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1.
Gómez-Raja J Andaluz E Magee B Calderone R Larriba G 《Fungal genetics and biology : FG & B》2008,45(4):527-541
Candida albicans is a diploid organism that exhibits high levels of heterozygosity. Although the precise manner by which this heterozygosity provides advantage for the commensal/pathogenic life styles of C. albicans is not known, heterozygous markers are themselves useful for studying genomic rearrangements, which occur frequently in C. albicans. Treatment of CAI-4 with UV light yielded histidine auxotrophs which could be complemented by HIS4, suggesting that strain CAI-4 is heterozygous for HIS4. These auxotrophs appeared to have undergone mitotic recombination and/or chromosome loss. As expected from a heterozygote, disruption of the functional allele of HIS4 resulted in a his4::hisG-URA3-hisG strain that is auxotrophic for histidine. Sequencing of random clones of the HIS4 ORF from CAI-4 and its precursor SC5314 revealed the presence of 11 SNPs, seven synonymous and four non-synonymous. Site-directed mutagenesis indicates that only one of those SNPs, T929G (Gly310Val), is responsible for the non-functionality of the encoded enzyme. HIS4 analysis of five commonly used laboratory strains is reported. This study provides a new, easily measured nutritional marker that can be used in future genetic studies in C. albicans. 相似文献
2.
Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism. 相似文献
3.
Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph. 总被引:5,自引:4,他引:1
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Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains. 相似文献
4.
Andaluz E Bellido A Gómez-Raja J Selmecki A Bouchonville K Calderone R Berman J Larriba G 《Molecular microbiology》2011,79(6):1462-1482
RAD52 is required for almost all recombination events in Saccharomyces cerevisiae. We took advantage of the heterozygosity of HIS4 in the Candida albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ~10(-3) , at least 100-fold higher than in Rad52(+) strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH -GL lab His(-)) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homologue. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies. 相似文献
5.
Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans 总被引:1,自引:0,他引:1
Background
Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.Results
Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.Conclusion
The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids. 相似文献6.
Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae 总被引:1,自引:0,他引:1
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In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes. 相似文献
7.
We performed an investigation of two unrelated cases with extremal variants of chromosome 21 without visible materials of the short arms (Christchurch or Ch1 chromosome). In the first case chromosome 21p- was initially detected during routine cytogenetic amniocentesis. Chromosomal variant was inherited from phenotypically normal father to phenotypically normal fetus (phenotypically normal boy after the birth). The second case of chromosome 21p- was detected in 7 years old boy, referred to cytogenetic analysis due to mental retardation and mild congenital malformation, including prenatal hypoplasia, microcephaly, low-set dysplastic ears, short nose, micrognatia, short neck. Molecular characterization of 21p-variant chromosomes was performed by the use of FISH with DNA probes specific to the short arm and centromeric region of chromosome 21 (telomeric, beta-satellite, ribosomal, classical satellite and alphoid DNA probes). Chromosomes 21p-hybridized positively only with telomeric DNA at both chromosomal ends and alphoid DNA probes at centromeric region of the first patient. In second case (de novo deletion of 21p), the Ch1 was associated with clinical phenotype and loss of telomeric and subtelomeric DNA in the p-arm of chromosome 21. Therefore, the complete absent of the short arm of chromosome 21 may be considered as abnormal. We propose that de novo deletion 21p- could have negative consequences due to absence of large portion of chromosomal DNA from the p-arm (telomeric, satellite or ribosomal DNAs) and following imbalance in organization and functioning of genome. 相似文献
8.
9.
Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life-threatening deep-tissue infections. Recent studies provide strong evidence that C. albicans possesses an arsenal of genetic mechanisms promoting genome plasticity and that it uses these mechanisms under conditions of nutritional or antifungal drug stress. Two microarray-based methods, single nucleotide polymorphism (SNP) and comparative genome hybridization arrays, have been developed to study genome changes in C. albicans . However, array technologies can be relatively expensive and are not available to every laboratory. In addition, they often generate more data than needed to analyze specific genomic loci or regions. Here, we have developed a set of SNP-restriction fragment length polymorphism (RFLP) (or PCR-RFLP) markers, two per chromosome arm, for C. albicans . These markers can be used to rapidly and accurately detect large-scale changes in the C. albicans genome including loss of heterozygosity (LOH) at single loci, across chromosome arms or across whole chromosomes. Furthermore, skewed SNP-RFLP allelic ratios are indicative of trisomy at heterozygous loci. While less comprehensive than array-based approaches, we propose SNP-RFLP as an inexpensive, rapid, and reliable method to screen strains of interest for possible genome changes. 相似文献
10.
A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast 总被引:48,自引:0,他引:48
We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control. 相似文献
11.
beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans 总被引:2,自引:0,他引:2
We have identified a novel 399 bp repetitive DNA element (which we designate beta ) 9 bp upstream of a seryl-tRNACAG gene in the genome of Candida albicans . There are two copies of the seryl-tRNACAG gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNACAG gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNAAsp , tRNAAla and tRNAIle . The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae . We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes. 相似文献
12.
Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system 总被引:1,自引:0,他引:1
The development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans. 相似文献
13.
Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans 总被引:14,自引:0,他引:14
By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans. 相似文献
14.
15.
Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage 总被引:4,自引:0,他引:4
Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV. 相似文献
16.
Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts. 相似文献
17.
18.
Prescott K Woodfine K Stubbs P Super M Kerr B Palmer R Carter NP Scambler P 《Human genetics》2005,116(1-2):83-90
The 22q11 deletion syndrome (22q11DS) is a developmental syndrome comprising of heart, palate, thymus and parathyroid glands defects. Individuals with 22q11DS usually carry a 1.5- to 3-Mb heterozygous deletion on chromosome 22q11.2. However, there are many patients with features of 22q11DS without a known cause from conventional karyotype and FISH analysis. Six patients with features of 22q11DS, a normal chromosomal and FISH 22q11 analysis, were selected for investigation by microarray genomic comparative hybridisation (array CGH). Array-CGH is a powerful technology enabling detection of submicroscopic chromosome duplications and deletions by comparing a differentially labelled test sample to a control. The samples are co-hybridised to a microarray containing genomic clones and the resulting ratio of fluorescence intensities on each array element is proportional to the DNA copy number difference. No chromosomal changes were detected by hybridisation to a high resolution array representing chromosome 22q. However, one patient was found to have a 6-Mb deletion on 5q11.2 detected by a whole genome 1-Mb array. This deletion was confirmed with fluorescence in-situ hybridisation (FISH) and microsatellite marker analysis. It is the first deletion described in this region. The patient had tetralogy of Fallot, a bifid uvula and velopharyngeal insufficiency, short stature, learning and behavioural difficulties. This case shows the increased sensitivity of array CGH over detailed karyotype analysis for detection of chromosomal changes. It is anticipated that array CGH will improve the clinicians capacity to diagnose congenital syndromes with an unknown aetiology. 相似文献
19.
A method is described for the formation of nearly 100% pseudohyphae populations of wild-type Candida albicans A72. The method employs fungal growth at 37 degrees C (ca. 5x10(6) cells/ml) in a glucose-proline-N-acetyl-glucosamine medium supplemented with up to 600 mM phosphate (KH(2)PO(4)/K(2)HPO(4) 1:1) at pH 6.5. Four other strains of C. albicans (MEN, 10261, SG5314 and CAI-4) also formed pseudohyphae under these conditions, although the phosphate response profiles differed in the concentration required for each strain to form pseudohyphae. 相似文献
20.
Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques. 总被引:18,自引:1,他引:17
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Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes. 相似文献