DNS-Replikation und Morphologie der X-Chromosomen während der Syntheseperiode bei Microtus agrestis

X-chromosome behaviour of female Microtus agrestis in interphase nuclei with and without large chromocenters was examined in cultured epithelial and fibroblast cells. By means of pulse-labeling, the configuration of the X-chromosomes in these nuclei can be illustrated; staining with pararosaniline-methylgreen seems to be most suitable. According to the replication behaviour, three types of chromatin can be discerned in the X-chromosomes: early replicating euchromatin, late replicating sex chromatin, and very late replicating heterochromatin. In fibroblasts only the sex chromatin forms a single, small chromocenter; in epithelial cells both the sex chromatin and the remaining heterochromatin form large chromocenters. Both types of heterochromatin replicate their DNA in the condensed state. It seems likely that the late replicating segments of the X-chromosomes (sex chromatin and remaining heterochromatin) are condensed in every cell, but they may not always be configurated or even visible as typical chromocenters; these segments could be distributed over a wide range of compact to more or less diffuse superstructures.     

Cloning and characterization of human and mouse SNRK sucrose non-fermenting protein (SNF-1)-related kinases

Topography of three genetic elements--dystrophin (dmd) exons 5-7 (E(1)), 46-47 (E(2)), and centromere of chromosome X (N(X)) were studied relative to cell nuclei and to chromosome X territories of spatially fixed human lymphocytes. Repeated three-dimensional (3D) dual color fluorescence in situ hybridization combined with high-resolution cytometry was used. In addition, the nuclear location of fluorescence weight centers (FWC), spatial volume, and maximal area per one section of chromosome-X territories were investigated. The larger (X(L)) and smaller (X(S)) homologous X-chromosomes were distinguished for each nucleus according to the 3D volume of their territories. The distributions of the [center of nucleus]-to-[genetic element] distances (radial distributions) of dmd exons E(1), E(2), centromere N(X) and FWC were very similar for both homologous X-chromosomes of female lymphocytes as well as for the chromosome X of the human male. On the other hand, larger average mutual distances between all pairs of signals (E(1), E(2), N(X), FWC) and larger average maximal area were observed for the larger chromosome (X(L)) in comparison with the smaller one (X(S)). The territory of the larger homologue showed also more irregular surface. The most significant differences between homologous X-chromosomes were found for N(X)-E(1), N(X)-E(2) and E(1)-E(2) distances that were in average about twice longer for X(L) as compared with X(S). These parameters correlate to each other and can be used for the reliable determination of more (de)condensed X-chromosome territory. The longer E(1)-E(2) distances for X(L) indicate more open chromatin structure of the dystrophin gene on this chromosome in contrary to closed structure on X(S). Substantially shorter distances of the dystrophin exons from the centromeric heterochromatin in X(S) as compared to X(L) can be explained by silencing effect of centromeres as described in Nature 1 (2000) 137.     

玉米rRNA基因的组织和表达模式

玉米(Zea mays)只有1对45S rDNA位点并在分裂期染色体形成次缢痕, 是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交(fluorescence in situ hybridization, FISH)、CPD(PI与DAPI组合)染色和银染技术, 分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号: 荧光强烈地位于核仁周边的纽, 而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出; 点的数目越多, 纽变得越小; 点的数目多少与细胞的活性呈正相关。研究结果表明, 纽代表了处于凝缩状态的非活性的rDNA染色质, 纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现; 不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示, 大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示, 同一间期细胞的2个同源rDNA位点的表达水平存在差异, 同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示, 早中期细胞的rDNA染色质相对解凝缩, 银染在所有早中期细胞和部分中期细胞显示了明显的核仁, 表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录, 其转录在晚中期才停止。     

玉米rRNA基因的组织和表达模式

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号：荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。     

Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro

Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera. Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy. Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium. The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion. The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape. On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type. These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped. At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters. The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm. The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria. Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes. Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads. These beads are doubtless interphase chromomeres.     

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome α-satellite probes to interphase cells of the various genotypes: the α-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation. Electronic Publication     

Spatial Distributions of Early and Late Replicating Chromatin in Interphase Chromosome Territories

The surface area of chromosome territories has been suggested as a preferred site for genes, specific RNAs, and accumulations of splicing factors. Here, we investigated the localization of sites of replication within individual chromosome territories.In vivoreplication labeling with thymidine analogues IdUrd and CldUrd was combined with chromosome painting by fluorescentin situhybridization on three-dimensionally preserved human fibroblast nuclei. Spatial distributions of replication labels over the chromosome territory, as well as the territory volume and shape, were determined by 3D image analysis. During late S-phase a previously observed shape difference between the active and inactive X-chromosome in female cells was maintained, while the volumes of the two territories did not differ significantly. Domains containing early or mid to late replicating chromatin were distributed throughout territories of chromome 8 and the active X. In the inactive X-chromosome early replicating chromatin was observed preferentially near the territory surface. Most important, we established that the process of replication takes place in foci throughout the entire chromosome territory volume, in early as well as in late S-phase. This demonstrates that activity of macromolecular enzyme complexes takes place throughout chromosome territories and is not confined to the territory surface as suggested previously.     

Condensed chromatin surface and NORs surface enhancement in mitogen-stimulated lymphocytes of Down syndrome patients

Mitogen-stimulated lymphocytes of 20 Down syndrome (DS) patients with regular trisomy 21 contain more condensed chromatin surface (11.28 +/- 2.64 % of the total nuclear surface: mean +/- SD) and more nucleolus organiser regions surface (13.21 +/- 3.45 %) than that of 12 healthy controls: (8.84 +/- 2.23 and 9.12 +/- 2.33 %, reciprocally). The source of this peculiarity has been investigated. A computer program was designed for the planimetric measurement of the condensed chromatin surface (CCs)/ total nuclear surface(TNs) and the nucleolus organiser regions surface (NORss) /TNs proportions in interphase nuclei. CCs/TNs and NORss/TNs of 100 maximally activated nuclei (MANs) were measured for each patient and control case. The difference was found highly significant (P<0.01). Nuclei with a diameter of >/= 17 micrometer measured on the slide (in flattened state) were considered as maximally activated nuclei (MANs). NORss/TNs enhancement and fluorescent in situ hybridisation (FISH) studies in MANs of DS patients indicate that this phenomenon is due to the over-expression (or lack of downregulative mechanism) of NORs (rDNA) to some extent, including the NOR of the supernumerary chromosome 21. No statistical difference was observed between 12 healthy controls and 5 Robertsonian translocation type of DS Patients (where the two involved NORs are missing) when the two parameters were considered.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Chromatin structure of active and inactive human X chromosomes

Nuclei from a variety of human cell lines and tissues were digested with gradually increasing levels of DNase I. The DNA was then purified, treated with restriction enzymes and subjected to Southern blot hybridization using a cloned cDNA probe to 3-phosphoglycerate kinase (PGK) a housekeeping enzyme. At relatively high levels of DNase I, a specific, slightly sensitive site in chromatin sequences encoding PGK was observed in all of the cell types examined. This slightly sensitive site resides on the active X-chromosome since cell lines with increased numbers of inactive X-chromosomes do not show an increase in the region of chromatin which is sensitive. Except for this restricted region of enhanced sensitivity on the active X-chromosome, the data suggest that, for PGK encoding sequences, chromatin configurations on the active and inactive X-chromosomes are similar.     

Distinct patterns of histone methylation and acetylation in human interphase nuclei

To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.     

Spätreplizierendes X-chromosom und Sexchromatinhäufigkeit beim Menschen

Zusammenfassung In Fibroblasten- und Leukocytenkulturen von normalen, gesunden Frauen wurde die Häufigkeit eines spätreplizierenden X-Chromosoms mit Hilfe der³H-Thymidinmarkierung untersucht. In den Fibroblastenkulturen wurde außerdem die Sexchromatinhäufigkeit in Interphasekernen bestimmt.In allen untersuchten Mitosen sowohl der Fibroblasten- (94 Metaphasen) als auch der Leukocytenkulturen (62 Metaphasen) konnte ein spätreplizierendes X-Chromosom nachgewiesen werden. Die Interphasekerne der Fibroblasten, die von derselben Passage stammten, an der auch die Untersuchungen über die Häufigkeit eines spätreplizierenden X-Chromosoms durchgeführt wurden, zeigen eine Sexchromatinhäufigkeit von durchschnittlich 54%.Die Ergebnisse bedeuten daß in weiblichen Zellen stets eines der beiden X-Chromosomen spätreplizierend ist. Dies gilt insbesondere auch für die weiblichen Zellen, in denen in der Interphase kein Sexchromatin nachweisbar ist. Daraus folgt, daß die Spätreplikation des heterochromatischen X-Chromosoms nicht notwendigerweise mit der Heteropyknose desselben verbunden ist.

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Late-replicating X-chromosome and sex chromatin frequency in man

Summary The frequency of the late-replicating X-chromosome was investigated with the aid of³H-thymidine in fibroblast- and leucozyte cell cultures derived from normal human females. In the fibroblast cell cultures also the sex chromatin frequency in interphase nuclei was determined.A late-replicating X-chromosome was found in all of the investigated metaphases of either the fibroblast (94 metaphases) or leucozyte (62 metaphases) cell cultures. The interphase nuclei of the fibroblasts, derived from the same passage from which the studies on the frequency of the late-replicating X-chromosome were performed, indicate an average sex chromatin frequency of 54%.The results imply that in female nuclei one of the two X-chromosomes is constantly latereplicating. This is also valid for those female cells, which show no sex chromatin in interphase. Hence it follows that the late-replication of the heterochromatic X-chromosome is not necessarily correlated with its heteropycnosis.

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Der Medizinischen Fakultät der Justus Liebig-Universität Gießen als Dissertation vorgelegt.     

Mechanisms of chromosome banding : VI. Whole mount electron microscopy of banded metaphase chromosomes and a comparison with pachytene chromosomes

Chinese hamster chromosomes, banded by exposure to actinomycin D during the G 2 period, were examined by whole mount electron microscopy. Bands of condensed chromatin were present in unstained preparations that were not fixed with methanol-acetic acid indicating that the differential condensation of chromatin plays a role in banding by this technique. There was a tendency for interdigitation of the chromatin of the homologous bands on sister chromatids. Since previous studies had shown that the bands of mitotic chromosomes matched the chromomeres of meiotic chromosomes, whole mount electron microscope preparations of pachytene chromosomes were also examined. These suggest that in addition to condensation the chromatin of the chromomeres may also have a higher density of attachment sites to the lateral element of the synaptonemal complex, and probably to the nuclear membrane in interphase cells.