Biomedical subjects

John H McCusker

Publications and source records attributed to John H McCusker.

13 recordsLinked to original sources

PubMed2006

Complex genetic interactions in a quantitative trait locus.

Whether in natural populations or between two unrelated members of a species, most phenotypic variation is quantitative. To analyze such quantitative traits, one must first map the underlying quantitative trait loci. Next, and far more difficult, one must identify the quantitative trait genes (QTGs), characterize QTG interactions, and identify the phenotypically relevant polymorphisms to determine how QTGs contribute to phenotype. In this work, we analyzed three Saccharomyces cerevisiae high-temperature growth (Htg) QTGs (MKT1, END3, and RHO2). We observed a high level of genetic interactions among QTGs and strain background. Interestingly, while the MKT1 and END3 coding polymorphisms contribute to phenotype, it is the RHO2 3'UTR polymorphisms that are phenotypically relevant. Reciprocal hemizygosity analysis of the Htg QTGs in hybrids between S288c and ten unrelated S. cerevisiae strains reveals that the contributions of the Htg QTGs are not conserved in nine other hybrids, which has implications for QTG identification by marker-trait association. Our findings demonstrate the variety and complexity of QTG contributions to phenotype, the impact of genetic background, and the value of quantitative genetic studies in S. cerevisiae.

3' Untranslated Regions↗

PubMed2006

Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo.

Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, pyruvate, and fatty acids. Using mutants unable to either transport or utilize these compounds, we demonstrated that no individual nitrogen source was essential, while glucose was the most significant primary carbon source for yeast survival in vivo. Hydrolysis of the storage carbohydrate glycogen made a slight contribution for in vivo survival compared with a substantial requirement for trehalose hydrolysis. The ability to sense and respond to low glucose concentrations was also important for survival. In contrast, there was little or no requirement in vivo in this assay for any of the nitrogen-sensing pathways, nitrogen catabolite repression, the ammonium- or amino acid-sensing pathways, or general control. By using auxotrophic mutants, we found that some nitrogenous compounds (polyamines, methionine, and lysine) can be acquired from the host, while others (threonine, aromatic amino acids, isoleucine, and valine) must be synthesized by the pathogen. Our studies provide insights into the yeast-host environment interaction and identify potential antifungal drug targets.

Amino Acids↗

PubMed2005

Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae.

We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae. The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5-fluorocytosine (5FC) is toxic to cytosine deaminase-producing cells, fcy1 strains are resistant to 5FC. FCY1MX and FCA1MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1, respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1PR, FCY1PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1- and FCA1MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter-selection on both synthetic and rich media.

Candida albicans↗

PubMed2004

DsdA (D-serine deaminase): a new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae.

Dominant drug resistance markers offer experimental flexibility in the study of Saccharomyces cerevisiae by eliminating the dependence on auxotrophic mutations and, because they are phenotypically neutral, avoid the deleterious effects of auxotrophic mutations. We have developed a new dominant resistance marker, dsdAMX4, for use in the genetic manipulation of S. cerevisiae. The dsdA gene, which is derived from Escherichia coli and encodes a D-serine deaminase, confers to S. cerevisiae resistance to D-serine and the ability to use D-serine as a nitrogen source. Here we describe the construction of a dsdAMX4 cassette, capable of expression in S. cerevisiae, and the characterization of this new marker for use in chromosomal gene disruption. The unique selection properties of the dsdAMX4 cassette make it an important addition to the existing array of S. cerevisiae genetic tools.

Escherichia coli↗

PubMed2004

Positive and negative selection LYS5MX gene replacement cassettes for use in Saccharomyces cerevisiae.

Novel MX cassettes are described that contain the open reading frames (ORFs) of Saccharomyces cerevisiae or Candida albicans LYS5. The LYS5MX and CaLYS5MX cassettes, the targeting efficiencies of which are equivalent to those of other MX cassettes, are positively selected for Lys+ in a lys5 background. Unlike most of the other MX cassettes, the LYS5MX cassettes are also negatively selectable (alpha-aminoadipate-resistant), which will allow the use of the LYS5MX cassettes in plasmid shuffling and will also greatly facilitate marker recycling.

Candida albicans↗

PubMed2004

Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 37 degrees C and in vivo.

Acetolactate synthase catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors. The Cryptococcus neoformans ILV2 gene, encoding acetolactate synthase, was identified by complementation of a Saccharomyces cerevisiae ilv2 mutant. C. neoformans is highly resistant to the commercially available acetolactate synthase inhibitor, sulfometuron methyl (SM). Expression of C. neoformans ILV2 in S. cerevisiae conferred SM resistance, indicating that the SM resistance of C. neoformans is due, at least in part, to C. neoformans Ilv2p. The C. neoformans ILV2 gene was disrupted. The ilv2 mutants were auxotrophic for isoleucine and valine and the auxotrophy was satisfied by these amino acids only when proline, and not ammonium, was the nitrogen source, indicating nitrogen regulation of amino acid transport. ilv2 mutants rapidly lost viability at 37 degrees C and when starved for isoleucine and valine. Consistent with these phenotypes, an ilv2 mutant was avirulent and unable to survive in mice. Because C. neoformans Ilv2p is required for virulence and survival in vivo, inhibitors of branched-chain amino acid biosynthesis may make valuable antifungal agents.

Acetolactate Synthase↗

PubMed2004

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2004

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

Cell Proliferation↗

PubMed2004

Gene conversion and crossing over along the 405-kb left arm of Saccharomyces cerevisiae chromosome VII.

Gene conversions and crossing over were analyzed along 10 intervals in a 405-kb region comprising nearly all of the left arm of chromosome VII in Saccharomyces cerevisiae. Crossover interference was detected in all intervals as measured by a reduced number of nonparental ditypes. We have evaluated interference between crossovers in adjacent intervals by methods that retain the information contained in tetrads as opposed to single segregants. Interference was seen between intervals when the distance in the region adjacent to a crossover was < approximately 35 cM (90 kb). At the met13 locus, which exhibits approximately 9% gene conversions, those gene conversions accompanied by crossing over exerted interference in exchanges in an adjacent interval, whereas met13 gene conversions without an accompanying exchange did not show interference. The pattern of exchanges along this chromosome arm can be represented by a counting model in which there are three nonexchange events between adjacent exchanges; however, maximum-likelihood analysis suggests that approximately 8-12% of the crossovers on chromosome VII arise by a separate, noninterfering mechanism.

Chromosome Mapping↗

PubMed2002

Dissecting the architecture of a quantitative trait locus in yeast.

Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult. As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes. This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae. A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases.

Alleles↗

PubMed2002

Calcineurin is essential for survival during membrane stress in Candida albicans.

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

Antifungal Agents↗

PubMed2002

The tRNA-Tyr gene family of Saccharomyces cerevisiae: agents of phenotypic variation and position effects on mutation frequency.

Extensive phenotypic diversity or variation exists in clonal populations of microorganisms and is thought to play a role in adaptation to novel environments. This phenotypic variation or instability, which occurs by multiple mechanisms, may be a form of cellular differentiation and a stochastic means for modulating gene expression. This work dissects a case of phenotypic variation in a clinically derived Saccharomyces cerevisiae strain involving a cox15 ochre mutation, which acts as a reporter. The ochre mutation reverts to sense at a low frequency while tRNA-Tyr ochre suppressors (SUP-o) arise at a very high frequency to produce this phenotypic variation. The SUP-o mutations are highly pleiotropic. In addition, although all SUP-o mutations within the eight-member tRNA-Tyr gene family suppress the ochre mutation reporter, there are considerable phenotypic differences among the different SUP-o mutants. Finally, and of particular interest, there is a strong position effect on mutation frequency within the eight-member tRNA-Tyr gene family, with one locus, SUP6, mutating at a much higher than average frequency and two other loci, SUP2 and SUP8, mutating at much lower than average frequencies. Mechanisms for the position effect on mutation frequency are evaluated.

Genes, Suppressor↗

PubMed2002

Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant.

The Cryptococcus neoformans MET3 cDNA (encoding ATP sulfurylase) was cloned by complementation of the corresponding met3 mutation in Saccharomyces cerevisiae. Sequence analysis showed high similarity between the deduced amino acid sequence of the C. neoformans Met3p and other fungal ATP sulfurylases. A C. neoformans met3 mutant was made by targeted insertional mutagenesis, which had the expected auxotrophic phenotype, and reconstituted the met3 mutant to Met(+). In vitro, the C. neoformans met3 mutant had a substantial defect in melanin formation, significantly reduced growth rate, and greatly increased thermotolerance. In the murine inhalation infection model, the met3 mutant was avirulent and was deficient in its ability to survive in mice. It is concluded that, in contrast to the yeast form of Histoplasma capsulatum, in C. neoformans the sulfate-assimilation arm of the methionine biosynthetic pathway plays an important role in vitro, even in the presence of abundant exogenous methionine, and is critical for virulence, and indeed for survival, in vivo.

Animals↗