The GM2 gangliosidoses are a group of recessive disorders, which lead to the accumulation of GM2 ganglioside in neuronal cells. The genes responsible for these disorders are HEXA (Tay-Sachs disease and variants), HEXB (Sandhoff disease and variants) and GM2A (AB variant of GM2 gangliosidosis). We have established three relational locus-specific databases recording allelic variation at the HEXA, HEXB and GM2A genes, and these can be accessed through the G M2 Gangliosidoses home page (http://data.mch.mcgill.ca/gm2-gangliosidoses/). The purpose of these databases is to collect and distribute information on mutations in the genes responsible for GM2 gangliosidosis. These databases are available online for users to search and retrieve information about specific mutations either by mutation, phenotype or author(s). In addition, submission forms are available for the addition of new mutations to the databases. / In order to provide information concerning the effects of mutations on the manifestations of disease, we proceeded to model on the theoretical model of the alpha subunit a few missense mutations. (Abstract shortened by UMI.)

This research was an attempt to identify and provide a phylogeny of Vanilla orchids using PCR sequencing/ fingerprinting.Traditional classification methods haved relied heavily on morphological traits and are often contradictory, subjective and incomplete. This research attempted to discover an objective and reliable method for identification and classification of selected tropical orchids species using molecular techniques. Vanilla orchids were chosen for their commercial importance in the scent and flavoring industry.Reconstruction of phylogenetic data is possible using relatively new molecular techniques: Polymerase Chain Reaction (PCR) fingerprinting and sequencing. PCR fingerprinting reveals restriction fragment length polymorphisms (RFLPs) within an organism's genome. Thesevariations can be used to construct genetic maps in a variety of species. PCR sequencing reveals genetic variation at an even greater level: nucleotide sequence.The literature suggests that these methods are fairly quick and simple; however, the crucial step is obtaining high molecular weight DNA digestable with restriction enzymes or amplifiable by the PCR. DNA isolation has proven to be difficult with Vanilla orchids due to their numerous phenolic compounds, tough fibrous tissue and high number of polysaccharides. This research developed a method of isolating high molecular weight DNA from orchids. This DNA was digestible with restriction enzymes. The DNA was subjected to DNA fingerprinting with primers specific to the 18S-26S ribosomal RNA gene and with RAPD primers. The DNA was also sequenced using the PCR technique.Variation between Vanilla species genomes was discovered and compared to traditional phylogenetic information. It was found that banding patterns and nucleotide sequences were almost identical for Vanilla planifolia and planifolia varegata. The banding patterns for Vanilla barbellata were similar to those of the two unknown Vanilla orchids. Vanilla humboltii exhibited a banding pattern far different from either barbellata or planifolia. These results confirm the hypothesis that PCR fingerprinting/ sequencing is a useful technique for the identification and phylogenetic analyses of Vanilla orchids. / Department of Biology

No description available.

Finally, two main extensions of this approach were considered - linkage approaches to examine more than two loci, and extending the method in this study to include a test of association.

p13^suc1 from schizosaccharomyces pombe is a member of a family of non-enzymatic cell cycle regulatory proteins called CKS for <strong>C</strong>yclin-<strong>D</strong>ependent kinases <strong>S</strong>ubunit. Other members of this family include CKS1 (S. cerevisiae</em?), CksHsl and CksHs2 (Homo sapiens). The CDKs (CDK1-CDK8) for <strong>C</strong>yclin-<strong>D</strong>ependent <strong>K</strong>inases are a class of Ser/Thr kinases that regulate the cell cycle. The suc1⁺ gene was initially identified as a seppresor of certain CDKl temperature sensitive mutations. Despite the wealth of crystallographic models available plus ample - albeit, sometimes conflicting - evidence from genetics and biochemical studies, an account of the exact physiological role of the CKS proteins remains an elusive goal. In a quest to identify the residues and hence the particular surface region involved in mediating protein-protein interactions with CDK2,1 employed the X-ray structure of Suc1 at 2.7A resolution as guide for a site-specific mutagenesis study. Comparative biochemical and biophysical characterisation of Suc1 and the mutants led to the conclusion that isoleucine-94 and Leucine-96 (located in the hydrophobic patch) are involved in mediating protein-protein interactions with GST-CDK2. This conclusion has since been confirmed by the publication of the X-ray structure of monomeric CksHs1l in a complex with CDK2 by Bourne et al., 1996 (Cell 84: 863-874). An extension of the mutational study to test the hypothesis that Suc1 may utilise conserved residues at the anion-binding site to mediate protein-protein interactions with the Anaphase Promoting Complex (APC) is described. X-ray data has been collected on wild type Suc1 crystals at 100K to 2.3Å resolution. The structure has been resolved and refined to a crystallographic R-factor of 22.6%. S. pombe Suc1 exists as a zinc-stabilised, non strand-exchanged dimer in both the 2.1Å and 2.3Å models. Structural analyses of two Suc1 folding mutants are also presented. The cyclins (A - H) are positive regulatory subunits of CDKs. They share a high degree of homology over a region of about 100 amino acid residues called the "cyclin box". The determination of the crystal structure of cyclin A3 (an N-terminal truncated version of bovine cyclin A) and a CDK2-cyclin A3 complex by other workers has revealed the mechanism of activation of CDKs by cyclins. In S. pombe, the CDKl-cyclin B heterodimeric complex constitutes the mitotic kinase. In order to understand the specificity underlying the CDK-cyclin interaction, I embarked on a structural study of S. pombe cyclin B and CDK1. Both full- length proteins have proven intractable to attempts to overproduce them in a soluble form in E. coli for crystallisation studies. A truncated version of cyclin B (similar to cyclin A3) was designed, cloned and overproduced in E. coli. The cyclin B3 protein was directed into inclusion bodies as insoluble aggregates. Extensive attempts - both in vivo and in vitro - to produce a soluble cyclin B3 proved unsuccessful. Finally, an E. coli co-expression system designed to overproduce CDK1-cyclin A3, CDK1-cyclin B3, CDK1-cyclin B and CDK1-Suc1 complexes is described.

There is growing evidence that susceptibility to many inflammatory and infectious diseases may be influenced by our genetic make up. Genetic variants in important immune genes may partially explain variation in susceptibility to common diseases. Interferon-γ (IFNγ) is one of the central mediators of the innate and adaptive immunity and has been implicated in a wide range of infectious and inflammatory disease processes. Severe disruptive mutations in coding regions of the IFN-γ receptor 1 gene (IFNGR1) have been found to be associated with fatal but very rare mycobacterial infections. This study looked at common polymorphisms in potentially regulatory non-coding regions of the IFNγ gene and the IFNGR1 gene and investigated their association with susceptibility to severe malaria, a disease for which there have been indications of a genetic component to susceptibility. Malaria is one of the major causes of childhood deaths in Africa. IFNγ and its receptor have been shown to be critically involved in the host response to the malaria parasites. The promoter regions of IFNGR1 and its neighbouring genes, located on chromosome 6q23, and IFNγ and its neighbours, on chromosome 12ql4, were screened for polymorphisms. Haplotypes and linkage disequilibrium maps were constructed, signatures of natural selection were investigated, haplotype tagging SNPs were dentified, and association with disease was analysed. One of these preliminary results was a putative association between the IFNGRl-470ddel allele and susceptibility to severe malaria in the Mandinka ethnic group. This allele was in strong linkage disequilibrium (LD) with markers which are a considerable distance away which might represent a signature of natural selection. To assess the potential functional significance of the IFNGR1-47Q polymorphism, its effects on DNA-protein interactions and gene expression was investigated further in various cell lines. Evidence of tissue-specific nuclear protein binding to this site which seems to be involved in transcriptional regulation was observed.

Plasmodium falciparum and Mycobacterium tuberculosis infections collectively cause as many as five million deaths world-wide each year. In the most afflicted populations, currently available drugs and vaccines appear inadequate. By offering insight into the pathophysiology of diseases, genetic studies provide options for new therapeutic approaches to major health problems. The results of case-control studies of genetic factors associated with disease outcomes in malaria and tuberculosis in an African setting are presented in this thesis. Glucose-6-Phosphate dehydrogenase (G6PD) deficiency, the commonest enzymopathy of humans, affects over 400 million people. The geographical correlation of its distribution with the historical endemicity of malaria suggests that this disorder has risen in frequency through natural selection by malaria. However, attempts to confirm that G6PD deficiency is protective in case-control studies of malaria have yielded conflicting results. Hence, for this X-linked disorder, it is unclear whether both male hemizygotes and female heterozygotes are protected or, as frequently suggested, only females. Furthermore, how much protection may be afforded is unknown. In two large case-control studies of over 2000 African children, I found that the common African form of G6PD deficiency (G6PD A-) is associated with a 46-58% reduction in risk of severe malaria for both female heterozygotes and male hemizygotes. A mathematical model incorporating the measured selective advantage against malaria suggests that a counterbalancing selective disadvantage, associated with this enzyme deficiency, has retarded its rise in frequency in malaria-endemic regions. There is some evidence that two T helper cell subsets, Thl and Th2, regulate the immune response and thus influence the course of infections in mammalian hosts. These T cell subsets are reciprocal and associated with distinct cytokine profiles. Th2 T cell differentiation is promoted mainly by interleukin-4. Analysis of an IL-4 promoter polymorphism indicates that homozygosity for a putatively upregulatory IL-4 promoter variant is associated with a signficantly increased risk for severe malaria whilst heterozygotes are protected against this condition. Epidemiological evidence implicates host genetic factors as major determinants of variable susceptibility to tuberculosis. Most attempts to define the genetic factor(s) have focused on the HLA genes but only one result, an association of HLA-DR2 with increased susceptibility to disease in Asian populations, has been reported with any consistency. The genetic component in tuberculosis is likely to be determined by multiple genes and, therefore, in this study, the role of both HLA and non-HLA candidate genes was investigated. No association was found with variants of the macrophage gene, NRAMP1, the homologue of which has been implicated in the regulation of genetic resistance in the mouse model. Examination of certain class I and class II HLA alleles as well as the -590 interleukin-4 promoter polymorphism also did not show any association with disease. However, heterozygotes for a promoter polymorphism at position -238 of the tumour necrosis factor gene and homozygotes for dysfunctional variants of the gene encoding the collectin, mannose binding protein, were both at increased risk of developing pulmonary tuberculosis.

Progressive myoclonus epilepsy type 1 (EPM1), also known as Unverricht-Lundborg disease, is one of the rare forms of epilepsy that shows a clear pattern of autosomal recessive inheritance. The gene defective in this disease was linked to the distal tip of chromosome 21, in band q22.3. In this study, we have collected 93 samples from 15 EPM1 families and 5 affected individuals as the basis for identifying the EPM1 gene. We have also constructed a 770 kb cosmid and bacterial artificial chromosome contig covering the candidate EPM1 region, and have isolated expressed sequences from this contig. For three of the genes that we isolated (GT335, GT334, and PWP2), we have identified and sequenced a full-length cDNA, identified the putative protein, assessed the expression pattern of the gene by Northern blot, determined the exon/intron structure of the gene, characterized basepair polymorphisms within each gene, and finally excluded each of these genes as the one defective in EPM1 patients. Using these new polymorphisms, and others that were available and that we had identified, we were able to construct detailed haplotypes on each of the affected EPM1 chromosomes, to help pinpoint, the location of the EPM1 gene, and help estimate the number of different mutations we might have in our collection. / While these studies were underway, another group identified the cystatin B (STFB) gene as that defective in EPM1. This allowed us to directly test this gene for mutations in our collection of EPM1 patients. We could identify four different mutations in the STFB gene, the most common of which consisted of a variable length insertion in the 5 ' flanking region of the gene, and which was previously undescribed. This mutation, which is found in 78% of unrelated EPM1 chromosomes we studied, showed some level of meiotic instability, and mapped to a polymorphic 12-bp GC-rich repeat. Using a combination of PCR and Southern blotting assays, we could accurately diagnose nearly 100% of all EPM1 patients. This represents a significant step forward in our ability to diagnose this disease at the molecular level, and should allow a more precise definition of the progressive myoclonus epilepsies, as a whole.

Mutations in the p53 gene are known to be associated with a wide range of human tumors. In some primary tumors and established cell lines, stable mutant p53 protein is expressed at high levels, whereas, in normal cells unstable wild-type p53 protein is expressed at very low levels. Sera from some patients with breast and colon tumors contain anti-p53 antibodies. It is unclear whether changes in p53 structure, or its increased level in tumors, causes p53 to become antigenic. In our study we tested sera from patients with various types of cancer for anti-p53 antibodies. Examination of the sera was made by Western blot, and the results were confirmed by rescreening sera with immunoprecipitation. Both techniques revealed the presence of anti-p53 antibodies in some sera from lung and ovary cancer patients, as well as in the sera from patients with breast or colon cancers. Clearly, patients with various cancer tumors are able to produce anti-p53 antibodies. It was unclear whether this humoral immune response is against mutant or wild type p53. To provide a better definition of this immune response, we have examined the anti-p53 response from cancer patients against mutant and wild type p53 in the native and denaturated state. Western blot and Immunoprecipitation analysis showed that the anti-p53 sera recognise both wild type and mutant p53 conformational and denaturation resistant epitopes. Taken together, these data demonstrate that the mutant p53 is not more antigenic than the wild type p53. This provides strong evidence that the antibody response is not directed solely against the altered conformation in mutant p53.

<p>Clubfoot is the most common congenital structural deformity that leads to physical impairments in children in many poor developing countries. Inadequately treated or neglected clubfoot has been found to be a common cause of ohysical disability globally among children and young growing adults. Many children are referred to the clinics for treatment but some parents do not comply with the treatment regimen whcih requires attending for consecutive treatment sessions. The purpose of this study was to investigate barriers to treatment attendance parents/caregivers of children with clubfoot encounter in complying with clubfoot treatment during the plaster csting phase in Uganda.</p>