A role for Sds3p, a component of the Rpd3p/Sin3p deacetylase complex, in maintaining cellular integrity in Saccharomyces cerevisiae.

The SDS3 gene was identified in a suppressor screen for mutations that enhance position-effect silencing in yeast. Cells that are defective in SDS3 have pleiotropic phenotypes, similar to those seen in the absence of the histone deacetylase components Rpd3p and Sin3p, including meiotic defects and improper regulation of the HO gene. To gain further insight into SDS3 function we undertook an epistasis analysis with other SDS genes. We found that sds3 is synthetically lethal in combination with a deletion of the SWI6 (SDS11) gene, which encodes a cell-cycle regulator. sds3 swi6 double mutants do not display a specific cell-cycle arrest phenotype, but instead die due to cell lysis. Constitutive expression of the G1 cyclin gene CLN2 restores viability to an sds3 swi6 strain, as does overexpression of SKT5/ CHS4, which encodes a regulatory subunit of chitin synthase III, and SSD1, a gene previously implicated in ensuring cell-cycle progression and cellular integrity. Significantly, growth in the presence of 1 M sorbitol or overexpression of PKC1 also partially suppresses the lethal phenotype of the sds3 swi6 strain. This lethality in the absence of SWI6 function most probably reflects an important or essential role for Sds3p in the Rpd3p/Sin3p histone deacetylase complex, since RPD3 and SIN3 mutations are also synthetically lethal in combination with swi6 and these phenotypes are also rescued by elevated dosage of SKT5/CHS4, SSD1, or PCK1. Taken together, these data indicate that the transcription factor Swi6p and the Rpd3p-based deacetylase complex act in parallel pathways to activate genes required for cell wall biosynthesis.

Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3[middle dot]Rpd3 histone deacetylase complex and is required for histone deacetylase activity.

SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background. In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex.

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.

Suppressors of defective silencing in yeast: effects on transcriptional repression at the HMR locus, cell growth and telomere structure.

To identify factors that affect transcriptional silencing at the HMR mating-type locus in yeast, we characterized a set of extragenic suppressor mutations that restore metastable repression in cells containing both a mutant silencer-binding protein (rap1s) and a mutated silencer element (hmr delta A). A total of 57 suppressors comprising 21 different complementation groups was identified. This report describes a detailed genetic analysis of these suppressors of defective silencing (sds) mutants. The sds mutants fall into several distinct categories based on secondary phenotypes, such as their ability to suppress the rap1s telomere lengthening phenotype, general effects on telomere length, temperature-dependent growth defects, and the ability to bypass the requirement for cis regulatory elements at the HMR-E silencer. One particular mutant, sds4-1, strongly suppresses the rap1s silencing defect, restores telomeres to nearly wild-type length, and displays a severe growth defect at all temperatures. SDS4 mutations also suppress the silencing defect caused by mutations in the RAP1-interacting factor RIF1. We cloned the SDS4 gene and show that it is identical to GAL11(SPT13), which encodes a component of a protein complex that mediates transcriptional activation. Possible mechanism(s) of suppression by sds4 and the other sds mutations is discussed.

Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae.

In this study, we used the ADE2 gene in a colony color assay to monitor transcription from the normally silent HMR mating-type locus in Saccharomyces cerevisiae. This sensitive assay reveals that some previously identified cis- and trans-acting mutations destabilize silencing, causing genetically identical cells to switch between repressed and derepressed transcriptional states. Deletion of the autonomously replicating sequence (ARS) consensus element at the HMR-E silencer or mutation of the silencer binding protein RAP1 (rap1s) results in the presence of large sectors within individual colonies of both repressed (Ade-, pink) and derepressed (Ade+, white) cells. These results suggest that both the ARS consensus element and the RAP1 protein play a role in the establishment of repression at HMR. In diploid cells, the two copies of HMR appear to behave identically, suggesting that the switching event, though apparently stochastic, reflects some property of the cell rather than a specific event at each HMR locus. In the ADE2 assay system, silencing depends completely upon the function of the SIR genes, known trans-acting regulators of the silent loci, and is sensitive to the gene dosage of two SIR genes, SIR1 and SIR4. Using the ADE2 colony color assay in a genetic screen for suppressors of rap1s, silencer ARS element deletion double mutants, we have identified a large number of genes that may affect the establishment of repression at the HMR silent mating-type locus.

Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.

In vivo RNA-RNA recombination of coronavirus in mouse brain.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.

Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA.

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.

[Study of left ventricular function in valvular cardiopathies (mitral insufficiency and aortic insufficiency]. / Etude de la fonction ventriculaire gauche dans les cardiopathies valvulaires (insuffisance mitrale et Insuffisance aortique)

A study of the left ventricular function based on the haemodynamic data combined with those provided by biplane cineangiography was performed in 35 cases with left ventricular volume overload (20 cases of mitral incompetence and 15 of aortic insufficiency). The importance of the haemodynamic changes and of the adaptation mechanisms set up were described. The more intense dilatation-hypertrophy of aortic incompetence than of mitral incompetence plays an essential part. The role of Starling's mechanism is underlined. Estimation of the contractile value of the myocardium, taken into account the mechanical overload and the conditions of late-diastolic lengthening of the fibre and of impedance to left ventricular ejection was determined. An obvious myocardial failure, demonstrated in approximately one third of the cases, by determination of some contractility indices estimated in the ejection phase, Vf sigma max in particular, the only one valid in the presence of valvular regurgitation. In the other cases, the moderate decrease of myocardial contractility was masked by compensatory mechanisms.