Inter- and intra-species crosses between Candida albicans and Candida guilliermondii

Hybridization was shown to occur both between strains of the imperfect diploid yeast Candida albicans and between C. albicans and the distantly related haploid yeast Candida (Pichia) guilliermondii. Prototrophic hybrids were selected from crosses of multiply marked auxotrophic mutants of the two species. In most cases, mild ultraviolet irradiation of the C. albicans partner was required. Examination of auxotrophic markers in segregants from the hybrids indicated that recombination, rather than heterokaryon formation, had occurred in these crosses. The DNA content of the hybrids varied from diploid or aneuploid (for crosses between C. albicans and C. guilliermondii) to triploid (for C. albicans x C. albicans). It seems possible that genetic exchange analogous to this may occur in nature.     

Cloning of the Candida albicans nucleoside transporter by complementation of nucleoside transport-deficient Saccharomyces

The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.     

A Candida albicans gene encoding a DNA ligase

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession Number X95001.     

白色念珠菌检测方法的研究进展

白色念珠菌是导致真菌感染的重要致病菌之一,文章对白色念珠菌的生物学特性和检测方法进行了综述。目前我国尚未出台食品中白色念珠菌检测的相关标准。本文通过对白色念珠菌检测技术发展历程的梳理,旨在为广大科研工作者、行业法规制定者提供一些参考依据。     

Large circular and linear rDNA plasmids in Candida albicans

Although plasmids containing rRNA genes (rDNA) are commonly found in fungi, they have not been reported in Candida. We discovered that the yeast opportunistic pathogen Candida albicans contains two types of rDNA plasmids which differ in their structure and number of rDNA repeats. A large circular plasmid of unknown size consists of multiple rDNA repeats, each of which includes an associated autonomously replicating sequence (ARS). In contrast, a linear plasmid, which is represented by a series of molecules with a spread of sizes ranging from 50-150 kbp, carries a limited number of rDNA units and associated ARSs, as well as telomeres. The number of linear plasmids per cell is growth cycle-dependent, accumulating in abundance in actively growing cells. We suggest that the total copy number of rDNA is better controlled when a portion of copies are on a linear extrachromosomal plasmid, thus allowing a rapid shift in the number of corresponding genes and, as a result, better adaptation to the environment. This is the first report of a linear rDNA plasmid in yeast, as well as of the coexistence of circular and linear plasmids. In addition, this is a first report of naturally occurring plasmids in C. albicans.     

The ALS6 and ALS7 genes of Candida albicans

ALS genes of Candida albicans encode a family of cell-surface glycoproteins that are composed of an N-terminal domain, a central domain of a tandemly repeated motif, and a relatively variable C-terminal domain. Although several ALS genes have been characterized, more ALS-like sequences are present in the C. albicans genome. Two short DNA sequences with similarity to the 5' domains of known ALS genes were detected among data from the C. albicans genome sequencing project. Probes developed from unique regions of these sequences were used to screen a genomic library from which two full-length genes, designated ALS6 and ALS7, were cloned. ALS6 and ALS7 encode features similar to other genes in the ALS family and map to chromosome 3, a chromosome previously not known to encode ALS sequences. ALS6 and ALS7 are present in all C. albicans strains examined. Additional analysis suggested that some C. albicans strains have another ALS gene with a 5' domain similar to that of ALS6. Characterization of ALS7 revealed a novel tandemly repeated sequence within the C-terminal domain. Unlike other ALS family tandem repeats, the newly characterized ALS7 repeat does not appear to define additional genes in the ALS family. However, our data and information from the C. albicans genome sequencing project suggest that there are additional ALS genes remaining to be characterized.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Specific identification of Candida albicans by hybridization with oligonucleotides derived from ribosomal DNA internal spacers

In order to develop DNA probes for rapid, sensitive and specific detection of the pathogenic yeast species Candida albicans, we carried out comparative sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) units of C. albicans and the closely related pathogenic species C. tropicalis. While overall sequence similarity between the two species was considerable (65–75%), both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific target sites for the identification of C. albicans. On the basis of these results one ITS1-derived ( ANAB1 ) and two ITS2-derived ( ANAB2 and ANAB3 ) oligonucleotides were selected, chemically synthesized, and used as hybridization probes. Their specificity and reliability were evaluated in dot-blot hybridization experiments with total genomic DNA from 13 strains of medically important Candida species, six strains of other yeast genera associated with man and animals, and ten strains previously identified as C. albicans by phenotypic criteria. Under well-defined hybridization conditions the three probes hybridized exclusively with DNA derived from strains belonging to the species C. albicans, thus demonstrating their potential clinical usefulness. The failure of four of the (presumed) C. albicans strains to show hybridization to the ITS probes sheds doubt upon their taxonomic classification, which is reinforced by other phenotypic aspects of these strains.     

MET15 as a visual selection marker for Candida albicans

To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied. Both MET15 alleles of C. albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene. Growth of this complemented strain on Pb(2+)-containing medium was associated with a colour shift of brown into white colonies. The MET15 alleles of C. albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting. A met15-deficient strain of C. albicans CAI-4 was generated using the ura blaster technique. This strain showed a brown colony colour on Pb(2+)-containing medium, which corresponded with the colony colour of a S. cerevisiae strain lacking the MET15 gene. Unexpectedly, the met15-deficient strain of C. albicans still grew on methionine-depleted medium. However, this growth was severely delayed. In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb(2+)-containing medium. These transformants grew very well on methionine-depleted medium. Colony sectoring was obtained with the replicative plasmid and not with the integrative one. This study demonstrates that the MET15 gene of C. albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability. GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275.     

Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.     

香辛料提取物对白色念珠菌的抑制及工艺优化研究

研究24种香辛料的抑菌作用,并比较其醇提取液的抑菌效果。通过大量的实验测定白色念珠菌抑菌圈,其中肉桂提取液对白色念珠菌抑制作用最佳。然后运用响应面(RSM)分析优化肉桂乙醇提取工艺条件。在单因素实验基础上,以白色念珠菌抑菌圈为指标,研究提取温度、液固比、提取时间及乙醇浓度对白色念珠菌抑菌圈的影响。结果表明,肉桂醇提法最佳提取工艺为提取温度69.24℃,液固比10:1(mL/g),提取时间5h,乙醇浓度95%,在此条件下提取液对念珠菌的抑菌圈是39.46mm。     

Sequence Analysis of Candida albicans Phosphoribosyl-aminoimidazole Carboxylase (ADE2) Gene

The nucleotide sequence of the Candida albicans ADE2 gene, which encodes phosphoribosylaminoimidazole carboxylase, has been determined. The sequence possesses an uninterrupted open reading frame of 1704 nucleotides corresponding to 568 amino acid residues. The deduced amino acid sequence shares a high degree of homology with ADE2 homologues in other fungal species including Saccharomyces cerevisiae, Pichia methanolica, Schwanniomyces occidentalis and Schizosaccharomyces pombe. Three regions of amino acid sequence were highly conserved among all reported ADE2 genes. The hexanucleotide TGACTC characteristic of genes involved in purine and amino acid biosyntheses is located in front of putative TATA boxes in the promoter region. The GenBank Accession Number of this gene is U75582. © John Wiley & Sons, Ltd.     

酚类化合物对白色念珠菌的抗真菌活性及其构效关系(英文)

目的寻找对白色念珠菌具有抗真菌作用或与氟康唑具有联合抗真菌作用的活性化合物。方法以美国临床标准化委员会建立的真菌敏感性测定液基稀释法(NCCLSM27-A)评价了8种天然酚类化合物和其6种半合成衍生物对18株临床新近分离的白色念珠菌的抗真菌活性。对具有抗真菌活性的实验化合物进一步进行了与氟康唑的联合抗真菌作用评价。结果化合物albicanylcaffeate对白色念珠菌表现出强的抗真菌活性,MIC为4μg/ml;而化合物大黄素、白藜芦醇、3,4'-二氟二苯乙烯、2,2'-二氟二苯乙烯及2,3',5'-三氟二苯乙烯显示出中等程度的抗真菌活性,MIC为16μg/ml;双联苄pakyonol、多酚类儿茶素、白藜芦醇二聚体野葡萄素A、3,4,4'-三羟基联苄、2,4'-二氟二苯乙烯、4-氟二苯乙烯及2-甲氧基-4'-氟二苯乙烯显示出较弱的抗真菌活性,MIC为32μg/ml。当与氟康唑联合时,albicanylcaffeate和3,4'-二氟二苯乙烯显示出协同抗真菌活性,而儿茶素与白藜芦醇显示出相加的抗真菌活性。结论所实验的酚类化合物对白色念珠菌具有较强的抗真菌活性,其中部分活性化合物与氟康唑呈现出协同或相加的联合抗真菌作用。在未来抗念珠菌感染的治疗中可能会起到重要的作用。