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

Molecular and Cellular Biology (Impact Factor: 4.78). 05/1982; 2(4):437-42. DOI: 10.1128/MCB.2.4.437
Source: PubMed


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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    • "Thymidine kinase of herpes simplex virus (HSV-TK), when expressed from appropriate promoters, is active in yeast and can also function as a thymidylate kinase, as it complements a loss of the CDC8 gene (25). In addition, HSV-TK converts 5-fluoro-2′-deoxyuridine (FUdR) into 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), which covalently binds and inhibits thymidylate synthetase (Cdc21p) (26,27). We used these characteristics of HSV-TK to examine whether telomerase was sensitive to nucleotide supply in vivo. "
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    ABSTRACT: An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both essential for de novo TTP synthesis. The effects of temperature-sensitive mutations in these genes have been characterized and, curiously, the phenotypes displayed by cells harboring them include shortened telomeric repeat tracts. This finding raised the possibility that the enzyme telomerase is very sensitive to TTP-pools. We tested this possibility in vivo by assessing telomerase-dependent extension in situations of lowered TTP supply. The results show that the above-mentioned short telomere phenotype is not a consequence of an inability of telomerase to elongate telomeres when TTP synthesis is impaired. Moreover, this telomere shortening was abolished in cells harboring a mutation in DNA polymerase α. Previously, this same mutation was shown to affect the coordination between conventional replication and telomerase-mediated extension. These results thus re-emphasize the importance of the interplay between conventional replication and telomerase-mediated addition of telomeric repeats in telomere replication.
    Full-text · Article · Feb 2005 · Nucleic Acids Research
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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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    ABSTRACT: The 7.8-kilobase HindIII insert in phage lambda NM589thyA [Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. & Murray, N. (1976) Mol. Gen. Genet. 146, 199] was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC of E. coli K-12 by using biochemical, structural, and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1- to 1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage lambda rho L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage lambda lysogen containing a thermolabile repressor, intracellular synthetase levels were increased about 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography, and some of its properties are described.
    Preview · Article · May 1983 · Proceedings of the National Academy of Sciences
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