Heterogeneity of constitutive heterochromatin in somatic Syrian hamster chromosomes.

Syrian hamster constitutive heterochromatin was analyzed for C-band distribution and for BrU late-replication pattern. Characteristic for this species is relatively large amounts of sex-chromosome and autosomal heterochromatin. The distribution of constitutive heterochromatin was determined. The long term of the X chromosome, the whole Y, the short arms of 8 autosomal pairs, the long arm of the smallest metacentric pair, and the centromeric regions of 12 pairs stained intensely dark on C-band preparations. In contrast to the heterochromatin in the centromeric regions, the autosomal short-arm heterochromatin has an increased susceptibility to the denaturation process, as indicated by prolonged exposure to NaOH or Ba(OH)2. Such further exposure to denaturing agents results in an intense dark stain only on the sex-chromosome heterochromatin and centromeric regions of the autosomes. The BrdU late-replication pattern demonstrated that the late-replicating regions correspond to C-bands. Centromeric regions replicate late in the S phase; however, no centromeric region is among the latest replicating segments of the complement. Centromeric and noncentromeric heterochromatin are two distinct categories of constitutive heterochromatin.     

Chromosome banding homologies in Swamp and Murrah buffalo

Silver staining of Swamp buffalo (2n = 48) metaphase chromosomes revealed telomeric nucleolus organizer regions (NOR's) located on five pairs of autosomes identified by R-banding as numbers 4 p (submetacentric), 8, 20, 22, and 23 (acrocentrics); interphase nuclei also showed no more than five nucleoli. The Murrah buffalo (2n = 50) was previously reported to have telomeric NOR's located on six pairs, -3 p and 4 p (submetacentric), 8, 21, 23, and 24 (acrocentrics). By comparing the two types of buffalo it was concluded that: all of the chromosomes are similar in banding patterns; chromosome 1 of Swamp results from a telomere-centromere tandem fusion between two chromosomes identified as 4 p and 9, respectively, in the Murrah karyotype, thus accounting for the reduced diploid number of Swamp buffalo; the fusion causes the loss of NOR's on the telomeres of chromosome 4, thus accounting for the reduced number of NOR chromosome pairs of Swamp; the presence of a pale C-band are in the region of junction between chromosome 4 and 9 involved in the fusion suggests that the centromeric region of the later is retained and altered.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.     

Chromosome restriction enzyme digestion in domestic pig (Sus scrofa) constitutive heterochromatin arrangement

The bimodal karyotype of pig appears to contain two types of constitutive heterochromatin, reflecting different satellite DNA families: GC-rich heterochromatin located mainly in the centromeric regions of the biarmed chromosomes, and less-GC-rich heterochromatin in the centromeric regions of the one-armed chromosomes. In order to better discriminate this constitutive heterochromatin, we treated pig chromosome preparations with eight different restriction endonucleases, followed by C-banding. This technique allowed an expedited characterization of the constitutive heterochromatin and demonstrated its great heterogeneity in pig chromosomes. Our work allowed the detection and identification of twenty-two heterochromatin subclasses (twelve centromeric, four interstitial, five telomeric, and the Yq band). Moreover, several cryptic interstitial and telomeric bands were revealed. The work presented here is useful not only for fundamental studies of chromosome banding and constitutive heterochromatin, but also offers a new approach for pig clinical cytogenetics.     

New C-band polymorphism in the White Park cattle of Great Britain

A chromosome analysis, using G- and sequential Q- and C-banding, of 133 cattle distributed among seven rare breeds of cattle in Great Britain, showed a new C-band polymorphism of chromosome 27. The polymorphism was demonstrated by the absence of a centromeric block of heterochromatin and was only seen in the White Park breed. The polymorphic chromosome had a frequency of 0.446 and was present in either a heterozygous or homozygous state in 75 percent of the White Park cattle studied. The animals carrying the polymorphic chromosome have been traced back to three bulls, two of which have been widely used throughout the breed. It seems likely that the polymorphism arose in one common ancestor before 1949.     

Complex sex chromosome system in Eigenmannia sp. (Pisces,Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10.     

小熊猫染色体异染色质的显示

以培养的小熊猫外周淋巴细胞为实验材料,结合Ｃ－显带技术及ＣＭＡ３／ＤＡ／ＤＡＰＩ三竽荧光杂色的方法,对小熊猫的染色体组型、Ｃ－带带型及ＣＭＡ３／ＤＡ／ＤＡＰＩ荧光带带型进行了研究,发现：（１）经Ｃ－显带技术处理,可在小熊猫染色体上呈现出一种极为独特的Ｃ－带带型。在多数染色体上可见到丰富的插入Ｃ－带及端粒Ｃ－带。而着丝区仅显示弱阳性Ｃ－带;（２）除着丝粒区外,ＣＭＡ３诱导的大多数强荧光带纹与Ｃ－阳性     

The origin of mitotic sex-chromosome association in the brush-tailed possum, Trichosurus vulpecula (marsupalia:phalangeridae)

Nonrandom associations between the sex chromosomes of the brush-tailed possum, Trichosurus vulpecula, were found to be due to association of nucleolar organizer regions (NOR's) on the X and Y chromosomes. NOR association was also observed between an autosome and the X chromosome. These findings, based on silver staining, are in contrast to the report of MURRAY (1977), who observed sex-chromosome association in this animal and indicated that these nonrandom associations may reflect an association between heterochromatic regions during interphase. We observed only two pairs of NOR's per cell in this animal, one autosomal and one on the sex chromosomes, rather than the several such regions observed by MURRAY, who used an N-banding technique. We discuss the problem of nonhomologous chromosome association in mammalian cells as influenced by heterochromatin and NOR's and find little support for nonhomologous chromosome associations at mitotic metaphase due to heterochromatin association.     

Nonradioactive in situ nick translation combined with counterstaining: Characterization of C-band and silver positive regions in mouse testicular cells

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.     

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。     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Chromosome markers in Mus musculus: Differences in C-banding between the subspecies M. m. musculus and M. m. molossinus

Quinacrine (Q-band) and centromeric heterochromatin (C-band) patterns of metaphase chromosomes of two subspecies of Mus musculus were compared. M. m. musculus (the laboratory mouse) and M. m. molossinus (a subspecies from Southeast Asia) had similar Q-band patterns along the length of the chromosomes, but differences were observed in the centromeric region of some chromosomes. The two subspecies had very different distributions of C-band material. Antibodies to 5-methylcytosine were bound to regions of the chromosome corresponding to the C-bands in each animal. These findings support the idea that satellite DNA, which is concentrated in the C-band region, changes more quickly than bulk DNA. The interfertility of these two subspecies permits the development of a musculus strain carrying normal marker chromosomes for genetic studies.     

DNA replication,3H-cRNA in situ hybridization and C-band patterns in the polycentric chromosomes ofLuzula purpurea Link

DNA late-replication,³H-cRNA in situ hybridization, and C-band distribution patterns were studied inLuzula purpurea Link chromosomes (2n=6). With each technique it was possible to identify homologous chromosomes. DNA late-replicating regions were present at the ends and in the middle of one chromosome pair (pair 1), on both ends of another chromosome pair with one end having more late-replicating regions than the other end (pair 2), and all along the length of the final pair (pair 3). The distribution of label following in situ hybridization of³H-cRNA complementary to Cot 1-reassociated DNA was similar to the DNA late-replication patterns. One chromosome pair had grains concentrated at the ends and in the middle of the chromosomes; another pair had grains at both ends with a greater grain concentration at one end; the final chromosome pair had grains distributed all along the length. C-band distribution patterns were also similar to the DNA late-replication and³H-cRNA in situ-hybridized ones. The results demonstrate that the constitutive heterochromatin ofL. purpurea polycentric chromosomes is similar to the constitutive heterochromatin of monocentric animal chromosomes in that it consists of highly repeated DNA sequences which are replicated late in the S stage of interphase.     

Molecular cytogenetic evidence for amplification of chromosome-specific alphoid sequences at enlarged C-bands on chromosome 6.

We have characterized variant centromeric regions of chromosome 6 segregating in two families. The heteromorphism, 6ph+, stains negatively with G- and Q-banding and darkly with C-banding. The variant C-band regions measure two to three times the length of their homologues. The centromeric regions of the variant chromosome 6 and its homologue are not significantly elongated by adding 5-azacytidine to culture. We determined that the amount of the alphoid centromeric repeat 308 (DZ61), which is chromosome 6 specific, is amplified two- to threefold in the genomes of individuals with the 6ph+ variants. In situ hybridization localized the increase in 308 repeats to the 6ph+ region. These results suggest an association between amplification of chromosome-specific alphoid sequences and enlargement of specific C-band regions.     

The staining of constitutive heterochromatin,and A-T and G-C rich DNA in lymphocytes and primary spermatocytes of the Chinese hamster

The staining of male Chinese hamster chromosomes at meiotic prophase with several banding techniques is described. C-banding results only occasionally in well-differentiated pachytene and diakinesis bivalents. Meiotic C-bands are small compared with those in somatic metaphase chromosomes. In mice C-bands mainly consist of highly repetitive satellite DNA, whereas in Chinese hamsters the majority of the DNA in C-bands is not or hardly repetitive. Especially in Chinese hamsters both the degree of chromatin despiralisation and the folding pattern of the chromatin drastically reduce the distinction of C-bands in late meiotic prophasc chromosomes. In contrast to the situation in mice, C-heterochromatin associations are never observed in Chinese hamster spermatocytes. It is assumed that the presence of satellite DNA rather than constitutive heterochromatin is the basis for the associations of the paracentromeric chromosome regions in mice. The location and behaviour of AT- and GC-rich DNA in Chinese hamster primary spermatocytes is studied with base-specific fluorochromes (H 33258 and Chromomycin A₃ for AT-and GC-rich DNA respectively), in combination with a pretreatment with base-specific non-fluorescent antibiotics (Actinomycin D and Netropsin for GC-and AT-rich DNA respectively). No indications are found for the clustering of AT-or GC-rich DNA in Chinese hamster pachytene nuclei. A comparison of banding patterns observed in somatic metaphases and in diakinesis gives some information about the partial homology of the X and Y chromosome. The results are conflicting. The short arm of the Y chromosome is homologous with a part of the X chromosome. According to the C-band pattern the long arm of the X chromosome is involved in the pairing with Y, whereas fluorescence banding patterns indicate that it is the short arm of X.     

C-band morphology of T(8;9)/8;9 in Blattella germanica

Summary Centromere position and each arm of the T(8;9)/8;9 quadrivalent at late pachytene can be recognized by C-banding. The chromosome 9 breakpoint lies immediately adjacent to the centromeric C-band; that of 8, in the general region of the centromere but the relationship to centromeric bands was not determined since the latter stain very faintly. Chromosome 9 differs from no. 8 in the presence of a complex series of intercalary bands, heavy centromeric bands and, overall, a larger amount of C-band material. Possible implications of these differences with respect to chromosome breakability and the nature and distribution of mutant loci are noted. Earlier identification of adjacent-1 and adjacent-2 metaphase I ring orientations, made on the basis of orientation pattern, was confirmed.     

Synaptonemal complex analysis of hybrid cattle. III. Meiotic pairing mechanisms in F1 Brahman x Hereford hybrids

The mechanisms of homologous chromosome pairing were studied in synaptonemal complex (SC) spreads of F1 Brahman (Bos indicus) x Hereford (Bos taurus) cattle. The most common SC abnormalities were bivalents with partial pairing failure and interlocks. While C-band polymorphisms could underlie most of the SC abnormalities observed in the full-blood cattle, other causes seem also to be contributing in the hybrids. The pattern of the abnormalities indicates that genic differences between the species were probably involved. Pachytene substaging data suggest that in some spreads, early pachytene bivalents with partial pairing failure may achieve complete synapsis or may be converted to interlocks by late pachytene.     

Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids

Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p 93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.