Evidence that the transcriptional regulators SIN3 and RPD3, and a novel gene (SDS3) with similar functions, are involved in transcriptional silencing in S. cerevisiae.

In a screen for extragenic suppressors of a silencing defective rap 1s hmr delta A strain, recessive mutations in 21 different genes were found that restored repression to HMR. We describe the characterization of three of these SDS (suppressors of defective silencing) genes. SDS16 and SDS6 are known transcriptional modifiers, SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively, while the third is a novel gene, SDS3. SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in haploid cells and is necessary for sporulation in diploid cells. However, sds3 mutations differ from sin3 and rpd3 mutations in that they do not derepress TRK2. These sds mutations suppress a variety of cis- and trans-defects, which impair the establishment of silencing at HMR. Any one of the sds mutations slightly increases telomere position effect while a striking synergistic increase in repression is observed in a rap 1s background. Epistasis studies suggest that SDS3 works in a different pathway from RPD3 and SIN3 to affect silencing at HMR. Together these results show that defects in certain general transcriptional modifiers can have a pronounced influence on position-effect gene silencing in yeast. Mechanisms for this increase in position effect are discussed.

Disturbance of normal cell cycle progression enhances the establishment of transcriptional silencing in Saccharomyces cerevisiae.

Previous studies have indicated that mutation of RAP1 (rap1s) or of the HMR-E silencer ARS consensus element leads to metastable repression of HMR. A number of extragenic suppressor mutations (sds, suppressors of defective silencing) that increase the fraction of repressed cells in rap1s hmr delta A strains have been identified. Here we report the cloning of three SDS genes. SDS11 is identical to SWI6, a transcriptional regulator of genes required for DNA replication and of cyclin genes. SDS12 is identical to RNR1, which encodes a subunit of ribonucleotide reductase. SDS15 is identical to CIN8, whose product is required for spindle formation. We propose that mutations in these genes improve the establishment of silencing by interfering with normal cell cycle progression. In support of this idea, we show that exposure to hydroxyurea, which increases the length of S phase, also restores silencing in rap1s hmr delta A strains. Mutations in different cyclin genes (CLN3, CLB5, and CLB2) and two cell cycle transcriptional regulators (SWI4 and MBP1) also suppress the silencing defect at HMR. The effect of these cell cycle regulators is not specific to the rap1s or hmr delta A mutation, since swi6, swi4, and clb5 mutations also suppress mutations in SIR1, another gene implicated in the establishment of silencing. Several mutations also improve the efficiency of telomeric silencing in wild-type strains, further demonstrating that disturbance of the cell cycle has a general effect on position effect repression in Saccharomyces cerevisiae. We suggest several possible models to explain this phenomenon.

Precise deletions in large bacterial genomes by vector-mediated excision (VEX). The trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts.

We have developed a simple and efficient method of vector-mediated excision (VEX) for in vivo generation of precisely defined deletions in large bacterial genomes. The system uses homologous recombination with small cloned fragments on specialized pVEX plasmids to insert directly repeated bacteriophage P1 loxP sites at positions flanking the region to be deleted. In the presence of Cre recombinase, the loxP sites are efficiently recombined to produce the deletion. Deletion endpoints can be directed to specific nucleotides because they are defined by the termini of small homology-bearing fragments cloned into the pVEX plasmids. We have used VEX to delete trfA, the only known replication initiator gene of the 56.4 kb broad host-range plasmid RK2. The RK2 delta trfA mutant was found to be conjugation proficient, but unable to replicate in the RK2 hosts Acinetobacter calcoaceticus, Caulobacter crescentus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Rhizobium meliloti, or Rhodobacter sphaeroides. These results show that trfA is essential for replication in these hosts and indicate that the broad host-range of RK2 does not involve multiple replicons.

An essential yeast gene encoding a TTAGGG repeat-binding protein.

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing.

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)