Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

Molecular and Cellular Biology (Impact Factor: 5.04). 05/1982; 2(4):437-42. DOI: 10.1128/MCB.2.4.437
Source: PubMed

ABSTRACT The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

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Available from: Reginald Storms, Jan 12, 2015
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    ABSTRACT: Received 3November 1983/Accepted 13February 1984 Deoxycytidylate deaminase activity inSaccharomyces cerevisiae hasbeenpartially characterized. The yeast enzymewasfound toexhibit properties similar tothose ofdCMPdeaminases isolated fromhigher eucaryotes. A mutant strain completely deficient indCMPdeaminase activity wasisolated byselection for resistance to5-fluoro-2'-deoxycytidylate followed byscreening forcross sensitivity to5-fluoro-2'-deoxyuri- dylate, apotent inhibitor oftheyeast thymidylate synthetase. We havedesignated this newallele dcdl. A strain exhibiting anauxotrophic requirement fordUMP wasisolated after mutagenesis ofadcdltup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted fromacombination ofthedcdl allele andasecond, unlinked, nuclear mutation thatwe designated dmpl.Thisallele, whichbyitself conveys noreadily discernable phenotype, presumably impairs efficient synthesis ofdUMPfromUDP.The auxotrophic requirement ofdcdldmpltup7strains alsocanbesatisfied byexogenous dTMPbutnot deoxyuridine. Inrecent years numerous studies, inbothprocaryotic and eucaryotic systems, haveshownthat disturbances indeoxy- nucleotide biosynthesis arecapable ofinducing various kinds ofgenetic change (15). Previous workinourlaboratory hasshownthat inhibition ofdTMPsynthesis inSaccharomy- cescerevisiae results inenhanced frequencies ofmitotic recombination events, bothgeneconversion andmitotic crossing over(1,16). Inviewofthese findings, particularly those involving pyrimidine deoxynucleotides,
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    Proceedings of the National Academy of Sciences 05/1983; 80(7):1858-61. DOI:10.1073/pnas.80.7.1858 · 9.81 Impact Factor
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    ABSTRACT: Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.
    Journal of Bacteriology 06/1984; 158(2):644-9. · 2.69 Impact Factor
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