968) Some aspects of amino acid biosynthesis in microorganism. Annu Rev Biochem 37:79–105
Yang RCA, Liz J, Wu R (1979) Recovery of DNA fragments from gels. Methods Enzymol 68:178–181
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Communicated by W. Gehring
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Eibel, H., Philippsen, P. Identification of the cloned S. cerevisiae LYS2 gene by an integrative transformation approach. Molec Gen Genet 191, 66–73 (1983). https://doi.org/10.1007/BF00330891
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DOI: https://doi.org/10.1007/BF00330891