Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.     

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.     

Deoxyribonucleic acid synthesis in isolated polytene nuclei of Drosophila hydei

DNA synthesis has been studied in polytene nuclei isolated from larval salivary glands of Drosophila hydei. The incubation conditions employed promote maximum incorporation of TTP-H³ and retention of normal polytene chromosome morphology. The chromosome structure is sensitive to the Mg²⁺ concentration; a normal banding pattern is observed between 4 and 10 mM Mg²⁺. At the optimum pH of 7.8, incorporation continues for over an hour. All four deoxyribonucleoside triphosphates are required for maximum incorporation. The reaction is stimulated by 0.6 mmATP and strongly inhibited at higher ATP concentrations. Competition experiments demonstrate that either TDP or TTP is the effective labeled precursor. The labeled product is sensitive to DNase and has a density identical to that of nuclear DNA. Autoradiographs prepared from spread chromosomes demonstrate that discontinuous and continuous labeling patterns observed in vivo are also produced with isolated nuclei in the absence of cytoplasmic factors. Incubation of the isolated nuclei results in a low level of uniform incorporation that is superimposed on the normal autoradiographic pattern obtained after in vivo labeling. This background incorporation can be greatly increased by prior irradiation of the glands. The presence of exogenous DNA during nuclear incubation stimulates total incorporation. These observations demonstrate that the isolated nuclei possess a reserve synthetic capacity. About 20% of the isolated nuclei are inactive in DNA synthesis.This investigation was supported by PHS Research Grant No. 5 R01 GM 16298 from the National Institute of General Medical Sciences.     

Sex chromatin and nucleolar analyses inRumex acetosa L.

Summary Rumex acetosa (sorrel) is a dioecious plant with a XX/XY₁Y₂ sex chromosome system. Both the Y chromosomes are nearly entirely heterochromatic and it has been hypothesised that they can persist as chromocenters in male interphase nuclei. Using specific antibodies against 5-methylcytosine and histone H4 acetylated at terminal lysine 5, global levels of DNA methylation and histone acetylation were studied on the sex chromosomes and autosomes of both sexes. The heterochromatic Y chromosomes did not display a higher methylation level compared to the autosomes. The only prominent hypermethylation signals were found at two nucleolar organising regions located on the autosome pair V, as confirmed by in situ hybridisation with 25S rDNA probe and staining. Immunoanalysis of DNA methylation on female and male interphase nuclei neither revealed any sex-specific differences. Two active (silverpositive) nucleoli and two likely inactive nucleolar organising regions (displaying prominent methylation signals) were found in both sexes. In a fraction of nuclei isolated from leaf cells, two peripheral bodies strongly positive for 4,6-diamidino-2-phenylindole were observed only in males, never in females. These heterochromatin regions were depleted in histone H4 acetylation at terminal lysine 5 and corresponded, according to in situ hybridisation with a Y-chromosome-specific repetitive probe, to the two Y chromosomes. We conclude that the peripheral condensed bodies observed exclusively in male nuclei represent the constitutive heterochromatin of the Y chromosomes which is characterised by a substantial histone H4 underacetylation.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.     

Polysaccharides associated with chromosomes and their behavior in the cell cycle

Summary The interphase nuclei, especially of the latest stages (G₂ or early prophase), in the mouse and rat livers were stained blue in the histochemical demonstration of acidic polysaccharide according to the method of Mowry, while the mitotic chromosomes (meta-, ana- and telophase) in the livers, sea urchin embryos as well as root tips of broad beans were stained red, suggesting the presence of neutral polysaccharide. The giant polytenic interphase chromosome of the salivary gland of Chironomus larvae was stained blue in the puffing and nucleolar regions while stained red in the condensed part of the chromosome. ³H-Glucosamine as well as ³H-glucose incorporations into the regenerating rat liver nuclei reached a peak at 30 h after partial hepatectomy when the highest mitosis is seen. These results suggest that the nuclear acid mucopolysaccharide present in the swollen chromosomes may be converted to or replaced with the neutral polysaccharide in the condensed chromosomes such as mitotic chromosomes or polytenic giant interphase chromosomes.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

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.     

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.     

Actin is a Constituent of the Nuclear Matrix and Chromosome Scaffold in Physarum poly cephalum

Nuclear matrices and chromosome scaffolds were obtained by digesting and extracting, respectively with DNase Ⅰ and 2 mol/L NaC1, the nuclei and chromosomes isolated from the plasmedia of Physarum polycephalum Schw. The results of the indirect immunofluorescence of tacit antiactin antibody as immunomarker indicated that the nuclear matrices and chromosome scaffolds both had positive reaction with the antibody. The results of the indirect immunodotting experiment further verified the presence of actin antibody in their constituent. Protein A-colloidal gold immunoelectron microscopy technique revealed that gold particles were distributed in the interphase nuclei and metaphase chromosomes. The above results showed that actin is a constituent of the nuclear matrix and chromosome scaffold of P. polycephalum.     

肌动蛋白是多头绒泡菌细胞核骨架和染色体骨架的组成成分

自多头绒泡菌(Physarum polycephalum Schw.)的原质团中分离细胞核和染色体,分别经DNaseⅠ消化和2 mol/L NaCl抽提后制备成细胞核骨架和染色体骨架。以抗肌动蛋白的抗体作一抗、FITC标记的羊抗兔IgG抗体作二抗进行的间接免疫荧光实验结果显示,细胞核骨架和染色体骨架都分别与抗体呈阳性反应。间接免疫斑点印迹实验结果进一步证实,细胞核骨架和染色体骨架的蛋白质成分中存在与肌动蛋白抗体呈阳性显色反应的抗原。以抗肌动蛋白的抗体作一抗、金颗粒标记的蛋白A作二抗的间接免疫电镜实验结果表明,在实验组间期细胞核的核仁、集缩染色质和核基质以及中期染色体上都有很多金颗粒分布。上述结果证明,肌动蛋白是多头绒泡菌细胞核和染色体及其骨架的组成成分。     

Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction.

While it is known that all chromosomes are susceptible to meiotic nondisjunction, it is not clear whether all chromosomes display the same frequency of nondisjunction. By use of multicolor FISH and chromosome-specific probes, the frequency of disomy in human sperm was determined for chromosomes 1, 2, 4, 9, 12, 15, 16, 18, 20, and 21, and the sex chromosomes. A minimum of 10,000 sperm nuclei were scored from each of five healthy, chromosomally normal donors for every chromosome studied, giving a total of 418,931 sperm nuclei. The mean frequencies of disomy obtained were 0.09% for chromosome 1; 0.08% for chromosome 2; 0.11% for chromosome 4; 0.14% for chromosome 9; 0.16% for chromosome 12; 0.11% for chromosomes 15, 16, and 18; 0.12% for chromosome 20; 0.29% for chromosome 21; and 0.43% for the sex chromosomes. Data for chromosomes 1, 12, 15, and 18, and the sex chromosomes have been published elsewhere. When the mean frequencies of disomy were compared, the sex chromosomes and chromosome 21 had significantly higher frequencies of disomy than that of any other autosome studied. These results corroborate the pooled data obtained from human sperm karyotypes and suggest that the sex chromosome bivalent and the chromosome 21 bivalent are more susceptible to nondisjunction during spermatogenesis. From these findings, theories proposed to explain the variable incidence of nondisjunction can be supported or discarded as improbable.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindle sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G₂ nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.Key words: mitotic chromosomes, Xenopus, egg extracts, intracellular scaling, spindle, embryogenesis, cell division     

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (2⁶–2⁷ chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 Å in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.     

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.     

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.