Background: Understanding of the firing time determination of replication origins in the entire genome will require a genome‐wide survey of replication origins and their mapping on chromosomes. A microarray technology was applied to obtain a genome‐wide profile of DNA replication and to classify early firing origins. Results: A total of 260 potential replication origins (PROs) were identified in the entire budding yeast genome: 247 as defined peaks on the replication profile and 13 as regions located in the chromosomal termini. Based on the firing time, the 247 PROs were classified into 143 early PROs and 104 late PROs, that were not randomly distributed on chromosomes but formed separated clusters. Most of the early PROs were found to fire in the presence of hydroxyurea, indicating that they were free from the control of the intra‐S‐checkpoint mediated by Mec1 and Rad53. Conclusions: The monitoring method of DNA replication and the analysis method of microarray data used in this study proved powerful for obtaining a genome‐wide view of the initiation and progression of DNA replication.

FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for synthesis of beta-1,3-glucan in the Saccharomyces cerevisiae cell wall. Disruption of FKS1 reduces the glucan content of the cell wall, increases chitin content and activates the expression of CWP1, which encodes a glycosylphosphatidylinositol (GPI)-dependent cell wall protein. These cellular responses have been regarded as compensating for cell wall damage in order to maintain cell wall integrity. Here, we report the identification, by genome-wide screening, of 22 genes that are transcriptionally up-regulated in fks1delta cells. Among them, five genes were found to encode GPI-attached proteins, three of which are covalently associated with the cell wall. Deletion and replacement analysis of the promoter regions identified Rlm1-binding sequences as being responsible for the up-regulation following disruption of FKS1. Using the rlm1delta tetOp-FKS1 strain, in which the expression of FKS1 can be repressed by doxycycline, we examined the requirement for Rlm1 for the transcriptional up-regulation of these five genes. Three of the five genes were not up-regulated by doxycycline, indicating that Rlm1 mediates their up-regulation when FKS1 is inactivated. The remaining two genes were up-regulated by doxycycline, suggesting that a transcription factor other than Rlm1 is involved in their response to disruption of FKS1.

During cell wall biogenesis in Saccharomyces cerevisiae, some glycosylphosphatidylinositol (GPI)-attached proteins are detached from GPI moieties and bound to ␤-1,6-glucan of the cell wall. The amino acid sequence requirement for the incorporation of GPI-attached proteins into the cell wall was studied by using reporter fusion proteins. Only the short -minus region composed of five amino acids, which is located upstream of the site for GPI attachment, determined the cellular localization of the GPI-associated proteins. Within the -minus region, amino acid residues at the -4 or -5 and -2 sites were important for the cell wall incorporation. Yap3p, a well characterized GPI-anchored plasma membrane aspartic protease, was localized in the cell wall when the -minus region was mutated to sequences containing Val or Ile at the -4 or -5 site and Val or Tyr at the -2 site.Mannoproteins, one of the components of the yeast cell wall, can be divided into three groups: SDS-extractable mannoproteins, reducing agent-extractable mannoproteins, and glucanase-extractable mannoproteins (1, 2). The glucanase-extractable mannoproteins are covalently bound to ␤-1,6-glucan of the cell wall and are released only by glucanase treatment (3-6). Many genes encoding glucanase-extractable cell wall mannoproteins have been isolated in Saccharomyces cerevisiae, and so far all of them have been identified as GPI-dependent 1 cell wall proteins (7-15).GPI-associated proteins have been isolated from various organisms from yeasts and protozoa to mammals (16 -18). These proteins are structurally related in that they all contain a signal sequence for secretion in the N terminus and a GPI signal for an attachment to a GPI in the C terminus. The GPI-signal region is composed of three domains: a GPI attachment region comprising , ϩ1, and ϩ2 sites; a spacer of 5-10 amino acids; and a hydrophobic stretch of 10 -15 amino acids. A protein containing the GPI-signal is cleaved at the site, and the resulting carboxyl terminus of the protein becomes covalently bound to a GPI moiety. This reaction occurs in the luminal face of the endoplasmic reticulum and, in yeast, requires GAA1 (19) and GPI8 (20) gene products. The GPI-attached proteins are then transported to the cell surface, where they are exposed on the extracytoplasmic face of the plasma membrane. During the transportation from the endoplasmic reticulum to the plasma membrane, the proteins are mannosylated and become GPI-anchored mannoproteins.In protozoa and mammals, the GPI-anchored mannoproteins remain on the plasma membrane and take biological functions related to cell-cell and cell-environment interactions (18,21,22). In addition to the above functions, some of the GPI-anchored mannoproteins in the yeast are further processed and are incorporated into the cell wall. The incorporation of GPIassociated proteins into the cell wall is thought to occur in two steps: detachment of a GPI moiety from the protein and linking of the GPI-detached protein to ␤-1,6-glucan of the cell wall (3). Our knowledge of t...

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.

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