Genetics

Degradation of functional TPI protein underlies sugarkill pathology.

Publication Date: 2008 May 5 PMID: 18458110
Authors: Seigle, J. L. - Celotto, A. M. - Palladino, M.
Journal: Genetics

TPI deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the triose phosphate isomerase protein (TPI) and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPIsugarkill mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.

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Genome Integrity Is Regulated by the C. elegans Rad51D homologue, rfs-1.

Publication Date: 2008 May 5 PMID: 18458109
Authors: Yanowitz, J.
Journal: Genetics

Multiple mechanisms ensure genome maintenance including DNA damage repair, suppression of transposition, and telomere length regulation. The mortal germ line (MRT) mutants in C. elegans are defective in maintaining genome integrity and result in a progressive loss of fertility over many generations. Here I show that the him-15 locus, defined by the deficiency eDf25, is allelic to rfs-1, the sole rad-51 paralog group member in C. elegans. The rfs-1/ eDf25 mutant displays a MRT phenotype and mutant animals exhibit features of chromosome fusions prior to the onset of sterility. Unlike other MRT genes, rfs-1 manifests fluctuations in telomere lengths and functions independently of telomerase. These data suggest that rfs-1 is a novel regulator of genome stability.

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FHA domain of yeast Xrs2, a homologue of human Nbs1, promotes non-homologous end joining through the interaction with a Ligase IV partner protein, Lif1.

Publication Date: 2008 May 5 PMID: 18458108
Authors: Matsuzaki, K. - Shinohara, A. - Shinohara, M.
Journal: Genetics

DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Yeast Xrs2, a homologue of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminus FHA domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase-IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of Serine-383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at Serine-383 is recognized by the Xrs2 FHA domain, which in turn may promotes recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in human; FHA domain Nbs1 interacts with Xrcc4, a Lif1 homologue of human.

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Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L.).

Publication Date: 2008 May 5 PMID: 18458107
Authors: Olsen, K. M. - Hsu, S. C. - Small, L. L.
Journal: Genetics

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest studied and best documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.

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Gene-centric Genomewide Association Study via Entropy.

Publication Date: 2008 May 5 PMID: 18458106
Authors: Cui, Y. - Kang, G. - Sun, K. - Qian, M. - Romero, R. - Fu, W.
Journal: Genetics

Genes are the functional units in most organisms. Compared to genetic variants located outside genes, genic variants are more likely to affect disease risk. The development of the human HapMap project provides an unprecedented opportunity for genetic association studies at the genomewide level for elucidating disease etiology. Currently, most association studies at the single-nucleotide-polymorphism (SNP) or the haplotype level rely on the linkage information between SNP markers and disease variants, with which association findings are difficult to replicate. Moreover, variants in genes might not be sufficiently covered by currently available methods. In this article, we present a gene-centric approach via entropy statistics for genomewide association study to identify disease genes. The new entropy-based approach considers genic variants within one gene simultaneously and is developed based on a joint genotype distribution among genetic variants for an association test. A grouping algorithm based on a penalized entropy measure is proposed to reduce the dimension of the test statistic. Type I error rates and power of the entropy test are evaluated through extensive simulation studies. The results indicate that the entropy test has stable power under different disease models with reasonable sample size. Compared to single SNP-based analysis, the gene-centric approach has greater power, especially when there is more than one disease variant in a gene. As the genomewide genic SNPs become available, our entropy-based gene-centric approach would provide a robust and computationally efficient way for gene-based genomewide association study.

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Hsp90/Hsp70 chaperone machine regulation of the Saccharomyces MAL-activator as determined in vivo using noninducible and constitutive mutant alleles.

Publication Date: 2008 May 5 PMID: 18458105
Authors: Ran, F. - Bali, M. - Michels, C. A.
Journal: Genetics

The Hsp90/Hsp70 chaperone machine is an essential regulator of cell growth and division. It is required for activation of select client proteins, chiefly protein kinases and transcription activators, and thus plays a major role in regulating intracellular signaling and gene expression. This report demonstrates, in vivo, the association of the Saccharomyces cerevisiae maltose-responsive transcription activator Mal63 (MAL-activator) with the yeast Hsp70 (Ssa1), Hsp90 (Hsp82), and Hop (Sti1) homologs using a collection of inducible, constitutive, and noninducible alleles. Each class of mutant activator forms a distinctly different stable multi-chaperone complex in the absence of maltose. Inducible Mal63p associates with Ssa1, Hsp82, and Sti1 and is released in the presence of maltose. Noninducible mal63 mutant proteins bind to Ssa1 alone and do not stably associate with Hsp82 or Sti1. Constitutive MAL-activators bind well to Hsp82 and poorly to Ssa1 and Sti1, but deletion of STI1 restores Ssa1 binding. Taken together, Mal63p regulation requires the formation of Hsp90/Hsp70 sub-complexes comparable to yet distinct from those observed with previously characterized Hsp90 clients including glucocorticoid receptor and yeast Hap1p. Thus, comparative studies of different client proteins highlight functional diversity in the operation of the Hsp90/Hsp70 chaperone machine.

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High rates of "unselected" aneuploidy and chromosome rearrangements in tel1 mec1 haploid yeast strains.

Publication Date: 2008 May 5 PMID: 18458104
Authors: Vernon, M. - Lobachev, K. - Petes, T. D.
Journal: Genetics

The yeast TEL1 and MEC1 genes (homologous to the mammalian ATM and ATR genes, respectively) serve partially redundant roles in the detection of DNA damage and in the regulation of telomere length. Haploid yeast tel1 mec1 strains were sub-cultured non-selectively for about 200 cell divisions. The sub-cultured strains had very high rates of chromosome aberrations: duplications, deletions, and translocations. The breakpoints of the rearranged chromosomes were within retrotransposons (Ty or delta repeats), and these chromosome aberrations non-randomly involved chromosome III. In addition, we showed that strains with the hypomorphic mec1-21 allele often became disomic for chromosome VIII. This property of the mec1-21 strains is suppressed by a plasmid containing the DNA2 gene (located on chromosome VIII) which encodes an essential nuclease/helicase involved in DNA replication and DNA repair.

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Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae.

Publication Date: 2008 May 5 PMID: 18458103
Authors: Coic, E. - Feldman, T. - Landman, A. S. - Haber, J. E.
Journal: Genetics

In wild type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but about 22% of recombinants had associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants were crossover-associated, with the largest class being half-crossovers in which the other participating chromatid was lost. We report that UV-irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in WT cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n-1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun's target, the repressor Crt1, whereas gene conversions events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.

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Caenorhabditis elegans genes required for the engulfment of apoptotic corpses function in the cytotoxic cell deaths induced by mutations in lin-24 and lin-33.

Publication Date: 2008 May 5 PMID: 18458102
Authors: Galvin, B. D. - Kim, S. - Horvitz, R.
Journal: Genetics

Two types of cell death have been studied extensively in C. elegans, programmed cell death and necrosis. We describe a novel type of cell death that occurs in animals containing mutations in either of two genes, lin-24 and lin-33. Gain-of-function mutations in lin-24 and lin-33 cause the inappropriate deaths of many of the Pn.p hypodermal blast cells and prevent the surviving Pn.p cells from expressing their normal developmental fates. The abnormal Pn.p cells in lin-24 and lin-33 mutant animals are morphologically distinct from the dying cells characteristic of C. elegans programmed cell deaths and necrotic cell deaths. lin-24 encodes a protein with homology to bacterial toxins. lin-33 encodes a novel protein. The cytotoxicity caused by mutation of either gene requires the function of the other. An evolutionarily conserved set of genes required for the efficient engulfment and removal of both apoptotic and necrotic cell corpses is required for the full cell killing effect of mutant lin-24 and lin-33 genes, suggesting that engulfment promotes these cytotoxic cell deaths.

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Chromosome 11 Region from Strain 129 Provides Protection from Sex Reversal in XYPOS Mice.

Publication Date: 2008 May 5 PMID: 18458101
Authors: Nikolova, G. - Sinsheimer, J. S. - Eicher, E. M. - Vilain, E.
Journal: Genetics

C57BL/6J (B6) mice containing the Mus domesticus poschiavinus Y chromosome, YPOS, develop ovarian tissue, whereas testicular tissue develops in DBA/2J or 129S1/SvImJ (129) mice containing the YPOS chromosome. To identify genes involved in sex determination, we used a congenic strain approach to determine which chromosomal regions from 129Sl/SvImJ provide protection against sex reversal in XYPOS C57BL/6J.129-YPOS mice. Genome scans using microsatellite and SNP markers identified a chromosome 11 region of 129 origin in C57BL/6J.129-YPOS mice. To determine if this region influenced testis development in XYPOS mice, two strains of C57BL/6J-YPOS mice were produced and used in genetic experiments. XYPOS adults homozygous for the 129 region had a lower incidence of sex reversal than XYPOS adults homozygous for the B6 region. In addition, many homozygous 129 XYPOS fetuses developed normal appearing testes, an occurrence never observed in XYPOS C57BL/6J-YPOS mice. Finally, the amount of testicular tissue observed in ovotestes of heterozygous 129/B6 XYPOS fetuses was greater than the amount observed in ovotestes of homozygous B6 XYPOS fetuses. We conclude that a chromosome 11 locus derived from 129Sl/SvImJ essentially protects against sex reversal in XYPOS mice. A number of genes located in this chromosome 11 region are discussed as potential candidates.

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Altered Dosage and Mis-localization of Histone H3 and Cse4p Lead to Chromosome Loss in Saccharomyces cerevisiae.

Publication Date: 2008 May 5 PMID: 18458100
Authors: Au, W. C. - Crisp, M. J. - Deluca, S. Z. - Rando, O. J. - Basrai, M. A.
Journal: Genetics

Cse4p is an essential histone H3 variant in Saccharomyces cerevisiae that defines centromere identity and is required for proper segregation of chromosomes. In this study, we investigated phenotypic consequences of Cse4p mis-localization and increased dosage of histone H3 and Cse4p, and established a direct link between histone stoichiometry, mis-localization of Cse4p, and chromosome segregation. Overexpression of the stable Cse4p mutant, cse4(K16R), resulted in its mis-localization, increased association with chromatin, and a high rate of chromosome loss, all of which were suppressed by constitutive expression of histone H3 (16H3). We determined that 16H3 did not lead to increased chromosome loss; however, increasing the dosage of histone H3 (GALH3) resulted in significant chromosome loss due to reduced levels of CEN-associated Cse4p and synthetic dosage lethality (SDL) in kinetochore mutants. These phenotypes were suppressed by GALCSE4. We conclude that the chromosome mis-segregation of GALcse4(K16R) and GALH3 strains are due to mis-localization and a functionally compromised kinetochore, respectively. Suppression of these phenotypes by histone 16H3 and GALCSE4 supports the conclusion that proper stoichiometry affects the localization of histone H3 and Cse4p, and is thus essential for accurate chromosome segregation.

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