Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

Electrophoretic Karyotype of the Obligate Biotrophic Parasite Plasmodiophora brassicae Wor.

Classical genetic analysis is not possible with the protist Plasmodiophora brassicae due to the intracellular life of this obligate biotrophic parasite . An electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis to facilitate gene mapping of P. brassicae. Using two different separation conditions 16 chromosomal bands of P. brassicae were distinguished ranging in approximate size from 2.2 Mb to 680 kb. According to this determination of chromosome number and size, the total genome size of P. brassicae was estimated to be 20.3 Mb. The chromosomal bands were further designated by their hybridization pattern with repetitive elements of P. brassicae . The repetitive element H4 (1800 bp) hybridized with 14 chromosomal bands, but the sequence of H4 showed no homology to known centromere or telomere structures and revealed no repetitive motifs.     

Evolutionary conserved chromosomal segments in the human karyotype are bounded by unstable chromosome bands

In this paper an ancestral karyotype for primates, defining for the first time the ancestral chromosome morphology and the banding patterns, is proposed, and the ancestral syntenic chromosomal segments are identified in the human karyotype. The chromosomal bands that are boundaries of ancestral segments are identified. We have analyzed from data published in the literature 35 different primate species from 19 genera, using the order Scandentia, as well as other published mammalian species as out-groups, and propose an ancestral chromosome number of 2n = 54 for primates, which includes the following chromosomal forms: 1(a+c(1)), 1(b+c(2)), 2a, 2b, 3/21, 4, 5, 6, 7a, 7b, 8, 9, 10a, 10b, 11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y. From this analysis, we have been able to point out the human chromosome bands more "prone" to breakage during the evolutionary pathways and/or pathology processes. We have observed that 89.09% of the human chromosome bands, which are boundaries for ancestral chromosome segments, contain common fragile sites and/or intrachromosomal telomeric-like sequences. A more in depth analysis of twelve different human chromosomes has allowed us to determine that 62.16% of the chromosomal bands implicated in inversions and 100% involved in fusions/fissions correspond to fragile sites, intrachromosomal telomeric-like sequences and/or bands significantly affected by X irradiation. In addition, 73% of the bands affected in pathological processes are co-localized in bands where fragile sites, intrachromosomal telomeric-like sequences, bands significantly affected by X irradiation and/or evolutionary chromosomal bands have been described. Our data also support the hypothesis that chromosomal breakages detected in pathological processes are not randomly distributed along the chromosomes, but rather concentrate in those important evolutionary chromosome bands which correspond to fragile sites and/or intrachromosomal telomeric-like sequences.     

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two -carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.     

Breakpoints in variant Philadelphia translocations in chronic myeloid leukemia

To date, 327 cases of variant Philadelphia translocations found in chronic myeloid leukemia have been described. A statistical analysis using the Monte Carlo simulation was performed to determine which chromosomal bands were preferentially involved in variant Philadelphia translocations. The results showed that twenty-eight bands were significantly rearranged (P less than 0.05).     

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.     

Asymmetric banding of Vicia faba chromosomes after BrdU incorporation

Growth of Vicia faba roots in BrdU for 17 h (about one cell cycle duration) followed by application of the FPG technique resulted in a characteristic pattern of asymmetric FPG bands, which occupy one or the other of the two sister chromatids of the metaphase chromosomes and are assumed to be indicative of clusters in chromosomal DNA with unequal Thd distribution between the complementary polynucleotide chains. Chromosomal position and visualization frequency of these bands have been established and compared with the banding patterns obtained after application of other techniques (fluorescence- and Giemsa technique, incorporation of azacytidine into chromosomal DNA, DNA late replication pattern). FPG bands and bands occurring after application of the techniques showed only limited positional coincidence. Some aspects of the FPG technique after BrdU incorporation have been used to make inferences with respect to the cell cycle parameters in the main root meristem of Vicia faba.     

A reliable method of quantifying G-band position in chromosomes

The locations of chromosomal bands in the muntajac (Muntiacus muntjak, Zimmerman) are shown to be constant, that is the bands occupy the same relative position regardless of the state of contraction of the chormosome. Each band can thus be assigned a precise location. Different banding techniques produce bands at identical locations and thus precisely similar patterns, with one notable exception in which certain bands disappear. It it proposed that this more exact procedure be used to identify chromosomal bands, especially in cases of chromosomal rearrangement. It may be of particular use in computer analyses of karyotypes.     

Electrophoretic karyotype of Saprolegnia monoica

Abstract The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Hansenula wingei , is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.     

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.     

Nineteen of 26 cellular oncogenes precisely localized in the human genome map to one of the 83 bands involved in primary cancer-specific rearrangements

Summary A total of 83 bands have been found to be specifically involved in primary structural chromosome rearrangements in human cancer. We have compared the distribution of these cancer-specific breakpoints with the chromosomal sites of the 26 cellular oncogenes currently mapped to individual bands within the human genome. Nineteen of the 26 oncogenes are localized in cancer-associated bands. This clustering of oncogene sites and cancer breakpoints is statistically highly significant (P=0.0000012).     

温室希蛛染色体的观察(蜘蛛目:球蛛科)

报道了温室希蛛的染色体数目、形态结构和性染色体组成。从目前的结果可见,温室希蛛的染色体数目是:雄性体细胞染色体数为2n=22,雌性为2n=24。其性决定机制属于X_1X_2O型。所有染色体似乎均为端或亚端着丝粒染色体,这个结论被对其C-带标本的分析所证实。两个(对)X-染色体是最短的和次最短的,温室希蛛染色体C-带标本的分析没有观察到染色体间有明显的结构差异。在染色体G-带标本中,获得了稳定的带纹。     

Chromosomal Bands Affected by Acute Oil Exposure and DNA Repair Errors

Background

In a previous study, we showed that individuals who had participated in oil clean-up tasks after the wreckage of the Prestige presented an increase of structural chromosomal alterations two years after the acute exposure had occurred. Other studies have also reported the presence of DNA damage during acute oil exposure, but little is known about the long term persistence of chromosomal alterations, which can be considered as a marker of cancer risk.

Objectives

We analyzed whether the breakpoints involved in chromosomal damage can help to assess the risk of cancer as well as to investigate their possible association with DNA repair efficiency.

Methods

Cytogenetic analyses were carried out on the same individuals of our previous study and DNA repair errors were assessed in cultures with aphidicolin.

Results

Three chromosomal bands, 2q21, 3q27 and 5q31, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed (p= 0.016).

Conclusion

The present study shows that breaks in 2q21, 3q27 and 5q31 chromosomal bands, which are commonly involved in hematological cancer, could be considered useful genotoxic oil biomarkers. Moreover, breakages in these bands could induce chromosomal instability, which can explain the increased risk of cancer (leukemia and lymphomas) reported in chronically benzene-exposed individuals. In addition, it has been determined that the individuals who participated in clean-up of the oil spill presented an alteration of their DNA repair mechanisms two years after exposure.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

Banding of Allium chromosomes protected against DNase digestion by DNA-binding drugs

Metaphase chromosome bands were induced in Allium flavum (Liliaceae) by protecting the chromosomal DNA with DNA-binding compounds of different base specificities against DNase digestion. The bands obtained represented different subsets of C-band heterochromatin.     

Human chromosomal bands: nested structure,high-definition map and molecular basis

In this paper, we report investigations on the nested structure, the high-definition mapping, and the molecular basis of the classical Giemsa and Reverse bands in human chromosomes. We found the rules according to which the approximately 3,200 isochores of the human genome are assembled in high (850-band) resolution bands, and the latter in low (400-band) resolution bands, so forming the nested mosaic structure of chromosomes. Moreover, we identified the borders of both sets of chromosomal bands at the DNA sequence level on the basis of our recent map of isochores, which represent the highest-resolution, ultimate bands. Indeed, beyond the 100-kb resolution of the isochore map, the guanine and cytosine (GC) profile of DNA becomes turbulent owing to the contribution of specific sequences such as exons, introns, interspersed repeats, CpG islands, etc. The isochore-based level of definition (100 kb) of chromosomal bands is much higher than the cytogenetic definition level (2-3 Mb). The major conclusions of this work concern the high degree of order found in the structure of chromosomal bands, their mapping at a high definition, and the solution of the long-standing problem of the molecular basis of chromosomal bands, as these could be defined on the basis of compositional DNA properties alone.     

Circular chromosomal DNA in the sulfur-dependent archaebacterium Sulfolobus acidocaldarius.

The shape of the chromosomal DNA of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius was analyzed by the pulsed-field gel electrophoresis(PFGE). S.acidocaldarius DNA digested with Notl showed two DNA bands at around 1.0 Mbp and 2.1 Mbp. Notl-linking clones were isolated from the library of S.acidocaldarius chromosomal DNA. It contained two Notl sites. Both 1.0 and 2.1 Mbp DNA band separated by PFGE were hybridized with the two independent Notl-linking fragment. Each right and left arms of two Notl-linking fragments were hybridized with one of the two DNA bands separated by PFGE. The results indicated that the chromosomal DNA of S.acidocaldarius is circular.     

Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.     

Human chromosomal polymorphism. VII. The distribution of chromosomal Q-polymorphic bands in different human populations

The distribution of chromosomal Q-polymorphic bands was studied in different human populations. The populations studied showed no differences in the relative amount of Q bands in all the 12 polymorphic loci of seven autosomes, but interpopulation differences did exist in the absolute amount of Q bands in all the 12 potentially polymorphic loci of seven autosomes, these differences consisting of uniform increases or decreases in this absolute amount. Comparisons of the mean number of Q-heterochromatin bands with fluorescence levels 4 and 5 per individual showed a consistent prevalence of this quantitative parameter of chromosomal Q polymorphism in females as compared to males in all the national groups. It is suggested that there is some dosage compensation of chromosomal Q-heterochromatin material in females due to the absence of a chromosome in their genome, which is able to "compensate" for the large Q band in chromosome Y which is present only in the karyotype of males.