Electrophoretic Karyotyping Research Articles

Association of Saccharomyces bayanus var. uvarum with some French wines: genetic analysis of yeast populations

Using genetic hybridization analysis, electrophoretic karyotyping and PCR-RFLP of the MET2 gene, we found that the yeast Saccharomyces bayanus var. uvarum is associated with certain types of wines produced in the Val de Loire, Sauternes, and Jurançon regions. The average frequency of appearance of this yeast in the three regions of France was 41, 7 and 77%, respectively. In contrast, we did not find S. bayanus var. uvarum in red wines produced in the Bordeaux area. The results of this study, as well as the findings already reported on Tokay (Slovakia), Muscat (Crimea, Ukraine) and Amarone (Italy) wines, lead us to consider that distribution of S. bayanus var. uvarum yeast is connected with low temperature climatic conditions and/or wine technologies in which must fermentation is at least partially carried out at low temperatures (10–15 °C).

Strain characterization of Candida parapsilosis fungemia by molecular typing methods.

The present study used two molecular typing methods to investigate a cluster of eight cases of Candida parapsilosis fungemia in a hospital in Rio de Janeiro, Brazil. Candida parapsilosis is an important opportunistic pathogen that is frequently involved in outbreaks of nosocomial fungemia. Identification of a common source of infection and determination of genetic relatedness among the strains involved in outbreaks are important for infection control. Candida parapsilosis strains were isolated from the bloodstream of patients housed in an intensive-care unit (n=5) and in individual rooms (n=3). An additional strain of Candida parapsilosis was isolated from a hyperalimentation infusion flask, which was implicated by molecular typing to be the source of infection. All strains were identified using morphological and biochemical methods. The genetic relationship between patients' strains and the hyperalimentation infusion strain was assessed by electrophoretic karyotype (EK) analysis and random amplification of polymorphic DNA (RAPD). Both methods resulted in patterns that allowed differentiation of the isolates. Candida parapsilosis fungemia, in three of the eight patients, resulted from a common source of infection, as demonstrated by molecular typing methods. Image analysis of EK patterns indicated that these strains were closest to Candida parapsilosis Group II, a grouping that is a less frequent clinical isolate than the major Group I strains.

Genetic diversity of isolates of the Leptosphaeria maculans species complex from Australia, Europe and North America using amplified fragment length polymorphism analysis

Amplified Fragment Length Polymorphism (AFLP) analysis has been used to analyse 100 Australian, European and North American isolates of Leptosphaeria maculans. All isolates had distinct AFLP profiles. They could be classified into five types, which had very few AFLP bands in common and corresponded to classifications made previously on the basis of ability to cause stem cankers on Brassica napus (A group), or inability to do so (B group), and on host range. Four isolates had AFLP profiles completely dissimilar to these groups and to each other. Genetic diversity and geographic differentiation were analysed separately within AFLP types 1 and 2. UPGMA analysis of the 66 AFLP type 1 (A group) isolates using 50 polymorphic bands did not provide evidence for clustering according to geographic origin. Non-metric multidimensional scaling (NMDS) analysis suggested that the Australian and European populations were separate adjacent clusters, while the North American population partially overlapped both the others. This geographic differentiation was supported by Wright's fixation index (Fst) analysis. Three measures of genetic variability between isolates within regions (effective number of alleles, gene diversity, and Shannon index) showed that the North American A group isolates were less diverse than those from Australia and Europe. The 21 AFLP type 2 (B group; NA1 sub-group) isolates did not cluster based on geographic region, which was confirmed by NMDS and Fst analysis. There was a similar degree of genetic diversity within A group and the NA1 sub-group of B group isolates. Unlike other techniques, AFLP analysis can readily discriminate between group B isolates of the L. maculans complex that were previously difficult to classify and also provides individual fingerprints for isolates. Isolates of the A group and of the NA1 sub-group of B group could be also distinguished readily by electrophoretic karyotyping, as the latter isolates had more bands smaller than 1.4 Mb than the A group isolates.

Electrophoretic karyotypes of the phytopathogenic Pyrenophora graminea and P. teres

Contour-clamped homogeneous electric field has been applied to analyse genome size and organisation of two important pathogens of barley, Pyrenophora graminea and P. teres. Electrophoretic karyotypes of four P. graminea isolates resolved 6–8 chromosomal bands, depending on the isolate, while the one P. teres isolate analysed contained six chromosomal bands. Chromosome length polymorphism was observed among P. graminea isolates. For both fungi the chromosomes ranged from 1.6 to more than 6 Mb, with chromosomes larger than 6 Mb migrating as a single band within the compression zone of the gel.

Oral colonization, phenotypic, and genotypic profiles of Candida species in irradiated, dentate, xerostomic nasopharyngeal carcinoma survivors.

The aim of this study was to investigate oral yeast colonization and oral yeast strain diversity in irradiated (head and neck), dentate, xerostomic individuals. Subjects were recruited from a nasopharyngeal carcinoma clinic and were segregated into group A (age, <60 years [n = 25; average age +/- standard deviation (SD), 48 +/- 6 years; average postirradiation time +/- SD, 5 +/- 5 years]) and group B (age, >/=60 years [n = 8; average age +/- SD, 67 +/- 4 years; average postirradiation time +/- SD, 2 +/- 2 years]) and were compared with age- and sex-matched healthy individuals in group C (age, <60 years [n = 20; average age +/- SD, 44 +/- 12 years] and group D (age, >/=60 years [n = 10; average age, 70 +/- 3 years]). Selective culture of oral rinse samples was carried out to isolate, quantify, and speciate yeast recovery. All test subjects underwent a 3-month comprehensive oral and preventive care regimen plus topical antifungal therapy, if indicated. A total of 12 subjects from group A and 5 subjects from group B were recalled for reassessment of yeast colonization. Sequential (pre- and posttherapy) Candida isolate pairs from patients were phenotypically (all isolate pairs; biotyping and resistotyping profiles) and genotypically (Candida albicans isolate pairs only; electrophoretic karyotyping by pulsed-field gel electrophoresis, restriction fragment length polymorphism [RFLP], and randomly amplified polymorphic DNA [RAPD] assays) evaluated. All isolates were Candida species. Irradiated individuals were found to have a significantly increased yeast carriage compared with the controls. The isolation rate of Candida posttherapy remained unchanged. A total of 9 of the 12 subjects in group A and 3 of the 5 subjects in group B harbored the same C. albicans or Candida tropicalis phenotype at recall. Varying degrees of congruence in the molecular profiles were observed when these sequential isolate pairs of C. albicans were analyzed by RFLP and RAPD assays. Variations in the genotype were complementary to those in the phenotypic characteristics for some isolates. In conclusion, irradiation-induced xerostomia seems to favor intraoral colonization of Candida species, particularly C. albicans, which appeared to undergo temporal modifications in clonal profiles both phenotypically and genotypically following hygienic and preventive oral care which included topical antifungal therapy, if indicated. We postulate that the observed ability of Candida species to undergo genetic and phenotypic adaptation could strategically enhance its survival in the human oral cavity, particularly when salivary defenses are impaired.

Electrophoretic characterization of Aspergillus nidulans strains with chromosomal duplications

Pulsed-field gel electrophoresis was used to characterize strains of Aspergillus nidulans with a chromosomal duplication Dp(I-II). Morphologically deteriorated and improved variants of these strains were also analyzed. The electrophoretic karyotype demonstrated that in two duplicated strains (A and B) the 4.2 Mb band, which corresponds to chromosome II, was absent and a new band was observed. Hybridization studies using the uapA (chromosome I) and wA (chromosome II) genes demonstrated that the new band corresponded to chromosome II plus the duplicated segment of chromosome I. The size of the chromosomal duplication was approximately 1.0 Mb. Analysis of the chromosomal bands of a morphologically improved strain showed that the duplicated segment of chromosome I was completely lost. The morphologically deteriorated variants V9 and V17 had the same karyotype as the duplicated strains. However, the deteriorated variant V5 lost part of chromosome I and had a rearrangement involving chromosome V. This rearrangement may have resulted from the mutagenic treatment used to obtain the genetic markers. Pulsed-field gel electrophoresis was found to be an excellent tool for locating chromosomal rearrangements.

Variation in cultural characteristics, pathogenicity, vegetative compatibility and electrophoretic karyotype within field populations of Stagonospora nodorum

Isolates of Stagonospora nodorum, the cause of septoria leaf and glume blotch of wheat, were sampled intensively from single fields in two consecutive years and compared for growth rate, colony morphology, pathogenicity, vegetative compatibility and electrophoretic karyotype. The two populations were highly variable for all characters, even though the isolates in each population originated from a small geographical area. Studies of vegetative compatibility within each population indicated the presence of many genotypes and suggested that clonal spread was very limited. Individual lesions contained a single genotype and therefore, presumably, originated from a single unit of inoculum. However, adjacent lesions, even on the same leaf, frequently contained different genotypes. Ten isolates from population II possessed six different karyotypes, demonstrating that polymorphism for this fundamental feature of the genome occurs within field populations. The population structure revealed by these studies supports the hypothesis that ascospores play a major role in the epidemiology of the disease. On this basis, the observed variation in all the characters, including karyotype, can be explained by recombination during sexual reproduction.

The ins and outs of DNA fingerprinting the infectious fungi.

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

High-frequency occurrence of chromosome translocation in a mutant strain of Candida albicans by a suppressor mutation of ploidy shift.

Significant occurrence of high-ploidy cells is commonly observed among many Candida albicans strains. We isolated two isogenic strains, STN21 and STN22, each from a half sector of a colony obtained after mild UV-irradiation of a Arg(-) derivative of CBS5736. The two strains were different from each other in ploidy states and chromosome organization. Although cells of STN22 were homogeneous in size and had a single nucleus, high-ploidy cells, with either a single large nucleus or several nuclei, were present together with apparently normal cells with a single nucleus in the cell population of STN21. Flow cytometry showed that STN22 was a stable diploid; however, STN21 seemed to be the mixture of different ploidy states, including diploid and tetraploid. The phenotype of STN21 containing high-ploidy cells is referred to here as the Sps(-) phenotype (suppressor of ploidy shift). STN22 showed a typical electrophoretic karyotype similar to strain 1006 in C. albicans. However, an extra chromosomal band appeared in some clones of STN21 at high frequency. By assignment of several DNA probes, this extra chromosome was shown to be a translocation of the 7F-7G portion of chromosome 7 with the 470 kb DNA segment containing H SfiI fragment from chromosome 4. Thus, this extra chromosome is a hybrid of 4H and 7F-7G. Since the isogenic Sps(+) strain STN22 exhibited no extra chromosome bands, a correlation is suggested between the Sps(-) phenotype and the occurrence of chromosome translocations.

High‐frequency occurrence of chromosome translocation in a mutant strain of Candida albicans by a suppressor mutation of ploidy shift

Significant occurrence of high-ploidy cells is commonly observed among many Candida albicans strains. We isolated two isogenic strains, STN21 and STN22, each from a half sector of a colony obtained after mild UV-irradiation of a Arg− derivative of CBS5736. The two strains were different from each other in ploidy states and chromosome organization. Although cells of STN22 were homogeneous in size and had a single nucleus, high-ploidy cells, with either a single large nucleus or several nuclei, were present together with apparently normal cells with a single nucleus in the cell population of STN21. Flow cytometry showed that STN22 was a stable diploid; however, STN21 seemed to be the mixture of different ploidy states, including diploid and tetraploid. The phenotype of STN21 containing high-ploidy cells is referred to here as the Sps− phenotype (suppressor of ploidy shift). STN22 showed a typical electrophoretic karyotype similar to strain 1006 in C. albicans. However, an extra chromosomal band appeared in some clones of STN21 at high frequency. By assignment of several DNA probes, this extra chromosome was shown to be a translocation of the 7F–7G portion of chromosome 7 with the 470 kb DNA segment containing H SfiI fragment from chromosome 4. Thus, this extra chromosome is a hybrid of 4H and 7F–7G. Since the isogenic Sps+ strain STN22 exhibited no extra chromosome bands, a correlation is suggested between the Sps− phenotype and the occurrence of chromosome translocations. Copyright © 2000 John Wiley & Sons, Ltd.

Evolution of drug resistance in experimental populations of Candida albicans.

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.

Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora

Abstract The homothallic fungus Sordaria macrospora produces perithecia with meiotically derived ascospores. In most cases, intraspecies crosses between strains from different culture collections generate fertile hybrid perithecia in the contact zone of two mycelia. However, in some of these crosses we observed a significant decrease in the fertility of the hybrid perithecia when strains of different origin were used for mating. Since we assumed that chromosome variability between the culture collection strains might contribute to this reduction in fertility, we performed pulsed-field gel electrophoresis. In the course of our study, we were able to identify two major groups of electrophoretic karyotypes in S. macrospora culture collection strains. A quantitative analysis revealed that polymorphic karyotypes contribute to a reduction of fertility in forced crosses between strains carrying differently sized chromosomes. The observed intraspecific chromosome length polymorphism might have consequences on the speciation process of a homothallic fungus capable of sexual but not of asexual spore formation.

Isolation, characterization, and chromosomal mapping of an ftsZ gene from the unicellular primitive red alga Cyanidium caldarium RK-1.

The FtsZ protein is involved in eukaryote plastid division, but there is little information on its involvement in the plastid-dividing apparatus. To investigate the relationship between FtsZ and the plastid-dividing ring, the ftsZ gene was isolated from the unicellular primitive red alga Cyanidium caldarium RK-1. Comparison of several prokaryotic and eukaryotic FtsZ proteins shows that there are six highly conserved domains in the core region of FtsZ. To determine the chromosomal location of ftsZ, we first determined the electrophoretic karyotype of C. caldarium RK-1. Southern-hybridization analysis combined with CHEF revealed the chromosomes on which the ftsZ gene exist. Northern-hybridization analysis indicated that the C. caldarium RK-1 ftsZ gene is transcribed as a 1.9-kb molecule, and that the transcripts specifically accumulate just before plastid division. Phylogenetic analysis indicated that C. caldarium RK-1 and other eukaryotic ftsZ genes are the descendants of cyanobacterial ftsZ genes, supporting the current agreement that FtsZ is involved in plastid division.

Electrophoretic karyotype analysis in some species of Bolbitius and Conocybe (Bolbitiaceae, Agaricales)

Electrophoretic karyotype analysis in some species of Bolbitius and Conocybe (Bolbitiaceae, Agaricales)

Taxonomic relationships among the taxa in the Candida guilliermondii complex, as revealed by comparative electrophoretic karyotyping.

Electrophoretic karyotypes of 15 type strains of the taxa in the Candida guilliermondii complex including Candida fukuyamaensis Nakase et al. and Candida xestobii Yarrow et S. A. Meyer were comparatively analysed by using the CHEF (contour-clamped homogeneous electric field) method of PFGE. Eighteen strains (isolated from various natural sources in China) which were originally identified as C. guilliermondii by conventional methods were also included. Six electrophoretic karyotype groups were recognized among the strains compared. The following type strains were grouped together with the type strains of C. guilliermondii (Castellani) Langeron et Guerra and Pichia guilliermondii Wickerham: Blastodendrion arztii Ota, Blastodendrion krausi Ota, Candida amidovorans Balloni et al., C. guilliermondii var. japonica Sugiyama et Goto, Candida mamillae S. Goto, Candida parapsilosis (Ashford) Langeron et Talice var. tokyoensis Suzuki et al., C. parapsilosis var. tuxtlensis Herrera et al. and six Chinese strains. The type strain of Torulopsis kestonii Scarr et Rose was classified into the group together with the type strain of Candida fermentati (Saito) Bai and seven Chinese strains. The group represented by the type strain of C. fukuyamaensis included five other strains isolated in China. The type strains of Candida xestobii, C. guilliermondii var. carpophila Phaff et M. W. Miller and Trichosporon appendiculare Batista et al. were separated into three different groups, respectively. Taxonomic relationships among the taxa studied are discussed.

Saccharomyces bulderi sp. nov., a yeast that ferments gluconolactone.

An unknown yeast species was isolated from maize silage and was determined to be novel on the basis of morphological and physiological characteristics, nucleotide sequence of domain D1/D2 of LSU rDNA and from its electrophoretic karyotype. The name for the proposed new species is Saccharomyces bulderi Middelhoven, Kurtzman et Vaughan-Martini (type strain CBS 8638, NRRL Y-27203, DBVPG 7127). S. bulderi is closely related to S. barnettii and S. exiguus from which it can be distinguished by having a double vitamin requirement of biotin and thiamine and by no or slow aerobic growth on raffinose, a sugar that on the contrary is fermented rapidly. Gluconolactone is rapidly fermented with ethanol, glycerol and carbon dioxide being the main products.

Reclassification of Pichia membranifaciens sensu Kurtzman.

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72-98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79-95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9-45.3 mol% for the P. membranifaciens group, 40.0-42.0 mol% for the P. manshurica group, and 27.2-44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.

Complementary mating types of Mucor circinelloides show electrophoretic karyotype heterogeneity.

Electrophoretic karyotypes of ten strains of Mucor circinelloides f. lusitanicus were generated by contour-clamped homogeneous electric field (CHEF) gel-electrophoresis. Most of the strains analyzed showed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Genome structure was further analyzed by gene assignment to the chromosome-sized DNAs. Nonradioactive hybridization techniques identified the chromosomal localization of seven cloned genes. The hybridization patterns confirmed the similarity between the mating-type (-) strains and showed some heterogeneity among the mating-type (+) strains. Linkage was found between genes pyrF and chs3 in all the strains, between the gene leuA and the rDNA in all mating-type (-) strains and ATCC 1216b (+), and between chs2 and the rDNA in CBS 969.68 (+). This is the first time that gene linkage in M. circinelloides has been reported, but in some cases the linkage relationships obtained are strain-dependent.

Oral Candida dubliniensis as a clinically important species in HIV-seropositive patients in the United States

Objective. Interest in Candida dubliniensis has led to renewed clinical investigations regarding incidence, drug resistance, pathogenesis, and epidemiology of fungal infections in patients with HIV. C dubliniensis phenotypically resembles Candida albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of this study was to prospectively evaluate the prevalence of C dubliniensis in clinical isolates and determine the clinical and demographic characteristics of patients harboring C dubliniensis . Study Design. Over a 6-week period, 24 yeast-positive isolates from HIV-positive dental patients were screened for C dubliniensis through use of phenotypic criteria. HIV viral load, CD4 count, and complete oral health evaluations were performed on each patient at the same visit during which the oral fungal surveillance culture was taken. Results. Six isolates from 24 HIV-seropositive and yeast-positive patients were shown to be consistent phenotypically and by electrophoretic karyotyping with the European reference strain of C dubliniensis . Dose-dependent susceptibility to fluconazole was shown in one of the C dubliniensis isolates. Five of the 6 patients demonstrated moderate to high viral loads. General oral health, as evidenced by the presence of advanced periodontal lesions and a high decayed, missing, and filled teeth index (>20), was poor in 3 of the 6 patients with C dubliniensis and 7 of the 18 patients with C albicans . A history of intravenous drug abuse was present in 50% of the C dubliniensis –positive patients, which is representative of the HIV-positive population at the hospital. Conclusions. In this small sample, C dubliniensis represented 25% of the yeast-positive cultures. The clinical significance of this interesting species in the United States may be related to high viral load, rapid AIDS progression, and/or concomitant oral disease, such as a high caries index or periodontal disease. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:573-80)