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

Journal Article · Thu Apr 01 04:00:00 EST 1982 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.2.4.437· OSTI ID:5878131

Taylor, G R; Barclay, B J; Storms, R K; Friesen, J D; Haynes, R H

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) 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/sup +/ 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/sup +/ transformants directly, it was found that all pTL1 transformants were phenotypically Thy/sup +/ 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-/sup 3/H)dUMP to (6-/sup 3/H)dTMP. In protein extracts from the thymidylate auxotroph (tmpl-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.

• Research Organization: Dept. of Biology, York Univ., Toronto, Ontario M3J 1P3
• OSTI ID: 5878131
• Journal Information: Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 2:4; ISSN MCEBD
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
AZINES
BACTERIA
CELL CONSTITUENTS
CELL CULTURES
CHIMERAS
CHROMATOGRAPHY
CLONING
DNA-CLONING
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
FUNGI
GENES
GENETIC ENGINEERING
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
METHYL TRANSFERASES
MICROORGANISMS
MOSAICISM
MUTANTS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHENOTYPE
PLANTS
PLASMIDS
PYRIMIDINES
RIBOSIDES
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
SENSITIVITY
SEPARATION PROCESSES
TEMPERATURE EFFECTS
THYMIDINE
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
YEASTS