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

Taylor, G. R.; Barclay, B. J.; Storms, Reginald; Friesen, James D.; Haynes, Robert H.

doi:10.1128/mcb.2.4.437

Cited by 27 publications

(18 citation statements)

References 34 publications

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“…The plasmids pTL1 (45) and YIp5 (44) were used, respectively, as sources of TMPI and URA3 gene sequences in the hybridization experiments. pTL221 and pBTAH were used in thymidylate synthase expression studies in Escherichia coli.…”

Section: Methodsmentioning

confidence: 99%

“…TMPI mRNA was identified and relative levels were determined by hybridization to the radiolabeled HindlIl to EcoRI fragment of the TMPI sequence (45). Autoradiographic analysis of the resulting Northern blot (Fig.…”

Section: Downloaded Frommentioning

confidence: 99%

“…(i) Synchronous populations of cells are easily generated. (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).…”

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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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Section: Methodsmentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

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

Storms¹,

Ord²,

Greenwood³

et al. 1984

Mol. Cell. Biol.

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“…In this regard, S. cerevisiae is particularly useful, since three of these genes, dihydrofolate reductase, thymidylate synthe-* Corresponding author. tase (TMPI), and thymidylate kinase (CDC8), have recently been cloned (23,42,57).In an effort to isolate additional S. cerevisiae genes involved in deoxynucleoside triphosphate metabolism, we developed a selection system suitable for cloning the dCMP deaminase (DCDI) gene (36). dCMP deaminase catalyzes the hydrolytic deamination of dCMP to yield dUMP and thus supplies the nucleotide substrate for thymidylate synthetase.…”

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confidence: 99%

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

McIntosh¹,

Haynes²

1986

Mol. Cell. Biol.

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The dCMP dminase gene (DCDI) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcdl dmpl haploids. Plasmid pDC3, contining a 7-kilobase (kb) Sau3A insert, restores dCMP dminase activity to dedl mutants and leads to an average 17. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCDI mRNA is a 1.15-kb poly(A)+ hnscript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCDI gene is not subject to the same cel cyde-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMPI).The yeast Saccharomyces cerevisiae has proved to be a useful organism for the analysis of both the cytological and biochemical events which occur during the cell division cycle (39,48). This lower eucaryote is particularly well suited for such studies, since synchronous populations can be generated by a variety of physical or biochemical methods (19,21,39) or by the use of conditional mutants in which growth is arrested at defined stages of the cell cycle (48). The recent development and application of gene manipulation techniques to yeasts (6), however, promises a greater understanding of the molecular events involved in the cell division process.One area of increasing interest is the regulation of enzymes involved in deoxynucleoside triphosphate synthesis. Over the years, numerous studies of a variety of eucaryotic systems, including S. cerevisiae, have found that many of these enzymes exhibit increased activity coincident with the onset of the S phase (13, 16-18, 20, 26, 29, 40, 43). Studies of dihydrofolate reductase and thymidylate synthetase in mammalian cells (20,43) and, more recently, thymidylate synthetase in yeast cells (54) indicate that the induction of these enzymes is mediated at the level of transcription. These and other previous studies suggested that genes encoding enzymes involved in deoxynucleoside triphosphate synthesis may generally be subject to enhanced transcription just prior to the onset of the S phase in eucaryotic cells. To corroborate this hypothesis, however, it is desirable to examine the expression of a number of these genes in one system. In this regard, S. cerevisiae is particularly useful, sin...

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“…Instead, yeast synthesize thymidine entirely de novo from dUMP using thymidylate synthase, encoded by CDC21 (Taylor et al 1982;Taylor et al 1987;Bisson and Thorner 1977). Williamson and Fennell (1981) used tritiated adenine to label DNA and assayed the incorporation by emulsion-based autoradiography (Williamson and Fennell 1981).…”

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Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae

Burke

2013

Chromosome Res

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The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate nonrandomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without motherdaughter bias in a common laboratory strain grown under routine laboratory conditions.

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

Cited by 27 publications

References 34 publications

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

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

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae

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