same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.Control of mating-type loci in Saccharomyces cerevisiae presents a striking example of position effect regulation or altered gene expression as a function of chromosomal position. Genes encoding regulatory proteins that determine the mating type of the cell are present at three different locations on chromosome III (2, 29, 45) ( Fig. 1). The set of genes resident at the MAT locus are transcribed, and their products establish the cell's mating type (7,10,20,27,28,40). The same genes resident at either end of the chromosome, at loci designated HML and HMR, are not transcribed and do not contribute to the establishment or maintenance of cell type (18,27,30). These silent loci serve solely as repositories of mating-type information that normally can be activated only by transposition of a copy of the genes into the MAT locus (11,14,15,17,22,29,45).Differential expression of MAT versus HML and HMR results from repression of expression of the genes residing at the silent loci HML and HMR. The products of three genes, SIR2 (or MARI) through SIR4, unlinked to each other or to the mating-type loci, are required to maintain the silent loci in a transcriptionally inactive state (8,16,31,32). In the absence of any one of these products, both silent loci are expressed at a level equivalent to that of MAT. A fourth gene, SIR], is required for complete repression of the silent loci, but its inactivation yields only partial expression of the otherwise silent genes (13, 31).Repression of the genes resident at HML and at HMR also requires the integrity of cis-acting sites adjacent to each locus. Deletion of a site designated E, which lies to the left of each locus as they are conventionally represented, yields full expression of the genes in the adjacent locus (1, 5). Both E sites are less than 250 base pairs (bp) in length and can function in cis in an orientation-independent manner to repress expression fr...

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase , f 3 genes (7SBl and 7SB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although 7SBl produces >90% of tryptophan synthase ,f3 mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase B activity. The trp2-1 mutation is complemented by the 7SBl transgene and is linked genetically to a polymorphism in the 7SBl gene, strongly suggesting that trp2-1 is a mutation in 7SB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, 7SB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (7RP1 and 7RP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus. We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus. In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity. Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression. This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression. Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, mediated independently through two c/s-acting sites, termed the E and I silencers. We have found that in the absence of the I silencer, deletion of any one of three distinct elements within E yields partial derepression of the mating-type genes resident at HML, whereas deletion of any two yields full derepression. These elements correspond to a binding site for the abundant DNA-binding protein RAP1, an autonomous replicating sequence (ARS), and an as yet undistinguished region. From detailed deletion analysis of the E site we conclude that the ARS element contributes to silencer function in a capacity distinct from its role as an initiator of DNA replication. In addition, we find that strains deleted for any one of these elements comprise two genetically identical but phenotypically distinct types of cells: Those with HML apparently fully derepressed, and those with HML apparently completely repressed. These results reinforce the notion that epigenetic inheritance is an intrinsic characteristic of silencer action.

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.