Multilocus sequence typing of Candida albicans isolates from animals

Multilocus sequencing strain types of a panel of 43 Candida albicans isolates from animals, including mammals and avian species, were compared with strain types for human isolates. The clade distribution of the animal isolates was significantly different from that of the human isolates, in both a comparison involving a total of 1580 isolates from multiple geographical sources and a comparison restricted to 675 human isolates from the same geographical regions as the animal isolates. A nearest-neighbour analysis involving the 43 animal isolates and 67 human isolates, randomly selected to give a proportionate distribution of geographical sources, showed a strong statistical trend towards genetic selection of different C. albicans strain types adapted to non-human animal hosts, but without complete genetic separation.     

Nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA as inferred from the gene sequence: secondary structure and evolutionary implications

Summary The nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA (SSU rRNA) gene has been determined. Sequence data indicate that the mature 19S SSU rRNA is 1,964 nucleotides long. A complete secondary structure model for P. polycephalum SSU rRNA has been constructed on the basis of the Escherichia coli 16S rRNA model and data from comparative analyses of 28 different eukaryotic sequences. A four-helix model is presented for the central domain variable region. This model can be applied both to vertebrate and most lower eukaryotic SSU rRNAs. The increased size of P. polycephalum SSU rRNA relative to the smaller SSU rRNAs from such other lower eukaryotes, as Dictyostelium, Tetrahymena or Saccharomyces is due mainly to three G+C-rich insertions found in two regions known to be of variable length in eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic SSU rRNA sequences, the acellular myxomycete P. polycephalum is seen to diverge before the appearance of the cellular mycomycete Dictyostelium discoideum.     

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa

Summary Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3-terminal protion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.     

Identification,characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.     

Organisation and sequence analysis of nuclear-encoded 5s ribosomal RNA genes in cryptomonad algae

Southern hybridisation, polymerase chain reaction (PCR), and nucleotide sequence, data indicate that the 5s ribosomal RNA (rRNA) gene is linked to the main rRNA gene repeat in the nuclear genome of four cryptomonad algae (Rhinomonas pauca, Storeatula major, Komma caudata, and isolate cs 134). The 5s gene is apparently transcribed in the same direction as the large and small subunit rRNA genes. The intergenic spacer between the 5s gene and the large subunit rRNA gene exhibits length and sequence polymorphism among the different species. Cryptomonads contain two different eukaryotic genomes: the host nucleus and the nucleus of a eukaryotic endosymbiont. Mapping experiments with isolated chromosomes of the host and endosymbiont genomes showed that the intergenic spacer between the large subunit and the 5s rRNA gene, which was amplified from total DNA by PCR, was derived from the host nuclear genome.     

Segregant-defective heterokaryons of Candida albicans

Summary Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one het nucleus to another. Het populations grown at 30 °C or 37 °C contain high frequencies (approx. 5%–10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 °C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C. albicans and other yeasts through protoplast fusions are considered.     

The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum,a symbiotic dinoflagellate

Summary The complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum. This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium. The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned. Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling. By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata. Among the dinoflagellates, C. cohnii was found to be more closely affiliated with the Apicomplexa than either P. micans or S. pilosum.     

Inflammation Induced by Inoculation of the Joint with Candida albicans

In humans Candida albicans is the most frequently isolated opportunistic fungal pathogen. In immunocompromized host the balance with the commensal fungus easily turns to life-threatening disseminated infection. The asymptomatic Candida persistence in organs and the recurrent infections suggest continuous circulation of yeast cells and their degradation products. Under certain conditions, joints might become one of the infectious sites. More easily a reactivation and destructive process can be provoked in individuals with established arthritis. We have investigated the joint inflammation caused by inoculation of the paw with live C. albicans, in intact mice and mice with collagen-induced arthritis (CIA). The results demonstrate that C. albicans infection when localized into the joints caused rapidly progressing septic arthritis. The effect was associated with a strong swelling, a rapid influx of polymorphonuclear (PMN) cells, and an elevated secretion of TNF-alpha and IFN-gamma by lymph node cells. Joint infection exacerbated the established CIA which correlated with an increased level of anti-collagen antibodies.     

Group-I intron family in the nuclear ribosomal RNA small subunit genes ofCenococcum geophilum isolates

A family of optional group-I introns was found near the 3 end of the nuclear small subunit rRNA genes in 61 out of 70 isolates of the deuteromycete mycorrhizal fungusCenococcum geophilum. DNA sequence polymorphisms among the introns (termedCgSSU introns) from ten of the isolates were studied. The sequences, ranging in size from 488 to 514 nucleotides, were from 93.2% to 99.6% similar to each other. Mutations were less common in predicted base-paired regions (33% of all mutations) than in free-standing regions (67%). The introns were self-spliced in vitro and were closest to subgroup ICI according to sequence and predicted secondary structure. Group-I intron pairing regions P1 through P10, including core regions P, Q, R and S, were present in all tenCgSSU introns studied. No lengthy open reading frames were found in any of the introns, indicating that the introns do not encode a protein, and therefore may not be mobile. It is likely that a single intron entered a progenote ofC. geophilum and changed as the species evolved.     

The isolation of osmotic-remedial conditional lethal mutants of Candida albicans

Summary We have developed a method for the isolation of a broad range of conditional lethal mutants of the pathogenic fungus Candida albicans. The method substantially alleviates the problems posed by the diploid nature, and the biased mutant spectrum, exhibited by most strains of this organism. We have used the method to isolate 560 temperature-sensitive mutants which grew at 30°C but not at 42°C. We identified amongst them a number of osmotic-remedial strains which, at the restrictive temperature, show phenotypes indicating defects in cell wall biosynthesis.     

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified. The sequence has been deposited in the EMBL data bank under the accession number Z 32848.     

Isolation and sequence analysis of the small subunit ribosomal RNA gene from the euryhaline yeast Debaryomyces hansenii

Summary The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced. After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D. hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis. It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.     

Temperature-dependent internuclear transfer of genetic material in heterokaryons of Candida albicans

Summary Heterokaryons (hets) of Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains and can be propagated continuously on minimal medium despite their tendency to assort nuclei into monokaryotic blastospores. Most mono-karyons have parental-type nuclei, but some are nuclear hybrids with DNA contents between one and two times that of their parental strains. Evidence is presented that hybrids arise by transfer of a portion of the genetic material of one bet nucleus to another, and that the amount of material conveyed during transfer increases with increasing het growth temperatures over the range 25°C to 41°C. This partial hybridization is a general property of hets and is not determined by the wild-type strain backgrounds of their parental components or by the kinds of auxotrophies forcing heterokaryosis. Frequencies of mitotic recombinants induced in partial hybrids by ultraviolet radiation indicate that nuclei of C. albicans are naturally diploid.     

The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation

We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37°C, to growth at 37°C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.     

Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinus cinereus

Summary We find three interesting characteristics of the genes encoding the ribosomal RNA (rRNA) in the basidiomycete Coprinus cinereus. First, there are only 60 to 90 copies of the genes, fewer than in other fungi. Second, the genes are organized in an unusual arrangement. The 5S rRNA genes are located in the repeat unit which encodes the other rRNAs and all four rRNAs are transcribed in the same direction. Third, meiotic recombination is inhibited within the ribosomal DNA.     

Production of heterokaryons of Candida albicans by protoplast fusions: Effects of differences in proportions and regenerative abilities of fusion partners

Production of heterokaryons of Candida albicans by polyethylene glycol-induced fusion of complementing auxotrophic protoplasts was assayed in two series of fusion crosses designed to test the significances of the in-put ratios and regenerative abilities of parental protoplasts. The findings indicate that productive fusions require interaction of multiple protoplasts of each complementing type and can occur equally well with either regenerable or non-regenerable protoplasts.     

Structure and evolution of the small subunit ribosomal RNA gene of Crithidia fasciculata

Summary We present the cloning and sequence analysis of the nuclear-encoded Crithidia fasciculata small subunit (SSU) rRNA gene, the longest (2,206 bp) such gene yet characterized by direct sequence analysis. Much of the sequence can be folded to fit a phylogenetically conserved secondary structure model, with the additional length of this gene being accommodated within discrete variable domains that are present in eukaryotic SSU rRNAs. On the basis of sequence comparisons, we conclude that Crithidia contains the most highly diverged SSU rRNA described to date among the eukaryotes, and therefore represents one of the earliest branchings within the eukaryotic primary kingdom.