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The genetic component of mammalian male fertility is complex and involves thousands of genes. The majority of these genes are distributed on autosomes and the X chromosome, while a small number are located on the Y chromosome. Human and mouse studies demonstrate that the most critical Y-linked male fertility genes are present in multiple copies, show testis-specific expression and are different between species. In the equine industry, where stallions are selected according to pedigrees and athletic abilities but not for reproductive performance, reduced fertility of many breeding stallions is a recognized problem. Therefore, the aim of the present research was to acquire comprehensive information about the organization of the horse Y chromosome (ECAY), identify Y-linked genes and investigate potential candidate genes regulating stallion fertility. To achieve theses goals, a direct cDNA (complementary DNA) selection procedure was used to isolate Y-linked genes from horse testes and 29 Y-specific genes were identified. All 29 genes were mapped to ECAY and their sequences were used to further expand the existing map. Copy number analysis identified 15 multicopy genes of which 9 were novel transcripts. Gene expression analysis on a panel of selected body tissues showed that some ECAY genes are expressed exclusively in testes while others show ubiquitous or intermediate expression. Quantitative Real-Time PCR using primers for 9 testis-specific multicopy genes revealed 5 genes with statistically significant differential expression in testis of normal fertile stallions and stallions with impaired fertility. Gene copy number analysis showed that the average copy number of 4 such genes was decreased in subfertile/infertile stallions compared to normal animals. Taken together, this research generated the first comprehensive physical gene map for the horse Y chromosome and identified a number of candidate genes for stallion fertility. The findings essentially expand our knowledge about Y chromosome genes in horses, open a new avenue for investigating the potential role of ECAY genes in stallion fertility which contribute to the development of molecular tools for the assessment of fertility in stallions.

A shotgun genome sequencing project was undertaken in the expectation that access to the entire protein coding potential of E. Ruminantium (Welgevonden) will facilitate the identification of vaccine candidate genes against heartwater. The 1,516,355 bp sequence is predicted to encode 888 proteins and 41 stable RNA species. The most prominent feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number. These repeats have mediated numerous translocation and inversion events and seem to be responsible for the generation of both new full and partial protein coding sequences. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Of the 13 members of the order Rickettsiales compared in this study, E. Ruminantium has the lowest coding capacity (62%), lowest GC content (27.5%), but the highest proportion of repetitive sequences, which comprise 8.5% of the genome. Metabolic reconstruction of E. Ruminantium revealed the metabolic and biosynthetic capabilities typical of an obligate intracellular organism. We identified a number of genes unique to E. Ruminantium, most of which are not functionally characterised in any organism, and those shared with 12 other members of the Rickettsiales. Bioinformatic tools were used to identify possible vaccine candidates from the annotated genome sequence. The protective properties of seven open reading frames (ORFs), which induced cellular immune responses in vitro, were tested in vivo Only 20% survival was obtained in sheep immunised with a DNA formulation consisting of three ORFs. We found that the levels of peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production did not correlate with each other, nor with the levels of protection, suggesting that the current assays are just not reliable and that IFN-γ expression alone is not an indicator of protection. Therefore more cytokines and different assays will have to be investigated to define in detail what constitutes a protective immune response against E. Ruminantium infection. However, the data generated from the genome sequence will continue to facilitate novel approaches to study the organism and to develop an efficacious vaccine against heartwater. / Thesis (PhD)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

To establish the gene expression profile and cascade subsequently on avian sex-determining candidate genes, seven avian sex-determining candidate genes including DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1 and SOX9 were analyzed at early embryogenesis. Quantitative reverse transcription PCR (Quantitative RT-PCR) technology was used to establish the gene expression profiles among these genes at four, five, six and seven days of embryos. The results of quantitative RT-PCR reveal that the DMRT1 was expressed in chicken embryos of both sexes. DMRT1 gene expressions were up-regulated at four, five and six days of chicken embryos. DMRT1 expression increased at 5-Dpc. of male embryos, however, expression was not signification different in females embryos. Gene expression of FET1, FOXL2, LHX9 and HINT1 were higher in females than in males. The SMC2L1 and SOX9 were expressed in both sexes. Also, to identify the novel sex-determination genes in early chicken subtractive embryos, cDNA libraries from male-minus-female and female-minus-male 3.5 Dpc. embryos cDNA were established. Gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA) and TIGR gene indices (The Institute for Genome Research, USA). A total 548 of colonies in male-minus-female library and 79 sequences were annotated. However, a total of 589 of colonies in female-minus-male library and 16 sequences were annotated. Sequences were homologous to the steroid 5£\-reductase protein (SRD5A1) using BLASTx in male-minus-female subtractive library. The SRD5A1 may play a sex-differentiation role in male chicken. We need more study to know function of steroid 5£\-reductase protein in future.

Bladder cancer is a common malignancy affecting the genitourinary system. Although a large number of studies have been carried out on these areas for a long time, little is know about the molecular events which may involve in tumorigenesis. Until now, no profound immunohistological or molecular markers have been identified to define clinically relevant subsets of bladder cancer. The purpose of this thesis is to identify a novel bladder cancer carcinogenesis related genes. Chapter 1 attempts to illustrate the background, molecular markers, chromosomal abnormalities and genetic instability related to bladder cancer. In Chapter 2, various bioinformatics methodologies were used to annotate and identify candidate genes. Twenty-one genes were identified 1.5-fold up- or down-regulated in mRNA expression from RT4, TSGH8301 and J82, three different stages of bladder cancer cell lines by microarray chips (Dr. Liu, personal communications). Another eight candidate tumor suppressor genes were preliminarily identified from suppression subtractive hybridization (SSH) cDNA library of RT4 cell line based on an isoflavones-treated minus non-treated and further subjected to quantitative RT-PCR analyses to confirm the mRNA expression level in different stages of bladder cancer cell lines. Chapter 3 studies on the ANXA10 gene with special emphasis on its cloning, protein expression, subcellular localization and the preparation of polyclonal antibody. The result suggests that ANXA10 is a cytoplasmic protein in N18 cells. Chapter 4 analyzes the CDK2AP1 gene in mRNA and protein level at different bladder cancer cell lines and various specimens. In our preliminary observations, there are lost of CDK2AP1 expressions at invasive TCCs specimens when compared to noninvasive TCCs specimens. The mechanism of the tumor-associated loss of the CDK2AP1 expression is currently not clear. In Chapter 5, bladder cancer cell lines TSGH8301, UB37, TCCSUP and J82 in SCID mice xenograft model were established for further in vivo studies.

Soybean [Glycine max (L.) Merr.] seed is a major source of edible oil in the world and the main renewable raw material for biodiesel production in North America. Oil, which on average accounts for 20% of the soybean seed weight, is a complex quantitative trait controlled by many genes with mostly minor effects and influenced by environmental conditions. Because of its quantitative nature, the seed oil concentration may have an indirect effect on other economically important and agronomic traits such as seed yield and protein concentration. Increasing the oil concentration in soybean has been given more attention in recent years due to increasing demand for both edible oil and feedstock. To achieve this objective, it is important to understand the genetic control of the oil accumulation and its relationship with other traits. The main objectives of this thesis were to identify quantitative trait loci (QTL) and genes involved in oil biosynthesis in soybean. Two recombinant inbred line (RIL) populations were developed from crosses between moderately high oil soybean cultivars with high seed yield and protein concentration. In a population of 203 F3:6 RILs from a cross of ‘OAC Wallace’ and ‘OAC Glencoe’, a total of 11 genomic regions located on nine different chromosomes were identified as associated with oil concentration using multiple QTL mapping (MQM) and single-factor ANOVA. Among the 11 oil-associated QTL, four QTL were also validated in a population of 211 F3:5 RILs from a cross of ‘RCAT Angora’ and ‘OAC Wallace’. There were six oil QTL identified in this study that were co-localized with seed protein QTL and four for seed yield QTL. The oil-beneficial allele of the QTL tagged by marker Sat_020, on Chromosome 9, was positively associated with seed protein concentration. The oil-enhancing alleles at markers Satt001 and GmDGAT2B were positively correlated with seed yield. In this study, three sequence mutations were also discovered in either the coding or non-coding regions of three DGAT soybean genes (GmDGAT2B, GmDGAT2C, and GmDGAT1B) between ‘OAC Wallace’ and ‘OAC Glencoe’ that showed significant effects on some of the traits evaluated. GmDGAT2B showed significant association with seed oil and yield across different environments. The oil-favorable allele of the gene GmDGAT2B from ‘OAC Glencoe’ was also positively correlated with seed yield. GmDGAT2C was associated with seed yield, whereas GmDGAT1B showed significant effects on seed yield and protein concentration. However, neither of these two genes showed any association with seed oil. The yield-enhancing allele of GmDGAT1B showed negative association with protein concentration. The identification of oil QTL that were either positively associated with seed yield and protein or neutral to both traits and the development of new gene-based markers will facilitate marker-assisted breeding to develop high oil soybean cultivars with high yield and minimal effect on protein concentration. / Generous funding to conduct this research was provided by the Alternative Renewable Fuels II Program of the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and by the Grain Farmers of Ontario.

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Because it is predicted to be durable and broad spectrum, quantitative trait loci (QTL)-based resistance is an important option for rice disease control. However, manipulation of this type of resistance requires knowledge of the contributing genes. This study demonstrates the contribution of two of three defense response (DR) genes to QTL-governed resistance, and identifies a third gene that negatively regulates resistance. The contribution to QTL-governed resistance of one of nine rice OsPAL genes, which encode phenylalanine ammonia-lyase, was determined using reverse genetics. Mutant ospal4 contains a 750 bp deletion in OsPAL4 and was identified using a PCR-pooling strategy. OsPAL4 underlies a QTL on chromosome 2, and is located in cluster with three other OsPAL members. Rice lines mutated in OsPAL4 are more susceptible to a virulent strain of Xanthomonas oryzae pv. oryzae (Xoo) than lines with the wild type allele. RNAi suppression was used to evaluate the contributions of genes encoding oxalate oxidase-like proteins (OsOXL) and a 14-3-3 protein (GF14-e) to disease resistance. Silencing of 12 OsOXL genes clustered on chromosome 8, varied from suppression of a few gene members to silencing of all expressed family members. Screening of transgenic lines by challenge with Magnaporthe grisea (Mg), the rice blast pathogen, revealed that the more chromosome 8 OsOXL genes suppressed, the more susceptible the plants were to Mg. GF14-e co-localizes with a disease resistance QTL on chromosome 2. Specific suppression of GF14-e by RNAi silencing did not result in enhanced susceptibility to Mg. Instead, the lines exhibited spontaneous HR-type lesions. The presence of this lesion mimic phenotype correlated with enhanced resistance to a virulent strain of Xoo, suggesting that the GF14-e encoded 14-3-3 protein functions as a negative regulator of plant cell death and bacterial resistance in rice. This study supports the hypothesis that DR genes, such as OsOXL and OsPAL4 contribute to disease resistance governed by QTL. The role of GF14-e is less clear, however its down regulation may contribute to QTL-governed resistance. Thus, incorporation of regions harboring the effective DR gene alleles into rice will enhance broad spectrum and durable resistance.

Magister Scientiae - MSc / Autism is a pervasive developmental disorder (PDD) that's incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman® SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman® study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples. / South Africa