RNA polymerase II (Pol II) is the enzyme responsible for the synthesis of all mRNAs in eukaryotic cells. As the central component of the eukaryotic transcription machinery, Pol II is the final target of regulatory pathways. While the role for different Pol II associated proteins, co-activators and general transcription factors (GTFs) in regulation of transcription in response to different stimuli is well studied, a similar role for some subunits of the core Pol II is only now being recognized. The studies reported in this thesis address the role of the fourth largest subunit of Pol II, Rpb4, in transcription and stress response using Saccharomyces cerevisiae as the model system. Rpb4 is closely associated with another smaller subunit, Rpb7 and forms a dissociable complex (Edwards et al., 1991). The rpb4 null mutant is viable but is unable to survive at extreme temperatures (>34ºC and <12ºC) (Woychik and Young, 1989). This mutant has also been shown to be defective in activated transcription and unable to respond properly in several stress conditions (Pillai et al., 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in four different parts below: i) Studies on Genetic and Functional Interactions of Rpb4 with SAGA/TFIID Complex to Confer Promoter- Specific Transcriptional Control To carry out transcription, Pol II has to depend on several general transcription factors, mediators, activators, and co-activators and chromatin remodeling complexes. In the present study, we tried to understand the genetic and functional relationship of Rpb4 with some of the components of transcription machinery, which will provide some insight into the role of Rpb4 during transcription. Our microarray analysis of rpb4∆ strain suggests that down regulated genes show significant overlap with genes regulated by the SAGA complex, a complex functionally related to TFIID and involved in regulation of the stress dependent genes. The analysis of combination of double deletion mutants of either the SAGA complex subunits or the TFIID complex with rpb4∆ showed that both these double mutants are extremely slow growing and show synthetic growth phenotype. Further studies, including microarray analysis of these double mutants and ChIP (chromatin immunoprecipitation) of Rpb4 and SAGA complex, suggested that Rpb4 functions together with SAGA complex to regulate the expression of stress dependent genes. ii) Study of Genome Wide Recruitment of Rpb4 and Evidence for its Role in Transcription Elongation Biochemical studies have shown that Rpb4 associates sub-stoichiometrically with the core RNA polymerase during log phase but whether recruitment of Rpb4 is promoter context dependent or occurs only at specific stage of transcription remains largely unknown. Having discovered that Rpb4 can recruit on both TFIID and SAGA dominated promoters, it was important to study the genome wide role of Rpb4. Using ChIP on chip experiments, we have carried out a systematic assessment of genome wide binding of Rpb4 as compared to the core Pol II subunit, Rpb3. Our analysis showed that Rpb4 is recruited on coding regions of most transcriptionally active genes similar to the core Pol II subunit Rpb3 albeit to a lesser extent. This extent of Rpb4 recruitment increased on the coding regions of long genes pointing towards a role of Rpb4 in transcription elongation of long genes. Further studies showing transcription defect of long and GC rich genes, 6-azauracil sensitivity and defective PUR5 gene expression in rpb4∆ mutant supported the in vivo evidence of the role of Rpb4 in transcription elongation. iii) Genome Wide Expression Profiling and RNA Polymerase II Recruitment in rpb4∆ Mutant in Non-Stress and Stress Conditions Structural studies have suggested a role of Rpb4/Rpb7 sub-complex in recruitment of different factors involved in transcription (Armache et al., 2003; Bushnell and Kornberg, 2003). Though only few studies have supported this aspect of Rpb4/Rpb7 sub-complex, more research needs to be directed to explore this role of Rpb4/Rpb7 sub-complex. To study if Rpb4 has any role in recruitment of Pol II under different growth conditions, we have studied genome wide recruitment of Pol II in the presence and absence of Rpb4 during growth in normal rich medium as well as under stress conditions like heat shock and stationary phase where Rpb4 is shown to be indispensable for survival. Our analysis showed that absence of Rpb4 results in overall reduced recruitment of Pol II in moderate condition but this reduction was more pronounced during heat shock condition. During stationary phase where overall recruitment of Pol II also goes down in wild type cells, absence of Rpb4 did not lead to further decrease in overall recruitment. Interestingly, increased expression levels of many genes in the absence of Rpb4 did not show concomitant increase in the recruitment of Pol II, suggesting that Rpb4 might regulate these genes at a post-transcriptional step. iv) Role of Rpb4 in Pseudohyphal Growth The budding yeast S. cerevisiae can initiate distinct developmental programs depending on the presence of various nutrients. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathways. Previous work from our group has shown that rpb4∆ strain shows predisposed pseudohyphal morphology (Pillai et al., 2003), but how Rpb4 regulates this differentiation program is yet to be established. In the present study, we found that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4∆/ rpb4∆ cells exhibit pseudohyphae even in the absence of a functional MAP kinase and cAMP-PKA pathways. Genome wide expression profile showed that several downstream genes of RAM signaling pathway were down regulated in rpb4∆ cells. Our detailed genetic analysis further supported the hypothesis that down regulation of RAM pathway might be leading to the pseudohyphal morphogenesis in rpb4∆ cells.

O uso de microrganismos na biotecnologia tem grande importância e interesse econômico no Brasil. Entre esses microrganismos a levedura Saccharomyces cerevisiae tem grande destaque nos processos fermentativos para produção de pães, bebidas e álcool combustível. Dimorfismo em S. cerevisiae (alteração na morfologia celular de células brotantes para estruturas filamentosas) tem sido observado em condições de deficiência de nitrogênio, carbono e presença de álcoois superiores. Isso pode ser um tipo de defesa da levedura, que ao encontrar um meio prejudicial ao seu desenvolvimento, através da alongação das células, crescimento hifal e da invasão do meio, cria novos mecanismos para encontrar alimento para o seu desenvolvimento. Neste trabalho, dezessete linhagens (haplóides e diplóides) de S. cerevisiae isoladas do processo fermentativo industrial para produção de etanol foram caracterizadas quanto à filamentação induzida por deficiência de carbono (crescimento invasivo), nitrogênio e presença de álcoois superiores, em meio de cultura sólido. Objetivou-se também avaliar a indução da filamentação por álcool isoamílico em condições de fermentação e seus efeitos sobre os parâmetros fermentativos. A maioria das linhagens apresentou filamentação em resposta aos álcoois isoamílico, butanol e isobutanol, não respondendo ao metanol, sendo mais marcante em linhagens haplóides. O álcool isoamílico foi o indutor mais eficiente em meio (YEPD), linhagens diplóides apresentaram crescimento invasivo, embora esse tipo de filamentação seja mais comum em linhagens haplóides. Resultados semelhantes foram observados quando se utilizou frutose e manose em substituição à glicose no meio de cultura. As linhagens não filamentaram em meio de cultura deficiente em nitrogênio (SLAD). A linhagem CCA193 (PE-02), extensivamente utilizada nas destilarias da região, foi escolhida para a indução da filamentação por álcool isoamílico em condições de fermentação (meio de caldo de cana e sistema de reciclo celular). A adição de 0,1% desse álcool superior afetou significativamente os parâmetros fermentativos, induzindo filamentação após o terceiro ciclo fermentativo, coincidindo com a recuperação da viabilidade celular, número de células viáveis, produção de etanol e diminuição do Brix residual, embora não se comparando aos valores alcançados ao final do sexto ciclo no tratamento sem álcool isoamílico. Os resultados obtidos indicaram que a filamentação induzida por álcoois superiores e deficiência de nutrientes (especialmente carbono) é um processo comum em linhagens industriais de S. cerevisiae e pode contribuir para a manutenção/sobrevivência das células em condições adversas. / The use of microorganisms in biotechnology is an important and economical area of interest in Brazil. Among these, the yeast Saccharomyces cerevisiae is specially remarkable in baking industry and alcohol fermentation. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and presence of fusel alcohoes. This can be a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal growth and medium invasiveness. In this work seventeen industrial strains of S. cerevisiae (haploid and diploid) isolated from the fermentative process for alcohol production were characterized for filamentation induced by carbon (invasive growth) and nitrogen deprivation and presence of fusel alcohol, in solid culture media. The induction of filamentation by isoamyl alcohol in fermentation condition was also aimed, evaluating its effects on the fermentative parameters. The majority presented filamentation induced by isoamyl alcohol, butanol and isobutanol, but not by methanol. The isoamyl alcohol was the most effective inducer in rich medium (YEPD), diploid strains showed invasive growth, although this kind of filamentous growth be common in haploid strains. Similar results were observed when fructose and mannose were utilized replacing glucose in the culture medium. In nitrogen-deficient medium (SLAD) the strains did not filament. The yeast strain CCA193 (PE-02), extensively used in the alcohol units, was chosen for filamentation induction by isoamyl alcohol in fermentation conditions (sugar cane juice medium and cell recycle system). The addition of 0.1% of this higher alcohol affected significantly the fermentative parameters, however filamentation was induced after the third fermentative cycle, coincident with the cell viability, viable cell number, ethanol production recuperation and residual Brix decrease, though the values were not comparable to the treatment without isoamyl alcohol by the sixth cycle.The results obtained indicate that the filamentation induced by higher alcohol and nutrient deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae and can contribute for the cell maintenance/survival in adverse conditions.

<p>In an attempt to gain more insight on functional evolution of cAMP-PKA pathway I have taken a comparative approach and examined functional interactions of cAMP-PKA signaling in well-studied yeast developmental programs and closely related <italic>Saccharomyces sensu stricto<italic/>. species. I have shown that variation in cAMP-PKA signaling contributes significantly to variation in developmental responses in <italic>S cerevisiae. Variation in pseudohyphal growth and sporulation, two inversely correlated developmental strategies to nutrient limitation in yeast, proportional to variation in intracellular cAMP levels. <italic>S. cerevisiae strains proficient in pseudohyphal growth have higher intracellular cAMP concentrations relative to strains that sporulate efficiently. Phenotypic, genetic and signaling data presented here suggest that the cAMP-PKA signaling underlies a phenotypic trade-off between sporulation and pseudohyphal growth in <italic>S. cerevisiae<italic/>.</p><p>Further investigation into the role of cAMP-PKA signaling in closely related <italic>S paradoxus<italic/> and <italic>S bayanus revealed an antagonistic function of cAMP-PKA signaling for developmental responses in <italic>S. bayanus. Unlike in <italic>S. cerevisiae, increased cAMP concentrations surprisingly inhibit pseudohyphal response in <italic>S. bayanus<italic/>. Another unanticipated finding in this work is that in <italic>S. bayanus<italic/>. Flo11, required for pseudohyphal differentiation in S. cerevisiae, is dispensable. Additionally, interactions of cAMP-PKA signaling and the general-stress response mechanism appear reversed in <italic>S. bayanus<italic/>. As shown by deletion mutation, gene expression and pharmacological treatment data, altered interactions and alternative targets downstream of cAMP-PKA could critically contribute to alternative regulation of nutrient-induced development in <italic>S. bayanus<italic/>.</p><p>Intracellular cAMP concentrations show decaying oscillations upon glucose replenishment in derepressed yeast cells. The quantitative characteristics of oscillations are distinct within and between Saccharomyces species. Given the tight regulation of cAMP levels and its critical role, the variation in cAMP oscillatory dynamics could be reflective of differential interactions of cAMP-PKA signaling that also underlie induction of developmental programs to changing environments. As such, intracellular cAMP levels and dynamics could potentially be used as molecular phenotypes.</p> / Dissertation

O uso de microrganismos na biotecnologia tem grande importância e interesse econômico no Brasil. Entre esses microrganismos a levedura Saccharomyces cerevisiae tem grande destaque nos processos fermentativos para produção de pães, bebidas e álcool combustível. Dimorfismo em S. cerevisiae (alteração na morfologia celular de células brotantes para estruturas filamentosas) tem sido observado em condições de deficiência de nitrogênio, carbono e presença de álcoois superiores. Isso pode ser um tipo de defesa da levedura, que ao encontrar um meio prejudicial ao seu desenvolvimento, através da alongação das células, crescimento hifal e da invasão do meio, cria novos mecanismos para encontrar alimento para o seu desenvolvimento. Neste trabalho, dezessete linhagens (haplóides e diplóides) de S. cerevisiae isoladas do processo fermentativo industrial para produção de etanol foram caracterizadas quanto à filamentação induzida por deficiência de carbono (crescimento invasivo), nitrogênio e presença de álcoois superiores, em meio de cultura sólido. Objetivou-se também avaliar a indução da filamentação por álcool isoamílico em condições de fermentação e seus efeitos sobre os parâmetros fermentativos. A maioria das linhagens apresentou filamentação em resposta aos álcoois isoamílico, butanol e isobutanol, não respondendo ao metanol, sendo mais marcante em linhagens haplóides. O álcool isoamílico foi o indutor mais eficiente em meio (YEPD), linhagens diplóides apresentaram crescimento invasivo, embora esse tipo de filamentação seja mais comum em linhagens haplóides. Resultados semelhantes foram observados quando se utilizou frutose e manose em substituição à glicose no meio de cultura. As linhagens não filamentaram em meio de cultura deficiente em nitrogênio (SLAD). A linhagem CCA193 (PE-02), extensivamente utilizada nas destilarias da região, foi escolhida para a indução da filamentação por álcool isoamílico em condições de fermentação (meio de caldo de cana e sistema de reciclo celular). A adição de 0,1% desse álcool superior afetou significativamente os parâmetros fermentativos, induzindo filamentação após o terceiro ciclo fermentativo, coincidindo com a recuperação da viabilidade celular, número de células viáveis, produção de etanol e diminuição do Brix residual, embora não se comparando aos valores alcançados ao final do sexto ciclo no tratamento sem álcool isoamílico. Os resultados obtidos indicaram que a filamentação induzida por álcoois superiores e deficiência de nutrientes (especialmente carbono) é um processo comum em linhagens industriais de S. cerevisiae e pode contribuir para a manutenção/sobrevivência das células em condições adversas. / The use of microorganisms in biotechnology is an important and economical area of interest in Brazil. Among these, the yeast Saccharomyces cerevisiae is specially remarkable in baking industry and alcohol fermentation. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and presence of fusel alcohoes. This can be a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal growth and medium invasiveness. In this work seventeen industrial strains of S. cerevisiae (haploid and diploid) isolated from the fermentative process for alcohol production were characterized for filamentation induced by carbon (invasive growth) and nitrogen deprivation and presence of fusel alcohol, in solid culture media. The induction of filamentation by isoamyl alcohol in fermentation condition was also aimed, evaluating its effects on the fermentative parameters. The majority presented filamentation induced by isoamyl alcohol, butanol and isobutanol, but not by methanol. The isoamyl alcohol was the most effective inducer in rich medium (YEPD), diploid strains showed invasive growth, although this kind of filamentous growth be common in haploid strains. Similar results were observed when fructose and mannose were utilized replacing glucose in the culture medium. In nitrogen-deficient medium (SLAD) the strains did not filament. The yeast strain CCA193 (PE-02), extensively used in the alcohol units, was chosen for filamentation induction by isoamyl alcohol in fermentation conditions (sugar cane juice medium and cell recycle system). The addition of 0.1% of this higher alcohol affected significantly the fermentative parameters, however filamentation was induced after the third fermentative cycle, coincident with the cell viability, viable cell number, ethanol production recuperation and residual Brix decrease, though the values were not comparable to the treatment without isoamyl alcohol by the sixth cycle.The results obtained indicate that the filamentation induced by higher alcohol and nutrient deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae and can contribute for the cell maintenance/survival in adverse conditions.

No description available.

Yeast Saccharomyces cerevisiae as an unicellular organism is one of the best-studied experimental organisms. It is an important model organism for the study of intracellular processes of eukaryotic cells. Yeasts are also social organisms with cell-to-cell communication able to form organized multicellular structures (colonies and biofilms). Yeast and other microorganisms in nature prefer to form colonies on solid substrates rather than to grow as "planktonic" single cells (Palková, 2004; Wimpenny, 2009). The yeast S. cerevisiae typically forms colonies, biofilms were described only rarely. Yeast colonies exhibit an organized morphological pattern characteristic of each particular yeast strain (Kocková-Kratochvílová, 1982). This work is focusing on morphology and differentiation of the S. cerevisiae colonies of common laboratory strains forming less structured colonies, and strains of the Σ1278b genetic background forming highly structured "fluffy" colonies. It shows that polarized budding pattern and especially cell ability to form aggregates enable development of structured morphology. During development of "fluffy" colonies two differently regulated events of dimorphic switch from yeast form to filamentous growth occur. One of these events is dependent on the surface glycoprotein, Flo11p flocculin. This...

No description available.