Levels of conservation and variation of heterochromatin and nucleolus organizers in the Bovidae

Chromomycin A3 banding of the mitotic sets of 10 species of Bovidae (cattle, wisent, yak, banteng, gaur, red buffalo, swamp buffalo, sheep, mufflon, and goat) serves to demarcate both centromeric constitutive heterochromatin and R-banding patterns capable of identifying all the chromosomes within a given complement. In all species significant amounts of chromomycin-bright heterochromatin are present at the centromeres of all autosomes, though there was a high degree of intra- and inter-individual variation in the size of the heterochromatic blocks. Marked interspecies differences in the centromeric patterns were evident. The X chromosomes contained appreciable amounts of centromeric heterochromatin only in the two buffaloes. All the animals studied lacked distamycin A - diamidinophenylindole type heterochromatin. AgNO3 staining was applied sequentially to detect the location of active nucleolus organizer regions (NORs). The distribution of NORs was reasonably conservative in most of the species. An exceptional situation was found in the two buffaloes, where only one NOR pair matched with the standard karyotype of the Bovidae.     

Cytogenetic variation in farmed stock of Dybowski's sika deer

The chromosome constitution of Dybowski's sika deer was studied on the basis of 15 samples obtained from farmed stock maintained in an enclosure. The diploid chromosome number was 2n=68, 2n=67 and 2n=66. The constitutive heterochromatin (C-bands) was located in the centromeric regions of all acrocentric chromosomes. Metacentric chromosomes were C-negative. Chromosomes of three pairs proved to be NORs carriers. The size polymorphism of silver deposits was identified in two animals. A cytogenetic analysis indicated that the farmed stock of Dybowski's sika deer demonstrates considerable variation. The chromosome polymorphism observed may be a valuable marker for the management and preservation of this species.     

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.     

Chromosome banding in Amphibia

The distribution and quantity of constitutive heterochromatin and of the nucleolus organizer regions (NORs) on the chromosomes of 22 species of bufonids and hylids (Amphibia, Anura) was investigated. Three different kinds of constitutive heterochromatin were found and the frequency of brightly fluorescing heterochromatic regions was remarkably high. On almost all chromosomes there is centric and telomeric heterochromatin. Quantitative estimates of heterochromatin demonstrate that large DNA differences among closely related species can not be attributed to differing quantities of constitutive heterochromatin. In all species investigated, only one homologous pair of NORs was found, which lies preferentially in the proximal and interstitial segments of the long chromosome arms. The NORs are always associated with constitutive heterochromatin on both sides. The size variability between homologous NORs is very high. In the euchromatic regions of the metaphase chromosomes, neither Q- nor G-bands can be demonstrated; this can be attributed to an extremely strong contraction of the anuran chromosomes. On the basis of these results various mechanism of the chromosomal evolution in Anura are discussed.     

Karyology of the Antarctic scallop Adamussium colbecki, with some comments on the karyological evolution of pectinids

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag–NOR, CMA₃, DA/MM and NOR–FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA₃ and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA₃. In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.     

Cytogenetic study of Anopheles albitarsis (Diptera: Culicidae) by C-banding and in situ hybridization

The C-banding pattern and the size and location of the nucleolar organizer regions (NORs) are described for the first time in Brazilian populations of Anopheles (Nyssorhynchus) albitarsis sensu lato. C-banding revealed variation in the size of the centromeric heterochromatic blocks in autosomal chromosomes and in the acrocentric (X) and puntiform (Y) sex chromosomes. Fluorescence in situ hybridization showed that the NORs were located in the pericentromeric region of the sex (XX/XY) chromosomes and that this coincided with the number and location of centromeric constitutive heterochromatin blocks previously revealed by C-banding. The NORs varied in size among the homologues of the three populations. These findings of the populations studied support the hypothesis that the stability of NORs in the A. albitarsis complex is characterized by the presence of clustered and conserved sites in a unique pair of chromosomes.     

白眉长臂猿(Hylobates hoolock leuconedys)的染色体研究

本文对两只雄性白眉长臂猿的染色体的C带、G带及Ag-NORs分布进行了较详细的分析,证实染色体数2n=38,并对该种的分类地位提出了一些新看法。     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

Classical and molecular cytogenetics of Atherinella brasiliensis (Teleostei, Atheriniformes) from South coast of Brazil

Cytogenetic studies were performed on specimens of Atherinella brasiliensis from Laranjeiras Bay (Paraná State, Brazil). All specimens had a diploid number of 48 chromosomes, with a karyotype constituted by 4m+14sm+18st+12a and fundamental number of 84. The C-positive heterochromatin was distributed over the nucleolar organizer regions (NORs) in the centromeric regions and on short arms of metacentric and submetacentric chromosomes. Most of this heterochromatin was AT-rich, except in the NORs, which were rich in GC, as detected by double staining with chromomycin A₃/4'-6-diamin-2-phenylindole. Single NORs were located at terminal positions of a submetacentric pair, as confirmed by fluorescent in situ hybridization with 18S rDNA probes. Both techniques showed a size heteromorphism between the homologous chromosomes. The 5S rDNA clusters were located in terminal positions on two chromosomal pairs and also displayed a size heteromorphism. Despite the conserved diploid number, the data on the karyotype microstructure help characterize the cytogenetic profile of this group.     

Chromosome homology and heterochromatin in goat,sheep and ox studied by banding techniques

Peripheral blood lymphocyte metaphase chromosomes of three Bovoidean species have been studied using Quinacrine fluorescence and Giemsa banding techniques to give Q-, G-, and C-banding patterns. Q- and G-banding characteristics, coupled with chromosome length, enabled all of the chromosomes in each of the chromosome complements to be clearly distinguished, although some difficulties were encountered with the very smallest chromosomes. A comparison of G-banding patterns between the species revealed a remarkable degree of homology of banding patterns. Each of the 23 different acrocentric autosomes of the domestic sheep (2n=54) was represented by an identical chromosome in the goat (2n=60) and the arms of the 3 pairs of sheep metacentric autosomes were identical matches with the remaining 6 goat acrocentrics. A similar interspecies homology was evident for all but two of the autosomes in the ox (2n=60). This homology between sheep metacentric and goat acrocentric elements confirms a previously suggested Robertsonian variation. The close homology in G-banding patterns between these related species indicates that the banding patterns are evolutionarily conservative and may be a useful guide in assessing interspecific relationships. —The centromeric heterochromatin in the autosomes of the three species was found to show little or no Q-or G-staining, in contrast to the sex chromosomes. This lack of centromeric staining with the G-technique (ASG) contrasts markedly with results obtained with other mammalian species. However, with the C-banding technique these regions show a normal intense Giemsa stain and the C-bands in the sex chromosomes are inconspicuous. The amount of centromeric heterochromatin in the sheep metacentric chromosomes is considerable less than in the acrocentric autosomes or in a newly derived metacentric element discovered in a goat. It is suggested that the pale G-staining of the centromeric heterochromatin in these species might be related to the presence of G-Crich satellite DNA.     

Cytogenetic and molecular evaluation of centromere-associated DNA sequences from a marsupial (Macropodidae: Macropus rufogriseus) X chromosome

The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.     

Characterization of heterochromatin by sequential counterstain-enhanced fluorescence in three domestic bird species: Meleagris gallopavo, Columba livia domestica, and Anser anser L

The karyotypes of three avian species--Meleagris gallopavo, Anser anser L., and Columba livia domestica--were investigated by means of counterstain-enhanced fluorescence techniques (chromomycin A3/distamycin A/DAPI followed by DAPI/actinomycin D staining). A heterochromatin characterization of macro- and microchromosomes was performed. CMA3-positive (GC-rich) regions in the turkey included the telomeres of chromosomes 1, 3, 4, and Z. In the goose, the chromosome 2 was also CMA3-bright at the telomeres. The W chromosome possessed large amounts of CMA3-bright material on the short arm in both the turkey and the goose. Two types of centromeric heterochromatin were distinguished on acro- to telocentric chromosomes 6 to 14 in the pigeon. The microchromosomal heterochromation of the turkey and goose was GC-rich but had a high degree of variation. In the pigeon, several microchromosomes possessed predominantly AT-rich heterochromatin.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

Chromosome banding studies in an Indian mullet: evidence of structural rearrangements from NOR locations

C-, G- and NOR bands have been studied in the female sex of Rhinomugil corsula. (Mugilidae, Pisces) by deploying the conventional methodologies with suitable modifications of minor nature. The diploid metaphase complements contained 48 acrocentric chromosomes. The localization of C-band heterochromatin was found to be mostly at or near the centromeric regions of the acrocentric chromosomes. The G-type bands were not so well defined, but some of the G-banded chromosomes also contained C-bands. Interestingly, silver-positive NORs were found at the telomeric ends of five acrocentric chromosomes, including one homologous pair having NORs in both chromatids, while one chromosome showed NORs in both of its chromatids and the other two had only one NOR localized at one of its chromatids. This would suggest that one homologue of the second pair of NOR-bearing chromosomes possibly underwent a chromatid exchange with a non-NOR bearing chromosome. This is quite a unique situation not reported earlier in any species of fish., though some other form of NOR-polymorphism/heteromorphism has rarely been reported. Therefore, further exploration in natural populations of this species to examine the other sex and to verify if there also exists other chromosomally polymorphic races (in respect of NOR-polymorphism) of this species, would be rewarding.     

大蹄蝠的核型分析

研究了大蹄蝠的核型、C-带和Ag-NORs。大蹄蝠的染色体数目是2n=32,NF=60,No.8染色体上有一明显的次缢痕,大蹄蝠有丰富的结构异染色质,主要以着丝粒带的形式存在;且有若干对染色体部分或全部异染色质化;一对Ag-NORs稳定地出现于No.8染色体。     

Distribution of silver-stained nucleolus-organizing regions in the chromosomes of the Equidae

The distribution of silver-stained nucleolus-organizing regions (NORs) in fibroblast chromosomes from all seven extant species of Equidae are described. There are variations in the number and locations of silver-stained NORs in different species but most of the cells in an individual of any speies had only 2 to 4 silver-stained NORs. In Equus przewalskii and E. caballus NORs were detected on up to three different chromosomes. In E. asinus 11 different chromosomes were observed to possess NOR sites. E. hemionus kulan and E. hemionus onager had NORs on 2 large metacentric pairs and a small acrocentric pair. In E. grevyi and E. burchelli NORs were located on 3 to 4 different pairs. E. zebra hartmannae had silver-staining over the telomere regions of the short arms of the chromosomes 1, 3 and 4. A comparison of G-banded chromosomes and silver-stained NORs has revealed one autosome with conserved G-band patterns and possessing a silver-staining NOR in all the species except E. asinus. Variations in the number and multichromosomal locations of NORs in various species could have evolved by pairing and exchanges between non-homologous chromosomal heterochromatin having similar satellite DNA sequences.     

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA(3) ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO (3) staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.     

Restriction enzyme and fluorochrome banding analysis of the constitutive heterochromatin of Saguinus species (Callitrichidae, Primates)

Metaphases of Saguinas fuscicollis fuscicollis and Saguinas mystax were subjected to restriction enzyme banding (Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I and Msp I) and sequenced C-banding, together with fluorochrome staining (CMA3 and DAPI). Both species showed large C-bands in the pericentromeric regions. S. f. fuscicollis also manifested distal C-bands in both arms of pair 5 and in the short arms of pairs 8-15. In each species the heterochromatin revealed different reactions to the restriction enzymes and fluorochromes. This was related to its location in the genome (centromeric, pericentromeric, distal), making possible the identification of distinct categories of constitutive heterochromatin. In S. f. fuscicollis there were at least five types, namely centromeric in bi-armed chromosomes, centromeric in acrocentrics, pericentromeric, distal, and cryptic bands, detected only with the Alu I. There were three types in S. mystax, viz centromeric in bi-armed chromosomes, centromeric in acrocentric, and pericentromeric chromosomes. Several aspects of their constitution and origin are discussed.     

Sequence of Centromere Separation: Role of Centromeric Heterochromatin

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.