Abstract
TheILV1 gene from Saccharomyces cerevisiae coding for the anabolic threonine deaminase was isolated from a cosmid-yeast DNA gene pool on a 35 kb insert by transformation of a haploidilv1 yeast mutant to isoleucine prototrophy. By subcloning, the functional gene was isolated on a 4.8 kb DNA fragment. Hyridization of radioactively labelled DNA fragments from the cloned region to electrophoretically separated yeast RNA showed that theILV1 mRNA is about 2,000 ribonucleotides long. This analysis revealed also the approximate position of the transcribed region. The location of theILV1 gene in the cloned region was confirmed by gene splitting: The transformation of a wild type strain with a plasmid containing an internal fragment of the cloned gene caused inactivation of the chromosomalILV1 gene. By RNA hybridization (Northern) analysis, a 5–10 fold decrease in the threonine deaminase mRNA level was observed in wild type cells when minimal medium with leucine was supplemented with isoleucine and valine. This indicates thatILV1 is under transcriptional control.
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Abbreviations
- kb:
-
kilo base (pairs)
- NPD:
-
non-parental ditype
- PD:
-
parental ditype
- T:
-
tetratype
References
Ahmed, S.I., A.P. Bollon, S.J. Rogers &P.T. Magee: Purification and properties of threonine deaminase from Saccharomyces cerevisiae. Biochimie 58, 225–232 (1976)
Alwine, J.C., D.J. Kemp &G.R. Stark: Method for the detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl paper and hybridization with DNA probes. Proc. Nat. Acad. Sci. USA 74, 5350–5354 (1977)
Beggs, J.D.: Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–108 (1978)
Betz, J.L., L.M. Hereford &P.T. Magee: Threonine deaminases from Saccharomyces cerevisiae mutationally altered in regulatory properties. Biochemistry 10, 1818–1824 (1971)
Birnboim, H.C. &J. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7, 1513–1523 (1979)
Blazey, D.L., R. Kim &R.O. Burns: Molecular cloning and expression of theilvGEDAY genes from Salmonella typhimurium. J. Bacteriol. 147, 452–462 (1981)
Bollon, A.P.: Fine structure analysis of a eukaryotic multifunctional gene. Nature 250, 630–634 (1974)
Bollon, A.P.: Regulation of theilvl multifunctional gene in Saccharomyces cerevisiae. Mol. Gen. Genet. 142, 1–12 (1975)
Bollon, A.P. &P.T. Magee: Involvement of threonine deaminase in multivalent repression of the isoleucine-valine pathway in Saccharomyces cerevisiae. Proc. Nat. Acad. Sci. USA. 68, 2169–2172 (1971)
Bollon, A.P. &P.T. Magee: Involvement of threonine deaminase in repression of the isoleucine-valine and leucine pathways in Saccharomyces cerevisiae. J. Bacteriol. 113, 1333–1344 (1973)
Broach, J.R., J.F. Atkins, C. McGill &L. Chow: Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2 μ circle. Cell 16, 827–839 (1979)
Bussey, H. &H.E. Umbarger: Biosynthesis of branched-chain amino acids in yeast: Regulation of synthesis of the enzymes of isoleucine and valine biosynthesis. J. Bacteriol. 98, 623–628 (1969)
Calhoun, D.H. &J.E. Gray: Cloning of theilvA538 gene coding for feedback-hypersensitive threonine deaminase from Escherichia coli K-12. J. Bacteriol. 151, 274–280 (1982)
Delforge, J., F. Messenguy &J. Wiame: The regulation of the arginine biosynthesis in Saccharomyces cerevisiae: The specificity ofargR mutations and the general control of amino acid biosynthesis. Eur. J. Biochem. 57, 231–239 (1975)
Guidelines for research involving recombinant DNA molecules. Federal Register U.S. 47 (77), 17180–17198 (1982)
Holmberg, S., J.G.L. Petersen, T. Nilsson-Tillgren &M.C. Kielland-Brandt: Molecular characterization of a Saccharomyces plasmid containing theHIS4 gene. Carlsberg Res. Commun. 44, 269–282 (1979)
Ito, H., Y. Fukuda, K. Murata &A. Kimura: Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983)
Jones, E.W. & G.R. Fink: Regulation of amino acid and nucleotide biosynthesis in yeast. In: The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. J.N. Strathern, E.W. Jones & J.R. Broach, eds. Cold Spring Harbor, New York, vol. 11B, pp. 181–299 (1982)
Katsunuma, T., S. Elsässer &H. Holzer: Purification and properties of threonine dehydratase from yeast. Eur. J. Biochem. 24, 83–97 (1971)
Katz, L., D.T. Kingsbury &D.R. Helinski: Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolic repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J. Bacteriol. 114, 577–591 (1973)
Kielland-Brandt, M.C., T. Nilsson-Tillgren, S. Holmberg, J.G.L. Petersen &B.A. Svenningsen: Transformation of yeast without the use of foreign DNA. Carlsberg Res. Commun. 44, 77–87 (1979)
Klapholz, S. &R.E. Esposito: A new mapping method employing a meiotic Rec mutant of yeast. Genetics 100, 387–412 (1982)
Kovaleva, S.V., A.I. Dorozhko, N.F. Belayeva &Z.S. Kagan: Comparative kinetic properties of structure-bound and solubilizedl-threonine dehydratase from brewers yeast. Biokhimiya (Moscow) 44, 1906–1911 (1979)
Lehrach, H., O. Diamond, J.M. Wozney &H. Boedtker: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16, 4743–4751 (1977)
Lessie, T.G. &H.R. Whiteley: Properties of threonine deaminase from a bacterium able to use threonine as sole source of carbon. J. Bacteriol. 100, 878–889 (1969)
Livingston, D.M. &H.L. Klein: Deoxy-ribonucleic acid sequence organization of a yeast plasmid. J. Bacteriol. 129, 472–481 (1977)
Mandel, M. &A. Higa: Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159–162 (1970)
Maniatis, T., E.F. Fritsch & J. Sambrook: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, New York, pp. 264–267 (1982)
McCorkle, G.M., T.D. Leathers &H.E. Umbarger: Physical organization of theilvGEDA genes of Escherichia coli K-12. Proc. Nat. Acad. Sci. USA 75, 89–93 (1978)
Mortimer, R.K. &D. Schild: Genetic map of Saccharomyces cerevisiae. Microbiol. Rev. 44, 519–571 (1980)
Nilsson-Tillgren, T., M.C., Kielland-Brandt, S. Holmberg, J.G.L. Petersen & C. Gjermansen: Is lager yeast a species hybrid? Utilization of intrinsic genetic variation in breeding. Proc. 4th Int. Symp. on Genetics of Industrial Organisms, Kyoto, pp. 143–147 (1982)
Orr-Weaver, T., J.W. Szostak &R.J. Rothstein: Yeast transformation: A model system for the study of recombination. Proc. Nat. Sci. USA 78, 6354–6358 (1981)
Perkins, D.D.: Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34, 607–626 (1949)
Petersen, J.G.L., M.C. Kielland-Brandt, S. Holmberg &T. Nilsson-Tillgren: Mutational analysis of isoleucine-valine biosynthesis in Saccharomyces cerevisiae. Mapping ofilv2 andilv5. Carlsberg Res. Commun. 48, 21–34 (1983)
Rigby, P.W.J., M. Dieckmann, C. Rhodes &P. Berg: Labeling of deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251 (1977)
de Robichon-Szulmajster, H. &P.T. Magee: The regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae. I. Threonine deaminase. Eur. J. Biochem. 3, 492–501 (1968)
Ryan, E.D. &G.B. Kohlhaw: Subcellular localization of isoleucine-valine biosynthetic enzymes in yeast. J. Bacteriol. 120, 631–637 (1974)
Satayanarayana, T., H.E. Umbarger &G. Lindegren: Biosynthesis of branched-chain amino acids in yeast: Regulation of leucine biosynthesis in prototrophic and leucine auxotrophic strains. J. Bacteriol. 96, 2018–2024 (1968)
Shortle, D., J. Haber &D. Botstein: Lethal disruption of the yeast actin gene by integrative transformation. Science 217, 371–373 (1982)
Souciet, J.-L., J.-C. Hubert &F. Lacroute: Cloning and restriction mapping of the yeastURA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex. Mol. Gen. Genet. 186, 385–390 (1982)
Southern, E.M.: Detection of specific DNA sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 (1975)
St John, T.P., S. Scherer, M.W. McDonell &R.W. Davis: Deletion analysis of the SaccharomycesGAL gene cluster. J. Mol. Biol. 152, 317–334 (1981)
Struhl, K., D.T. Stinchcomb, S. Scherer &R.W. Davis: High-frequency transformation of yeast: Autonomous replication of hybrid molecules. Proc. Nat. Acad. Sci. USA 76, 1035–1039 (1979)
Thuriaux, P., M. Minet, A.M.A. ten Berge &F.K. Zimmermann: Genetic fine structure and function of mutants at theilvl gene locus of Saccharomyces cerevisiae. Mol. Gen. Genet. 112, 60–72 (1971)
Ulm, E., R. Böhme &G. Kohlhaw: α-isopropylmalate synthase from yeast: Purification, kinetic studies and effect of ligands on stability. J. Bacteriol. 110, 1118–1126 (1972)
Umbarger, H.E.: Amino acid biosynthesis and its regulation. Ann. Rev. Biochem. 47, 533–606 (1978)
Wolfner, M., D. Yep, F. Messenguy &G.R. Fink: Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J. Mol. Biol. 96, 273–290 (1975)
Yang, R.C.-A., J. Lis &R. Wu: Elution of DNA from agarose gels after electrophoresis. In: Methods in Enzymology, R. Wu, ed. Academic Press, New York, vol. 68, pp. 179–182 (1979)
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Litske Petersen, J.G., Holmberg, S., Nilsson-Tillgren, T. et al. Molecular cloning and characterization of the threonine deaminase (ILV1) gene of Saccharomyces cerevisiae. Carlsberg Res. Commun. 48, 149–159 (1983). https://doi.org/10.1007/BF02907764
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DOI: https://doi.org/10.1007/BF02907764