Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Cynthia Kenyon.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Jennifer C. Fung.

<p> Forward progress in empirical population genetics is closely tied to the development of theory which can accomodate and keep pace with the production of genetic data. In recent years, the ability to survey genetic variation at increasingly greater resolution, across the genomes of a variety of species, has prompted new approaches to use this data for population genetic inference. While many models have historically relied on assuming independence among genetic variants in a sample of chromosomes, there are now a variety of methods which can use the non-independence among variants as a source of information. In particular, the unique combination and co-inheritance of variants on a chromosome can be used to define "haplotypes" of linked genetic variation associated with specific populations, individuals, or variants from which they are descended. The work presented here is a contribution to this class of population genetic models which describes: (1) a method to estimate the timing of adaptation for a beneficial allele, including several applications to recent human evolution, (2) an application of the same method to infer the timing of introgression for coat color alleles in North American wolves and high-altitude adaptation in Tibetans, (3) a model to infer the action of purifying selection against genetic incompatibilities in a hybrid zone, and (4) a reanalysis of genomic data from <i>Heliconius</i> butterflies which confirms the role of hybridization in transfering mimicry phenotypes between species.</p><p>

Most eukaryotic protein-coding genes are split into exons and introns, and introns need to be spliced for the production of mature mRNA by pre-mRNA splicing. Pre-mRNA splicing is very important for eukaryotic gene expression, because it is not only a key step in producing mature mRNA, but can also affect transcription and translation. The purpose of this study is to investigate the relationship between gene expression and splicing efficiency since the relationship has not been studied systematically from a bioinformatic approach. In this thesis research, we focus on the question of how gene expression would constrain the evolution of three principal splicing signals: the donor splice site, the acceptor splice site, and the branchpoint sequence (BPS). We chose yeast, Saccharomyces cerevisiae, as the model organism in this study due to its many research advantages such as relatively simple splicing mechanism and extensive genome-wide characterization of gene expression at both transcript and protein levels. We first studied the relationship between gene expression and the strength of the donor and acceptor splice sites in the yeast, with the latter being characterized by position weight matrix scores. We found that donor and acceptor splice sites in highly expressed genes have significantly higher mean, but smaller variance, of splicing strength than that in lowly expressed genes. In addition, genes with extremely low splice site strength tend to be spliced by non-spliceosomal mechanisms, and the last nucleotide on the exon side in the donor splice site (immediately upstream of the 5'GU dinucleotide) is important for splicing. We further studied the relationship between gene expression and BPS in the yeast. The results revealed that the BPS strength of highly expressed yeast intron-containing genes (ICGs) is significantly higher than that of lowly expressed yeast ICGs. Moreover, highly expressed yeast ICGs have significantly longer distance between the donor splice site and BPS (Si distance) and slightly longer distance between BPS and the acceptor splice site (S2 distance) than lowly expressed yeast ICGs. The long Si distance of highly expressed yeast ICG does not indicate the potential of enhancing splicing efficiency through forming secondary structure in the region between the donor splice site and BPS.

Myoclonus dystonia (DYT11, OMIM 159900) (MD) is a movement disorder characterized by bilateral alcohol responsive myoclonic jerks often seen in combination with dystonia. MD is inherited as an autosomal dominant trait with reduced penetrance upon maternal transmission. Patients with this disorder are not diminished in their intellectual capacity and have a normal life span. In 2001, mutations in the epsilon-sarcoglycan gene on human chromosome 7q21 were implicated in causing this disorder. Our laboratory identified a 2nd locus on chromosome 18p11 that co-segregates with this disorder, however, a disease causing mutation has not been identified. To establish the function of epsilon-sarcoglycan within the mammalian brain, I generated a conditional knock-out (CKO) mouse of Sgce and utilized bacterial recombineering to generate a cassette that contained a "floxed" exon 1 of epsilon-sarcoglycan. ES cells that had correctly incorporated the CKO targeting cassette were selected and used to generate 3 chimeric male mice by blastocyst injection or morula aggregation. Once germline transmission of the CKO allele has been established, these mice will be bred to Cre expressing mice to generate an sgcenull, and recapitulate the phenotype of MD patients. In addition, I also developed an in vitro model of MD using RNAi directed against epsilon-sarcoglycan in the mouse neuroblastoma cell line, N1E-115. Silencing of epsilon-sarcoglycan in this cell line resulted in a decrease in the neuronal nitric oxide synthase (nNos) and an increase in cell proliferation. This data will provide insight and direction during the characterization of the sgcenull mice.

Variations in the expression of drug metabolizing enzymes of the Cytochrome P450 superfamily are a principal cause of atypical reactions to therapeutics. The molecular mechanisms by which the metabolizing enzymes of the drug are regulated, and the effects of genetic variation on this regulation, are not completely understood. Cytochrome P450 1A2 (CYP1A2) is one such enzyme. The APN mouse strain has low expression of the CYP1A2 enzyme, relative to the C3H/HeJ strain. It was hypothesized that this difference in expression of the CYP1A2 was occurring as a result of a single nucleotide polymorphism at the Cyp1a2 locus. This work has demonstrated that this variation in CYP1A2 expression occurs at the level of transcription and that a single nucleotide change 76 base pairs upstream of the transcriptional start site was critical for promoter function. The mechanism of constitutive CYP1A2 expression involves a previously unidentified cis-acting element in this region.

The transcriptional coactivator p300 displays an intrinsic histone acetyltransferase activity. It contains an evolutionarily conserved bromodomain serving as a specific acelyl-lysine binding module for histones or transcription factors to facilitate chromatin remodeling and transcriptional activation. The function of p300 is required by a diverse set of promoters. However, roles of bromodomain in the function of p300 are yet to be fully determined. In this study, we utilize cell lines expressing either wild-type or bromodomain truncated p300 to examine the expression of several p300-dependent genes that are involved in cell cycle regulation. The effects of histone acetylation on the expression of these genes were also examined by utilizing histone deacetylase inhibitors. Our results suggest that p300 regulates genes through different mechanisms and that bromodomain has roles in the recruitment of p300 to target promoters, providing an indication that the role of bromodomain in p300-dependent transcription is determined by individual promoter context.

Myoclonus Dystonia (MD) is an autosomal dominant disease with high but incomplete penetrance and is characterized by both involuntary myoclonic jerks and dystonic posturing. Our group has found that mutations within the epsilon sarcoglycan (SGCE) gene on chromosome 7q21 are associated with MD in 30-40% of affected individuals in 31 families studied, supporting the basis for genetic heterogeneity. Novel mutations have been found in SGCE by screening these families for point mutations and large deletions and duplications through the use of sequencing, high performance liquid chromatography (HPLC) and multi&dot;ligation probe amplification (MLPA) analysis. A 10cM genome wide linkage analysis of a large Canadian family provided significant LOD scores for microsatellite markers within the 18p11 region, now designated as the DYT 15 locus. Further haplotype analysis has narrowed a non-recombinant region associated with the disease phenotype to a 3.18 Mb region in this locus. Since the current understanding of Myoclonus Dystonia is poor, it is difficult to predict genes that could be responsible for MD. Sarcoglycans are essential constituents of the dystrophin-glycoprotein complex and are involved in linking the extracellular lamanin matrix to the actin filaments within the cytoplasm; therefore focus is given to the examination of potentially related structural genes that are expressed in the brain. By analyzing such candidate genes in a panel of affected individuals, we believe that a novel gene will be elucidated and provide insight into the mechanism of Myoclonus Dystonia.

Systems biology is an interdisciplinary field that combines engineering and molecular biology to better understand the 'design principles' of biological systems. One of the main goals in systems biology is to understand and map complex biological networks. In order to achieve this goal, tools able to process the non-linearity and high dimensionality of biological systems are urgently needed. To develop, test and benchmark tools for investigation of biological processes, it is important to utilize a model system that is able to provide a glimpse of complexity found in higher organisms, and be simple enough such that detailed studies can be performed rapidly, accurately, and reproducibly. Here, we have developed a system-identification framework for the extraction of quantitative and mechanistic information about causal relationship among genes using the canonical galactose utilization pathway in Saccharomyces cerevisiae. This framework, referred to as s&barbelow;ystematic g&barbelow;ene p&barbelow;erturbation a&barbelow;nalysis (SGPA), is based on the effects of systematic pair-wise gene deletions. In essence, the method establishes dynamical models of the regulatory network from single-cell measurements of steady-state input-output relationships, in systematically perturbed networks. SGPA framework is successful in identifying the network structure for the GAL system. This strategy leads the way to a better application of available resources and provides a scalable framework for system-identification and reverse-engineering of biological networks based on in vivo systematic data generation.

<p> Diabetes mellitus is the 7^th leading cause of death in the United States, with the most prevalent form being type II diabetes (T2D). T2D has a strong genetic component, but environmental factors like poor diet and lack of physical activity are major risk factors. T2D is characterized by the inability of insulin sensitive cell to respond to insulin, which leads to the reduction of glucose uptake. As a result diabetic patients excrete large amounts of glucose in their urine, but they also excrete large amount of <i>myo</i>-inositol (MI). MI is a six-carbon sugar alcohol that is a precursor to the phospholipid component, phosphatidylinositol. MI is also involved with other cellular functions as an insulin mimetic, a carbon/energy source, a phosphate storage molecule, and as an osmolyte. MI levels are regulated by synthesis, transport, recycling, and catabolism. Diabetic tissues have been found to have lower levels of MI. Moreover, the enzyme that catabolizes MI, myo-inositol oxygenase (MIOX), has been shown to be up-regulated in the renal tissue of diabetic patients. This study focuses on MI catabolism. Over the past decade <i>Drosophila melanogaster</i> has emerged as good organism to model diabetes and its associated complications. However, the <i> MIOX</i> gene has not been characterized in drosophilids. In this study the <i>MIOX</i> gene was characterized for seven drosophilid species, and the presence of the MIOX protein was established by demonstrating the ability of each species to utilize MI as the sole energy source. The steady-state levels of MIOX transcript in each of the species was shown to be higher in larvae grown with dietary inositol. Wild-type larvae grown under diabetic conditions (with high levels of sucrose) have decreased steady-state levels of <i>MIOX</i> transcript. Reducing the expression of MIOX results in larvae with a higher body density yet more triacylglycerides. Additionally, when expression of MIOX is knocked-down under diabetic conditions, abnormal lipid droplet storage is observed. These studies may contribute to the understanding of the role of MIOX in diabetic conditions such as obesity.</p><p>