Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements.GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT a genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences within the (Cl_3A)-repeat region at yeast telomeres.Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCAN NCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR 1, and HML I and were associated with ARS1, ARS2, and the 2,um plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DEDI genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.Eucaryotic chromosomes appear to be organized in domains governing such processes as gene expression, DNA replication, and chromosome condensation. In an effort to define the biochemical components directing these events, we have been attracted to the features of the mating type loci of Saccharomyces cerevisiae (reviewed in reference 33). Haploid yeast cells of the a or a mating type express different genes at MAT, in the middle of chromosome III (Fig. 1). The alternate forms of this locus, MATa and MA To, encode distinct regulatory factors al and a2 or al and oa2, but have extensive regions of homology, referred to as W, X, and Z (2, 61). There are, in addition, silent copies of genes specifying each mating type stored on the left and right arms of chromosome III at HMLa and HMRa, respectively. In homothallic strains, the mating type of successive generations switches in a tightly regulated manner (29,35,80). Switching is accomplished by gene conversion whereby sequences at MAT are replaced by those at HML or HMR (33,47). This process is initiated with the cleavage of MAT DNA in Z by a specific endonuclease that is the product of the HO gene (49, 50). The silent copies of the mating type loci are controlled by negative regulatory sequences located over 1,000 base pairs (bp) away from the promoters for these genes. Deletion analysis of HML and HMR has revealed cis-acting regulatory elements, E and I, that flank each locus on the left and right sides, respectivel...

The HMR E silencer is required for SIR‐dependent transcriptional repression of the silent mating‐type locus, HMR. The silencer also behaves as an origin of replication (ARS element) and allows plasmids to replicate autonomously in yeast. The replication and segregation properties of these plasmids are also dependent on the four SIR genes. We have previously characterized two DNA‐binding factors in yeast extracts that recognize specific sequences at the HMR E silencer. These proteins, called ABFI (ARS‐Binding Factor) and GRFI (General Regulatory Factor), are not encoded by any of the SIR genes. To investigate the biological roles of these factors, single‐base‐pair mutations were constructed in both binding sites at the HMR E silencer that were no longer recognized by the corresponding proteins in vitro. Our results indicate that the GRFI‐binding site is required for the efficient segregation of plasmids replicated by the HMR E silencer. SIR‐dependent transcriptional repression requires either an intact ABFI‐binding site or GRFI‐binding site, although the GRFI‐binding site appears to be more important. A double‐mutant silencer that binds neither ABFI nor GRFI does not mediate transcriptional repression of HMR. The replacement of HMR E with a chromosomal origin of replication (ARS1) allows partial SIR‐dependent transcriptional repression of HMR, indicating a role for replication in silencer function. Together, these results suggest that the SIR proteins influence the properties of the HMR E silencer through interactions with other DNA‐binding proteins.

In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the al-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARSI, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.The genes HML and HMR encode cryptic copies of the MAT genes. HML, located on the left arm of chromosome III, encodes an unexpressed copy of the MATa genes (HMLo). HMR, located distal to MAT on the right arm of chromosome III, contains an unexpressed copy of the MATa genes (3,23,44,59). These duplicated genes serve as donor templates in HO-mediated mating-type interconversion (reviewed in reference 24). The expression of the genes at HML and HMR is repressed by the products of SIR], SIR2 (MARI), SIR3 (CMT), and SIR4 (21,33,35,47,48). Both HML and HMR are flanked by cis-acting sites, called E and I, that are also required for transcriptional repression. Deletion and insertion analyses indicate that the E site is contained within a 220-base-pair (bp) region and the I site within an 85-bp region at HMR (1, 19). Presumably, E and I are the sites through which the SIR proteins mediate transcriptional repression of HML and HMR. Indeed, DNA sequence analysis of the E and I elements has revealed several short conserved blocks of homology, some of which may be sites of SIR action (19).In addition to their role in SIR-mediated transcriptional repression, the E and I elements allow plasmids to replicate autonomously in yeasts (1,10,19 chance and may represent a mechanistic link between the processes of DNA replication and transcriptional repression. In this regard, the onset of SIR-mediated repression at HMRa requires transit through the S phase of the cell cycle (42). It is not known whether the requirement ...

A PCR-based sequence-tagged site (STS) content mapping strategy has been used to generate a physical map with 90% coverage of the 120-Mb euchromatic portion of the Drosophila genome. To facilitate map completion, the bulk of the STS markers was chosen in a nonrandom fashion. To ensure that all contigs were localized in relation to each other and the genome, these contig-building procedures were performed in conjunction with a large-scale in situ hybridization analysis of randomly selected clones from a Drosophila genomic library that had been generated in a PI cloning vector. To date, the map consists of 649 contigs with an STS localized on average every 50 kb. This is the first whole genome that has been mapped based on a library constructed with large inserts in a vector that is maintained in Escherichia coli as a single-copy plasmid.

Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements. GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT alpha genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences with the (C1-3A)-repeat region at yeast telomeres. Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCANNCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR I, and HML I and were associated with ARS1, ARS2, and the 2 micron plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DED1 genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.