Biomedical subjects
Publications and source records attributed to M D Kaytor.
Three papers published recently in Cell bring the power of human genetics, Drosophila genetics, and genomics to bear on the understanding of fragile X syndrome. They provide further support for the importance of local protein synthesis within a neuron as a determinant of proper synaptogenesis and the development of cognitive abilities.
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Spinocerebellar ataxia type 7 (SCA7) belongs to a group of neurological disorders caused by a CAG repeat expansion in the coding region of the associated gene. To gain insight into the pathogenesis of SCA7 and possible functions of ataxin-7, we examined the subcellular localization of ataxin-7 in transfected COS-1 cells using SCA7 cDNA clones with different CAG repeat tract lengths. In addition to a diffuse distribution throughout the nucleus, ataxin-7 associated with the nuclear matrix and the nucleolus. The location of the putative SCA7 nuclear localization sequence (NLS) was confirmed by fusing an ataxin-7 fragment with the normally cytoplasmic protein chicken muscle pyruvate kinase. Mutation of this NLS prevented protein from entering the nucleus. Thus, expanded ataxin-7 may carry out its pathogenic effects in the nucleus by altering a matrix-associated nuclear structure and/or by disrupting nucleolar function.
Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1[82] with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1[77] containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.
Nucleotide repeat instability is associated with an increasing number of cancers and neurological disorders. The mechanisms that govern repeat instability in these biological disorders are not well understood. To examine genetic aspects of repeat instability we have introduced an expanded CAG trinucleotide repeat into transgenic mice. We have detected intergenerational CAG repeat instability in transgenic mice only when the transgene was maternally transmitted. These intergenerational instabilities increased in frequency and magnitude as the transgenic mother aged. Furthermore, triplet repeat variations were detected in unfertilized oocytes and were comparable with those in the offspring. These data show that maternal repeat instability in the transgenic mice occurs after meiotic DNA replication and prior to oocyte fertilization. Thus, these findings demonstrate that advanced maternal age is an important factor for instability of nucleotide repeats in mammalian DNA.
We screened for rad52 suppressors against temperature-sensitive (ts), missense, nonsense, and deletion rad52 mutations. Except for the deletion strain all mutants yielded suppressor candidates, indicating that suppressors completely bypassing the need for RAD52 are rare. Characterization of seven, recessive extragenic suppressors from our screen and two previously identified suppressors revealed that nearly all exhibit allele specificity. The allele specificity is positional in that suppressors that suppress a ts mutation in the C-terminal third of the coding region do not suppress three ts mutations in the N-terminal third. Conversely, suppressors against one of the three N-terminal mutations suppress more than one of these mutations but not the C-terminal mutation.
We have identified a gene, GSG1 (general sporulation gene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and alpha haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4-6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication.
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We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to gamma-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.