The goal of this project is to identify QTL involved in Salmonella -susceptibility in the chicken. Salmonellosis is one of the most common causes of food poisoning in humans and is frequently caused by the ingestion of Salmonella-contaminated poultry products. Identification of QTL responsible for Salmonella-susceptibility may lead to more efficient control strategies, and selection against genes that may lead to increased risk of transmission. The genome scan was performed on a selection of 425 backcross progeny, (W1 x C) F1 x C, derived from C Salmonella-susceptible and W1 Salmonella-resistant chickens. The 425 backcross birds were phenotyped for their susceptibility to infection with Salmonella Typhimurium. A collection of 160 fluorolabelled microsatellite markers (FAM, TET, and HEX) was used to scan the chicken genome, which is 3800cM in size (1.2 x 109 bp) and consists of 39 linkage groups, 9 chromosomes and 30 microchromosomes. We first tested the available microsatellite markers for informativeness in C and W1 chicken lines. In addition, we also determined the allele lengths and PCR product intensity (qualitatively) to facilitate pooling of markers during electrophoresis. PCR reaction mixtures were prepared using a Packard MultiProbe II Robotic System and Minitrak to pipette DNA-PCR Master mix and primer mix into microtiter plates. PCR products were then pooled and analyzed on Perkin-Elmer ABI Prism 3700 DNA Analyzers. Analysis of informative microsatellite markers on the backcross panel resulted in the detection of two loci, one on Chromosome 7 carrying NRAMP1, and the other on microchromosome E41W17 carrying TLR4, linked to resistance to Salmonella infection in chickens.

Tissue- and gene-specific DNA methylation patterns are hallmarks of vertebrate genomes and have been suggested to play a critical role in regulating genome functions. There is remarkable diversity of DNA methylation patterns. However, it is yet unclear what is responsible for this diversity. / In this dissertation, Chapters Two and Three, we test the hypothesis that multiple forms of DNA (cytosine-5) methyltransferase are generated from a single DNA methyltransferase gene (Dnmt1) in vertebrates in vivo. We show that diversification of the N-terminus of Dnmt1 occurs by two mechanisms, multiple transcription initiations and alternative splicing. The rat Dnmt1 initiates transcription from three sites at least: a distal promoter, and a downstream promoter in exon 3 and exon 4. In addition to alternative initiations, alternatively spliced N-terminus domains are also present in rat brain. Similarly, we show that the DNA methyltransferase domain of rat Dnmt1 is alternatively spliced in vivo, generating different in-frame variants of DNA methyltransferase in specific tissues. This process is developmentally regulated, and is induced in PC12 cells by nerve growth factor (NGF). / Using this PC12 differentiation model, in Chapter Four, we further test the hypothesis that the expression of Dnmt1 is regulated with the developmental state of neuronal cells. We show that DNA methyltransferase (Dnmt1) activity is sharply reduced 4 days after NGF treatment. Similarly, the adult brain expresses reduced levels of Dnmt1 activity. The abundance of Dnmt1 mRNA as well as the Dnmt1 polypeptide is down regulated. Downregulation of Dnmt1 parallels other indicators of withdrawal from the cell cycle such as p21 and prolife rating cell nuclear antigen (PCNA), and may not be required for initiation of PC12 differentiation. This temporal pattern is different from myotube differentiation where down regulation of DNA methyltransferase and demethylation is an early event and is proposed to play a causal role in differentiation.

The mouse Splotch phenotype is characterized by specific defects in neural tube closure, neural crest cell migration, and skeletal myogenesis, and results from loss-of-function mutations in Pax-3, a transcription factor expressed within a subset of neural and mesodermal lineages. A hypomorphic allele termed Splotch-delayed produces milder neural tube defects and neural crest cell deficiencies than other Splotch locus mutations. We found that this unique phenotype is associated with a glycine to arginine mutation of the ninth amino acid of the Pax-3 paired domain. We also identified and characterized two alternatively spliced Pax-3 isoforms termed Pax-3/Q+ and Pax-3/Q--. These isoforms differ solely by the presence or absence of a single glutamine residue in the linker which joins the two DNA-binding subdomains of the Pax-3 paired domain, and are co-expressed throughout the early stages of mouse embryogenesis. Preliminary biochemical studies suggested that the novel Pax-3/Q-- isoform possessed enhanced C-terminal subdomain-mediated DNA-binding activity. In follow-up studies, we generated derivatives of a classical paired domain binding site (CD19-2/A) in order to further characterize the distinct DNA-binding properties of the two naturally occurring Pax-3 isoforms. We found that the presence of a 5 '-TT-3' dinucleotide at positions 15 and 16 of the paired domain recognition sequence promoted high affinity binding by both Pax-3 isoforms. However, with a guanine at position 15, only the Q-- isoform retained high affinity binding to the derivatives tested, suggesting that the presence of the additional glutamine residue in the Q+ isoform restricts the sequence specificity of the Pax-3 paired domain. Altogether, these studies demonstrate that the C-terminal subdomain makes an important contribution to the DNA-binding activity of the Pax-3 paired domain, and highlight the importance of the paired domain linker region in modulating the interaction of Pax proteins with t

The first part of my thesis dealt with the physical mapping of human chromosome 2 employing the yeast artificial chromosome (YAC) cloning system. To generate a chromosome 2 YAC sublibrary, over 1,000 interspersed repetitive sequence (IRS)-PCR probes were generated and used to screen the CEPH midi YAC library and approximately 2,000 chromosome 2-specific midi YACs were identified. These YACs were divided into 223 YAC groups, i.e., sets of unordered overlapping YACs, and using publicly available contig analysis software, a tentative order of YACs within each YAC group could be established. To order YAC groups, the chromosome 2 YAC sublibrary was screened with 87 genetically mapped microsatellites and cytogenetically mapped expressed sequence tags (ESTs), and 44 YAC groups were localized along the genetic map of chromosome 2q. In addition, 16 known genes were physically linked with microsatellites within YAC groups, thus providing integration points for genetic, cytogenetic and YAC-based physical maps of chromosome 2q. In a subsequent step of the analysis, the chromosome 2 YAC mapping data created by the Whitehead Institute (WI)/MIT Genome Center were integrated into our dataset. The integrated dataset consisted of 240 YAC groups, of which 14 large groups containing both our and WL/MIT Genome Center YAC groups were located on chromosome 2q. These 14 groups consisted of 1,195 YACs, which will form the backbone for the construction of a complete YAC contig for human chromosome 2q. / The second part of my thesis dealt with the identification of genetic markers within or in the vicinity of NRAMP1, a candidate tuberculosis susceptibility locus. The human NRAMP1 gene was mapped to chromosome 2q35 by PCR analysis in a monochromosomal hybrid panel and by YAC contig analysis. Nine sequence variants and polymorphisms were identified within the NRAMP1 gene by single strand conformation analysis (SSCA), DNA sequencing and Southern analyses. Furthermore, two highly informative microsatellites, D2S104 and D2S173 were shown to be linked to NRAMP1 within a 1.5 Mbp YAC contig. Together, these markers provide molecular tools for further genetic analysis of inherited susceptibility to tuberculosis and related diseases of the macrophage.

Bipolar disorder is a major psychiatric condition that affects up to 1% of the general population and results in episodes of mania and depression. Genetic epidemiologic studies have consistently shown that genetic factors play an important role in the etiology of bipolar disorder, however, the precise mechanisms that increase the susceptibility to this condition are not known. In the past three decades, several studies have been carried out hoping to identify major genes. However, conflicting results between and within studies that do not replicate previous findings have constituted a major problem. It is believed that part of this problem is related to genetic heterogeneity. We have attempted to reduce heterogeneity by selecting patients responsive to longterm lithium treatment. There is compelling evidence that selecting patients according to lithium response may help define a more genetically homogeneous subgroup of bipolar patients. This evidence is based on the fact that lithium is more effective in bipolar patients with classical symptomatology and the absence of comorbidity. Similarly, nonresponders to lithium treatment are different with respect to neuroendocrine responses and family history from responders, who are more likely to have relatives affected with bipolar disorder than lithium nonresponders. Using association and linkage designs, we have studied 31 lithium responsive bipolar families and 138 unrelated lithium responsive bipolar patients and 163 normal controls. We used a candidate gene as well as a whole-genome scan approach. In the genome scan study, significant evidence for linkage was found with a locus on chromosome 15q14, and suggestive linkage with a locus on 7q11. In the candidate gene studies, we found preliminary evidence suggesting that a variant of PLCG1, a gene that codes for phospholipase C gamma 1, is associated with bipolar disorder. Our results provide evidence that these loci may confer susceptibility to lithium responsive bip

Carcinoembryonic antigen (CEA), a widely used clinical tumor marker, functions in vitro as an adhesion molecule and is the prototype for a family of glycoproteins, which includes nonspecific crossreacting antigen (NCA) and biliary glycoprotein (BGP). This study examines the control processes responsible for the overproduction of the CEA family in carcinomas and identifies the trans-acting factors which mediate CEA and BGP gene transcription. During differentiation and $ gamma$-interferon treatment, CEA and NCA are regulated at both transcriptional and post-transcriptional levels and transcriptional control is markedly different between these two corresponding genes. Characterization of the CEA gene promoter revealed both negative and positive regulatory elements and tissue-specific, differentiation-dependent interactions with USF, Sp1, Sp1-like, AP-2-like and novel silencer transcription factors. Characterization of the BGP promoter identified one positively-acting element binding both USF and HNF-4/LF-A1, thereby contributing to distinct BGP gene expression. The Sp1, Sp1-like and silencer factors are specific to CEA gene transcription.

Coronary artery disease (CAD) is a major health concern for both developed and developing countries. With a heritability estimated at around 50%, there is a strong rationale to better define the genetic contribution of CAD. In order to do so, my thesis project consists in the genetic analysis of over 1400 individuals from the Saguenay Lac St-Jean region using 1536 single nucleotide polymorphisms in 103 candidate genes for CAD. Using this data, suggestive linkage for HDL cholesterol was found on chromosome 1 and several significant associations were observed with lipoprotein-related traits as well as adiponectin plasma concentration, including two novel associations.

Chromosome 11p15.5 harbors a gene or genes involved in Beckwith Wiedemann Syndrome (BWS) and that confer(s) susceptibility to Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma. PowerBLAST of P1 artificial chromosome clones from this region identified two novel transcripts with open reading frames encoding putative proteins of 253 and 424 amino acids. The larger of the transcripts was shown by Northern blot to be predominantly expressed in the fetal and adult liver and kidney. This transcript shares homology with integral membrane organic cation transporters, such as the tetracycline resistance proteins and bacterial multidrug resistance proteins; and was therefore designated ORCTL2 (organic cation transporter-like 2). An expressed sequence polymorphism provided evidence that the ORCTL2 gene exhibits "leaky" imprinting in both human fetal kidney and human fetal liver. Given the expression pattern of ORCTL2, it is possible that this gene may have a role in the development of phenotypes associated with BWS, including Wilms' tumor (WT). SSCP analysis on 51 sporatic WT samples did not identify any mutations in ORCTL2 which would implicate it in disease. Investigation of the transport properties of ORCTL2 show that this protein can confer resistance to chloroquine and quinidine when overexpressed in bacteria. Immunohistochemistry performed with anti-ORCTL2 polyclonal antibodies on human renal sections indicate that ORCTL2 is localized on the apical membrane surface of the proximal tubules. These results suggest that ORCTL2 may play a role in the transport of chloroquine and quinidine related compounds within the kidney.

Terminal regions of chromosomes are frequently involved in structural chromosomal abnormalities and are prone to rearrangements. They are also gene rich, and have been shown to be associated with a number of clinical conditions. This study focuses on telomere-specific fluorescence in situ hybridization (FISH) probes. It addresses the technical aspects of their preparation and application, and examines whether FISH using these probes is a valuable tool for detection of aberrations in the terminal regions of chromosomes. A set of telomere specific probes was generated from half yeast artificial chromosome (YAC) and cosmid clones by DNA preparation via Alu-PCR, alkaline lysis, cesium chloride-ethidium bromide centrifugation, or using a "Qiagen" kit. Probe quality and optimal FISH conditions were established by hybridizing half-YACs specific to telomeres 21q, 18q, 10p, and cosmids for telomeres 2q/8p, 13q, 14q and 20p to normal metaphases. Probes for 10p, 13q, 14q, 18q and 21q yielded clear and specific hybridization signals, present in over 90% of metaphases analyzed. Interphase analysis using the probes was not accurate. The ability of the telomeric probes to characterize balanced and unbalanced abnormalities was established by hybridization to patients with previously diagnosed chromosomal aberrations. The probes identified and confirmed partial trisomies 21q, 18q, and balanced translocations t(8;14), t(6;14), t(8;18), t(5;10). In three of the cases, partial monosomy 18q, translocation t(10;13), and t(1;10), the probes were able to provide information which was not revealed by banding analysis. This study demonstrates the ability of telomere specific probes to characterize aberrations in the terminal regions of chromosomes, and concludes that FISH using the telomeric probes is a valuable tool for clinical cytogenetics.

<p> The prevalence of sequencing experiments in genomics has led to an increased use of methods for count data in analyzing high-throughput genomic data to perform analyses. The importance of shrinkage methods in improving the performance of statistical methods remains. A common example is that of gene expression data, where the counts per gene are often modeled as some form of an overdispersed Poisson. In this case, shrinkage estimates of the per-gene dispersion parameter have lead to improved estimation of dispersion in the case of a small number of samples. We address a different count setting introduced by the use of sequencing data: comparing differential proportional usage via an overdispersed binomial model. Such a model can be useful for testing differential exon inclusion in mRNA-Seq experiments in addition to the typical differential gene expression analysis. In this setting, there are fewer such shrinkage methods for the dispersion parameter. We introduce a novel method that is developed by modeling the dispersion based on the double exponential family of distributions proposed by Efron (1986), also known as the exponential dispersion model (Jorgensen, 1987). Our methods (WEB-Seq and DEB-Seq) are empirical bayes strategies for producing a shrunken estimate of dispersion that can be applied to any double exponential dispersion family, though we focus on the binomial and poisson. These methods effectively detect differential proportional usage, and have close ties to the weighted likelihood strategy of edgeR developed for gene expression data (Robinson and Smyth, 2007; Robinson <i>et al.,</i> 2010). We analyze their behavior on simulated data sets as well as real data for both differential exon usage and differential gene expression. In the exon usage case, we will demonstrate our methods' superior ability to control the FDR and detect truly different features compared to existing methods. In the gene expression setting, our methods fail to control the FDR; however, the rankings of the genes by p-value is among the top performers and proves to be robust to both changes in the probability distribution used to generate the counts and in low sample size situations. We provide implementation of our methods in the R package DoubleExpSeq available from the Comprehensive R Archive Network (CRAN).</p>