Expansion of the number of CAG repeats in the human ataxin-1 gene are the mutational cause of Spinocerebellar Ataxia Type 1 (SCA1). This proposal describes studies aimed at understanding when and how the CAG repeats change in repeat number. We will examine sperm DNA from ataxia patients and from normal controls to measure the frequency and size changes in the CAG repeats during male gametogenesis when most changes are known to occur. We will also be examining CAG repeat lengths in lymphoblastoid cells made from ataxic patients' lymphocytes to learn if some individuals are more prone to repeat instability than others. These studies will be extended to transgenic mice carrying the human ataxin-1 gene to learn whether CAG repeats which are unstable in humans are also unstable in this mammal. A major portion of the study is the establishment of two in vitro systems for examining CAG repeat instability. One system will be in cultured primate and human cells and the other in yeast cells. In both systems we will create a gene with a CAG repeat cassette and vary the number of CAG repeats in the cassette. After transfection or transformation of the cassette-containing gene into cells, we will screen or select for mutational changes in repeat length. By varying the number of repeat copies, the sequence of repeat copies and the placement of repeat copies, we will learn when repeat sequences become unstable. Furthermore, the yeast cell system will be used to find mutations of yeast genes which alter the stability of trinucleotide repeats. Such mutations define genes whose identity can help in determining the mechanism by which trinucleotide repeats expand.
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