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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
No abstract available.
Nuclear pore complexes (NPCs) orchestrate cargo between the cytoplasm and nucleus and regulate chromatin organization. NPC proteins, or nucleoporins (Nups), are required for human immunodeficiency virus type 1 (HIV-1) gene expression and genomic integration of viral DNA. I utilize the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because retrotransposons are the progenitors of retroviruses and have conserved integrase (IN) enzymes. Ty1-IN targets Ty1 elements into the genome upstream of RNA polymerase (Pol) III-transcribed genes such as transfer RNA (tRNA) genes. Evidence that S. cerevisiae tRNA genes are recruited to NPCs prompted my investigation of a functional role for the NPC in Ty1 targeting into the genome. I find that Ty1 mobility is reduced in multiple Nup mutants that cannot be accounted for by defects in Ty1 gene expression, complementary DNA (cDNA) production or Ty1-IN nuclear entry. Instead, I find that Ty1 insertion upstream of tRNA genes is impaired. I also identify Nup mutants with wild type Ty1 mobility but impaired Ty1 targeting. The NPC nuclear basket, which interacts with chromatin, is required for both Ty1 expression and nucleosome targeting. Deletion of components of the NPC nuclear basket causes mis-targeting of Ty1 elements to the ends of chromosomes. The mis-targeting suggests that nuclear basket Nups are required directly or indirectly, perhaps as global architects or regulators of chromatin organization to orchestrate Ty1 targeting upstream of Pol III-transcribed genes.
The budding yeast Saccharomyces cerevisiae is used as a model organism forunderstanding cellular dynamics such as cell cycle regulation. Separase endopeptidases areessential to these events, being responsible for cleavage of cohesin – the molecular glueholding sister chromatids together. To examine the function of separases in a systematicfashion, a temperature sensitive mutant of the yeast homologue, Esp1, was subjected to ahigh-throughput technique known as synthetic genetic array to identify genetic interactions.Examination of the list of alleles confirmed to cause a synthetic lethal or synthetic dosagelethal phenotype in the query esp1-1 mutant (“hits”) established the legitimacy of thesescreens, as many related to known Esp1 functions. Surprisingly, categorization of these hitsby biological process revealed an enrichment for genes involved in RNA metabolicprocesses. Concurrent affinity immunoprecipitation of separase followed by massspectrometry further showed a physical interaction between Esp1 and the integrase portion ofTy1 retrotransposons. Evidence that this interaction was indicative of a role for separase inretrotransposition was attained when the esp1-1 mutant was found to have transpositiondefects. Similar defects were also present in mutants of both the cohesin loader, SCC2, andthe structural maintenance of chromosome, SMC3, genes. Interestingly, cohesin loads at locitranscribed by RNA polymerase III, while hotspots of Ty1 integration are upstream of thesame sites. I propose that separase acts as a bridge to aid in targeting the Ty1 pre-integrationcomplex to these hotspots. The aforementioned categorization of esp1-1 genetic interactorsalso indicated that separase may somehow be involved in mRNA biogenesis. As esp1-1mutants have intact translational machinery, my data suggests that separase functions either directly in transcription or, more likely, post-transcriptionally. In all, screening for esp1-1genetic interactions has revealed several new avenues for separase studies.
No abstract available.
No abstract available.
Master's Student Supervision (2010 - 2018)
Wine fermentation presents a unique environment in which strains of the budding yeast Saccharomyces cerevisiae have evolved with superior tolerance to a multitude of stressors. Ethanol toxicity has one of the greatest impacts in reducing cellular viability and metabolic function and thus poses a threat in causing slow, stuck and incomplete fermentations. In pursuit of optimizing commercial strains of S. cerevisiae, identification of genes involved in ethanol tolerance has been of recent interest. Genomic resources such as the S288C deletion collection and microarray analysis have been widely utilized and have provided a foundation, albeit incomplete, for understanding ethanol toxicity in yeast. As a new approach, the recently developed molecular barcoded yeast open reading frame (MoBY-ORF) library, in which all S. cerevisiae genes along with native promoter and terminator sequences have been cloned into barcoded high copy 2μ plasmid vectors, has been utilized. Both the S288C laboratory strain and the M2 wine strain of S. cerevisiae were transformed with the MoBY ORF library and genes were identified by quantitation of molecular barcodes after 48 hours in 12% ethanol stressed library pools. Five genes were highly ranked in both S288C and M2 screens, two of which, RCN1 and RSA3, improved tolerance to high (16-21% v/v) ethanol toxicity over 1-3 hour incubation periods in both strain backgrounds. RCN1 is a regulator of the stress signalling protein calcineurin whereas RSA3 has a role in ribosome maturation. Additional fitness advantages conferred upon overproduction of RCN1 and RSA3 include increased resistance to cell wall degradation, heat, osmotic and oxidative stress. Neither RCN1 nor RSA3 over-expression in M2 during model fermentations of synthetic wine medium significantly increased the fermentation rate or final ethanol yield. Regulation of calcineurin and ribosomal assembly processes during ethanol stress, however, may still be key targets for improving tolerance to the stressful conditions of wine fermentation.