RESUMO
Although the diploid fungus Candida albicans, a human pathogen, has been thought to have no sexual cycle, it normally possesses mating-type-like orthologs (MTL) of both of the Saccharomyces cerevisiae mating-type genes (MAT) a and alpha. When strains containing only MTLa or MTLalpha were constructed by the loss of one homolog of chromosome 5, the site of the MTL loci, MTLa and MTLalpha strains mated, but like mating types did not. Evidence for mating included formation of stable prototrophs from strains with complementing auxotrophic markers; these contained both MTL alleles and molecular markers from both parents and were tetraploid in DNA content and mononucleate.
Assuntos
Candida albicans/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Recombinação Genética , Candida albicans/metabolismo , Cromossomos Fúngicos , DNA Fúngico , Ploidias , Saccharomyces cerevisiae/genética , Sorbose/metabolismo , Especificidade da EspécieRESUMO
Candida dubliniensis and Candida albicans, the most common human fungal pathogen, have most of the same genes and high sequence similarity, but C. dubliniensis is less virulent. C. albicans causes both mucosal and hematogenously disseminated disease, C. dubliniensis mostly mucosal infections. Pulse-field electrophoresis, genomic restriction enzyme digests, Southern blotting, and the emerging sequence from the Wellcome Trust Sanger Institute were used to determine the karyotype of C. dubliniensis type strain CD36. Three chromosomes have two intact homologues. A translocation in the rDNA repeat on chromosome R exchanges telomere-proximal regions of R and chromosome 5. Translocations involving the remaining chromosomes occur at the Major Repeat Sequence. CD36 lacks an MRS on chromosome R but has one on 3. Of six other C. dubliniensis strains, no two had the same electrophoretic karyotype. Despite extensive chromosome rearrangements, karyotypic differences between C. dubliniensis and C. albicans are unlikely to affect gene expression. Karyotypic instability may account for the diminished pathogenicity of C. dubliniensis.
Assuntos
Candida albicans/genética , Candida/genética , Aberrações Cromossômicas , Cromossomos Fúngicos/genética , Southern Blotting , Antígenos CD36/genética , Candida/classificação , Candida/patogenicidade , Candida albicans/classificação , Candida albicans/patogenicidade , DNA Fúngico/genética , DNA Ribossômico/genética , Eletroforese em Gel de Campo Pulsado , Cariotipagem/métodos , Técnicas de Tipagem Micológica , VirulênciaRESUMO
By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.
Assuntos
Candida albicans/genética , Mapeamento Cromossômico , Genes Fúngicos , Clonagem Molecular , Eletroforese , CariotipagemRESUMO
The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans.
Assuntos
Candida albicans/genética , Cromossomos Fúngicos , Genoma Fúngico , Mapeamento Físico do Cromossomo , Saccharomyces cerevisiae/genética , Sintenia , Sequência de Aminoácidos , Pareamento de Bases , Sequência de Bases , DNA Fúngico , Ligação Genética , Íntrons , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta , Análise de Sequência de DNARESUMO
As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.
Assuntos
Candida albicans/genética , Cromossomos Fúngicos/genética , Centrômero/genética , Mapeamento Cromossômico , Biblioteca Gênica , Genes Fúngicos , Marcadores Genéticos , Genoma Fúngico , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
The DNA sequence of a Candida albicans genomic fragment known to complement the arginine mutation designated arg57 in strain 1006 contains an ORF of 1404 nucleotides (nt) predicting a protein of 468 amino acids (aa). Database searches indicated that the deduced protein shares 75% identity and 85% similarity with the ARG4 protein of Saccharomyces cerevisiae. Analysis of the percent aa identity between C. albicans and S. cerevisiae sequences included in available databases suggested these values are within the range expected for biosynthetic enzymes from the two organisms which share similar function. Experiments to isolate C. albicans ARG4 by complementation in an arg4 strain of S. cerevisiae yielded a plasmid (pARG4-1) with a restriction map identical to that of the sequenced clone. From these data, we conclude that the gene previously designated ARG57 is in fact ARG4 encoding the enzyme argininosuccinate lyase (ASL). These results were unexpected, since ARG57 had been localized to chromosome 7, while a mutation causing an ASL deficiency had been linked to ade1, which is on chromosome R. Transformation of C. albicans strains with pARG4-1 indicated it complemented the arginine auxotrophy in strains TMSU221 and 1435, a derivative of 1006. Examination of commonly utilized C. albicans arginine auxotrophs by spheroplast fusion analysis indicated these strains comprise two complementation groups: one consisting of 1006 and TMSU221, which are arg4, and the other of A642, hOG318, hOG357, FC18-6 and WC-5-4, which possess an undefined defect in the arginine biosynthetic pathway which we designate arg100.
Assuntos
Argininossuccinato Liase/genética , Candida albicans/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/enzimologia , Mapeamento Cromossômico , Cromossomos Fúngicos , Clonagem Molecular , Sequência Consenso , Teste de Complementação Genética , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
We report the molecular cloning, nucleotide (nt) sequence and chromosomal assignment of the Saccharomyces cerevisiae gene GLP1. This gene encoded a 15-kDa protein that was synthesized at a low level during growth on glucose and was induced ninefold upon glucose deprivation. When glucose withdrawal was accompanied by the addition of fatty acids the induction was enhanced an additional two- to threefold. The GLP1 gene product was identified as a soluble protein and purified using a combination of gel permeation and ion exchange chromatography. Using oligodeoxyribonucleotides as hybridization probes we have isolated the GLP1 gene and sequenced the single, long open reading frame which is 351 nt in length and is not interrupted by introns. The GLP1 gene directed the transcription of a 700-nt mRNA in response to glucose deprivation. The accumulation of the mRNA was further enhanced twofold by the addition of oleate. We have localized the GLP1 gene to S. cerevisiae chromosome VI.
Assuntos
Proteínas de Caenorhabditis elegans , Ácidos Graxos/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Glicoproteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Clonagem Molecular , Biblioteca Gênica , Genes Fúngicos , Íntrons , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Receptores Notch , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Epidemiologic studies in women with recurrent Candida vaginitis have been hampered in the past by the lack of a reproducible typing system. Several molecular probes have now been developed that have the ability to differentiate strains of Candida albicans and give reproducible results. In this investigation, 24 women with Candida vaginitis were studied in a longitudinal fashion for 30 days following short-course antifungal therapy. Seven women with either recurrent vaginitis or with multiple culture-positive sites with C. albicans were included in an epidemiological study. A total of 18 isolates of C. albicans (12 vaginal and six rectal) were typed utilizing restriction fragment length polymorphisms of rDNA. This technique was able to differentiate five different strains of C. albicans. Our epidemiologic study revealed that vaginal and rectal strains recovered from the same women were usually different. None of our patients had a similar vaginal and rectal strain prior to treatment, and only one patient had the same strain isolated from both the rectum and the vagina at the time of recurrence. On the other hand, we found that the same strain of C. albicans was initially and later recovered from the vagina in four of five women who failed treatment or developed recurrent vaginitis. These results suggest that recurrent episodes of C. albicans vaginitis, following short-course antifungal therapy, are often due to relapse of the original infecting strain and not due to autoinoculation from the rectum.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Candida albicans/classificação , Candidíase Vulvovaginal/microbiologia , DNA Fúngico/análise , DNA Ribossômico/análise , Polimorfismo de Fragmento de Restrição , Candida albicans/genética , Candidíase Vulvovaginal/tratamento farmacológico , Candidíase Vulvovaginal/epidemiologia , Doença Crônica , Clotrimazol/uso terapêutico , Sondas de DNA , Eletroforese em Gel de Ágar , Feminino , Humanos , Hibridização de Ácido Nucleico , Recidiva , Reprodutibilidade dos Testes , Mapeamento por Restrição , ComprimidosRESUMO
We have cloned the first glucose transporter CaHGT1 (Candida albicans high-affinity glucose transporter) of a pathogenic yeast, Candida albicans. The DNA sequence (GenBank accession number Y16834) analysis revealed an ORF encoding a novel protein of 545 amino acids with a predicted molecular mass of 60.67 kDa. The putative protein with 12 transmembrane domains has 51% identity with Kluyveromyces lactis high-affinity glucose transporter, HGT1. The protein signatures which are conserved and distinctive of the sugar transporters belonging to the major facilitator superfamily (MFS) were also found in CaHgt1p. When heterologously expressed, the ORF functionally complemented a mutant strain of Saccharomyces cerevisiae RE700A which was deleted in seven hexose transporter genes and thus was unable to grow or transport glucose. The expression of CaHGT1 in C. albicans showed a transcript of 1.6 kb which was enhanced in response to the human steroid hormone progesterone. Interestingly, the transcript levels were also enhanced in the presence of drugs, e.g. cycloheximide, chloramphenicol and benomyl. The results suggest that CaHGT1, which encodes a MFS protein, could be linked to the drug resistance phenomenon in C. albicans.
Assuntos
Candida albicans/genética , Clonagem Molecular , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Candida albicans/metabolismo , DNA Fúngico/análise , Proteínas Fúngicas , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/química , Reação em Cadeia da Polimerase/métodos , Progesterona/farmacologia , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNAAssuntos
Cromatina , DNA , Sítios de Ligação , Linhagem Celular , Cromatina/ultraestrutura , Etídio , Cinética , Conformação de Ácido NucleicoRESUMO
BACKGROUND: The 10.9x genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined. RESULTS: Supercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome. CONCLUSION: Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.
Assuntos
Candida albicans/genética , Cromossomos Fúngicos/química , Genoma Fúngico , Sequência de Aminoácidos , Centrômero/química , Mapeamento de Sequências Contíguas , Dados de Sequência Molecular , Fases de Leitura Aberta , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Sintenia , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genéticaRESUMO
Candida albicans is the most common fungal pathogen of humans. The recent discovery of sexuality in this organism has led to the demonstration of a mating type locus which is usually heterozygous, although some isolates are homozygous. Tetraploids can be formed between homozygotes of the opposite mating type. However, the role of the mating process and tetraploid formation in virulence has not been investigated. We describe here experiments using a murine model of disseminated candidiasis which demonstrate that in three strains, including CAI-4, the most commonly used strain background, tetraploids are less virulent than diploids and can undergo changes in ploidy during infection. In contrast to reports with other strains, we find that MTL homozygotes are almost as virulent as the heterozygotes. These results show that the level of ploidy in Candida albicans can affect virulence, but the mating type configuration does not necessarily do so.
Assuntos
Candida albicans/genética , Candida albicans/patogenicidade , Genes Fúngicos Tipo Acasalamento/genética , Genes Fúngicos Tipo Acasalamento/fisiologia , Ploidias , Animais , Candida albicans/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Poliploidia , Virulência/genéticaRESUMO
The electrophoretic karyotypes of five Candida albicans isolates and of five other Candida species have been determined, using orthogonal field alternating gel electrophoresis (OFAGE). None of the C. albicans isolates had the same electrophoretic karyotype. By comparing all five strains, we arrived at a chromosome number of nine to ten, but since the organism is diploid, we cannot distinguish genetically different chromosomes from homologues which resolve. We determined minimal chromosome numbers of 9 for Candida stellatoidea, 10 for C. glabrata and 6 for C. guilliermondii.
Assuntos
Candida/genética , Cromossomos/classificação , Candida albicans/genética , DNA Fúngico/classificação , Eletroforese em Gel de Ágar/métodos , Cariotipagem , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMO
Candida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.
Assuntos
Aneuploidia , Candida albicans/genética , Candida albicans/citologia , Mapeamento Cromossômico , Rearranjo Gênico , Genoma Fúngico , Morfogênese/genética , Mutação , Fenótipo , Recombinação Genética , Translocação GenéticaRESUMO
This paper reports that the opaque and white phenotypes of Candida albicans constitute a true high-frequency reversible transition system. The rDNA restriction fragment and orthogonal field alternating gel electrophoresis profiles of opaque and white phenotypes are indistinguishable, and a genetic marker introduced into a white strain is present in all opaque derivatives of this strain. Opaque and white derivatives appear markedly different on a bismuth indicator medium and differ in a number of other respects. We have used bismuth medium to examine the spontaneous and temperature-induced frequencies of transition from opaque to white. The temperature-induced transition from opaque to white does not occur when opaque cells are held in water.
Assuntos
Candida albicans/citologia , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Meios de Cultura , Enzimas de Restrição do DNA , DNA Fúngico/genética , DNA Ribossômico/genética , Marcadores Genéticos , Cariotipagem , Mutação , Fenótipo , TemperaturaRESUMO
Chromosomes from Cryptococcus neoformans, an encapsulated yeast pathogen, were separated by contour-clamped homogeneous field gel electrophoresis. Seven strains representing all four serotypes were studied. It was found that each strain had a unique, reproducible pattern of chromosome bands which could potentially be used for strain polymorphism studies. There were between 10 and 12 chromosomes in the strains studied, with an approximate genomic size of 15,000 to 17,000 kilobases. Chromosome separation also could be used to assign locations for cloned genes, and the ribosomal DNA genes were found on one of the larger C. neoformans chromosomes. The technique of electrophoretic karyotyping should be helpful for genetic and molecular investigations into the biology of C. neoformans.
Assuntos
Cromossomos , Cryptococcus neoformans/genética , Cryptococcus/genética , DNA Fúngico/isolamento & purificação , Candida albicans/genética , Mapeamento Cromossômico/métodos , Eletroforese em Gel de Ágar/métodos , Genes Fúngicos , Cariotipagem , Saccharomyces cerevisiae/genéticaRESUMO
Candida albicans and Candida stellatoidea are two closely related imperfect yeasts. Some isolates characterized as C. stellatoidea are in fact C. albicans, while others differ with respect to virulence and to karyotype, containing extra small chromosomes. Experiments in this study allowed us to infer that a typical C. stellatoidea isolate, Y2360, has 12 chromosomes rather than the 7 previously shown for C. albicans. The majority of cloned sequences tested hybridized to analogous chromosomes in C. albicans and in C. stellatoidea, although there were exceptions, and a repeated element isolated as specific for C. albicans hybridized to most of the chromosomes of C. stellatoidea. Several genes tested hybridized to one of the smaller, C. stellatoidea-specific chromosomes as well as to a larger one. The arrangement of restriction enzyme sites around the gene was the same in both the large and small chromosomes. For ADE2 and LYS2, the arrangements were identical to those of a typical C. albicans strain, FC18, suggesting a high degree of sequence conservation between the two species. Spheroplast fusion and segregation experiments showed that the ADE2 genes on both the large and small chromosomes of C. stellatoidea are active, implying that the organism is functionally at least triploid for this gene and probably for any others duplicated on the smaller chromosomes.
Assuntos
Candida/genética , Cromossomos Fúngicos , Genes Fúngicos , Família Multigênica , Candida albicans/genética , Mapeamento Cromossômico , Polimorfismo de Fragmento de RestriçãoRESUMO
The binding of ethidium bromide, as monitored by fluorescence enhancement, to chromatin prepared by nuclease digestion has been compared with the binding of the dye to sheared chromatin. The nuclease preparation (native chromatin) is characterized by a high affinity region of the Scatchard plot (r = 0-0.025, K1 = 1 X 10(6) M-1), a transition (r = 0.025-0.05), and a low affinity region (r = 0.05-0.12, K2 = 3 X 10(5) M-1). The final amount of ethidium bromide bound per base is 0.12 as compared with 0.20 for free DNA. Sheared chromatin has the two regions of high and low affinity (K1 = 2 X 10(6) M-1, K2 = 5 X 10(5) M-1) as originally shown by Angerer and Moudrianakis (1972), but the transition is much reduced or absent. Binding of the dye to native chromatin is independent of salt at concentrations ranging from 0.2 mM EDTA to 10 mM Tris-Cl, 10 mM NaCl, 0.2 mM EDTA, while sheared chromatin and DNA both bind ethidium bromide electrostatically as well as by intercalation at the low salt concentration, leading to extensive energy transfer. Thus the phosphate groups in native chromatin are unavailable to external cations even at very low salt. Polarization of fluorescence of ethidium bromide intercalated into native chromatin at low r is very high, indicating a highly rigid structure. As r approaches 0.02, there is a very rapid depolarization; at r = 0.03, the polarization is no greater than that of the dye intercalated into DNA. Depolarization is not due to energy transfer. The Scatchard plot derived for the bulk preparation of native chromatin is very similar to the one derived for the monomer nu body. These results indicate that the DNA in native chromatin is in a very rigid form, with its phosphate anions neutralized by structural components, not by free salt. Ethidium bromide intercalation appears partially to disrupt this structure, perhaps by unwinding, leading to slight changes in its properties.
Assuntos
Cromatina/ultraestrutura , Etídio , Sítios de Ligação , Linhagem Celular , Cromatina/metabolismo , DNA/metabolismo , Etídio/metabolismo , Cinética , Desnaturação de Ácido NucleicoRESUMO
When chromatin prepared from WI-L2 lymphocytes by low salt extraction and shearing is centrifuged on a glycerol gradient, one area of the gradient yields chromatin enriched in template activity for Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) as compared to Saccharomyces cerevisiae RNA polymerase II (or B). Another area yields chromatin preferred by the eukaryotic enzyme. Kinetic studies indicate that the differences in activity cannot be explained by differences in affinity of the enzymes for the various templates. The DNA isolated from either fraction has a molecular weight of 8.5 X 106. The "yeast active" fraction seems enriched in proteins. Mixing experiments indicate that the yeast enzyme does not alter the template in such a way as to improve it for the bacterial enzyme.
Assuntos
Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Linfócitos/metabolismo , Linhagem Celular , Escherichia coli/enzimologia , Cinética , Saccharomyces cerevisiae/enzimologia , Espectrofotometria Ultravioleta , Moldes Genéticos , Transcrição GênicaRESUMO
The opportunistic fungal pathogen, Candida albicans, is diploid as usually isolated and has no apparent sexual cycle. Genetic analysis has therefore been very difficult. Molecular genetics has yielded important information in the past few years, but it too is hampered by the lack of a good genetic map. Using the well-characterized strain 1006 and strain WO-1, which undergoes the white-opaque phenotypic transition, we have developed a genomic restriction map of C. albicans with the enzyme SfiI. There are approximately 34 SfiI restriction sites in the C. albicans genome. Restriction fragments were separated by pulsed-field electrophoresis and were assigned to chromosomes by hybridization of complete and partial digests with known chromosome-specific probes as well as by digestion of isolated chromosomes. Telomeric fragments were identified by hybridization with a telomere-specific probe (C. Sadhu, M.J. McEachern, E.P. Rustchenko-Bulgac, J. Schmid, D.R. Soll, and J.B. Hicks, J. Bacteriol. 173:842-850, 1991). WO-1 differs from 1006 in that it has undergone three reciprocal chromosomal translocations. Analysis of the translocation products indicates that each translocation has occurred at or near an SfiI site; thus, the SfiI fragments from the two strains are similar or identical. The tendency for translocation to occur at or near SfiI sites may be related to the repeated sequence RPS 1, which contains four such sites and could provide homology for ectopic pairing and crossing over. The genome size of both strains is about 16 to 17 megabases, in good agreement with previous determinations.