Absence of true interchromosomal connectives in microsurgically isolated chromosomes

Mitotic chromosomes of the Indian muntjac were isolated from cultured fibroblast-like cells by microsurgery. The entire complement of seven chromosomes could be obtained with the radial array of chromosomes on the mitotic spindle intact. The center of the radial array was occupied by a fibrous network which stained with tubulin antiserum. This network was absent when cells were treated with colchicine or vinblastine sulfate prior to chromosome isolation, and probably represents a remnant of the mitotic spindle. Most isolated chromosomes were connected to the spindle by fibers attached to the centromeres. Such fibers did not stain for DNA and were resistant to DNases but sensitive to proteases. No interconnections were found to run from chromosome to chromosome except for occasional artifactual adhesions resulting from collisions between chromosomes which occurred during micromanipulation. We therefore found no evidence that chromosomes of the Indian muntjac are interconnected at mitosis.     

Direct evidence for the non-random localization of mammalian chromosomes in the interphase nucleus

Indirect immunofluorescence staining with human anti-centromere autoantibodies from a patient (LU 851) suffering from the CREST form of scleroderma was used to analyse chromosome topology in interphase nuclei of rat-kangaroo (PTO) and Indian muntjac (IM) cells. In some cells, centromeres were arranged in pairs suggesting association of homologous chromosomes. Clustering of centromeres at one pole of the nucleus (Rabl configuration) and other patterns suggesting higher order organization were also observed. In one fifth of the IM cells it was possible to identify the intranuclear location of each single chromosome on the basis of the morphology of the immunostained centromeres. In 30% of the IM cells in which centromeres could be identified, homologous chromosomes occupied adjacent territories within the interphase chromatin.     

大麦G—显带核型的研究

本文报道了 ASG 法处理的三个栽培大麦(Hordeum Vulgare)品种 G-带的核型研究。结果表明无论是早中期或中期染色体都显示出了密切邻近的、多重的 G-带带纹。在有丝分裂过程中染色体愈浓缩带纹数目愈少。同源染色体之间带纹分布的位置、染色深浅以及带纹数目都基本一致,可以较为准确地进行配对。同一分裂时期不同染色体的 G-带带纹各具一定的特点,可以作为鉴别的标记。讨论了显带技术和中期染色体的 G-带等问题。     

Studies on G-Banding Karyotype in Barley (Hordeum vulgare)

G-banding karyotypes of three cultivars in barley were analyzed. Multiple closely adjacent G-bands were able to be observed in each early metaphase or metaphase chromosome treatted by an ASG method. The more concentrated the chromosome, the less was the number of G-bands during mitosis. The position of band distribution, staining degree and band numbers between homologous chromosomes were basically identical. Chromosome pairing for karyotype analysis could be carried out more accurately. G-banding patterns of different chromosome pairs were not the same, they could be used as the markers to distinguish one from another chromosome pair. During the same mitotic stage the banding patterns including number, relative position and staining degree of the bands between different cultivars were basically the same, but they had differences in the size and staining degree of some bands near centromeres. G-banding technique and G-banding of metaphase chromosomes were discussed.     

Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division

Chromosome segregation requires centromeres on every sister chromatid to correctly form and attach the microtubule spindle during cell division. Even though centromeres are essential for genome stability, the underlying centromeric DNA is highly variable in sequence and evolves quickly. Epigenetic mechanisms are therefore thought to regulate centromeres. Here, we show that the 359-bp repeat satellite III (SAT III), which spans megabases on the X chromosome of Drosophila melanogaster, produces a long noncoding RNA that localizes to centromeric regions of all major chromosomes. Depletion of SAT III RNA causes mitotic defects, not only of the sex chromosome but also in trans of all autosomes. We furthermore find that SAT III RNA binds to the kinetochore component CENP-C, and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. In conclusion, our data reveal that SAT III RNA is an integral part of centromere identity, adding RNA to the complex epigenetic mark at centromeres in flies.     

Light and electron microscopy of rat kangaroo cells in mitosis

Chromosome orientation and behavior during prometaphase of mitosis in PtK₁ rat kangaroo cells were investigated by cinémicrography and electron microscopy. The first chromosome movements occur soon after the nuclear envelope begins to break down in the region near each pole. Initial chromosome behavior is primarily determined by the distance from the kinetochore region to the spindle poles. The predominant pattern is a movement to and/or association with the proximal pole. Movement to and association with the more distant pole, or direct alignment at or near the spindle equator (direct congression) are less frequent patterns. Except for rare cases, pole-associated chromosomes congress sooner or later and most congressed chromosomes oscillate about the equator. — Ultrastructural observations suggest that pole-associated chromosomes are oriented only to the proximal pole (monotelic or syntelic orientation) and they demonstrate that the sister-kinetochores of congressing or oscillating chromosomes are oriented to opposite poles (amphitelic orientation). — Based on the structure of the early prometaphase spindle and four assumptions concerning the formation of kinetochore fibers and their force-producing interaction with complementary elements, the different patterns of chromosome behavior observed can be explained as a result of synchronous or asynchronous formation of sister-kinetochore fibers. The few chromosomes whose kinetochore region is approximately equidistant from the poles amphi-orient immediately because their sister-kinetochores form fibers synchronously and they congress directly because of the bidirectional forces to which they are subjected. The kinetochore region of most chromosomes is not equidistant from the poles. Therefore, they form a functional fiber first to the nearer pole and move to, or associate with, it because of the unidirectional force. Eventually, however, these chromosomes achieve amphitelic orientation and congress. Once established, amphitelic orientation is stable. Re-orientations do not occur during congression or oscillatory movements.     

Comparative Chromosome Painting in Mammals: Human and the Indian Muntjac (Muntiacus muntjak vaginalis)

We have used human chromosome-specific painting probes forin situhybridization on Indian muntjac (Muntiacus muntjak vaginalis,2n= 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.     

Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei

Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G₁-phase of the cell cycle exhibit a distinctly non-random chromosome organization: centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G₁ to G₂ cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G₂ nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.by L. Manuelidis     

Localization of the repetitive telomeric sequence (TTAGGG)n in two muntjac species and implications for their karyotypic evolution

It has been suggested that the chromosome set of the Indian muntjac, Muntiacus muntjak vaginalis (female, 2n = 6; male, 2n = 7), evolved from small acrocentric chromosomes, such as those found in the complement of the Chinese muntjac, M. reevesi (2n = 46), by a series of tandem fusions and other rearrangements. The location of the highly conserved human telomeric sequence (TTAGGG)n in the metaphase chromosomes of M.m. vaginalis and its close relative, M. reevesi, was investigated by non-radioactive in situ hybridization. The (TTAGGG)n repeat was found adjacent to the centromeres in the short arm and at the telomeres in the long arm of M. reevesi acrocentric metaphase chromosomes. Tandem fusions present in the karyotype of M.m. vaginalis chromosomes were not reflected by interstitial signals of the telomere repeat, as these chromosomes displayed hybridization signals only at the ends of the chromatids. Mechanisms that might have played a role in the evolution of the reduced karyotype of the Indian muntjac are discussed.     

Molecular cytogenetic analysis of intergeneric chromosomal translocations between wheat (Triticum aestivum L.) and Dasypyrum villosum arising from tissue culture.

Fluorescence in situ hybridization (FISH) was applied with total genomic DNA extracted from Dasypyrum villosum (L.) Candargy as a probe to characterize chromosome translocations arising from tissue culture in hybrids of Triticum aestivum x (T. durum - D. villosum, amphiploid). Chromosome translocations between wheat and D. villosum occurred in callus cells at an average frequency of 1.9%. Translocations existed not only in callus cells but also in regenerants. Three plants with translocation chromosomes were characterized among 66 regenerants of T. aestivum 'Chinese Spring' x 'TH1W' and 'NPFP' x 'TH1'. One of them proved to be a reciprocal translocation with an exchange of about one third of a wheat chromosome arm with about one half of a chromosome arm of D. villosum. The breakpoints of the other two translocations were located at, or near centromeres. The results are similar for both callus cells and regenerants and provide further evidence that translocations take place in tissue culture. Other structural chromosomal changes, for example, fragments, telocentrics, dicentromeres, and deletions, as well as numerical alterations including aneuploidy and polyploidy were recorded both in callus cells and regenerants.     

几种动物染色体超微结构的研究

应用表面舒展技术、原位培养表面舒展技术和临界点干燥以及空气干燥等方法制备染色体标本,用FESEM和SEM观察了CHO、IB-RS-2哺乳动物细胞以及黄鳝肾细胞和鲫鱼血淋巴细胞的染色体。看到了染色体处于不同舒展状态的染色质纤维。在染色质纤维未完全展开排列紧密时,染色体臂的染色质纤维,缠绕排列有序,垂直于染色体纵轴,螺旋盘绕形成疏密程度不同的横纹。在纤维较为松散和完全松敌的状态下,可以看见直径约为300(?)的染色质纤维从有序到不完全有序到无序,弯扭、螺旋、缠绕,有些似“辐射环”状结构。在着丝点处可清楚地看到有二条纤维平行分别通连二染色单体臂,未见有染色体膜。初步比较了鱼类和哺乳类的染色质纤维,二者基本一致,但鱼类染色质纤维排列较哺乳动物的松散,类似“辐射环”状的结构较为明显。     

The effect of immobilized fibronectin on karyotypic variability in the skin fibroblast cell subline of indian muntjac

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblast cell subline MT on cultivating cells on the fibronectin-coated surface. In cell subline MT, cultivated on the fibronectin-coated surface for 1 and 2 days, the character of cell distribution for the chromosome number did not change. In 3, 4 and 8 days, the character of cell distribution for the chromosome number changed. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population. Detachment of cells from the fibronectin-coated surface, followed by a 1 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for the chromosome number, but cultivation in these conditions for 5 days restore control distribution. The frequency of chromosomal aberrations on cultivation on the fibronectin-coated surface for 3 and 4 days significantly increases, mainly at the expence of dicentrics (telomeric association). On prolongating the time of cultivation up to 8 days on the fibronectin-coated surface the frequency of chromosomal aberrations approaches the control value. Structural instability of chromosomes at cultivation on the fibronectin-coated surface demonstrates nonspecific reaction of "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability in cell lines of the Indian muntjac skin fibroblasts on cultivating on laminin and fibronectin.     

甜菜夜蛾细胞分裂期染色体的观察

以精巢组织为材料,采用空气干燥法制备染色体标本,对甜菜夜蛾Spodopteraexigua有丝分裂和减数分裂染色体形态行为进行了研究。结果表明：甜菜夜蛾的染色体数目为n=31;染色体为弥散着丝粒染色体,2条染色体上存在次缢痕;晚偶线期出现染色体互锁现象;从早粗线期到晚粗线期联会复合体逐渐伸长;终变期同源染色体形成环状、端部交叉、尾尾相对的结构。     

Nuclear and territorial topography of chromosome telomeres in human lymphocytes

Nuclear and territorial positioning of p- and q-telomeres and centromeres of chromosomes 3, 8, 9, 13, and 19 were studied by repeated fluorescence in situ hybridization, high-resolution cytometry, and three-dimensional image analysis in human blood lymphocytes before and after stimulation. Telomeres were found on the opposite side of the territories as compared with the centromeres for all chromosome territories investigated. Mutual distances between telomeres of submetacentric chromosomes were very short, usually shorter than centromere-to-telomere distances, which means that the chromosome territory is nonrandomly folded. Telomeres are, on average, much nearer to the center of the cell nucleus than centromeres; q-telomeres were found, on average, more centrally localized as compared with p-telomeres. Consequently, we directly showed that chromosome territories in the cell nucleus are (1) polar and (2) partially oriented in cell nuclei. The distributions of genetic elements relative to chromosome territories (territorial distributions) can be either narrower or broader than their nuclear distributions, which reflects the degree of adhesion of an element to the territory or to the nucleus. We found no tethering of heterologous telomeres of chromosomes 8, 9, and 19. In contrast, both pairs of homologous telomeres of chromosome 19 (but not in other chromosomes) are tethered (associated) very frequently.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.     

DNA duplication and chromosome structure in the Dinoflagellates

Summary Members of theDinopbyceae are characterized by having permanently condensed chromosomes throughout the cell cycle. At interphase the chromosomes appear to have bands perpendicular to the long axis of the chromosome with a periodicity of 127 nm. Each band is composed of 2.5 nm fibers and 9.0 nm granules coiled into a helix around a central core of 9.0 nm fibers. Chromosome uncoiling has been correlated with the uptake of³[H]-thymidine. As chromosomes enter the uncoiling phase of the cell cycle they appear less dense and reveal a number of fibrous extensions. At later stages chromosomes completely uncoil into elongate fibers 127 nm in width. Chromosome unwinding corresponds to the peak in the uptake of³[H]-thymidine. Chromosomes observed on either side of the peak possess the typical interphase banding. This study demonstrates, for the first time, the fine structural details of chromosome uncoiling during a specific phase of the cell cycle. A new model of the Dinoflagellate chromonema has been derived from this study.     

Amplification of the major satellite DNA family (FA-SAT) in a cat fibrosarcoma might be related to chromosomal instability

Most mammalian chromosomes have satellite DNA sequences located at or near the centromeres, organized in arrays of variable size and higher order structure. The implications of these specific repetitive DNA sequences and their organization for centromere function are still quite cloudy. In contrast to most mammalian species, the domestic cat seems to have the major satellite DNA family (FA-SAT) localized primarily at the telomeres and secondarily at the centromeres of the chromosomes. In the present work, we analyzed chromosome preparations from a fibrosarcoma, in comparison with nontumor cells (epithelial tissue) from the same individual, by in situ hybridization of the FA-SAT cat satellite DNA family. This repetitive sequence was found to be amplified in the cat tumor chromosomes analyzed. The amplification of these satellite DNA sequences in the cat chromosomes with variable number and appearance (marker chromosomes) is discussed and might be related to mitotic instability, which could explain the exhibition of complex patterns of chromosome aberrations detected in the fibrosarcoma analyzed.     

CENP-G: a new centromeric protein that is associated with the α-1 satellite DNA subfamily

A new constitutive centromere-specific protein (CENP) has been identified as a result of its recognition as an autoantigen by serum from a patient with gastric antral vascular ectasia disease. Conventional immunoblotting and two-dimensional double blotting with both this antiserum and a known anti-centromere antiserum showed that this antiserum predominantly recognized a M _r 95,000 protein that is different from all known CENPs. We have named this new protein CENP-G. This protein was detected at the centromeric region throughout the cell cycle. In mitosis, it was restricted to the kinetochore inner plate as shown by immunogold labeling and electron microscopy. The centromeres of some human chromosomes are known to contain two subfamilies of α-satellite DNA. Using immunofluorescence combined with fluorescent in situ hybridization with subfamily-specific DNA probes, we revealed that CENP-G was specifically associated with one of the subfamilies, which we have named α-1, but not the other. The localization and the α-1-specific association suggested that CENP-G may play a role in kinetochore organization and function. Like CENP-B and C, but unlike CENP-A, this protein remained with the nuclear matrix after intensive extraction. While CENP-B is absent from the human Y chromosome, the existence of CENP-G on the Y chromosome has been proven by immunofluorescence and whole chromosome painting. CENP-G was also detected in CHO, Indian muntjac and Chinese muntjac cells, suggesting that it is conserved in evolution. Received: 23 March 1998 / Accepted: 2 April 1998     

Assignment of rainbow trout linkage groups to specific chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.