For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCRbased identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.
By observing isolated long DNA chains with fluorescence microscopy, the collapsing transition of individual single DNAs induced by polyethylene glycol with pendant amino groups (PEG-A) was compared to that induced by neutral polyethylene glycol (PEG). The main results are as follows: (1) DNA is collapsed by PEG-A at concentrations 105 times lower than those required for PEG. (2) In PEG-A, the concentration which induces collapse decreases with an increase in the salt concentration. This trend is similar to that in the collapse induced by PEG. (3) The transition induced by PEG is all-or-none; individual DNA chains exhibit either elongated coil or compacted globule states. On the other hand, segregation of a collapsed region along a single DNA chain occurs as an intermediate state in the collapsing transition induced by PEG-A.
Cells of the dimorphic yeast Candida albicans are easily induced to germinate in synchrony. Using germinating cells of strain FC18, we examined the effects of several drugs that are known to affect the cytoskeleton on growth and School o f Medicine, Showaku, Nagoya 466, Japan cytoskeletal organization. Cytochalasin A (CA), an inhibitor of actin function, inhibited the germination of the yeast cells and changed the cylindrical expansion of the apex of the germ tube to swelling growth. Effects of CA on the organization of actin were examined with rhodamine-phalloidin (Rh-Ph), which specifically stains F-actin. In CA-untreated cells, Rh-Ph staining resulted in condensed dot-like fluorescence a t the growing tip, as well as filamentous fluorescence (actin cables) that ran from the apex t o the basal region. In CA-treated cells, condensed dot-like fluorescence was still observed a t the swelling tip, but actin cables had disappeared completely. This result indicates that CA does not affect the asymmetrical distribution of actin, and suggests that the actin cables are not required for maintenance of the polarized localization of actin. Benomyl, an anti-microtubule drug, inhibited the germination of yeast cells and the apical growth of germinated cells. Benomyl not only disrupted microtubules (MTs), but also affected the distribution of actin. In benomyl-treated cells, actin dots were randomly dispersed all over the cell. This result indicates that benomyl destroyed the mechanism that maintains the asymmetrical distribution of actin, and suggests that MTs are involved in such a mechanism. The polarized localization of organelles is one of the most important factors associated with dimorphism. Our data suggest that the cytoskeleton, composed of actin and MTs, is involved in the control of polarity in the hyphal growth of C. ahicans, and that actin and MTs are interrelated in the establishment of polarity.
The SLPI gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, has been cloned from a yeast genomic library by complementation of the sip)-) mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slpl-l mutation. The fragment was integrated at the chromosomal SLPI locus, indicating that it contains an authentic SLPI gene. By DNA sequencing of the SLPI gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (MA, 79,270) All eucaryotic cells have highly integrated membrane organizations in which various biochemical and physiological reactions take place. For example, the yeast Saccharomyces cerevisiae possesses membrane-bound organelles including endoplasmic reticulum (ER), Golgi apparatus, mitochondria, and a nucleus. The vacuole in S. cerevisiae is the most prominent organelle, occupying about a quarter of the cell volume. This organelle constitutes a large compartment with two major functions: storage of primary and secondary metabolites and digestion of macromolecules (21).We have found a number of solute transport systems in the vacuolar membrane: an H+ ATPase (2, 15, 41) that generates a proton motive force across the vacuolar membrane, antiport systems for amino acids (24, 35) and calcium ions (25) driven by the proton motive force, and a membrane potential-dependent ion channel for monovalent cations (43). These transport systems arrayed in the vacuolar membrane suggest that the vacuole is actively maintaining ionic homeostasis in the cytosol by regulating the transport of metabolites and ions (2,16,43). The inside of the vacuole is maintained at an acidic pH by H+ influx driven by the H+ ATPase (15). The vacuole contains various hydrolases for proteins, polysaccharides, and polynucleotides (21). The yeast vacuole is thus considered a counterpart of lysosomes as a digestive and lytic compartment with an acidic interior environment (21).We have recently reported the isolation of yeast mutants with defective vacuolar functions (17). The mutant carrying an slpl-l mutation (for small lysine pool) shows the following pleiotropic phenotypes. The slpl-l mutant does not have large prominent vacuoles. The vacuolar pool for basic amino * Corresponding author.acids is significantly reduced, probably because the volume of the vacuoles is reduced by the slpl-l mutation. The mutant is hypersensitive to lysine, histidine, and calcium, which are known to be sequestered in the vacuole by the mechanism of substrate/nH+ antiport (24,25,35). The activities of vacuolar proteases, including proteinase A, proteinase B, and carboxypeptidase Y, are lower than those in a wild-type strain. These pleiotropic phenotypes caused by the slpl-l mutation suggest that the SLPI gene is required for the expression of vacuolar functions through the biogenesis of the vacuole in yeas...
A clinical isolate of the pathogenic yeast Candida albicans varied in its colony morphology from smooth (o-smooth) to semi-rough type (SRT) and concomitantly lost its virulence for mice. In terms of DNA content, the smooth parent was near triploid when Saccharomyces cerevisiae strains of known ploidy were used as references. The SRT variant showed several features characteristic of polyploidy. From the SRT variant, revertant-like smooth (r-smooth) variants with recovered virulence were derived at a frequency of 5 x The results of pulsed-field gel electrophoresis on chromosomal DNA showed changes in patterns of chromosome-sized DNA bands in the SRT variant as well as in r-smooth variants, which correlated with these variations. Correlations between colony morphology, state of ploidy and virulence of this asporogenous yeast are considered.
We have described a unique binding system between Candida albicans yeast-form cells and the marginal zone of mouse spleen (16). The chemical nature of the fungal adhesin(s) involved in this binding phenomenon was examined. A fraction obtained by 2-mercaptoethanol extraction (2-ME extract) of fungal cells caused a dose-response inhibition of yeast cell adherence to splenic marginal zone sites and also to subcapsular and medullary sinuses of mouse popliteal lymph nodes. Latex beads coated with the 2-ME extract showed a pattern of spleen and lymph node tissue binding identical to that observed with yeast cells. The extracted adhesins retained their binding activity in vivo. When 0.5 mg of the 2-ME extract was given intravenously to mice, spleen tissue removed up to 3 h later showed over 80% inhibition of yeast cell binding to the spleen marginal zone, and over 50% inhibition was retained for at least 24 h. The adhesins bound to a concanavalin A affinity column and were eluted by 0.5 M ac-methyl-D-mannopyranoside, and the eluted adhesins were designated Fr.II. Fr.II was further fractionated by DEAE-Sephacel ion-exchange column chromatography, and one especially active and abundant fraction was designated Fr.IIa. The adhesin moiety appeared to be carbohydrate, because the activity of Fr.IIa was destroyed by 20 mM sodium periodate or by 5 U of ac-mannosidase, but boiling (30 min) or proteinase K (100 ,ug/ml) treatments had no effect. Chemically, whereas the 2-ME extract contained significant amounts of protein and mannose, Fr.IIa consisted of over 98% mannose and less than 0.5% protein. These data strongly suggest that the mannan portion within a mannoprotein is responsible for the binding of yeast cells to splenic marginal zone and to subcapsular and medullary sinuses of mouse lymph node tissue.
Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.
It is well-known that polyamines, such as spermidine, have the effect of condensing DNA molecules into a toroidal morphology with an outer diameter of 80−100 nm. We have investigated the collapse of long duplex T4 DNA induced by spermidine with use of fluorescence and electron microscopy. We found that giant toroids with an outer diameter of ∼200 nm are generated from single T4 DNA under a high concentration of spermidine in the buffer solutions with rather high salt.
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