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W L Whelan

Publications and source records attributed to W L Whelan.

At least 19 recordsLinked to original sources
PubMed1990

Candida albicans in patients with the acquired immunodeficiency syndrome: absence of a novel of hypervirulent strain.

To determine whether Candida albicans in patients with AIDS represents a unique strain, C. albicans isolated from 24 patients with AIDS was compared with Candida isolated from 23 healthy adults. Resistance to 5-fluorocytosine, synthesis of amino acids and nucleotides, sugar use, and enzyme activity patterns were similar among isolates from the two groups. Molecular analysis revealed similar banding patterns of EcoRI restriction fragments of DNA between 2.5-3 and 6-7 kb. In addition, the frequency of a dimorphic 3.7-versus 4.2-kb band, identified by agarose gel electrophoresis and by probing Southern transfers of EcoRI digests with a cloned fragment of C. albicans DNA encoding 25S ribosomal RNA, was not significantly different between the AIDS-derived and control C. albicans. C. albicans isolated at different time points in the course of disease and from different sites in individual patients showed identical DNA fingerprints. The similarity in isolates of C. albicans that cause disease in AIDS patients and those present in healthy subjects suggests that the candidiasis associated with AIDS is not due to the presence of a unique or particularly virulent strain but is likely the consequence of a defect in host defense mechanisms.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Isolation and characterization of cell surface mutants of Candida albicans.

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.

Agglutinins↗
PubMed1989

Genetic differences between type I and type II Candida stellatoidea.

Genetic similarities and differences between type I and type II Candida stellatoidea were studied. The electrophoretic karyotype, mitochondrial DNA (mtDNA) restriction patterns, and midrepeat sequence of nuclear DNA in type I C. stellatoidea were clearly distinguishable from those of a reference culture of Candida albicans. The karyotype and the major bands of the midrepeat sequence of type II C. stellatoidea were indistinguishable from those of the reference C. albicans. The mtDNA restriction patterns of four type I isolates were homogeneous regardless of the endonucleases and probes used. The mtDNA restriction patterns of type II C. stellatoidea varied from strain to strain. Some of them were identical to that of C. albicans, while others were the same as that of type I C. stellatoidea. Immunofluorescence with C. albicans serotype A-specific monoclonal antibody indicated that the four isolates of type I C. stellatoidea were serotype B (non-A), whereas all three type II isolates studied were serotype A. Taken together, these results support the hypothesis that the isolates of C. stellatoidea type II studied are sucrose-negative mutants of serotype A C. albicans. Since C. stellatoidea type I differs from C. albicans in several major genetic characteristics, it cannot be viewed as a simple mutant derived from C. albicans. Hybrids produced by protoplast fusion of type I and type II cells were capable of assimilating sucrose, indicating that the sucrose-negative phenotypes of the parents are due to different mutations.

Candida↗
PubMed1988

Auxotrophic heterozygosities and the ploidy of Candida parapsilosis and Candida krusei.

Evidence is presented that the type strain of Candida parapsilosis, ATCC 22019, is heterozygous MET/met and that Candida krusei Y 10930 is heterozygous URA/ura. This URA gene determines orotidine-5'-phosphate decarboxylase activity. Because each of these strains is demonstrably disomic for at least one chromosome, they are not haploid but must be diploid or aneuploid.

Candida↗
PubMed1987

Ploidy and DNA content of Candida stellatoidea cells.

Three isolates of Candida stellatoidea contained approximately the same amount of DNA per blastospore (38.3 to 41.9 fg) as did a known diploid isolate of Candida albicans and about twice as much as did a haploid isolate of Saccharomyces cerevisiae. The diploidy of C. stellatoidea was supported by demonstration of mitotic segregation of an ade marker.

Candida↗
PubMed1987

Flow cytometric analysis of the DNA synthetic cycle of Candida species.

The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.

Candida↗
PubMed1987

Parasexual genetics of Torulopsis glabrata.

Prototrophic hybrids were generated in the asexual yeast Torulopsis glabrata by the fusion of spheroplasts derived from parent strains which bore complementing auxotrophic markers. The DNA content (per cell) of two hybrids was essentially that predicted by summing the corresponding parental values. UV irradiation of these two hybrids resulted in the formation of sectored colonies with genetic properties consistent with their origin by either mitotic recombination or chromosomal nondisjunction.

Candida↗
PubMed1987

The genetics of medically important fungi.

The present review is concerned with recent progress in basic genetic investigations with a variety of fungi which are pathogenic for man and animals. The principles and strategies involved in undertaking genetic investigations of sexual species and of asexual species are discussed. Progress in genetic analysis of Cryptococcus neoformans made possible by the discovery of its sexual phase is described in detail, as is progress in development of parasexual methods of analysis in Candida albicans. The genetic bases of virulence and drug resistance are discussed for those few species in which these phenotypes have been investigated. Suggestions for future research, including the application of recent advances in molecular biology to the study of pathogenic fungi, are presented.

Alleles↗
PubMed1987

The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans.

In terms of genetically determined susceptibility to the clinical antifungal agent 5-fluorocytosine (5-FC), Candida albicans may be homozygous sensitive (FCY/FCY), homozygous resistant (fcy/fcy), or heterozygous (fcy/FCY). Although heterozygotes are only slightly resistant, they occur at significant frequency among clinical strains and carry preexisting resistance determinants which may be responsible, following homozygosis, for treatment failures. There are two resistance genes (FCY1 and FCY2) known. Resistance in fcy1/fcy1 strains was associated with decreased UMP pyrophosphorylase activity, whereas resistance in fcy2/fcy2 strains was associated with decreased cytosine deaminase activity. These results were confirmed and extended in a 19F nuclear magnetic resonance study of 5-FC uptake and metabolism in genetically defined strains. By means of hybridization via spheroplast fusion, a complementation test was devised to test allelism of resistance determinants. Resistance to 5-FC was employed as a useful genetic marker in basic studies. In tetraploid hybrids which bore appropriate fcy markers, it was possible to select for reduction in ploidy by selecting for increased resistance to 5-FC; a novel parasexual system was thus generated (2n x 2n----4n----2n). In linkage studies, the gene FCY1 was shown to be linked to the gene HIS. Reciprocal mitotic recombination was demonstrated repeatedly with fcy1 and his alleles in cis and in trans configurations and evidence for nonreciprocal recombination (mitotic gene conversion) was also obtained. In Cryptococcus neoformans, mutation in either of two genes (FCY1, FCY2) is sufficient to confer resistance. These genes behave as simple Mendelian determinants which recombine freely. Diploid C. neoformans heterozygous for resistance (FCY/fcy) provided useful strains in which to develop genetic mapping methodology based on mitotic recombination.

Candida↗
PubMed1986

A 19F nuclear magnetic resonance study of uptake and metabolism of 5-fluorocytosine in susceptible and resistant strains of Candida albicans.

The metabolism of the antifungal drug 5-fluorocytosine (5-FC) was studied in intact viable cells of Candida albicans by 19F nuclear magnetic resonance (NMR). The uptake of the drug and its conversion to the deaminated product 5-fluorouracil (5-FU) were easily observed by NMR analysis of both the cells and the supernatants of the incubation mixture. In the 5-FC-resistant mutant D14 of C. albicans, which lacked cytosine deaminase activity, the resonance peak of 5-FU was not observed. In intact cells of all 5-FC-susceptible strains the metabolism of 5-FU progressed to the formation of other fluorinated derivatives which were visualized as a single, broad resonance band at a lower field with respect to 5-FC and 5-FU. This band was resolved into three distinct peaks in the acid extract of treated cells, one of these peaks being attributable to 5-fluoro-dUMP (5-FdUMP). In strain 72R of C. albicans, which is 5-FC resistant because of a low level of UMP-pyrophosphorylase activity, the broad, low-field resonance band was detected later and with much less intensity than in the 5-FC-sensitive strains. This suggests that, besides 5-FdUMP, this band is also contributed to by 5-FUMP and possibly other phosphorylated derivatives. 19F NMR analysis also revealed that a significant amount of 5-FU is secreted into the external medium, the rate of secretion being higher in 5-FC-resistant strain 72R than in 5-FC-sensitive strain 72S. Although not all resonances were definitely identified, this study shows that 19F NMR spectroscopy may be an important tool for noninvasive analysis of the metabolism of fluorinated drugs in yeasts.

Candida albicans↗
PubMed1986

Complementation analysis of resistance to 5-fluorocytosine in Candida albicans.

A complementation test was devised to study allelism among the genetic determinants of resistance to 5-fluorocytosine in Candida albicans. Complementation was demonstrated in control hybrids produced by crossing a resistant strain that was deficient in cytosine deaminase activity with four other resistant strains deficient in UMP pyrophosphorylase activity. This complementation test was used to test allelism of the resistance determinants present in five clinical isolates. All were found to bear recessive alleles of the locus (FCY1) that determined 5-fluorocytosine resistance associated with low levels of UMP pyrophosphorylase activity.

Alleles↗
PubMed1986

Genetic complementation in Cryptococcus neoformans.

A complementation test was devised for the fungus Cryptococcus neoformans. Complementation was signalled by the growth of prototrophic heterokaryons generated in crosses of the type aB X Ab, where a and b represent any two of the genetic markers ilv1, cys1, cys2, and cys3. The cloned complementing heterokaryons formed characteristic hyphal colonies that contained both hyphae and yeast cells. The heterokaryon-derived yeasts were of three kinds: parental haploids, recombinant haploids, and diploids.

Cloning, Molecular↗
PubMed1985

UV-induced instability in Candida albicans hybrids.

Auxotrophic variants were obtained following UV-irradiation of Candida albicans hybrids which were heterozygous (+/+/-/-/) for various genetic markers (met, ade, his, lys). Some variants contained less DNA (per cell) than did the hybrids from which they originated; such variants were considered to arise in a process which resulted in generalized reduction in ploidy. These results provide the basis for a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis in this amictic diploid species.

Candida albicans↗
PubMed1985

Instability of Candida albicans hybrids.

Total cellular DNA content, determined by a colorimetric method, was used as an index of ploidy in Candida albicans. Mononucleate hybrids were formed by fusion of spheroplasts derived from diploid parent strains. Five hybrids, of six studied, were taken to be tetraploid on the basis of estimated DNA content. One hybrid was taken to be hexaploid or near-hexaploid. Selection for increased resistance to 5-fluorocytosine in the hybrids, which were heterozygous for resistance, resulted in isolation of variants which were of lower ploidy than the hybrids from which they originated. Variants were obtained which corresponded (in measured DNA content) to aneuploid, triploid, and diploid states. These results may form the basis of a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis of this asexual species.

Candida albicans↗
PubMed1979

The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates.

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.

Alleles↗
PubMed1976

Students' rights.

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Civil Rights↗