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

Abstract

Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMPI. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMPI. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMPI MIuI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.

SWI6 is a regulatory subunit of two different cell cycle START-dependent transcription factors in Saccharomyces cerevisiae

“…7). This is surpris ing because T M P l transcription is com pletely dependent upon the M CBs w ithin its prom oter (M cIntosh et al, 1991). In contrast, we do see effects on the RNR1 gene ( A ddressing the regulatory role o f SW I6 is com plicated by sw i6 m utants being grossly altered in their cell cycle control.…”

“…The best characterized M C Bs are the tw o required for efficient TMP1 transcription (M cIntosh et al, 1991). W e therefore chose as a probe for D N A binding a natural oligonucleotide from the TMP1 prom oter (M CB -TM P1) that contains both these M C Bs.…”

Comprehensive Identification of Cell Cycle–regulated Genes of the YeastSaccharomyces cerevisiaeby Microarray Hybridization

“…Carbon sources are indicated in the descriptions of each experiment and were used at a Lowndes et al, 1991;McIntosh et al, 1991;Koch et al, 1993 Mcm1p Mcm1p MCM1 TTACCNAATTNGGTAA Acton et al, 1997 SFF SFF SFF GTMAACAA Althoefer et al, 1995 Ace2p Ace2p SWI5 ACCAGC Dohrmann et al, 1996 Swi5p Swi5p SWI5 ACCAGC Knapp et al, 1996 Fitch et al, 1992 P.T. Spellman et al…”

Hydroxyurea Arrests DNA Replication by a Mechanism That Preserves Basal dNTP Pools

“…Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the genetic advantages of S. cerevisiae, the relative simplicity of its dNTP synthesis pathway, and the well studied transcriptional control of its dNTP-synthesizing genes (11)(12)(13)(14), relatively few analyses of dNTP levels in yeast have been done. Three studies have reported dNTP measurements in asynchronously growing yeast (15)(16)(17).…”