Prometaphase banding of human chromosomes with basic fuchsin

Summary A technique is described for the production of detailed and richly contrasting G-band patterns in human prometaphase chromosomes with the aid of the triphenylmethane dye basic fuchsin. The usefulness of this method is illustrated by its application for the precise analysis of two chromosome 11 rearrangements. It is also demonstrated that high-resolution banding with basic fuchsin can reveal bands not present in the international standard idiogram of human prophase chromosomes (ISCN 1981). The technique described can also be used for easy recognition of the late replicating X chromosome, which stains darker than its early replicating homologue. A preliminary analysis of the late replicating X chromosomes in a 49,XXXXY individual suggests that the three supernumerary X chromosomes do not necessarily replicate synchronously.     

黑眉锦蛇的有丝分裂染色体和减数分裂联会复合体的观察

本工作以C带、硝酸银染色、对黑眉锦蛇(Elaphe taeniura)的有丝分裂染色体进行了显微观察。其二倍体染色体数目2n=36,核型组成为16(8m+6sm+2t)大染色体+20微小染色体。C带显现于几乎所有染色体的着丝粒区,有一对插入型C带位于第6对端着丝粒染色体。一个银染核仁组织区(NORs)位于No.12小染色体。同时以界面铺张——硝酸银染色技术,对黑届锦蛇减数分裂精母细胞联会复合体(SC)的结构进行了亚显微观察。发现黑眉锦蛇的SC结构与其他动物的SC相似,是由两股平行的侧线组成,SC组型与有丝分裂染色体组型有较好的一致性。     

Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes

Aldehyde fixation followed by staining with phosphotungstic acid produces differential contrast between the synaptonemal complex and the chromatin of maize pachytene bivalents. Centromeres, heterochromatic knobs and large chromomeres are easily recognised. With this and other staining techniques the nucleolus organizer region can be differentiated into two components. — Microsporocyte nuclei at pachytene were serially sectioned and all ten bivalents reconstructed in five nuclei. An idiogram was derived from the mean chromosome (= synaptonemal complex) lengths, the arm ratios, positions of knobs and the nucleolus organizer region. The idiogram agrees well with that published from light microscopic analyses. However, bivalent lengths are only two thirds of those observed by light microscopy of squash preparations. Many telomeres of the bivalents are connected via chromatin to the nuclear envelope, but a varying number of free bivalent ends are observed in all five reconstructed nuclei. — Bivalents heterozygous for inversion 3b were reconstructed. In the presence of abnormal chromosome 10 (K10) the lateral components of the synaptonemal complex of chromosome 3 formed a typical inversion loop, while in one of the nuclei having no K10 the two lateral components of the long arms of chromosome 3 remained unpaired in the region of inversion heterozygosity. The presence of K10, which increases crossing-over frequencies and promotes intimate pairing at the light microscopic level, was thus found to permit formation of complete synaptonemal complexes in the inverted region. The extra terminal portion of the K10 chromosome folded back on itself and formed a morphologically normal synaptonemal complex in this — possibly non-homologously paired — region. The chromatin of centromeres and knobs from different bivalents were sometimes found to fuse, but the synaptonemal complexes transversing the fused centromeres or knobs retained their individuality.     

水稻染色体G—带的研究

用改良的ASG法首次在籼稻(O.sativa subsp.indica)品种珍汕97和粳稻(O.subsp.iaponica)品种秀岭的有丝分裂染色体上显示了G-带,并作了相应的G-带核型分析。就同一材料来说,随着有丝分裂时期的推进,染色体上带纹数目逐渐减少。籼、粳亚种间相对应的同源染色体上G-带带纹特征彼此相似。讨论了水稻G-带带型与染色体不同区域分化的关系;G-带带型与籼、粳稻分歧的关系;以及G-显带的方法。     

Pachytene studies in 2n=14 species of Medicago

Four annual species of Medicago (M. constricta Dur., M. polymorpha L., M. praccox D.C. and M. rigidula [L.] All.) were analysed at pachytene and idiograms prepared. Considerable differences in chromosome lengths and arm ratios occurred between species. In three of the species the longest chromosome of the complement was much longer than the second chromosome in the karyotype. In this respect they were similar to 2n=14 M. murex Willd., and similar types of origins by rranslocations from 2n=16 ancestors are probable. In M. constricta chromosome 1 was not exceptionally long, and different kinds of translocations may have been involved in its origin.The M. constricta idiogram was most like the M. rigidula idiogram, and the M. praecox idiogram was most like the M. polymorpha idiogram. These similarities support present taxonomic classification. However, the great similarity of the M. polymorpha idiogram to that previously deseribed for 2n=14 M. murex was unexpected, as these two species are usually classified in different subsections of the genus. A closer relationship is suggested. Evidence was found in M. polymorpha of variations in chromosome contraction and differences in karyotype between accessions.Part of Ph.D. Thesis at Dept. of Genetics, University of Alberta, Edmonton, Alberta, Canada.     

A carmine-Giemsa staining technic for meiotic prophase chromosomes of the genus Beta L.

For a detailed study of chromosome morphology in meiotic prophase stages of Beta species, a special double staining technic has been developed. It consists of combined maceration-staining in an ethanol-hydrochloric acid-carmine mixture followed by poststaining of the squashed material in a diluted Giemsa solution. The technic yields well-spread prophase meiotic nuclei showing detailed structures both in weaker stained chromosome segments and in threadlike chromatin structures. This technic proved to be especially favorable for stages which are difficult to interpret, such as pachytene, schizotene and diffuse stages.     

甜瓜花粉母细胞减数分裂及雄配子体的细胞学研究

以薄皮和厚皮类型甜瓜为试材,采用改良的染色体制片方法,系统观察了甜瓜花粉母细胞的减数分裂及雄配子体发育的过程,结果表明:(1)甜瓜细胞核减数分裂的同步性较高,细胞质是同时型分裂,在细胞核分裂的过程中,核仁在前期Ⅰ到中期Ⅰ逐渐消失,在前期Ⅱ再次出现,随后消失,染色体在前期Ⅰ到中期Ⅰ逐渐收缩,变得清晰,至末期Ⅰ变得模糊,在前期Ⅱ再次清晰;(2)2种类型甜瓜终变期的染色体构型都以环状二价体为主;(3)在后期Ⅱ,观察到染色体的垂直和平行2种分离方式;(4)在前期Ⅰ和前期Ⅱ,伽师瓜"形成了多个较小的核仁,呈现一定的特殊性;(5)雄配子体发育经历了单核期和双核期,最后形成了成熟的花粉粒.研究表明,薄皮和厚皮类型甜瓜减数分裂的染色体行为基本一致,没有明显差异;伽师瓜"的核仁数量表现特殊可能与其长期的生态适应性有关.     

A Carmine-Giemsa Staining Technic For Meiotic Prophase Chromosomes Of The Genus Beta L

For a detailed study of chromosome morphology in meiotic prophase stages of Beta species, a special double staining technic has been developed. It consists of combined maceration-staining in an ethanol-hydrochloric acid-carmine mixture followed by poststaining of the squashed material in a diluted Giemsa solution. The technic yields well-spread prophase meiotic nuclei showing detailed structures both in weaker stained chromosome segments and in threadlike chromatin structures. This technic proved to be especially favorable for stages which are difficult to interpret, such as pachytene, schizotene and diffuse stages.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods

Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n=32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x=8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.     

An indirect test for a role of the synaptonemal complex in the establishment of sister chromatid cohesiveness

The meiotic behavior of a special maize trisome was quantitatively observed at pachytene, metaphase I, anaphase I, prophase II, metaphase II and anaphase II. The data obtained are consistent with (but do not prove) the model that sister chromatid cohesiveness at anaphase I may be established during pachytene synapsis of the chromosome regions involved. The data suggest, however, that the normal prophase II maintenance of dyad integrity by cohesiveness of sister chromatid centromere regions does not depend upon prior synapsis of these regions, although monads separated from each other on the anaphase I spindle may be delivered to the same prophase II daughter nucleus. — The strands which some of the time connect sister chromatids which are separating equationally at anaphase I show a positive Feulgen staining reaction.     

Prophase chromosome unique band sequences: definition and utilization

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. The feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole-chromosome recognition. Through the use of prophase idiograms at the 850-band stage (Francke, 1981) and a systematic comparison system, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences, each of which has a banding pattern that is recognizable and distinct from any other nonhomologous chromosome portion. The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information.     

Bcl-2 is an integral component of mitotic chromosomes

We previously demonstrated that phospho-Thr56 Bcl-2 colocalizes with Ki-67 and nucleolin in nuclear structures in prophase cells and is detected on mitotic chromosomes in later mitotic phases. To gain insight into the fine localization of Bcl-2 on mitotic chromosomes, we further investigated Bcl-2 localization by immunostaining of Bcl-2 with known components of metaphase chromosomes and electron microscopic immunocytochemistry. Immunofluorescence analysis on HeLa mitotic cells together with chromatin immunoprecipitation assays showed that Bcl-2 is associated with the condensed chromatin. Co-immunostaining experiments performed on mitotic chromosome spreads demonstrated that Bcl-2 is not localized on the longitudinal axis of chromatids with the condensin complex, but partially colocalizes with histone H3 on some regions of the mitotic chromosome. Finally, most of the Bcl-2 staining overlaps with Ki-67 staining at the chromosome periphery. Bcl-2 localization at the periphery and over the mitotic chromosome was confirmed by immunoelectron microscopy on mitotic cells.Our results indicate that Bcl-2 is an integral component of the mitotic chromosome.     

The fine structure of chickpea (Cicer arietinum L.) chromosomes as revealed by pachytene analysis

A standard pachytene karyotype of chickpea (Cicer arietinum L.) is presented for the first time. Individual pachytene chromosomes were identified and described in detail. An idiogram was prepared on the basis of chromosome length, arm ratio, and distribution of heterochromatin and euchromatin. Chickpea pachytene chromosomes belong to the differentiated type with darker staining heterochromatin proximal to and lighter staining euchromatin distal to the centromeres. Chromosomes were numbered from 1 to 8 following a descending order of length. The total length of the chromosome complement at pachytene was 335.33 , and chromosome size ranged from 58.05 to 30.53 .     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

Dense bodies in silver-stained spermatocytes of the chinese hamster: behavior and cytochemical nature

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, thread-like axes seen at midpachytene, and (3) multistranded, branched, and anastomosed axes seen at late pachytene.Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body.Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.     

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G₂-early prophase. The 250–400 nm thick compact chromatin threads in G₂ nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1–1.5 m) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120–250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.     

Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch

During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).