Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2072. Adviser: Hiten D. Madhani.

<p> The vacuole in <i>Saccharomyces cerevisiae</i> serves as a model for the mammalian lysosome. In a genome wide screen for mutants with severe growth <u>h</u>ypersensitivity to <u>h</u>ygromycin B, our lab identified 14 <i>HHY</i> genes. Each of the <i> hhy</i> mutants is defective in vacuolar trafficking and/or function and also sensitive to rapamycin and caffeine, suggesting a compromised target of rapamycin (TOR) kinase pathway. My research divides the <i>hhy</i> mutants into two groups based on quantitative growth analyses in the presence of hygromycin B. (1) a super affected group (<i>s-hhy's</i>) and (2) a dose-dependent group (<i>d-hhy's</i>). The <i>s-HHY</i> genes include <i>CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52,</i> and <i>VPS54</i>. Evaluation of the known functions of <i> s-HHY</i> gene products reveals vesicular trafficking function at the trans-Golgi and late endosome interface to be a common factor. In yeast, the TORC1 complex localizes to the vacuole. Because a compromised TORC1 complex signaling is suggested in <i>hhy</i> mutant strains due to their caffeine or rapamycin sensitivity, I hypothesized that compromised TORC1 signaling in <i>HHY's</i> may be due to defects in the vacuolar localization of Tor1 kinase. To assess Tor1 kinase localization, we utilized a strain expressing endogenously tagged Tor1-GFP and assayed localization to the vacuolar membrane in each of the s-hhy mutants using confocal microscopy. In wild-type cells, Tor1-GFP co-localizes with the vacuolar membrane marker FM4-64 while s-hhy deletion strains fail to localize Tor1-GFP to the vacuolar membrane when treated with hygromycin B. Our results implicate that Tor1p is transported to the vacuole membrane via the late endosome (CPY pathway) and not the ALP (Vam3) pathway. Additionally, <i>s-hhy</i> mutants are unable to recover growth after a 4-hour treatment with hygromycin B, similar to EG0 mutants, which fail to exit from G0 after treatment with the TORC1 inhibiting drug rapamycin. Based on our data, we propose a model in which the <i>s-HHY </i> gene functions in vesicular trafficking at the trans-Golgi/late endosome interface are involved in recruitment and subsequent transport of Tor1p to the vacuolar membrane, and that interface is hypersensitive to hygromycin B. We also propose that Tor1 kinase localization at the vacuole is essential for its cell cycle regulatory function.</p>

<p>The success of Drosophila as a system for genetic analysis is closely linked to its amenability to genetic manipulation. Part 1 of the dissertation elucidates a novel scheme for long-range targeted manipulation of genes. We integrated an 80-kb genomic fragment at its endogenous locus, utilizing a targeted attP attachment site for the phiC31 integrase. We achieved single-copy reduction of the resulting region duplication by inducing recombinational DNA repair. We showed that this two-step scheme of integration and reduction is efficient and useful for delivering modifications. We established a vector configuration that facilitates the recovery of modifications. The integrating genomic fragment allowed for delivery of a new attachment site at 70 kb from the existing <i>attP</i> into a new locus, making it susceptible to targeted mutagenesis. We extrapolate that with this scheme, only 1 200 lines bearing att-sites throughout the genome would suffice to render all Drosophila genes amenable to targeted mutagenesis. Excitingly, this method should be readily applicable to other systems. </p><p> In Part 2 of the dissertation, I explored the question of telomere organization in Drosophila. Telomeres demarcate the ends of linear chromosomes to distinguish them from broken ends. In yeast, they cluster at the periphery of the nucleus establishing a compartment of silent chromatin. To bring insight into telomere organization in a higher organism, we followed EGFPlabeled Drosophila telomeric protein HOAP <i>in vivo</i> and found that the 16 telomeres cluster into 4-6 foci per nucleus in somatic tissues. Interestingly, HOAP signal intensity in the clusters doubles in interphase, potentially due to loading of HOAP to newly replicated telomeres. We tested several predictions about rules governing clustering. First, by inspecting mutant embryos that develop as haploids, we found that clustering is not mediated by associations between homologs. Second, by demonstrating clustering capability for a telomere of novel sequence, we eliminated DNA sequence homology and identity as important factors. Third, by marking both ends of a chromosome, we ruled out predominance of intra-chromosomal interactions. We propose that clustering is indiscriminate of sequence and is likely maintained by a yet undetermined factor. </p>

<p>Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsa??) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdca??) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10a?? mutation was introduced into five different wdchsa?? strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between WdCDC10 and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10a??. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases.

<p> Tumorigenesis of relatively late onset occurring in patients with no family history of cancer syndromes is assumed to be driven by somatic mutations. The advent of high-throughput sequencing allows unbiased probing for genomic aberrations on an unprecedented scale. Somatic mutations, insertions and deletions, and copy number variations are able to be identified by parallel sequencing of tumor DNA and normal DNA from an individual patient. Somatic aberrations identified are classified as either passenger mutations that do not contribute to tumorigenesis or pathogenic driver mutations. Driver mutations are able to be identified due to their recurrence across multiple affected patients at a frequency greater than would be expected by chance.</p><p> Tumors occurring in the same tissue and from the same cell type often display diverse phenotypes with distinct mutational signatures. Therefore I applied high-throughput sequencing to probe for somatic mutations in two very specific endocrine tumor types - parathyroid-producing adenomas and insulin-producing adenomas (insulinomas). Prior to this study, neither tumor type had been probed for somatic mutations in a large-scale, unbiased manner. Though a limited number of mutated genes had been identified to play a role in familial and sporadic tumorigenesis in these tumor types, the majority of pathogenesis remained unexplained.</p><p> In order to maximize detection of variation in coding regions of the genome, an exome capture array was applied to the DNA prior to sequencing. In both tumor types, exome sequencing was applied to a small number of tumor-normal tissue pairs. Additional targeted sequencing of candidate driver mutations was then performed using Sanger sequencing on larger validation cohorts of tumors.</p><p> Exome sequencing revealed few somatic, protein-altering mutations in each tumor type (average &lt;4 per tumor), therefore any recurrent variation was highly probable to be tumorigenic. Exome sequencing of the parathyroid adenomas revealed that four of eight tumors harbored a frameshift deletion or nonsense mutation in <i>MEN1</i>, which was always accompanied by loss of heterozygosity (LOH) of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N missense mutation of the histone methyltransferase <i>EZH2</i> gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in an additional 185 parathyroid adenomas revealed somatic <i>MEN1</i> mutations in a large number of tumors (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic <i>EZH2</i> mutation that was found by exome sequencing. This confirms the frequent role of LOH of chromosome 11 and <i>MEN1</i> gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, <i>EZH2</i>, in endocrine tumorigenesis. </p><p> Exome sequencing identified an identical somatic, heterozygous mutation in Yin Yang 1 transcription factor (<i>YY1</i>) in two of seven insulinomas. Targeted sequencing of an additional 36 insulinomas revealed twelve more insulinomas that harbored this identical T372R missense mutation in <i>YY1.</i> This mutation occurs at a highly-conserved residue in a highly-conserved zinc finger DNA-binding domain. ChIP-Seq demonstrated that this mutation changes the DNA motif bound by YY1. This altered binding likely drives pathogenesis due to aberrant regulation of genes not regulated by YY1^WT. With the goal of identifying differentially-expressed genes in <i>YY1^T372R</i> tumors, I performed gene expression analysis on eleven tumors; six that were <i>YY1^WT</i> and five that were <i>YY1^T372R.</i> This demonstrated that YY1^T372R imparts a unique expression signature. Interestingly, several differentially-expressed genes were involved in key pathways regulating insulin secretion, including <i>ADCY1</i> (an adenylyl cyclase) and <i>CACNA2D2</i> (a Ca²⁺ channel pore-forming subunit), both of which were upregulated in <i>YY1^T372R</i>-tumors. Importantly, <i>in vitro</i> studies using the INS-1 rat insulinoma cell line demonstrated that upregulation of each of gene is sufficient to markedly increase insulin secretion. Furthermore, both genes harbored specific YY1^T372R binding sites that may account for their significantly altered expression.</p><p> Both studies identify novel driver mutations that shed light on the mechanisms of endocrine tumorigenesis. Furthermore, my findings reinforce the notion that common somatic mutations within the exome account for the majority of instances of sporadic tumorigenesis.</p>

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6408. Adviser: Jeffery S. Cox.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007. / Title from home page (viewed Nov. 14, 2008). Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5708. Adviser: William Saxton.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-07, Section: B, page: 4220. Adviser: Thomas B. Kornberg.

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-02, Section: B, page: 0687. Adviser: Brian Black.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-02, Section: B, page: 0677. Adviser: Herwig Baier.