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

Abstract

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...

Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae.

“…The pattern of 5' ends observed in the experiment was generally consistent for the two primers and also for poly(A)+ versus plasmidamplified RNA. Heterogeneity in transcript 5' ends is common for yeast mRNAs (17,18), and so our findings for TMPI are not unusual. Notably, each of the TMPJ transcript ends lay downstream of a TATA sequence at -99 (Fig.…”

“…The arrested cells were harvested by centrifugation, washed three times with 5 ml of water, and then resuspended in 200 ml of fresh leucine omission medium. Samples of the culture (15 ml) were harvested at 10-min intervals by rapid filtration as described previously (17). The quality of the synchrony and progression through the cell cycle were monitored by determining the proportion of unbudded cells present at different times following release from a-factor-induced arrest.…”

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability.

“…Thus, another candidate for a damage-inducible gene would be CDC21, which encodes thymidylate synthase and is cell cycle regulated (35). Conversely, the DCDJ gene, which encodes dCMP deaminase, is not cell cycle regulated (23) and would not be expected to be DNA damage inducible.…”

Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

“…Molecular weights were determined with the 18S and 25S rRNAs, the 2.9-kb GAL4 mRNA (27), and the 3.0-kb LAC4 RNA (10; R. C. Dickson, unpublished data) as molecular weight standards. Transcription start sites were determined by a primer extension procedure (34). An autoradiogram of a DNAsequencing gel on the right side of the figure shows the products of the primer extension reaction (STARTS) and the products of the DNA-sequencing reactions (A, G, T, C).…”

“…Transcription start sites were determined by a primer extension procedure (34). The primer 5'-GGCAGTAACG TTTCCGCC-3' was labeled at its 5' end with [y-32PIATP and T4 bacteriophage polynucleotide kinase.…”