Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.     

Chromosome banding in the genusPinus

Somatic chromosomes (2n=24) ofPinus luchuensis Mayr at metaphase were observed by fluorescent banding methods with chromomycin A₃ (CMA) and DAPI. CMA-bands appeared at the interstitial and/or proximal regions of nearly all chromosomes. DAPI-bands appeared at the interstitial and/or centromeric regions of nearly all chromosomes, and pairs of DAPI-dots appeared at the centromeric regions. Each homologous pair of chromosomes in the chromosome complement was identified by the CMA and DAPI fluorescent banding patterns. The interstitial CMA-bands were mostly localized at the secondary constrictions of the Feulgen-stained chromosomes. The fluorescent banding pattern ofP. luchuensis was very similar to that ofP. thunbergii, but was different from that ofP. densiflora.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

The Karyotype of Chukotka Chars from the Estikhed Lake (Eastern Chukotka)

The karyotype of chars from the Estikhed Lake (Eastern Chukotka) was examined. This karyotype comprises 78 chromosomes, NF = 98. Marker chromosomes include one pair of submetacentrics, one pair of large acrocentrics, and one pair of large subtelocentrics with very short second arms. Nucleolus organizer regions are located in telomeric regions of short arms of marker submetacentric chromosomes. Small heterochromatin blocks are observed in centromeric regions of most chromosomes. The Chukotka char karyotype is very similar to that of Taranetz charSalvelinus taranetzi from the Achchen Lake: these karyotypes differ only in stability of the chromosome number.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

Human (Homo sapiens) and chimpanzee (Pan troglodytes) share similar ancestral centromeric alpha satellite DNA sequences but other fractions of heterochromatin differ considerably

The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. © 1995 Wiley-Liss, Inc.     

A natural triploid in Trichomycterus davisi (Siluriformes,Trichomycteridae): mitotic and meiotic characterization by chromosome banding and synaptonemal complex analyses

Within a total of 50 analyzed specimens a male individual of Trichomycterus davisi has been recorded with 81 chromosomes including 60 metacentric, 18 submetacentric and three subtelocentric chromosomes. When compared with diploid individuals (2n = 54) and the morphological standard of chromosomes, this male is a triploid with 3n = 81 chromosomes. Since staining with silver nitrate indicates three active nucleolar organizer regions (NORs), the three NOR-bearing chromosomes in this individual are genetically active. Analysis of the synaptonemal complex (SC) by electronic microscopy shows that there is an incomplete pairing of the third set of chromosomes in the triploid individual.     

Constitutive heterochromatin distribution in monocentric and polycentric chromosomes

Monocentric chromosomes of Vicia faba (2n=12) and polycentric chromosomes of Luzula purpurea (2n=6) and L. multiflora (2n=36) were studied by the C-banding technique. C-positive regions exhibited a restricted distribution in V. faba nuclei and were located near the centromeres of the chromosomes. Each chromosome had both a characteristic number and distribution of C-positive regions permitting homologue identification. L. purpurea and L. multiflora C-bands were much more numerous than those of V. faba and were widely distributed throughout both nuclei and chromosomes. Three distinct constitutive heterochromatin distribution patterns were present in L. purpurea metaphase chromosomes permitting homologous chromosome identification. One of three C-band distribution patterns was also evident in L. multiflora chromosomes.     

Localization of high level of sequence conservation and divergence regions in cotton

In a previous study, we observed that the variations in chromosome size are due to uneven expansion and contraction by comparing the structures and sizes of a pair of homoeologous high-resolution cytogenetic maps of chromosomes 12A and 12D in tetraploid cotton. To reveal the variation at the sequence level, in the present paper, we sequenced two pairs of homoeologous bacterial artificial chromosomes derived from high- to low-variable genomic regions. Comparisons of their sequence variations confirmed that the highly conserved and divergent sequences existed in the distal and pericentric regions, e.g., high- and low-variable genome size regions in these two pairs of cotton homoeologous chromosomes. Sequence analysis also confirmed that the differential accumulation of Gossypium retrotransposable gypsy-like element (Gorge3) accounted for the main contributions for the size difference between the pericentric regions. By fluorescence in situ hybridization analysis, we found that Gorge3 has a bias distribution in the A_T/A proximal regions and is associated with the heterochromatin along the chromosomes in the entire Gossypium genome. These results indicate that, between A_T/A and D_T/D genomes, the distal and pericentric regions usually possess high level of sequence conservation and divergence, respectively, in cotton.     

The karyotype of Nodipecten nodosus (Bivalvia: Pectinidae)

Earlier karyotypical work on Nodipecten nodosus embryos indicated that this species has a diploid number of 38, with six pairs respectively of metacentric and submetacentric chromosomes and seven pairs of subtelocentric chromosomes, although there were some difficulties in obtaining complete metaphases. The present work provides additional results on specific regions of the chromosomes in N. nodosus and, by meiotic studies, confirms the chromosome number with more reliability. Active nucleolar organizer regions (NOR), detected in mitotic metaphases from embryos, can be characterized in N. nodosus by a high level of heteromorphism of NOR-sites, indicating that these regions are not appropriate as chromosomal markers in this species. The procedure for detecting constitutive heterochromatin of chromosomes allowed us to observe most of the heterochromatic blocks at a pericentromeric position and some at telomeric and interstitial positions. The analysis of meiotic chromosomes from gonad tissue revealed the presence of 19 bivalents during metaphase I, all homomorphic and isopicnotic, confirming the previously described diploid chromosomal number of 38 for N. nodosus. From these results, some evolutionary aspects of the Pectinidae are briefly discussed.     

Distribution of genes and recombination on wheat homoeologous group <Emphasis Type="Italic">6</Emphasis> chromosomes: a synthesis of available information

Presence of genes in gene-rich regions on wheat chromosomes has been widely reported. However, there is a lack of information on the precise characterization of these regions with respect to the distribution of genes and recombination. We attempted to critically analyze the available data to characterize gene-rich regions and to study the distribution of genes and recombination on wheat homoeologous group 6 chromosomes which are a reservoir of several useful genes controlling traits of economic importance. Consensus physical and genetic linkage maps were constructed for homoeologous group 6 using physical and genetic mapping data. Five major gene-rich regions were identified on homoeologous group 6 chromosomes, with two on the short and three on long arm. More than 90% of marker or gene loci were present in these five gene-rich regions, which comprise about 30% of the total physical chromosomal length. The gene-rich regions were mainly present in the distal 60% regions of the chromosomes. About 61% of the total loci map in the most distal regions which span only about 4% of the physical length of the chromosome. A range of sub-microscopic regions within each gene-rich region were also identified. Comparisons of the consensus physical and genetic linkage maps revealed that recombination occurred mainly in the gene-rich regions. Seventy percent of the total recombination occurred in the two most distally located regions that span only 4% of the physical length of the chromosomes. The relationship of recombination to the gene-rich region is not linear with distance from the centromere, especially on the long arm. The kb/cM estimates for group 6 chromosomes ranged from 146 kb in the gene-rich to about 10 Mb in the gene-poor region. The information obtained here is vital in understanding wheat genome structure and organization, which may lead in developing better strategies for positional cloning in wheat and related cereals.This revised version was pubished online in April 2005 with corrections to the page numbering.     

Centromere organization in meiotic chromosomes of Parascaris univalens

The chromosomes of Parascaris univalens possess a continuous centromeric structure spanning their entire length in gonial cells. A cytological and ultrastructural analysis of P. univalens meiotic chromosomes was performed. The results show that during meiosis the holocentric germline chromosomes of male P. univalens undergo restriction of kinetic activity to the heterochromatic terminal regions. These regions lack kinetochore structures and interact directly with spindle microtubules.     

Karyomorphology of six taxa in Chrysanthemum sensu lato (Anthemideae) in Egypt and their genetic relationships by Giemsa C‐banding

Abstract Giemsa C‐banding was applied to the chromosome complements of six diploid species belonging to six genera in Chrysanthemum sensu lato (Anthemideae) distributed in Egypt. Four types of C‐banding distribution were observed in the taxa as follows: (i) negative C‐banding in Anacyclus monanthos (L.) Thell.; (ii) all bands in terminal regions in Achillea fragrantissima (Forssk.) Sch. Bip, which showed 32 bands on 18 chromosomes; (iii) all eight bands at centromeric regions on eight chromosomes in Matricaria recutita L.; and (iv) bands at terminal and centromeric regions in Brocchia cinerea Vis. (12 terminal and six centromeric bands on 12 chromosomes), Cotula barbata DC. (four terminal, six centromeric, and eight short arm bands on 16 chromosomes), and Glebionis coronaria (L.) Cass. ex Spach. (eight terminal on the short arms and four large bands in centromeric regions on 12 chromosomes).     

NOR Sites Detected by Ag-DAPI Staining of an Unusual Autosome Chromosome of Bradysia Hygida (Diptera:Sciaridae) Colocalize with C-banded Heterochromatic Region

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.     

Early replication banding in <Emphasis Type="Italic">Leporinus</Emphasis> species (Osteichthyes,Characiformes) bearing differentiated sex chromosomes (ZW)

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2′-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.     

Cytological and biochemical characterization of the in situ endonuclease digestion of fixed Tenebrio molitor chromosomes

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.by E.R. Schmidt