MCB-mediated regulation of cell cycle-specific cdc22(+) transcription in fission yeast

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe , encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed

Cell cycle, DNA damage and heat shock regulate <i>suc22</i>⁺ expression in fission yeast

The &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; gene of Schizosaccharomyces pombe encodes the small subunit of ribonucleotide reductase. Two transcripts that hybridise to &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; have previously been described: a constitutive transcript of 1.5 kb, and a transcript of approximately 1.9 kb that is induced when DNA replication is blocked by hydroxyurea. In this paper we show that both transcripts derive from the suc22+ gene, are polyadenylated, and have transcription initiation sites separated by approximately 550 nucleotides. The absence of translation initiation codons and predicted intron splice sites within this 550 nucleotide region suggests strongly that both transcripts encode the same protein. Under normal growth conditions, the larger &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript is present at a very low level. This low level expression is periodic during the cell cycle, showing a pattern similar to that of other genes under regulation by MCB elements with a maximum in G1/S phase. Consistent with this, there are MCB elements upstream of the initiation site of the transcript. This pattern of expression contrasts with the continuous expression, at a much higher level, of the smaller &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript. The larger &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript is induced by exposure of cells to 4-nitroquinoline oxide (4-NQO),a UV-mimetic agent that causes DNA damage. The transcriptional response to 4-NQO is observed in cells previously arrested in G2 by a &lt;i&gt;cdc2&lt;/i&gt;&lt;sup&gt;ts&lt;/sup&gt; mutation, demonstrating that induction can occur outside S phase. We show that the rad1+ gene, part of the mitotic checkpoint, is required for induction of the large transcript. Exposure of cells to heat shock also induces the &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; large transcript: a consensus heat shock element has been identified upstream of the large transcript start site

Positive and negative roles for cdc10 in cell cycle gene expression

In this paper we describe properties of the cdc10-C4 mutant of the fission yeast Schizosaccharomyces pombe. The cdc10&lt;sup&gt;+&lt;/sup&gt; gene encodes a component of the DSC1&lt;sup&gt;Sp&lt;/sup&gt;/MBF transcription complex, which is required for cell-cycle regulated expression at G1-S of several genes via cis-acting MCB (MIuI cell cycle box) elements. At permissive temperatures cdc10-C4 causes expression of MCB-regulated genes through the whole cell cycle, which in asynchronously dividing cells is manifested in overall higher expression levels. This overexpression phenotype is cold sensitive: in cdc10-C4 cells, MCB genes are expressed offprogressively higher levels at lower temperatures. In heterozygous cdc10-C4/cdc10&lt;sup&gt;+&lt;/sup&gt; diploid strains, MCB-regulated genes are not overexpressed, suggesting that loss, rather than alteration, of function of the cdc10-C4 gene product is the reason for unregulated target gene expression. Consistent with this, the cdc10-C4 mutant allele is known to encode a truncated protein. We have also overexpressed the region of the cdc10 protein absent in cdc10-C4 under the control of an inducible promoter. This induces a G1 delay, and additionally causes a reduction of the overexpression of MCB genes in cdc10-C4 strains. These results suggest that DSC1&lt;sup&gt;Sp&lt;/sup&gt;/MBF represses, as well as activates, MCB gene expression during the cell cycle

Hardware and software requirements for quantitative analysis of comparative genomic hybridization

Applied Science

SpSld3 Is Required for Loading and Maintenance of SpCdc45 on Chromatin in DNA Replication in Fission Yeast

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1–S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41(+) encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing