Abstract In the yeast Saccharomyces cerevisiae, glucose repression of SUC2 transcription requires the SSN6-TUP1 repressor complex. It has been proposed that the DNA-binding protein MIG1 secures SSN6-TUP1 to the SUC2 promoter. Here we show that a mig1 deletion does not cause nearly as dramatic a loss of repression as ssn6: glucose-grown mig1 mutants display 20-fold lower SUC2 expression than ssn6 mutants. Thus, repression by SSN6-TUP1 is not mediated solely by MIG1, but also involves MIG1-independent mechanisms. We report that mig1 partially restores SUC2 expression in mutants lacking the SNF1 protein kinase and show that mig1 is allelic to ssn1, a mutation selected as a suppressor of snf1. Other SSN genes identified in this selection were therefore candidates for a role in repression of SUC2. We show that mig1 acts synergistically with ssn2 through ssn5, ssn7, and ssn8 to relieve glucose repression of SUC2 and to suppress the requirement for SNF1. These findings indicate that the SSN proteins contribute to repression of SUC2, and the pleiotropic phenotypes of the ssn mutants suggest global roles in repression. Finally, the regulated SUC2 expression observed in snf1 mig1 mutants indicates that signals regarding glucose availability can be transmitted independently of the SNF1 protein kinase.
Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration). Several genes which complemented the original snf3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snf3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.
SPT6 is an essential gene of Saccharomyces cerevisiae that appears to play a role in transcription. Mutations in the SPT6 (SSN20, CRE2) gene suppress delta insertion mutations in the 5' regions of HIS4 and LYS2 and mutations in cis- and/or trans-acting elements that are required for expression of SUC2 and ADH2. We report here that SPT6 encodes a 170-kilodalton highly charged protein with an extremely acidic amino terminus. By use of an epitope-tagged SPT6 protein, we have determined by indirect immunofluorescence that the SPT6 protein is located in the nucleus.
Drosophila melanogaster is an ideal organism for the study of the structure and function of heterochromatin because it has only four chromosomes and 25% of its genome is heterochromatic. All of the heterochromatin is constitutive and much of it is located in the sex chromosomes, which have been well characterized genetically and cytogenetically (Cooper 1959; Ashburner and Novitski 1976). The study of Drosophila heterochromatin is also attractive at the molecular level since the bulk of the DNA of these regions can be isolated as several discrete satellite DNAs in CsCl equilibrium gradients (Peacock et al. 1974; Endow et al. 1975; Brutlag et al. 1977a). These unusual DNAs consist of short nucleotide sequences (5–378 base pairs [bp]) repeated in tandem arrays over 1,000,000 bp long (Goldring et al. 1975; Brutlag et al. 1977a).
The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.
The yeast SNF-SWI complex is required for transcriptional activation of diverse genes and has been shown to alter chromatin structure. The complex has at least 10 components, including SNF2/SWI2, SNF5, SNF6, SWI1/ADR6, and SWI3, and has been widely conserved in eukaryotes. Here we report the characterization of a new component. We identified proteins that interact in the two-hybrid system with the N-terminal region of SNF2, preceding the ATPase domain. In addition to SWI3, we recovered a new 19-kDa protein, designated SNF11. Like other SNF/SWI proteins, SNF11 functions as a transcriptional activator in genetic assays. SNF11 interacts with SNF2 in vitro and copurifies with the SNF-SWI complex from yeast cells. Using a specific antibody, we showed that SNF11 coimmunoprecipitates with members of the SNF-SWI complex and that SNF11 is tightly and stoichiometrically associated with the complex. Furthermore, SNF11 was detected in purified SNF-SWI complex by staining with Coomassie blue dye; its presence previously went unrecognized because it does not stain with silver. SNF11 interacts with a 40-residue sequence of SNF2 that is highly conserved, suggesting that SNF11 homologs exist in other organisms.
We performed positional cloning of genes carried on yeast artificial chromosomes that span a human translocation breakpoint associated with a human disease and isolated by chance human and bovine genes with strong homology to the S.cerevisiae genes, SNF2/SWI2 and STH1, and the D.melanogaster gene brahma.We report here sequence analysis, expression data, and functional studies for this human SNF2-like gene (hSNF2L) and its bovine homolog (bovSNF2L).Despite strong homology at the amino acid level, hSNF2L is not capable of complementing the yeast mutations snf2 or sthl in S.cerevisiae.Furthermore, in contrast to SNF2 itself, a fusion protein consisting of the DNA binding domain of LexA and hSNF2L did not transactivate a reporter gene downstream of LexA binding sites in a yeast expression system.The strong similarity between hSNF2L and these yeast and drosophila genes suggest that the mammalian genes are part of an evolutionarily conserved family that has been implicated as global activators of transcription in yeast and fruitflies but whose function in mammals remains unknown.
Activated transcription by eukaryotic RNA polymerase II requires general transcription factors and enhancer-binding activator proteins. Activators are thought to have roles in stabilizing or accelerating the assembly of the general transcription factors and in counteracting the repressive effects of histones (for review, see Kornberg and Lorch 1992; Zawel and Reinberg 1993). Other classes of intermediary proteins termed coactivators and co-antirepressors are often required (see Croston et al. 1992; Gill and Tjian 1992).
