Estimation of chromosome number and size by pulsed-field gel electrophoresis (PFGE) in medically important Candida species.

The chromosomal DNAs of eight medically important Candida species, C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata, were analysed by pulsed-field gel electrophoresis under various conditions. The corresponding bands in the gels were assigned by three kinds of DNA probe which hybridized to DNA of all the species: rDNA, TUB2 and PEP4. The best conditions for separating the chromosomal DNAs were investigated and the numbers and molecular sizes of the chromosome bands were determined for each species. The chromosomal DNAs of the species were separated into 5-14 bands ranging in size from 0.5 to 4.5 Mb. Based on the quantification of the chromosome band intensities using a laser fluorescent gel scanner, the chromosome numbers were estimated. The apparent average total number of chromosomes per cell was 16 for C. albicans, 16 for C. stellatoidea, 12 for C. tropicalis, 14 for C. parapsilosis, 8 for C. krusei, 8 for C. guilliermondii, 18 for C.kefyr, and 14 for C. glabrata; the total chromosomal DNA size of each species per cell was calculated at about 31 Mb, 33 Mb, 31 Mb, 26 Mb, 20 Mb, 12 Mb, 29 Mb and 14 Mb, respectively.     

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

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.     

Electrophoretic karyotypes and chromosome numbers in Candida species

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.     

Clonal variation of chromosome size derived from the rDNA cluster region in Candida albicans.

Of the eight Candida albicans chromosomes, chromosome 2, assigned by the MGL1 probe, is more variable in size than the other chromosomes among strains. We found that the clonal variation of chromosome 2, which carries a rDNA gene, occurred at a frequency of up to 10% of the progeny clones. After total chromosomal digestion with XhoI, which has no recognition sites within the rDNA repeat unit, the fragments containing the rDNA cluster were detected by Southern hybridization. The difference in fragment sizes corresponded to the clonal size variation of chromosome 2. The intensity of hybridization with rDNA also correlated with the difference in size. In addition, there was no size change in the non-rDNA region as detected by NotI digestion of chromosome 2, and there was no observed change in the individual rDNA basic repeat unit size. From these lines of evidence, we confirmed that the clonal size variation of chromosome 2 which occurs at high frequency is derived from the size change of the rDNA cluster.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

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.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

Extensive chromosome rearrangements distinguish the karyotype of the hypovirulent species Candida dubliniensis from the virulent Candida albicans

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.     

Chromosomes of Blastocystis hominis

Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to> 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.     

Assignment of cloned genes to electrophoretically separated chromosomes of the yeast Pachysolen tannophilus

An electrophoretic karyotype of Pachysolen tannophilus has been obtained using Pulsed Field Gel Electrophoresis. Seven chromosomal bands were separated with one of the bands migrating probably as a doublet. The sizes of the chromosomes were estimated to be between 1 and 3.1 megabase pairs. Eleven loci have been assigned to chromosomal bands, including four involved in the metabolism of D-xylose.     

Electrophoretic karyotypes of some species of the genus Leptomonas (kinetoplastida, trypanosomatidae), homoxenous trypanosomatids parasites of insects

Electrophoretic karyotypes of homoxenous trypanosomatids Leptomonas peterhoffi, L. mycophilus, L. nabiculae and Leptomonas sp. have been studied by transverse alternating-field electrophoresis under varying electrophoretic conditions. From 12 to 17 chromosomal DNA bands, ranging from 370 to more than 1500 kb were detected in the karyograms of the species compared. In each pattern, some intensely stained bands could represent more than one chromosome. Taking into account the number of intensely stained bands, the karyotype of L. peterhoffi was estimated to contain at least 18 chromosomes, the karyotypes of L. mycophilus and L. nabiculae, at least 21 chromosome each, and the karyotype of Leptomonas sp. up to 20 chromosomes. Interclonal variations of electrophoretic karyotypes of 10 clones of Leptomonas sp. (cfmI-cfmX) were studied. Seven of ten clones had identical electrophoretic patterns. In the karyograms of three clones (cfmI, cfmVI, cfmVII), additional chromosomal DNA bands were observed. The obtained results suggest, that electrophoretic karyotypes cannot be used as reliable markers of species of homoxenous trypanosomatids, since intraspecies variability does occur in these parasites.     

Construction of an SfiI macrorestriction map of the Candida albicans genome.

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.     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

克鲁斯假丝酵母及其近似种的脉冲电泳核型分析

用钳位均匀电场脉冲电泳(CHEF)系统分析了克鲁斯假丝酵母(Candida krusei),郎比可假丝酵母(C. lambica)和粗状假丝酵母(C. valiad)的模式菌株的电泳核型,发现这三种表型相似的假丝酵母却具有互不相同的染色体DNA分子带型,为其分类学研究提供了可靠的鉴别依据。在常规分类学研究的基础上,测定了AS 2.75(原定种名为(C. incospicua),AS2.1182(原定种名为 C. lambica)和AS 2.1772(未定种)等三株假丝酵母的G+C含量和脉冲电泳核型。通过对已报道的C. inconspicu的G+C含量及上述三种假丝酵母模式菌株的脉冲电泳核型的比较分析证明,AS 2.75和AS 2.1772为粗状假丝酵母(C. valida),AS 2.1182为克鲁斯假丝酵母(C. krusei)。     

An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.     

Genetic characterization of the yeast Pichia anomala (strain K), an antagonist of postharvest diseases of apple

AIMS: To obtain information about the genomic organization of Pichia anomala (strain K) and about its genomic diversity at species and intraspecies level. METHODS AND RESULTS: The PFGE karyotype of strain K was composed of four bands ranging in size from 1.1 to 3.2 Mb. The number of chromosomes was estimated at six since bands 2 and 3 seemed to result from the comigration of two chromosomes with similar size. A comparison of strain K and Hansenulawingeii migration profiles led to the estimate of K strain genome size at 11.7 Mb. Comparison with isogenic strains, resulting from the sporulation of strain K, highlighted some major karyotypic differences. Two segregants (KH6 and KH7) showed supernumerary chromosomes and one (KH9) displayed chromosomal length polymorphism. This genomic instability was confirmed by molecular hybridization with four probes, consisting of URA3, LEU2, PAEXG1 and PAEXG2 genes of P. anomala. URA3 and LEU2 probes showed second hybridization signals on supernumerary chromosomes of strain KH7 and on chromosome 6 of strain K for LEU2 only. Karyotypic comparison of seven non-isogenic P. anomala strains revealed chromosomal length polymorphism, a sign of intraspecies variation. CONCLUSIONS: This work has supplied information about genome size and chromosome number of strain K of P. anomala. The strain seems to be aneuploid because of the presence of supernumerary chromosomes and additional hybridization signals for URA3 and LEU2 probes in the chromosomal profile of some segregants. The work also highlighted genomic diversity within the P. anomala species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained here increase information about the aneuploidy of P. anomala (strain K). Information about the genomic diversity of the segregants will be of great interest for further studies on strain K mode of action. The genome size and chromosomal profile of P. anomala presented here are different from the results obtained elsewhere for Hansenula anomala, while Hansenula is included as a synonym of Pichia. This warrants further studies to investigate this taxonomic relationship.     

Chromosomal localisation of five genes in Perkinsus olseni (Phylum Perkinsozoa)

The molecular karyotype of Perkinsus olseni, a pathogenic protist that infects the clam Ruditapes decussatus, comprises nine chromosomes, ranging in size from 0.15 Mb to 6.5 Mb, representing a haploid genome of about 28 Mb. In order to establish chromosome specific markers, PCR-amplified DNA sequences belonging to five conserved genes (18S rRNA, actin type I, hsp90, β-tubulin and calmodulin) were hybridised to chromosomal bands separated by pulsed-field gel electrophoresis. Three of those probes (actin type I, hsp90 and calmodulin) hybridised to only one chromosome and the remaining two (18S rRNA and β-tubulin) hybridised to two chromosomes. In the first place, the hybridisation pattern obtained serves to dispel any doubt about the nuclear location of the smallest chromosome observed in the molecular karyotype of Perkinsus olseni. Additionally, it will be a reference for further analysis of karyotype polymorphisms in the genus Perkinsus.     

几种念珠菌DNA总含量的流式细胞分析

应用流式细胞术(FCM)对处于稳定生长阶段的念珠菌属(Candida)的7种8株念珠菌进行了DNA总含量的流式细胞(FCM)分析。这8株念珠菌是:白念珠菌(C.albicans)2株,热带念珠菌(C.tropicalis),克柔念珠菌(C.krusei),近平滑念珠菌(C.parapsiolosis),乳酒念珠菌(C.kefyr),白念珠菌星形变种(C.stellatoidea),即血清B型白念珠菌,季也蒙念珠菌(C.guilliermondii)各一株。应用EB一步插入法染色,用鸡红细胞(CRBC)作为内参标准进行DNA总含量测定。分析结果表明:稳定生长阶段的组方图上,大多数念珠菌细胞处于DNA合成周期的G_0/G_1期;DNA总含量有明显的种间和种内差异。     

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.