Contour-clamped Homogeneous Field Electrophoresis Research Articles

Chromosome length polymorphism in clinical isolates of Candida parapsilosis

Chromosome length polymorphism among 24 clinical isolates of Candida parapsilosis obtained from several human sources was analysed using pulsed-field gel electrophoresis. The isolates, from both superficial and deep infections, comprised a miscellaneous collection from the oral cavity, blood cultures, ear infections, wound secrete, a venous catheter and peritoneal dialysis fluid. Contour-clamped homogenous field electrophoresis using a hexagonal electrode was used for pulsed field gel electrophoresis. The chromosome numbers varied from seven to nine and their sizes ranged from 0.75 to 2.6 Mb. According to the electrophoretic karyotype patterns the 24 isolates could be divided into 9 profiles. However, the majority (18 isolates) fell into 3 groups comprising 7, 8 and 9 chromosomes, containing 5, 11, and 2 isolates, respectively. The remaining six isolates, all of which were from either an oral or another superficial site of isolation, could be categorized into a further six groups. These data confirm previous observations on the genomic heterogeneity of clinical isolates of C. parapsilosis, and illustrate the possible commonality in strains from related clinical habitats.

DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis

The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on biotypes of C. parapsilosis.

Yeast gene SRP1 (serine-rich protein) : Intragenic repeat structure and identification of a family of SRP1-related DNA sequences

We have isolated and sequenced a yeast gene encoding a protein ( M r 24,875) very rich in serine (SRP) and alanine residues that accounted for 25% and 20% of the total amino acids, respectively. The SRP1 gene is highly expressed in culture conditions leading to glucose repression (Marguet & Lauquin, 1986), the amount of SRP1 mRNA representing about 1 to 2% of total poly(A) + RNA. A repetitive structure of eight direct tandem repeats 36-base long, also reflected in the amino acid sequence, was found in the second half of the open reading frame. The consensus amino acid sequence of the repeat was Ser-Ser-Ser-Ala-Ala-Pro-Ser-Ser-Ser-Glu-Ala-Lys. Replacing the genomic copy of the cloned gene with a disrupted SRP1 gene indicated that the SRP1 gene was not essential for viability in yeast, but several SRP1 -homologous sequences were found within the yeast genome, raising the possibility that the disrupted SEP1 gene is rescued by one of the other SRP-homologous sequences. Complete separation of yeast chromosomes by contour-clamped homogeneous field electrophoresis indicated that, apart from chromosome V, which carries the SRP1 gene, 12 chromosomes have SRP-related sequences with various degrees of homology. These sequences were located on chromosomes XV, VII and XI under stringent conditions of hybridization ( t m−20 ° C), and observed on chromosomes I, II, III, IV, VI, VIII, X, XI and XII, only under low-stringency conditions ( t m −40 ° C). Northern blot analysis of both the wild type and SAP1-disrupted strains indicated that along with SRP1 at least one more member of the SRP family was transcribed to a 0.7 kb (1 kb = 10 3 bases) polyadenylated RNA species clearly distinct from the SRP 1-specific mRNA (1 kb long). Analyses of the SRP1 repeat domain suggested a model for the divergent evolution of the repeats in the SRP1 sequence.