The overall goal of this project is to clone and characterize the gene for the HLA-linked form of autosomal dominant spinocerebellar ataxia (SCA1). SCA1 is a neurodegenerative disorder with onset of symptoms in the mid-adulthood. The disease is progressive and results in death 10-15 years after onset. Studies from our laboratory using multilocus linkage analysis and somatic cell lybridiazation techniques have mapped the SCA1 gene locus centromeric to the HLA loci. Detailed linkage and deletion mapping of the relevant region of chromosome 6p is in progress. The first phase in this proposal is to isolate additional DNA clones that are more closely linked to SCA using a genomic DNA library that will be prepared from a hybrid cell line retaining 6p as the only human chromosome. In addition a more enriched source for markers close to SCA1 will be generated by isolating hybrid clones retaining fragments of chromosome 6p. Once the SCA1 gene region is saturated with polymorphic DNA markers, chromosomal hopping and walking techniques as well as analysis of recombinants will be used to narrow the region to 1-2 million base pairs (mb). the second phase will involve physical mapping of the SCA1 region and the identification of transcribed sequences in that region. Pulsed field gel electrophoresis will be performed and cosmid clones within the 1-2 mb surrounding the SCA1 locus will be used to establish a set of overlapping contiguous DNA segments. DNA sequences employing a variety of approaches including the identification of HpaII tiny fragments (HTF) islands, examination for evolutionary sequence conservation, and Northern blotting analysis. Alternative strategies to clone the SCA1 gene will be pursued using cerebellar cDNA clones that map to the relevant region on chromosome 6p. The last phase of the proposal will be to prove whether a candidate sequence represents the SCA! genomic sequence or gene product. This will be achieved using a variety of novel and standard studies including comparative Southern blot analysis using DNA fractionated by conventional and pulsed field gel electrophoresis, Northern blotting analysis, ribonuclease cleavage studies and sequencing of the wild type and mutant SCA1 cDNAs. If the SCA1 gene is identified efforts will focus on characterizing the structure and biological function of the SCA1 gene to elucidate the mechanism of pathogenesis in this disease.
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