Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.
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The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination. (+info)
Fus3p and Kss1p control G1 arrest in Saccharomyces cerevisiae through a balance of distinct arrest and proliferative functions that operate in parallel with Far1p.
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In Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition. (+info)
Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans.
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In the filamentous fungus Aspergillus nidulans, germination of an asexual conidiospore results in the formation of a hyphal cell. A key feature of spore germination is the switch from isotropic spore expansion to polarized apical growth. Here, temperature-sensitive mutations are used to characterize the roles of five genes (sepA, hypA, podB-podD) in the establishment and maintenance of hyphal polarity. Evidence that suggests that the hypA, podB, and sepA genes are required for multiple aspects of hyphal morphogenesis is presented. Notably, podB and sepA are needed for organization of the cytoskeleton at sites of polarized growth. In contrast, podC and podD encode proteins that appear to be specifically required for the establishment of hyphal polarity during spore germination. The role of sepA and the pod genes in controlling the spatial pattern of polarized morphogenesis in germinating spores is also described. Results obtained from these experiments indicate that the normal pattern of germ-tube emergence is dependent upon the integrity of the actin cytoskeleton. (+info)
Potential variance affecting homeotic Ultrabithorax and Antennapedia phenotypes in Drosophila melanogaster.
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Introgression of homeotic mutations into wild-type genetic backgrounds results in a wide variety of phenotypes and implies that major effect modifiers of extreme phenotypes are not uncommon in natural populations of Drosophila. A composite interval mapping procedure was used to demonstrate that one major effect locus accounts for three-quarters of the variance for haltere to wing margin transformation in Ultrabithorax flies, yet has no obvious effect on wild-type development. Several other genetic backgrounds result in enlargement of the haltere significantly beyond the normal range of haploinsufficient phenotypes, suggesting genetic variation in cofactors that mediate homeotic protein function. Introgression of Antennapedia produces lines with heritable phenotypes ranging from almost complete suppression to perfect antennal leg formation, as well as transformations that are restricted to either the distal or proximal portion of the appendage. It is argued that the existence of "potential" variance, which is genetic variation whose effects are not observable in wild-type individuals, is a prerequisite for the uncoupling of genetic from phenotypic divergence. (+info)
Thrombopoietin-induced conformational change in p53 lies downstream of the p44/p42 mitogen activated protein kinase cascade in the human growth factor-dependent cell line M07e.
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Thrombopoietin is a cytokine with potent megakaryocytopoietic and thrombopoietic activities in vivo. Wild-type p53 is a conformationally flexible, anti-oncogenic transcription factor that plays a principal role in mediating growth factor withdrawal-induced apoptosis in factor-dependent hematopoietic cells. We recently reported that Tpo induces a conformational change in and functional inactivation of p53, coincident with its anti-apoptotic effects, in the human factor-dependent cell line M07e. In an effort to identify potential signaling cascades through which Tpo illicits these effects on p53, we report here that treating M07e cells with MAPK kinase inhibitor PD98059 dramatically suppressed Tpo-induced conformational change in p53 as well as Tpo-enhanced viability in M07e cells in a p53-dependent manner. Furthermore, the expression of constitutively active Raf1 in M07e cells induced conformational change in p53 independent of Tpo stimulation. Inhibition of the JAK/STAT pathway revealed that JAK/STAT signaling plays an insignificant role in conformational modulation of p53 and apoptosis suppression. Inhibition of phosphatidylinositol-3 kinase did not have a significant effect on p53 conformation but did have a weak but significant effect on Tpo-enhanced viability. Cytokine-induced activation of the MAPK pathway and the subsequent functional neutralization of p53, may be an event by which apoptosis is commonly suppressed in hematopoiesis. (+info)
Polymorphisms in PTEN in breast cancer families.
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Germline mutations in PTEN are the underlying genetic defect in Cowden disease, which is associated with a lifetime risk of 25-50% of developing breast cancer. To investigate the role of PTEN in inherited breast cancer in the absence of manifestations of Cowden disease, we screened 177 unrelated subjects with breast cancer who also had a family history of breast cancer in at least one relative. We found no disease associated PTEN mutations in this cohort, supporting previous studies suggesting that PTEN mutations do not contribute to inherited susceptibility to breast cancer without associated manifestations of Cowden disease. We did identify an association between a common polymorphism in intron 4 and lower mean age of diagnosis of breast cancer. While preliminary, these findings suggest that further study is warranted to determine whether this allelic variant of PTEN could function as a low penetrance breast cancer susceptibility allele. (+info)
Age of onset in Huntington disease: sex specific influence of apolipoprotein E genotype and normal CAG repeat length.
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Age of onset (AO) of Huntington disease (HD) is known to be correlated with the length of an expanded CAG repeat in the HD gene. Apolipoprotein E (APOE) genotype, in turn, is known to influence AO in Alzheimer disease, rendering the APOE gene a likely candidate to affect AO in other neurological diseases too. We therefore determined APOE genotype and normal CAG repeat length in the HD gene for 138 HD patients who were previously analysed with respect to CAG repeat length. Genotyping for APOE was performed blind to clinical information. In addition to highlighting the effect of the normal repeat length upon AO in maternally inherited HD and in male patients, we show that the APOE epsilon2epsilon3 genotype is associated with significantly earlier AO in males than in females. Such a sex difference in AO was not apparent for any of the other APOE genotypes. Our findings suggest that subtle differences in the course of the neurodegeneration in HD may allow interacting genes to exert gender specific effects upon AO. (+info)
Mutation screening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis.
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21-hydroxylase deficiency is a recessively inherited disorder of steroidogenesis, resulting from mutations in the CYP21 gene. This 3.5 kb gene and a highly related CYP21P pseudogene reside on tandemly duplicated 30 kb segments of DNA in the class III HLA region, and the great majority of pathogenic mutations result from sequence exchanges involving the duplicated units. We now describe a comprehensive survey of CYP21 mutations in the British population, encompassing a screen for 17 different mutations in a total of 284 disease chromosomes. The most common mutations were as follows: large scale deletions/conversions (45% of the affected chromosomes), the intron 2 splice mutation (30.3%), R357W (9.8%), and I172N (7.0%). Mutations were detected in over 92% of the chromosomes examined, suggesting that accurate DNA based diagnosis is possible in most cases using the described strategy. In order to extend highly accurate prenatal diagnosis to all families where samples are available from a previously affected child, we have developed a linkage analysis approach using novel, highly informative microsatellite markers from the class III HLA region. (+info)