Isolation and Characterization of Mutants of Saccharomyces cerevisiae Auxotrophic and Conditionally Auxotrophic for 5′ -dTMP

Brendel, Martin; Fäth, Wolfgang W.

doi:10.1515/znc-1974-11-1214

Cited by 36 publications

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“…Typical enzymatic activities of mitochondria1 and cytoplasmic enzymes in wild-type, temp3 and ade3 segregants of the tmp3 mutants have lost the activities of the three mitochondrial enzymes, serine transhydroxymethylase, dihydrofolate reductase and thymidylate synthetase, while their cytoplasmic counterparts and others mitochondrial and cytoplasmic C1 enzymes such as 5,l O-methylenetetrahydrofolate dehydrogenase, 5,lOmethylenetetrahydrofolate reductase and 10-formyltetrahydrofolate synthetase are fully active ( Table 1). The tmpl mutant that requires only thymidylate [27] has lost the thymidylate synthetase activity assayed in whole cell homogenate (unpublished results).…”

Section: Enzyme Activities Of Mitochondria and Cytoplasm In Tmp3 And mentioning

confidence: 92%

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Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer

Zelikson

Luzzati

1977

European Journal of Biochemistry

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The study of the subcellular distribution of the enzymes involved in folate-coenzyme-mediated transfer of one-carbon groups in Saccharomyces cerevisiae suggests the existence of two parallel enzyme systems, one mitochondrial and the other cytoplasmic. All the enzymes assayed in the present study, serine transhydroxymethylase, dihydrofolate reductase, 5,lO-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,lO-methylenetetrahydrofolate reductase and thymidylate synthetase were found to be in both cytoplasm and mitochondria. Two of the mitochondrial enzymes, serine transhydroxymethylase and dihydrofolate reductase, were found to be different by several criteria from their cytoplasmic counterparts.By the use of three nuclear gene mutations ade3, tmp3 and serl, and different combinations of these three mutations, the physiological role of the cytoplasmic enzymes for the biosynthesis of adenine, histidine, methionine, thymidylate and serine was studied. The response of auxotrophes to an external source of tetrahydrofolate, i. e. 5-formyl tetrahydrofolic acid, was also examined. The mitochondrial set of enzymes might be responsible for the de novo formation of tetrahydrofolate, since the loss of the three mitochondrial enzyme activities, serine transhydroxymethylase, thymidylate synthetase and dihydrofolate reductase, due to the mutation tmp3, leads to a generalized requirement for thymidylate, methionine, adenine and histidine. The ade3 mutation in which the cytoplasmic activities of the 10-formyltetrahydrofolate synthetase, 5,lO-methenyltetrahydrofolate cyclohydrolase and 5,lO-methylenetetrahydrofolate dehydrogenase are lost, confers only adenine and histidine auxotrophy, showing that the ade3 proteins are synthesized in, and only function in, the cytoplasm. The mitochondrial counterparts of these enzymes may function solely in the formation of the 10-formyl tetrahydrofolate needed for the transformylation of Met-tRNA involved in mitochondrial protein synthesis.A study of a nuclear mutation in Saccharomyces cerevisiae, tmp3, leading concomittantly to multiple nutritional requirements for thymidylate, methionine, adenine and histidine and to the petite phenotype, was found to have a decreased activity of serine transhydroxymethylase, but a wild-type level of two other tetrahydrofolate-linked enzymes, 10-formylte- trahydrofolate-linked enzymes, 10-formyltetrahydrofolate synthetase and 5,1O-methylenetetrahydrofolate dehydrogenase [l]. Further study showed that wildtype strains of yeast have two forms of serine transhydroxymethylase, one cytoplasmic and the other mitochondrial, separable by TEAE-cellulose chromatography. The mutant tmp3 has lost the mitochondrial form [2].Serine transhydroxymethylase may be the major physiological source of 5,lO-methylene tetrahydrofolate needed for the formation of methionine, thymidylate and adenine. Loss of the mitochondrial enzyme, in tmp3, which leads to that multiple requirement, implies that either the mitochondrial serine transhydroxymethylase is the...

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Section: Enzyme Activities Of Mitochondria and Cytoplasm In Tmp3 And mentioning

confidence: 92%

“…p-Aminobenzoic acid does not replace the nutritional requirements of the mutant. tmp3 mutants complement tmpl -3 (obtained from M. Brendel [27]) which only requires thymidylate.…”

Section: Tmp3 Mutationmentioning

confidence: 99%

Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer

Zelikson

Luzzati

1977

European Journal of Biochemistry

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“…(ii) The yeast gene encoding thymidylate synthase (TMPI) has been previously isolated (45). (iii) Mutant alleles (both temperature sensitive and nutritional) of the TMPI gene have been isolated, and methods have been developed for generating additional mutants (10,30). (iv) Temperature-sensitive mutants af-* Corresponding author.…”

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confidence: 99%

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

Storms¹,

Ord²,

Greenwood³

et al. 1984

Mol. Cell. Biol.

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Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

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“…A possible explana-tion for this observation is that thymine nucleotides or their metabolic derivatives may be implicated in regulating the meiotic pathway in yeasts (25). These studies have been greatly facilitated by the availability of yeast mutants defective (7,20) or conditionally defective (5,13,16) in thymidylate synthetase.…”

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confidence: 99%

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

Taylor¹,

Barclay²,

Storms³

et al. 1982

Mol. Cell. Biol.

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The structural gene (TMPI) 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 (cdc2l) 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 condi-

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Isolation and Characterization of Mutants of Saccharomyces cerevisiae Auxotrophic and Conditionally Auxotrophic for 5′ -dTMP

Cited by 36 publications

References 0 publications

Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer

Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

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

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