2. The long range DNA organisation of the class II and class III regions in eight HLA homozygous cell lines has been analysed using PFGE. Comparison of the size of the BssHII restriction fragment observed for these cell lines and five individuals possessing one to three C4 genes, shows that the organisation of the C4 genes on each chromosome can be deduced from a single PFGE experiment. Outside of the C4 and 21-OHase loci the class III region shows a highly invariant structure, with no detectable differences in the amount of DNA present. Moreover the class III region is rich in CpG-islands, one of which has been characterised, and contains at least thirteen new genes. However, in the class II region, two differences between common haplotypes have been found. The DRw52-related haplotypes have the same DNA organisation. DR2 haplotypes possess 20-30 kb more DNA in the DRB region. DRw53 haplotypes have 100-130 kb more DNA than DRw52-related haplotypes in the region containing the DRB and DQA genes.

Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. The disorder shares features with Huntington's disease and McLeod syndrome (MLS), and can sometimes be difficult to distinguish clinically from the latter. In 1997, ChAc was linked to a 6-cM region on chromosome 9q21-22. A novel gene, >em>CHAC, was identified in the critical region. CHAC (now renamed VPS13A) encodes a large protein called chorein, with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in VPS13A were screened for mutations in 83 unrelated ChAc patients. We identified 88 different VPS13A mutations in 72 probands, comprising six deletions of entire exons, 22 nonsense, 36 frameshift, 19 splice-site and five missense mutations. This disorder therefore shows substantial allelic heterogeneity: however, evidence for common inheritance of the EX70_73del mutation in four French Canadian pedigrees indicates a possible founder effect in this population. Expression of VPS13A appears to be ubiquitous, as determined by tissue-specific analysis of mRNA and chorein distribution. However, chorein expression was markedly reduced or undetectable in lymphoblasts, fibroblasts and erythrocyte membranes from 14 ChAc patients. In contrast, MLS cells showed chorein expression similar to control levels, suggesting that loss of chorein expression is a diagnostic feature of ChAc. Yeast two-hybrid analysis of six different -600 amino-acid chorein fragments was used to screen a human brain cDNA library for proteins that may interact with chorein. One fragment interacted weakly with constructs derived from transcription factor NF-κB, putative protein phosphatase PP2Cη and TAB2, a protein implicated in the mitogen-activated kinase cascade. Although exogenously expressed chorein and TAB2 did not appear to colocalise, co-immunoprecipitation experiments supported an interaction between the two proteins, suggesting an avenue for future research into chorein function.

No description available.

Potato is one of the most important crops grown in Canada and all over the world. Late blight caused by P. infestans is one of the major diseases of potato and is mainly managed by fungicides application. The extensive use of fungicides not only causes adverse effects on the environment but also accelerates the development of resistance in this pathogen. Horizontal resistance is considered as the best choice to control P. infestans as it is durable over years. Breeding for durable resistance requires evaluation of hundreds of breeding lines in greenhouses and in the field. This is usually done by testing several epidemiological parameters such as infection efficiency, lesion size, latent period, and area under disease progress curve (AUDPC). These methods are time-consuming and expensive. The present study reports standardization of metabolic profiling protocols and exploration of metabolic profiling based on GC/MS as an additional tool to discriminate resistance in potato against late blight. Potato cultivars varying in horizontal resistance against late blight have been inoculated with water or the pathogen and more than 100 metabolites have been tentatively identified by GC/MS. Univariate analysis has been used to identify several pathogenesis related (PR) and defense related (DR) metabolites that have potential for application as resistance biomarker metabolites. Multivariate analysis of the abundances of metabolites (the mass spectral (MS) ion trap detector outputs were obtained using Saturn Lab Software Version 5.52 and these abundances are positively proportional to the concentration of mass ions of metabolites) in cultivars were mainly used to identify pathogenesis and resistance functions. Following pathogen inoculation, several metabolites such as amino acids, organic acids, fatty acids and sugars, were significantly increased in abundances, especially in the resistant cultivar. Other metabolites such as phenylalanine, tyrosine, shikimic acid and malonic acid detected here are well known for their direct participation in the shikimic acid, the phenylpropanoid, and the malonic acid metabolic pathways. These pathways lead to the production of several defense metabolites including antimicrobial compounds including phenolics, flavonoids and phytoalexins. The metabolic profiling technology developed here has the potential application for screening of potato breeding lines for horizontal resistance against late blight.

Lettuce mosaic virus (LMV) is an economically important pathogen with worldwide distribution. LMV infection in L. sativa can cause significant yield losses. Resistance to LMV in L. sativa is conferred by the recessive gene mo. We attempted to position the mo gene on the L. sativa map. The ultimate goal is a better understanding of plant-virus interactions. To do so, Random Amplified Polymorphic DNA (RAPD) markers were screened in the near isogenic lines (NILs) Vanguard and Vanguard 75. These NILs differ in the presence of the mo gene in Vanguard 75. Polymorphic markers were screened for linkage to mo in two F$ sb2$ populations segregating for resistance to LMV. The F$ sb2$ populations used were derived from 2 crosses, the first one between the L. sativa cultivars Dwarf 2 (resistant to LMV via the presence of mo) and Saffier and the second one between two breeding lines 87-25M-1 (momo) and 87-1090M-1 (MoMo). In order to develop a highly stringent antibody detection system to phenotype plants infected with LMV, a plasmid construct was developed which overproduces LMV coat protein. This construct will be used in the future to produce enough recombinant LMV coat protein for antibody production. To further characterize mo, a selection of cultivars resistant and susceptible to LMV according to the literature were subjected to various temperature changes to determine the environmental influences on virus movement.

In lettuce (Lactuca sativa), the dominant gene Tu confers resistance to turnip mosaic virus (TuMV) infection. In order to eventually clone and characterize the Tu gene using a map-based cloning strategy, the chromosome region in which Tu is located needs to be saturated with molecular markers. Random polymorphic DNA (RAPD) markers were screened using bulked segregant analysis. Nine new RAPD markers, UBC431$ rm sb{420}, UBC431 sb{940}, UBC434 sb{360}, UBC434 sb{1000}, UBC439 sb{520}, UBC448 sb{685:750}, UBC135 sb{240}, OP108 sb{410} and OP108 sb{1305},$ were identified as linked to Tu. Each marker was mapped relative to Tu using F$ sb2$ individuals previously known to be recombinant in the area surrounding the Tu locus. Three new markers, UBC431$ rm sb{420}, UBC439 sb{520} and UBC135 sb{240}$ are within a 5 cM area of Tu. As the number of DNA markers on the map increased map expansion and difficulties in determining a unique order were encountered. To increase the confidence in the estimate of genetic distances, a population of 500 F$ sb2$ plants was screened in order to identify more recombinant individuals around the Tu locus. The population was screened using markers UBC431$ sb{420}$ and UBC135$ sb{240}.$ Thirty-three recombinants were identified in an interval of 6.6 cM. Two markers, UBC346$ sb{1067}$ and OP108$ sb{634},$ tightly flanking Tu were converted to sequence characterized amplified regions (SCAR 346 and SCAR L08). No polymorphism was detected among the SCARs generated. The area surrounding Tu now includes 24 RAPD markers in an interval of 44 cM.

Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of three mitochondrial and one cytosolic forms of biotin-dependent carboxylases in humans. Patients suffering from this autosomal recessive disease have Multiple Carboxylase Deficiency (MCD) with symptoms of life-threatening metabolic acidosis which, in almost all cases, can be successfully treated with pharmacologic doses of oral biotin. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS maybe targeted to the cytoplasm and mitochondria or that carboxylases are biotinylated in the cytoplasm prior to import into the mitochondria. In order to resolve the compartmentalization of HCS, 5' HCS cDNA sequences have been examined for a targeting signal and a candidate sequence was tested for its capacity to target mitochondria. Analysis of 5' cDNA reveals complex alternative splicing, none of which appear to contain mitochondrial targeting sequences. In addition, antibodies have been developed in order to perform immunochemical analysis of the subcellular distribution of HCS. Polyclonal antisera were raised against full length HCS as well as two peptides corresponding to a 20 amino acid region in the N-terminus and to the 20 amino acids preceding the stop codon. Immunohistochemical staining of human fibroblasts with the antibody to full length HCS gives cytosolic, mitochondrial and nuclear localization. Interestingly, analysis with the N-terminal antiserum reveals a large punctate staining pattern exclusively localized to the nucleus. The corresponding C-terminal antiserum reveals solid nuclear staining with some mitochondrial co-localization. Taken together, these results indicate the ubiquitous nature of HCS in human cells and also allude to a potential role for HCS in the nucleus of human cells.

The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders. To date, ten SCA loci have been described (SCA1-SCA8, SCA10 and SCA11), with six genes having been cloned (SCA1, SCA2, SCA3/MJD, SCA6, SCA7 and SCA8) and shown to contain CAG/CTG repeats. / This study investigated various aspects of the SCA2, SCA6, and SCA7 subtypes. Haplotype analysis in our panel of SCA2 families identified multiple ancestral mutation events to be responsible for disease in this group. Screening for the newly identified SCA6 and SCA7 mutations in our large collection of SCA families and patients revealed that these mutations are rare in our panel, each accounting for less than 1% of our ataxia samples. Finally, the CAG repeat-containing locus hGT1 was found to be associated with residual age at onset variability in our SCA2 families. / Together, these results add to our growing understanding of the SCAs, and bring us a few steps closer to effective diagnoses of, and treatments for, these devastating diseases.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 202-237). / Microfiche. / xvi, 237 leaves, bound ill., photos. 29 cm