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

Abstract

We have previously shown that transcript levels expressed from the yeast TMPI gene fluctuate periodically during the yeast cell cycle. However, it was not known whether periodic expression resulted from a regulatory mechanism acting at the level of transcription or a regulatory mechanism acting at the level of cell cycle stage-dependent changes in the stability of the TMPI transcript. In this report we now show that the periodic expression of TMPI transcript is primarily controlled at the level of its transcription by sequences which are upstream of its transcription initiation sites. We also localized the upstream sequences necessary for periodic transcription to a 150-base-pair region and show that this region encodes an element(s) with the properties of a periodic upstream activating sequence. The regulatory region defined in this study apparently does not contain consensus sequences similar to those reported for the cell cycle-regulated HO endonuclease or for the histone H2A and H2B genes of Saccharomyces cerevisiae.One approach to investigating the processes which control eucaryotic cell division is to identify and study those genes which are differentially regulated during the cell cycle. If progression through the cell division cycle results from the sequential transient expression of dormant genes, then identifying the factors which govern the expression of these genes is of considerable interest. In the yeast Saccharomyces cerevisiae, only a few genes are currently known to exhibit differential expression during the cell cycle. These include the thymidylate synthase gene TMPJ (25), the histone H2A and H2B genes of the TRTJ locus (12), the HO endonuclease gene (20), the thymidylate kinase gene CDC8 (28), and the DNA ligase gene CDC9 (23).Previous studies have shown that the regulation of the periodically expressed yeast histone genes results from complex controls acting at both the transcriptional and posttranscriptional levels (14,21,22). In addition, their proper expression may also involve the placement of these genes adjacent to an origin of DNA replication (21). Similarly, expression of the HO gene appears to be governed by complex controls acting transcriptionally and posttranscriptionally (4,20). Nasmyth (20) has identified a 12-base-pair (bp) consensus sequence which is repeated many times within the URS2 region of HO and has been shown to be critically involved in the periodic expression of this gene.We found that TMPJ mRNA is transiently expressed during the cell cycle, with peak amounts occurring during late G1 and early S phase just after the cdc28la-factor arrest point (25). In a recent study, White et al. (28) demonstrated that the periodic expression of TMPJ, CDC8, and CDC9 mRNA was coincident, while the histone H2A gene was expressed distinctly later in the cell cycle. This was shown by analyzing the times at which these genes were expressed after release from G1 arrest and also by showing that only the periodicity of the histone genes was influenced by the cdc4-3 mutation. These results ...

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

“…Analysis of the 5' noncoding region of CDC21, which contains two 5'-ACGCGT-3' repeats, indicates that a 37-bp deletion, removing the region in between and reconstituting a single hexamer, severely affects the level of CDC21 transcript and possibly impairs its fluctuation during the cell cycle (23). We are now investigating whether this 6-bp sequence has any role in mediating transcriptional control of POLl, PRII, and PRI2.…”

“…The 520-bp sequence upstream of the PRI2 coding region contains the hexanucleotide 5'-ACGCGT-3', which is present at least once in the 5' noncoding regions of the genes listed above (23,26). Analysis of the 5' noncoding region of CDC21, which contains two 5'-ACGCGT-3' repeats, indicates that a 37-bp deletion, removing the region in between and reconstituting a single hexamer, severely affects the level of CDC21 transcript and possibly impairs its fluctuation during the cell cycle (23).…”

Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae.

“…Plasmid pEM54 is a YEp13 derivative containing the TMPI gene and has been described previously (14). Plasmid pEM80 contains a 2.7-kb HindIII-to-Asp718 fragment containing the TMPI gene inserted into the HindlIl and Asp718 sites of pEMBL19+.…”

“…For each of these genes, this pattern of expression involves cell cycle stage-dependent changes in steady-state mRNA levels, with maximal levels occurring near the Gl/S-phase boundary. For TMPI, this fluctuation in mRNA levels is largely due to a transcriptional process (14). Studies by other groups have shown that TMPI, CDC8, CDC9, and POLI transcripts are induced at virtually the * Corresponding author.…”

Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.

“…This regulation could occur at the level of mRNA synthesis or degradation, or both. In yeast, the only genes that have been examined so far, HO (Nasmyth 1985), CDC21 (McIntosh et al 1988), and HTA1 and HTB1 (Osley et al 1986), are regulated at the level of mRNA synthesis, although post-transcriptional regulation is important for histone regulation in higher eukaryotic cells (Schumperli 1986). Positive-and negativeacting sequences are involved in cell-cycle regulation of the histone HTA1 and HTB1 genes (Osley et al 1986), and mutations in the HIR genes have been identified that alter the negative regulation (Osley and Lycans 1987).…”