Chromosome Length Polymorphism Research Articles

Inheritance of chromosome-length polymorphisms in Ophiostoma ulmi (sensu lato).

We have investigated the mitotic and meiotic transmission of chromosome-length polymorphisms in Ophiostoma ulmi s.l., the causal agent of Dutch elm disease. The North-American aggressive (NAN) strain CESS16K has an atypical electrophoretic karyotype, carrying two chromosome-sized DNAs (chDNAs) that have not been observed in other members of the NAN biotype. Independent CESS16K chDNA preparations, even after repeated inoculation and recovery from the elm host, and analysis of 16 progeny strains after a cross between the NAN strains FG245Br-O and CESS16K, demonstrated that these unique chDNAs are integral components of the CESS16K genome. Analysis of the progeny, by electrophoretic karyotyping and hybridizations with probes specific to individual chDNAs, presented evidence that genome rearrangements can occur as a consequence of meiosis. Even though novel electrophoretic karyotypes and a novel-sized chromosome were observed in the karyotypes of the progeny strains, the low level of reassortment between the chromosomes carrying length polymorphisms presented evidence that there are constraints to genome plasticity for this fungus.

Inheritance of chromosome length polymorphisms in Coprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F1 progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F1 progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F1 progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F1 progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F1 progeny.

Chromosome-length polymorphism in fungi.

The examination of fungal chromosomes by pulsed-field gel electrophoresis has revealed that length polymorphism is widespread in both sexual and asexual species. This review summarizes characteristics of fungal chromosome-length polymorphism and possible mitotic and meiotic mechanisms of chromosome length change. Most fungal chromosome-length polymorphisms are currently uncharacterized with respect to content and origin. However, it is clear that long tandem repeats, such as tracts of rRNA genes, are frequently variable in length and that other chromosomal rearrangements are suppressed during normal mitotic growth. Dispensable chromosomes and dispensable chromosome regions, which have been well documented for some fungi, also contribute to the variability of the fungal karyotype. For sexual species, meiotic recombination increases the overall karyotypic variability in a population while suppressing genetic translocations. The range of karyotypes observed in fungi indicates that many karyotypic changes may be genetically neutral, at least under some conditions. In addition, new linkage combinations of genes may also be advantageous in allowing adaptation of fungi to new environments.

Characterization of PCR-ribotyping for Burkholderia (Pseudomonas) cepacia.

Ribotyping, a method of genotyping bacterial isolates for epidemiologic study, uses rRNA as a probe to detect chromosomal restriction fragment length polymorphisms. Although ribotyping is accurate, its utility is limited by the labor and time necessary for Southern blot analysis. PCR-ribotyping uses PCR to amplify the 16S-23S intergenic spacer region of the bacterial rRNA operon. Length heterogeneity in the spacer region has previously been found to be useful as an alternative to standard ribotyping in a study of Burkholderia (Pseudomonas) cepacia. To further analyze the accuracy of PCR-ribotyping, three groups of previously characterized isolates of B. cepacia were investigated. PCR-ribotyping grouped 90 isolates recovered from seven well-defined epidemics into the correct outbreak group with a mean concordance of 93%. Both standard ribotyping and PCR-ribotyping separated 15 unrelated isolates into 14 types. In an analysis of 83 B. cepacia isolates from chronically colonized cystic fibrosis patients, the concordance of PCR-ribotyping with standard ribotyping ranged from 83 to 100%, with a mean of 98%. One isolate from a chronically colonized patient had a different type by standard ribotyping but was identical to the other isolates from this patient by PCR-ribotyping. Thus, PCR-ribotyping is a rapid and accurate method for typing B. cepacia and is less labor intensive than standard ribotyping.

Inheritance of chromosome-length polymorphisms in Coprinus cinereus.

We have investigated the inheritance of chromosome-length polymorphisms in the basidiomycete Coprinus cinereus. The electrophoretic karyotypes of interfertile strains of C. cinereus are strikingly different, and crosses between strains with different karyotypes yield progeny with chromosomes of new sizes. Repeated backcrossing of a mutant to one parent often stabilizes the mutant chromosome at a unique size; this then becomes a chromosome-length polymorphism marker for that mutant gene. A comparison of mutant strains, their wild-type progenitor, and backcrossed strains revealed that these marker chromosomes are not caused by the initial mutagenic treatment and are found only in progeny of crosses between strains with polymorphic chromosomes. Thus, they are most likely formed by meiotic recombination. For the rad12 gene, the marker chromosome can further recombine to become the size of the homolog of the backcross parent. For the rad3 gene, both ectopic and homologous recombination events are likely involved in the generation of the marker chromosomes. As predicted by a recombination model, a cross to a new wild-type parent can change the size of a mutant marker chromosome. Therefore, changes in chromosome length are a common and prominent feature of the genome of this sexual fungus, and a variety of karyotypes is tolerated by the organism.

Sequence organization of the human chromosome 2q telomere.

The terminal 240 kb of a human 2q telomere region was cloned in two overlapping yeast artificial-chromosomes (YACs). This DNA contains a region of low-copy subtelomeric repeats (within 50 kb of the 2q telomere), a segment of DNA duplicated on distal 8p23 (100 kb from the 2q telomere), and a region of single-copy DNA (230 kb from the 2q telomere). Two CpG islands are present in the DNA segment duplicated on distal 8p23. RecA-assisted restriction endonuclease cleavage of genomic DNA samples revealed a potential 55 kb chromosome length polymorphism at the 2q telomere. This work provides telomeric closure of maps for human chromosome 2q, demonstrates a novel, subtelomere-specific DNA duplication, and will permit detailed molecular and cytological studies of this human telomere region.

Characterization of genome plasticity in Ustilago hordei.

Southern-blot hybridization analysis was used to identify and quantify chromosome-length polymorphisms for ten linkage groups of 14 races of Ustilago hordei. The bands identified by the probes were shown to vary as much as hundreds of kilobase pairs, but the magnitude of the variability was typically 5-15% of the average size of all bands to which a particular probe hybridized. A filamentous morphology mutant, recovered following heat-shock treatment of a strain with the greatest number of chromosome bands, was shown to have suffered a 50-kb deletion in a 940-kb chromosome. The mutation to filamentous morphology, designated fil1-1, and the deletion, were shown to invariably cosegregate 2:2 with the wild-type (sporidial) morphology in an ordered tetrad. Genetic and physical analyses place the Fil1 locus and the deletion near the terminus of one arm of the 940-kb chromosome. These results suggest that deletions of this type may be one of the causes of chromosome-length polymorphisms observed in field isolates of U. hordei.

Source of Pseudomonas cepacia: Ribotyping of isolates from patients and from the environment

Ribotyping, an analysis of bacterial strain identity based on chromosomal restriction fragment length polymorphisms, was performed on 68 isolates of Pseudomonas cepacia recovered from patients receiving care at the two children's hospitals in Philadelphia and from environmental samples. Twenty different ribotypes were identified. Ribotype R3 predominated among isolates from all three sources. These findings are consistent with the acquisition of P. cepacia from the environment or by person-to-person transmission.

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans.

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.

Use of electrophoretic karyotyping and DNA-DNA hybridization in yeast identification

Contour-clamped homogenous electric field (CHEF) gel electrophoresis was used to separate intact, chromosome-size DNA of different species of Saccharomyces and Zygosaccharomyces. Strains of the same Saccharomyces species had similar electrophoretic karyotypes. However, reproducible differences between individual bands indicated that strain-specific chromosome length polymorphism (CLP) is widely spread in these organisms. Strains of Zygosaccharomyces spp. showed karyotype differences beyond chromosome length polymorphism. A new, DNA-DNA hybridization technique was developed to test conspecificity, using individual chromosomes as templates to prepare randomly primed, radioactive probes. Under our conditions of stringency, species and chromosome-specific hybridization reactions were achieved with these probes. Using isolated chromosomes of Sacch. cerevisiae, Sacch. bayanus, and Zygosacch. rouxii as templates for probe preparation, conspecificity of strains was established, and closely related yeast species were distinguished with high reproducibility. This method is an efficient tool for studying genetic diversity of a given yeast species. Also, it can assist in yeast species identification and taxonomy.

Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus, Magnaporthe grisea

We have analyzed the karyotype of the rice blast fungus, Magnaporthe grisea, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.

Genetic diversity among Borrelia burgdorferi isolates: more than three genospecies?

In order to assess the genetic relationships among isolates of Borrelia burgdorferi, we have determined chromosomal DNA restriction fragment length polymorphisms (RFLP) of 27 isolates. Using five restriction enzymes and seven DNA probes, we recorded 246 different hybridizing bands. The resulting matrix was subjected to factorial correspondence analysis and to phylogenetic analysis using a parsimony program. These analyses demonstrate two closely related genospecies and three individual strains. RFLP variations among these three strains are much more pronounced than among the members of the other two genospecies. This may indicate the existence of more than three genospecies. Plasmid analysis did not contribute significantly to the genotypic differentiation.

Chromosomal rearrangements during vegetative growth of a wild strain of Saccharomyces cerevisiae.

Electrophoretic karyotypes of two strains of Saccharomyces cerevisiae, a haploid laboratory strain and a wild strain known to be at least diploid, have been checked during vegetative growth. The karyotype of the haploid strain was very stable; however, the diploid strain underwent frequent modifications. In most cases the number of bands was reduced, but occasionally we observed one band splitting into two. In one case, chromosomal rearrangements took place between differently sized copies of chromosomes I and VI. We concluded that the chromosome length polymorphism observed among wild strains of S. cerevisiae could be explained partly by chromosomal structure reorganization occurring during mitosis.

Genetic diversity of the yeast Candida utilis.

The electrophoretic karyotypes and some mtDNA restriction fragment patterns of 13 strains of Candida utilis and one strain of Hansenula jadinii were compared. PFGE separations revealed remarkable chromosome length polymorphisms between two groups of strains suggesting that perhaps they do not belong to the same species. However, all strains had the same or similar EcoRI, HindIII and BamHI mtDNA restriction patterns. The mtDNA genomes had an average size range of 55 kb. These results support the supposition that C. utilis is a yeast with a highly variable electrophoretic karyotype as already known for another imperfect yeast species, Candida albicans.

Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping).

Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.

Variable electrophoretic karyotypes of members of Aspergillus Section Flavi

Contour-clamped homogeneous electric field gel electrophoresis was used to establish karyotypes for fungi of Aspergillus Section Flavi. Under identical electrophoretic conditions, five to eight chromosomal bands were separated in Aspergillus flavus isolates and five to seven chromosomal bands in A. parasiticus isolates. Each distinct chromosomal band contained one or more chromosomes. Other members of Aspergillus Section Flavi (A. oryzae, A. sojae, and A. tamarii) had similar karyotypes to those of A. flavus and A. parasiticus. A related species, A. versicolor, showed six chromosomal bands. With the exception of small chromosomes present in some isolates, the estimated sizes of chromosomes for all six species range from approximately 3.0 to ≥7.0 Mb. It is likely that all isolates of these species contain the same number of large (>3 Mb) chromosomes; however, not all of the chromosomal bands could be resolved into separate chromosomes for each isolate due to chromosome length polymorphisms. This variability, observed in A. flavus and A. parasiticus, generated unique chromosomal band patterns within these species. The total genome sizes of these fungi were at least as large as those reported for A. nidulans and A. niger (31–38.5 Mb). Conserved genes were mapped to analogous chromosomes of A. flavus and A. parasiticus by gene hybridization.

Electrophoretic karyotyping of yeasts, and southern blotting using whole chromosomes as templates for the probe preparation.

The technique of Contour-clamped Homogenous Electric Field (CHEF) gel electrophoresis has been used to separate intact chromosomal DNA of different species of the yeast genera Saccharomyces and Zygosaccharomyces. Various strains of the same species had similar chromosomal band patterns. However, differences between individual bands indicated that strain-specific chromosome length polymorphism is common in these organisms.To test conspecificity, a new, DNA-DNA hybridization technique was developed using individual whole chromosomes as templates to prepare randomly primed, radioactive probes. Under our conditions of stringency, species-specific hybridization reactions were achieved with these probes. The method is an efficient tool to study the genetic diversity of a given yeast species. Also, it can assist yeast species identification.

Analysis of hybrids obtained by rare-mating of Saccharomyces strains

Rare-mating of closely related Saccharomyces cerevisiae and S. diastaticus strains led to the formation of different hybrids. Mating-type switching and chromosome losses could be observed by means of classical genetic analysis and pulsed field gel electrophoresis of intact chromosomes. The latter was facilitated by extensive chromosome length polymorphism in both strains. When crossing the two haploid strains S. cerevisiae 41 α and S. diastaticus ATCC 28339 α, two different types of hybrids occurred. Both types showed complete addition of both parental genomes, one aα-status and the other αα-status. The αα-status could be explained by assuming a transient premutational lesion in MAT α. Usually lesions are repaired after a mating event and the α-mating type is restored. When crossing a diploid S. diastaticus strain, isogenic to the one previously mentioned, with the haploid S. cerevisiae strain, three different types of hybrids could be distinguished regarding their mating-types. It was possible to prove that the haploid S. diastaticus strain ATCC 28339 is disomic and the diploid hybrid, named 41ATCC-b, is trisomic for chromosome I. This could be shown by means of electrophoretic karyotyping of the hybrid and of the four single-spore cultures from one ascus of the hybrid.

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35-36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.

Linkage analysis in a large family with nonspecific X-linked mental retardation.

We report on a large 5-generation family with "nonspecific" X-linked mental retardation. Nine living affected males have an IQ between 50 and 70 but have normal stature, facial appearance, and testicular volumes and no other abnormalities. Two obligate carrier females had borderline intellectual abilities and visual-psychomotor difficulties similar to those seen in affected males. Results of chromosome studies, including fragile X, were normal in males and females. Linkage analysis was undertaken, with 19 X-specific chromosomal restriction fragment length polymorphisms (RFLPs), giving a maximal LOD score of 1.60 at a 0.10 recombination fraction for F9, suggesting a localization to distal Xq for the mutant gene in this family.