DNA content of the B chromosomes in grasshopper <Emphasis Type="Italic">Podisma kanoi</Emphasis> Storozh. (Orthoptera,Acrididae)

A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database () was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telometric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.     

Microdissection and chromosome painting of X and B chromosomes in Locusta migratoria

Acquisition of knowledge of the nature and DNA content of B chromosomes has been triggered by a collection of molecular techniques, one of which, microdissection, has provided interesting results in a number of B chromosome systems. Here we provide the first data on the molecular composition of B chromosomes in Locusta migratoria, after microdissection of the B and X chromosomes, DNA amplification by one (B) or two (X) different methods, and chromosome painting. The results showed that B chromosomes share at least two types of repetitive DNA sequences with the A chromosomes, suggesting that Bs in this species most likely arose intraspecifically. One of these repetitive DNAs is located on the heterochromatic distal half of the B chromosome and in the pericentromeric regions of about half of the A chromosomes, including the X. The other type of repetitive DNA is located interspersedly over the non-centromeric euchromatic regions of all A chromosomes and in an interstitial part of the proximal euchromatic half of the B chromosome. Chromosome painting, however, did not provide results sufficiently reliable to determine, in this species, which A chromosome gave rise to the B; this might be done by detailed analysis of the microdissected DNA sequences     

Chromosomal study of a lamprey (<Emphasis Type="Italic">Lampetra zanandreai</Emphasis> Vladykov, 1955) (Petromyzonida: Petromyzontiformes): conventional and FISH analysis

Karyotype and other chromosomal characteristics in the Adriatic brook lamprey Lampetra zanandreai, representative of one of the most ancestral group of vertebrates, were examined using conventional (Ag-staining, C-banding as well as CMA₃ and DAPI fluorescence) and molecular (FISH with 18/28S rDNA and EcoRI satDNA as probes) protocols with metaphase chromosomes derived from whole blood cultures. The chromosome complement had a modal diploid chromosome number of 2n = 164, as in other petromyzontid lamprey species. Ag-staining and CMA₃ fluorescence, as well as FISH with 18/28S rDNA probes, detected nucleolar organizer regions (NORs) close to the centromeres of the biarmed chromosomes of pairs 1 and 2, the largest chromosome pairs of the complement. In addition to NORs, CMA₃ fluorescence revealed positive signals in approximately 40 other chromosomes. DAPI stained mostly centromeric regions of many chromosomes as well as conspicuously massive blocks overlapping NOR sites. C-banding evidenced a large amount of constitutive heterochromatin in somatic chromosomes, with approximately 40 C-positive acrocentric elements completely heterochromatic, corresponding with the 40 CMA₃+ chromosomes and positive heterochromatic blocks in pericentromeric regions of chromosome pairs 1 and 2. Polymerase chain reaction (PCR)-based cloning of satellite DNA with primers derived from Petromyzon marinus centromeric sequences was successful for L. zanandreai genomic DNA. The sequence was AT-rich (59%) and characterized by short consensus motifs similar to other centromeric satellite motifs. FISH using satDNA clones as a probe produced a fluorescent signal on a single pair of small chromosomes. This sequence was PCR-amplified also in L. planeri and P. marinus genomic DNA, and the evolution of this repetitive element in the above species was analysed.     

Interstitial colocalization of two cervid satellite DNAs involved in the genesis of the Indian muntjac karyotype

A number of repetitive DNA clones were generated from PCR amplifications of Indian muntjac genomic DNA using primer sequences derived from a white tailed deer satellite II DNA sequence. One clone (Mmv-0.7) was characterized and shown to be a cervid satellite II DNA clone. Multiple colored FISH studies with cervid satellite I (C5) and this satellite II clone (Mmv-0.7) to Chinese muntjac metaphase chromosomes localized both satellite DNAs at the pericentromeric regions of all chromosomes except for chromosome 3 and the Y chromosome, whereas chromosome 3 exhibited pericentromeric satellite II DNA only. Where distinguishable, the pericentromeric satellite II signals appeared terminally oriented with respect to satellite I. Six pairs of Chinese muntjac autosomes had interstitial satellite I sites with four of these autosomal pairs (chromosomes 1, 2 and two other smaller autosomal pairs) also exhibiting interstitial satellite II signals. An interstitial site on the X chromosome was found to have satellite II signals. For the Indian muntjac chromosomes, FISH studies revealed a pericentromeric hybridization for satellites I and II as well as 27 distinct interstitial hybridization sites, each having at least one of the satellite DNAs. These data were used to more precisely define the chromosome fusion-associated breakpoints that presumably led to the formation of the present-day Indian muntjac karyotype. It further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a 2n=70 ancestral karyotype rather than from an intermediate 2n=46 Chinese muntjac-like karyotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species

Cytogenetic maps of common bean chromosomes 3, 4 and 7 were constructed by fluorescence in-situ hybridization (FISH) of BAC and a few other genomic clones. Although all clones were selected with genetically mapped markers, mostly with single-copy RFLPs, a large subset of BACs, from 13 different genomic regions, contained repetitive sequences, as concluded from the regional distribution patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Pericentromeric repeats were present in all 11 chromosome pairs with different intensities, whereas subtelomeric repeats were present in several chromosome ends, but with different signal intensities depending on the BAC, suggesting that the terminal heterochromatin fraction of this species may be composed of different repeats. The correlation of genetic and physical distances along the three studied chromosomes was obtained for 23 clones. This correlation suggests suppression of recombination around extended pericentromeric regions in a similar way to that previously reported for plant species with larger genomes. These results indicate that a relatively small plant genome may also possess a large proportion of repeats interspersed with single-copy sequences in regions other than the pericentromeric heterochromatin and, nevertheless, exhibit lower recombination around the pericentromeric fraction of the genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complex structure of B-chromosomes in two mammalian species: Apodemus peninsulae (Rodentia) and Nyctereutes procyonoides (Carnivora)

B-chromosomes (Bs) of two mammalian species, raccoon dog (Nyctereutes procyonoides, Carnivora) and Asian wood mouse (Apodemus peninsulae, Rodentia) were investigated using chromosome segment microdissection and double-colour FISH. In the raccoon dog, all B-chromosomes showed homology with each other but not with the A-chromosomes. Two segment-specific probes (from proximal and distal parts of B) have been localized in corresponding chromosome parts, with significant variation in their sizes. In Asian wood mice, two types of B-specific chromatin were revealed – B1 and B2. Most Bs were either B1 or B2 specific; furthermore, some Bs were found to be composed of both types of chromatin. B-chromosome-specific libraries of A. peninsulae contain sequences homologous to the heterochromatic regions of sex and some A-chromosomes and dispersed repeated sequences. B1-specific probes gave signals on sex chromosomes of Apodemus speciosus and Apodemus agrarius. The origin and evolution of B-chromosomes in mammals are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mapping nonrecombining regions in barley using multicolor FISH

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.     

Classical and Molecular Cytogenetics of the Pufferfish Tetraodon Nigroviridis

Because of its highly compact genome, the pufferfish has become an important animal model in genome research. Although the small chromosome size renders chromosome analysis difficult, we have established both classical and molecular cytogenetics in the freshwater pufferfish Tetraodon nigroviridis (TNI). The karyotype of T. nigroviridis consists of 2n = 42 biarmed chromosomes, in contrast to the known 2n = 44 chromosomes of the Japanese pufferfish Fugu rubripes (FRU). RBA banding can identify homologous chromosomes in both species. TNI 1 corresponds to two smaller FRU chromosomes, explaining the difference in chromosome number. TNI 2 is homologous to FRU 1. Fluorescence in-situ hybridization (FISH) allows one to map single-copy sequences, i.e. the Huntingtin gene, on chromosomes of the species of origin and also on chromosomes of the heterologous pufferfish species. Hybridization of total genomic DNA shows large blocks of (species-specific) repetitive sequences in the pericentromeric region of all TNI and FRU chromosomes. Hybridization with cloned human rDNA and classical silver staining reveal two large and actively transcribed rRNA gene clusters. Similar to the situation in mammals, the highly compact pufferfish genome is endowed with considerable amounts of localized repeat DNAs.     

Possible origin of a B chromosome deduced from its DNA composition using double FISH technique

Double fluorescentin situ hybridization (FISH) with two DNA probes (a 180 bp tandemly repeated DNA and ribosomal DNA) was performed in embryo cells of the grasshopperEyprepocnemis plorans. Repetitive DNA was present in most standard chromosomes (excepting 7, 8 and 10) and in the proximal two-thirds of the B chromosome, which was its major location in the complement. Ribosomal DNA was present distally on the B, and in the active nucleolar organizer regions (NORs) of the X, 9, 10 and 11 chromosomes. A small number of rRNA gene clusters was also observed in the pericentromeric regions of chromosomes 1–8. The double FISH technique showed that the B chromosome (B₂ type) is mainly composed of a 180 bp tandem repeat and ribosomal DNA, the minute short arm being the only region that does not hybridize with them. The location and order of the centromere and both the DNA sequences on the B chromosome coincide only with those in the X chromosome, indicating that the B most probably derives from the X.     

Cosmid clones from microdissected human chromosomal region 15q11–q13

Summary A human chromosomal region, 15q11–q13, was microdissected, its DNA was amplified with the primer-linker PCR method, and the PCR products were cloned into a plasmid vector to construct a microclone library. Of 193 microclones analyzed with Southern blot hybridization on hybrid cell panels, 26 (13.5%) were either single-copy (unique) or low-repetitive fragments. By screening of a cosmid library of human genomic DNA using the 26 microclones as probes, 47 positive cosmids were obtained and underwent regional mapping with chromosome fluorescencein situ hybridization (FISH). Sixteen cosmids gave FISH signals at 15p-cen, 5 at 15q11–q13, 6 at 15q22–q26, 3 at other chromosomes, and 17 no signal. These 27 cosmids mapped to chromosome 15 are useful additions to the inventory of DNA markers of this chromosome including the much interested Prader-Willi/Angelman syndrome region.     

Cytogenetic map of common bean (Phaseolus vulgaris L.)

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.     

Analysis of rye B-chromosome structure using fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomicin situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A–B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.     

Isolation of genomic clones homologous to transcribed sequences from human X chromosome

Several X chromosome DNA clones homologous to transcribed sequences were isolated from a human X chromosome library. The clones were selected for their ability to hybridize either with³²P -labeled human cDNA in the presence of an excess of unlabeled human repetitive DNA or with mouse fibroblast cDNA. The X chromosome specificity of these sequences was demonstrated by two criteria: A dosage effect was seen when the clones were hybridized to Southern blots of DNA from 1X and 5X cells, and they hybridized to DNA from mouse-human hybrid cells containing only the human X chromosome. The presence of transcribed sequences in these X clones was detected by hybridization with mouse cDNA or with human cDNA in the presence of unlabeled human repetitive sequences, by identifying restriction fragments which hybridize with cDNA but not with human repetitive DNA, and by hybridization with poly A⁺ RNA on Northern blots. These clones were mapped on the human X chromosomes using a panel of mouse-human somatic cell hybrids carrying various translocated human X chromosomes.     

Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers

Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.2 kb/μm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping. This revised version was published online in July 2006 with corrections to the Cover Date.     

PRINS tandem labeling of satellite DNA in the study of chromosome damage

Tandem labeling of satellite DNA was proposed a few years ago (1) for evaluating preferential chromosome breaks in the pericentromeric regions of mammalian chromosomes, and (2) for distinguishing chromosome breaks from chromosome segregation errors in interphase cells. In the presence of primers and modified nucleotides, primed in situ labeling (PRINS) tags repetitive DNA sequences, and serves as a useful alternative to fluorescence in situ hybridization (FISH). We developed a two‐color method for PRINS tandem labeling of centromeric and pericentromeric sequences. The method, which appears to be more sensitive than FISH, was used to assay micronuclei in mouse splenocytes and early spermatids, and it provided insight into mechanisms of induction of chromosome damage in these cells. We compared the sensitivity of this method and of a different two‐color approach, based on simultaneous labeling of centromeric and telomeric sequences. © 2001 Wiley‐Liss, Inc.     

FISH to mitotic chromosomes and extended DNA fibres of Beta procumbens in a series of monosomic additions to beet (B. vulgaris)

The physical localization and organization of a Procumbentes-specific repetitive DNA sequence, PB6-4, on the chromosomes of Beta procumbens (2n=18) were studied, using FISH (fluorescence in situ hybridization) to mitotic chromosomes and extended DNA fibres. The chromosomes of B. procumbens were studied in metaphase complements of the species itself, as well as in preparations of a series of eight different B. procumbens-derived monosomic additions to B. vulgaris (2n=18). FISH to chromosome spreads of B. procumbens revealed that PB6-4 hybridizes to all chromosomes, predominantly in the pericentromeric regions, but with differences in size and brightness of the signals. Hybridization of PB6-4 to metaphase complements of B. vulgaris revealed no signals, indicating that cross-hybridization with the genome of this species was negligible. Consequently, hybridization of PB6-4 to metaphase complements of the monosomic additions yielded fluorescent signals on the alien chromosomes only. The previously observed differences in size and brightness of the fluorescent spots were confirmed using the single alien chromosomes. FISH of PB6-4 to extended DNA fibres of the monosomic additions indicated differences in the fluorescent track lengths between the alien chromosomes. Measurements of the fluorescent tracts allowed classification into discrete groups, varying from one to three groups per B. procumbens chromosome. The data revealed that the brightness or size of the signal at mitotic metaphase and the length of the fluorescent tracks on the DNA fibres were correlated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of B chromosomes on rDNA organization in rye interphase nuclei

Patterns of rye rDNA organization in interphase nuclei were studied through the use ofin situ hybridization in spreads of root meristem cells from plants with and without B chromosomes (Bs). In cells from plants without Bs each rDNA locus is organized as a single perinucleolar knob of condensed chromatin with decondensed chromatin inside the nucleolus. In plants with Bs there is a marked modification of the pattern, found in more than 23% of nuclei, which involves several regions of condensed chromatin interspersed with decondensed chromatin inside the nucleolus. This B-induced alteration in rDNA interphase organization suggests a change in expression of the rRNA genes located on the A chromosomes probably related to the reduction in nuclear RNA observed previously in plants with Bs. The influence of the Bs on the expression of A chromosome genes, through rearrangement of interphase chromatin, could provide the basis of an explanation for some of the known phenotypic effects of B chromosomes in rye.     

Modification of wheat rDNA loci by rye B chromosomes: a chromatin organization model

The rDNA loci, and their associated NORs, on chromosomes 1B and 6B of the hexaploid wheat cv. Lindstr?m have been used as a chromatin marker to investigate the functional basis of the phenotype effects of introgressed supernumerary B chromosomes (Bs) of rye. The rye Bs themselves lack genes, other than those which determine their mitotic drive mechanism, and the way in which they can modulate characters determined by the A chromosome background has always been a puzzle. An isogenic line of Lindstr?m plants carrying different numbers of Bs was used as the experimental system to see how different doses of Bs (from 0 to 6) affected the activity of the wheat NORs and the organization of their rDNA loci at interphase. Silver staining on metaphase chromosomes was used to evaluate the previous activity of the NORs, and to reveal variations in their size; and the pTa71 FISH probe from wheat was used to visualize structural modifications to the interphase rDNA loci. A single B had no measurable effect, but, as the B-number increased, there were significant changes in the physical dimensions of the metaphase NORs, reflecting reduced levels of their activity earlier in the cell cycle, and also in the condensation patterns of the interphase rDNA loci. In addition, the higher B-numbers caused a size heteromorphism between the homologous NORs. A model is discussed which interprets the phenotypic effects of Bs generically, in nucleotypic terms, based of their being 'genetically inert' but 'chromosomally active'.     

Construction of radiation-reduced hybrids and their use in mapping of microclones from chromosome 10p11.2-q11.2

Summary Radiation-reduced hybrids for mapping of DNA markers in the pericentromeric region of chromosome 10 were developed. A Chinese hamster/human somatic cell hybrid (762-8A) carrying chromosomes 10 and Y as the only human material were exposed to 40,000 rads of irradiation and then rescued by fusion with non-irradiated recipient Chinese hamster cells (GM459). Southern hybridization analyses revealed that 10 of 128 HAT-resistant clones contained human chromosomal fragments corresponding to at least one marker locus betweenFNRB (10p-11.2) andRBP3 (10q11.2). These hybrids were then used to map microdissection clones previously isolated and roughly mapped to this chromosomal region by fluorescencein situ hybridization (FISH). Two of the six microclones studied could be mapped to the proximity of the D10-S102 locus. These radiation hybrids are useful for the construction of refined genetic maps of the pericentromeric region of chromosome 10.