Overexpression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotypes associated with absence of the largest, nonessential subunit Rpb4

The easily dissociable subcomplex of Rpb4 and Rpb7 subunits of yeast RNA polymerase II has been considered, for long, to play a role in stabilizing Pol II under stress. On the basis of previous genetic and biochemical observations, it was proposed that within the subcomplex one of the functions of Rpb4p is to stabilize the interaction between Rpb7p and the rest of Pol II. We took a direct approach to test the latter possibility by overexpression and mutagenesis of RPB7 in absence of Rpb4p. We report here the results, which support the latter hypothesis. While it has been previously reported that absence of Rpb4p results in reduction in overall transcription by Pol II, our comparative analysis of RNAs from RPB4 and rpb4d cells suggests that there are indeed several genes differentially expressed between the two cells. We propose that the qualitative differences in overall transcription in presence and absence of Rpb4p imply a more active role for Rpb4p in transcription of at least a subset of genes

Genome-wide expression profile of RNA polymerase II subunit mutant of yeast using microarray technology

Rpb4, the non-essential core subunit of RNA polymerase II has been assigned a function of regulating stress response in S. cerevisiae based mainly on phenotypes associated with its deletion. The actual mechanism has been elusive, although various hypotheses have been put forth. We have shown previously that it plays a significant role in activation of a subset of genes, rather than causing generalized defect in transcription. We used the microarray technology to look at the effect of this RNA polymerase subunit on the expression pattern of the entire S. cerevisiae genome. Many surprises emerged when we compared the genome-wide expression patterns of wild type and a mutant lacking the RPB4 gene (rpb4Δ) subjected to heat shock. The initial analysis of genes downregulated in the mutant showed that the co-regulation of genes is not position-dependent, although the locus carrying the deletion had unexpectedly a large cluster of down-regulated genes. We also found that among the known down-regulated genes, a majority is involved in hexose uptake and utilization. We speculate that this could potentially contribute to the slow growth rate of the mutant. Compared to the other components of the transcription machinery, the Rpb4 subunit affects a unique set of genes

A stable hybrid containing haploid genomes of two obligate diploid Candida species

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future

Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression

Histone acetylation is a diagnostic feature of transcriptionally active genes. The proper recruitment and function of histone acetyltransferases (HATs) and deacetylases (HDACs) are key regulatory steps for gene expression and cell cycle. Functional defects of either of these enzymes may lead to several diseases, including cancer. HATs and HDACs thus are potential therapeutic targets. Here we report that garcinol, a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind, is a potent inhibitor of histone acetyltransferases p300 (IC50≈7 μM) and PCAF (IC50≈5 μM) both in vitro and in vivo. The kinetic analysis shows that it is a mixed type of inhibitor with an increased affinity for PCAF compared with p300. HAT activity-dependent chromatin transcription was strongly inhibited by garcinol, whereas transcription from DNA template was not affected. Furthermore, it was found to be a potent inducer of apoptosis, and it alters (predominantly down-regulates) the global gene expression in HeLa cells

Whole genome expression profiles of yeast RNA polymerase II core subunit, Rpb4, in stress and nonstress conditions

Organisms respond to environmental stress by adopting changes in gene expression at the transcriptional level. Rpb4, a nonessential subunit of the core RNA polymerase II has been proposed to play a role in non-stress-specific transcription and in the regulation of stress response in yeast. We find that in addition to the temperature sensitivity of the null mutant of Rpb4, diploid null mutants are also compromised in sporulation and show morphological changes associated with nitrogen starvation. Using whole genome expression analysis, we report here the effects of Rpb4 on expression of genes during normal growth and following heat shock and nutritional starvation. Our analysis shows that Rpb4 affects expression of a small yet significant fraction of the genome in both stress and normal conditions. We found that genes involved in galactose metabolism were dependent on the presence of Rpb4 irrespective of the environmental condition. Rpb4 was also found to affect the expression of several other genes specifically in conditions of nutritional starvation. The general defect in the absence of Rpb4 is in the expression of metabolic genes, especially those involved in carbon metabolism and energy generation. We report that various stresses are affected byRPB4 and that on overexpression the stress-specific activators can partially rescue the corresponding defects

Modulation of polymerase II composition: A possible mode of transcriptional regulation of stress response in eukaryotes

Regulation of stress response in prokaryotes is mainly achieved at the transcriptional initiation level. Prokaryotes use alternative holoenzymes, consisting of the core polymerase associated with different sigma factors, which confer on it altered specificity of transcriptional initiation. Stress response being probably one of the most inevitable features of life, it would be interesting to find if eukaryotes also use a similar strategy at this level of regulation. Since the yeastSaccharomyces cerevisiae is a model system for studying many different phenomena in eukaryotes we review the transcriptional regulation of stress in this system. Based on published observations in the literature and our own studies, we have analysed the regulation of stress response, in the yeastS. cerevisiae. Two of the core subunits of the yeast RNA polymerase II, which show altered stoichiometry within the polymerase under different conditions appear to be involved specifically in regulating the stress response. In a very broad sense then, the altered subunit composition of the core polymerase or a different holoenzyme, appears to correlate with gene expression specific to stress response inS. cerevisiae and probably reflects the scenario in other eukaryotes

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens

Overexpression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotypes associated with the absence of the largest nonessential subunit

Abstract The easily dissociable subcomplex of Rpb4 and Rpb7 subunits of yeast RNA polymerase II has been considered, for long, to play a role in stabilizing Pol II under stress. On the basis of previous genetic and biochemical observations, it was proposed that within the subcomplex one of the functions of Rpb4p is to stabilize the interaction between Rpb7p and the rest of Pol II. We took a direct approach to test the latter possibility by overexpression and mutagenesis of RPB7 in absence of Rpb4p. We report here the results, which support the latter hypothesis. While it has been previously reported that absence of Rpb4p results in reduction in overall transcription by Pol II, our comparative analysis of RNAs from RPB4 and rpb4Á cells suggests that there are indeed several genes differentially expressed between the two cells. We propose that the qualitative differences in overall transcription in presence and absence of Rpb4p imply a more active role for Rpb4p in transcription of at least a subset of genes. [Sharma N. and Sadhale P. P. 1999 Overexpression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotypes associated with absence of the largest, nonessential subunit Rpb4

Identification and characterization of DdRPB4, a subunit of Dictyostelium discoideum RNA polymerase II

Rpb4, the fourth largest subunit of the eukaryotic RNA polymerase II (RNAPII), is required for growth at extreme temperatures and for an appropriate response to nutrient starvation in yeast. Sequence homologs of Rpb4 are found in most sequenced genomes from yeast to humans. To elucidate the role of this subunit in nutrient starvation, we chose Dictyostelium discoideum, a soil amoeba, which responds to nutrient deprivation by undergoing a complex developmental program. Here we report the identification of homolog of Saccharomyces cerevisiae RPB4 in D. discoideum. Localization and complementation studies suggest that Rpb4 is functionally conserved. DdRPB4 transcript and protein levels are developmentally regulated. Although DdRPB4 could not be deleted, overexpression revealed that the Rpb4 protein is essential for cell survival and is regulated stringently at the post-transcriptional level in D. discoideum. Thus maintaining a critical level of Rpb4 is important for this organism