Chromosome-sized DNA Molecules Research Articles

Pulsed-Field Gel Electrophoresis: A Versatilef Tool or Analysis of Fungal Genomes

The separation of chromosome-size DNA molecules by pulsed-field gel electrophoresis (PFGE) has become a well-established technique in recent years. Although it has very wide-ranging applications, it made a real breakthrough for fungal genome analysis. Because of the small size of fungal chromosomes, their investigation was not possible earlier. Different PFGE approaches allowed the separation of DNA molecules larger than 10 megabase pairs in size, and electrophoretic karyotypes for numerous previously genetically uncharacterized fungal species could be established. This review discusses the applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation.

Electrophoretic karyotype of two Micromucor species.

Electrophoretic karyotype analysis was applied to obtain information on the organisation and intrageneric variability of the nuclear genome in three Micromucor isolates of two different species (M. isabellina and M. ramanniana). A protoplast formation protocol, conditions for the preparation of highly-intact chromosome-size DNA molecules and for the separation of DNA molecules were established. The chromosomal banding patterns revealed substantial variability among the isolates: 11 to 14 chromosomal mobility groups were resolved. The DNA in the Micromucor chromosomes were rather small; their estimated sizes were calculated to be between 2.60 and 0.4 Mb. Using Saccharomyces cerevisiae and Schizosaccharomyces pombe as size standard, the minimum total genome sizes were estimated to be between 24.19 and 24.9 Mb.

A Simple and Rapid PCR-based Method to Isolate Complete Small Macronuclear Minichromosomes from Hypotrich Ciliates: 5S rDNA and S26 Ribosomal Protein Gene of Oxytricha (Sterkiella) nova

Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This "End-End-PCR" method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.

Variation in the electrophoretic karyotype of Brazilian strains of Metarhizium anisopliae

Pulsed-field gel electrophoresis (PFGE) was used to separate chromosome-sized DNA molecules of four strains of Metarhizium anisopliae from Brazil. Metarhizium anisopliae isolates from Japan have been reported as possessing seven chromosomes. Variation was observed among the Brazilian strains and the chromosomal DNA was resolved into eight bands for strain CG46. Densitometric analysis of PFGE gels suggested that the other three Brazilian strains also possess eight chromosomes, with two chromosomes migrating as doublets under the electrophoretic conditions used. The genome size was estimated as varying between 23.39 to 31.88 Mb, not including possible doublet chromosomes.

Electrophoretic karyotypes and genome sizing of the pathogenic fungus Paracoccidioides brasiliensis.

Here we present the karyotype analysis and genome sizing of Paracoccidioides brasiliensis, a pathogen refractory to conventional genetic analysis. We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates of P. brasiliensis. Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp. Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found. Different technical approaches were employed to estimate the DNA content and to define the ploidy of P. brasiliensis. An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4',6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy). The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separated P. brasiliensis chromosomal band is diploid.

Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule.

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning, sequencing and chromosomal localization of its TRP1 gene.

The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF). The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules. The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae. The chromosomal bands were estimated to be within the range 0.7-2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb. The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E. coli. The DNA sequence was determined (EMBL accession No. Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5'-phosphoribosyl) anthranilate isomerase (PRAI) activity. The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified.

Molecular typing of Candida albicans in oral candidiasis: karyotype epidemiology with human immunodeficiency virus-seropositive patients in comparison with that with healthy carriers

Candida albicans organisms isolated from the oral cavities of healthy carriers (26 individuals) and compromised hosts (40 human immunodeficiency virus [HIV]-seropositive patients, all showing symptomatic oral candidiasis) were compared by resolving chromosome-sized DNA molecules into electrophoretic karyotypes. Seven- to 10-band electrophoretic patterns were obtained, with significant and reproducible differences in the distributions of the DNA bands. Seven distinct classes were identified and were designated type a (8 bands), type b (8 bands), type c (7 bands), type d (9 bands), type x (10 bands), type y (10 bands), and type z (9 bands). Four of these (types a to d) were the most representative within all of the isolated strains (95.5%), and the other three (types x to z) were observed only once in three HIV-seropositive individuals (4.5%). Only types b and c were isolated from healthy carriers, with the percentage of their isolation being 61.5 and 38.5%, respectively, while all the described karyotypes were isolated from HIV-seropositive patients, with type b being the most frequent (45%); this was followed by types c (25%), a (15%), and d (7.5%). The prevalence of type b and c karyotypes in HIV-infected individuals, as well as in healthy carriers, suggests that commensal strains in the oral cavities of healthy individuals may become the prevalent agents of subsequent oral candidiasis in compromised hosts. However, replacement of the original, commensal strain, if there is one, cannot be excluded in a compromised host, although strain replacement may be more reasonably hypothesized for types a and d, since only these types were isolated at a relative high percentage from the oral lesions of HIV-infected individuals.

Electrophoretic Karyotyping of Fusarium oxysporum.

Chromosome-sized DNAs of Fusarium species were prepared for pulsed-field gel electrophoresis without first generating protoplasts. The technique involves pre-treatment of intact, agarose-solidified microconidia with endo-β-1, 3-glucanase before treatment with protease in the presence of EDTA and SDS. By using four different electrophoretic conditions for the separation of four size ranges of chromosomes, we have succeeded in separating the chromosomes of 6 formae speciales (lycopersici, radicislycopersici, melonis, cucumerinum, fragariae and spinaciae) or Fusarium oxysporum in an agarose gel matrix by crossed field gel electrophoresis. The total number of chromosome-sized DNA molecules of these fungi varied from 9 to 15, depending on the formae speciales and, in some case, individual strains. The sizes of chromosomes ranged from 0.8 to approximately 7.4Mb which gave estimates of genome size of between 36.0 and 56.3Mb.

Occurrence of chromosome rearrangements during the fusion process in the imperfect yeast Candida albicans

Auxotrophic derivatives of three strains of the pathogenic yeast Candida albicans of different origins, including 1006 derived from CBS5736, A5153 derived from FC18 and NARA2 derived from NUM961, were used in spheroplast fusion experiments. The DNA content of the prototrophic fusion product obtained following fusion between strains 1006 and A5153 approximated to the sum of those of the parents, but was variable when NARA2 was used as the parent for fusion. Chromosome-sized DNA molecules of the fusion derivatives were separated by pulsed-field gel electrophoresis to examine whether either or both of the chromosome-sized DNA molecules of each parent were transferred into the fusion derivatives. In the fusion derivatives obtained following fusion between strains 1006 and A5153, nearly the full complement of chromosomes was shown to be transferred, but partial transfer of chromosomes occurred in the fusion derivatives that were obtained following fusion between strains NARA2 and A5153. Results indicated that chromosome loss also occurred when these two strains were fused. Variations in the size of R chromosomes, the rDNA-containing chromosomes, were observed in all fusion derivatives tested, indicating high-frequency recombination between R chromosomes during the fusion process.

Electrophoretic karyotyping is a sensitive epidemiological tool for studying Trypanosoma evansi infections

Thirty-six isolates of trypanozoon trypanosomes collected from camels in Northern Kenya during the dry season sporadic infections of 1986 and during the wet season epidemic infections of 1987 were identified as Trypanosoma evansi by the homogeneity of their kinetoplast DNA minicircles. Although the minicircles of all the isolates were indistinguishable, polymorphism in chromosome-sized DNA molecules detected by electrophoresis was extensive. The isolates could be grouped into eight distinct electrophoretic karyotypes which could be distinguished from three additional karyotypes identified among earlier T. evansi isolates. In one camel herd with a long history of trypanocide application, which was continued during the present study, all isolates bar one belonged to one karyotype group. From a second herd, in which trypanosomosis management was by individual treatment of proven parasitaemic cases, isolates with diverse karyotypes were obtained. Some of the karyotypes identified during the dry season sporadic infections were re-isolated in the subsequent wet season epidemic. These observations indicate that distinguishing T. evansi isolates by molecular electrophoretic karyotypes is more discriminating than kDNA analysis. Observations of karyotype patterns recurring in isolates from herds kept under chemoprophylaxis could help in the identification of drug-resistant parasites.

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella.

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.

Pulsed-Field Gel Electrophoresis of DNA from Four Microsporidian Isolates

Pulsed-field gel electrophoresis is used to visualize chromosome-sized DNA molecules of four microsporidian isolates from pest insects. Vavraia oncoperae isolated from Wiseana spp. caterpillars had 16 resolvable bands and the same species isolated from grass grubs had 14 resolvable bands. Chromosomal bands were more difficult to resolve for Nosema costelytrae and a Vairimorpha species from cabbage white butterflies, but electrophoresis revealed that these species had a minimum of 8 bands. The microsporidian DNA molecules resolved by this method are estimated to be between 130 and 1930 kilobases in size.

Investigation of human giardiasis by karyotype analysis.

The patterns of transmission of Giardia lamblia and the potential contribution of strain differences to pathogenicity of infection is poorly understood. We used pulsed field gradient gel electrophoresis (PFGE) to separate chromosome-sized DNA molecules of 22 stocks of G. lamblia isolated from 13 individuals (6 symptomatic, 7 asymptomatic) living in Jerusalem. PGFE gels run under a variety of conditions revealed up to nine ethidium bromide-stained bands per isolate ranging in size from 0.7 to greater than 3 megabasepairs. Relative staining intensities indicated that some bands contained multiple chromosomes. Major differences in the number, size, and intensity of bands allowed a clear differentiation of the karyotypes of isolates from each of the different individuals. This is in contrast to previous studies where the karyotype of different isolates have been strikingly homogeneous. Hybridization of Southern blots with surface antigen, beta-tubulin, and ribosomal RNA genes revealed that these gene families were distributed to different sized chromosomes amongst the different isolates. PFGE thus revealed major differences in the karyotypes of different G. lamblia isolates that were obtained over a short period of time from a relatively confined geographic area. In contrast, karyotypes of isolates established either by direct cultivation of duodenal trophozoites or by excystation of stool cysts from the same individuals were almost identical. Also, isolates from the same individuals obtained over a prolonged period of time revealed only minor differences in their karyotype, suggesting that recurrent infection can be caused by genetically similar organisms. We conclude that chronic giardiasis can result from recurrence of occult infection or reinfection from a common source.

Rapid estimation of chromosomal damage in yeast due to the effects of environmental chemicals using pulsed field gel electrophoresis

We present a procedure to rapidly estimate the damage to yeast chromosomes by toxic chemicals. This procedure employs the following steps: incubation of yeast cells with the chemicals, DNA preparation in an agarose matrix, separation of chromosome-sized DNA molecules into reproducible band patterns by pulsed field gel electrophoresis, and quantification of the intensity of chromosomal bands by densitometry. Saccharomyces cerevisiae cells have been treated with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and cis-Platinum(II) diamminedichloride (cisPT), both of which are known to interact with DNA, and trichlorethylen (TCE), for which such an effect has not been shown in yeast. Treatment of cells with MNNG and cisPt led to an impairment of the intensity of the band pattern to an extent dependent on the concentration of the chemicals applied. For TCE a similar effect could not be discerned. This procedure will be useful as a screening test for the estimation of the biological hazards of toxic chemicals.

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva)

Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.

The repair of double‐strand breaks and S1 nuclease‐sensitive sites can be monitored chromosome‐specifically in Saccharomyces cerevisiae using pulsed‐field gel electrophoresis

Repair under non-growth conditions of DNA double-stranded breaks (DSBs) and S1 nuclease-sensitive sites (SSSs; e.g. DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by [60Co]-gamma-rays (200 Gy; anoxic conditions) was monitored in a diploid repair-competent strain of Saccharomyces cerevisiae. We used pulsed-field gel electrophoresis (PFGE), which allows the separation of chromosome-sized yeast DNA molecules, to determine the number of DSBs and SSSs in individual chromosome species of yeast. Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair-proficient diploid strain of yeast. The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism. Both types of repair kinetics are different for different chromosome species.

Electrophoretic karyotype for Dictyostelium discoideum.

This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.

Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.

We have identified a family of dispersed repetitive DNA sequences in the genome of Magnaporthe grisea, the fungus that causes rice blast disease. We have named this family of DNA sequences "MGR" for M. grisea repeat. Analysis of five MGR clones demonstrates that MGR sequences are highly polymorphic. The segregation of MGR sequences in genetic crosses and hybridization of MGR probes to separated, chromosome-size DNA molecules of M. grisea shows that this family of sequences is distributed among the M. grisea chromosomes. MGR sequences also hybridize to discrete poly(A)+ RNAs. Southern blot analysis using a MGR probe can distinguish rice pathogens from various sources. However, MGR sequences are not highly conserved in the genomes of M. grisea field isolates that do not infect rice. These results suggest that host selection for a specific pathogen genotype has occurred during the breeding and cultivation of rice.