Longitudinal differentiation of metaphase chromosomes of Indian muntjac as studied by restriction enzyme digestion,in situ hybridization with cloned DNA probes and distamycin A plus DAPI fluorescence staining

The longitudinal differentiation of metaphase chromosomes of the Indian muntjac was studied by digestion with restriction enzymes, in situ hybridization with cloned DNA probes and distamycin A plus DAPI (4-6-diamidino-2-phenylindole) fluorescence staining. The centromeric regions of chromosomes 3 and 3 + X of a male Indian muntjac cell line were distinct from each other and different from those of other chromosomes. Digestion with a combination of EcoRI^* and Sau3A revealed a pattern corresponding to that of C-banding. Digestion with AluI, EcoRII or RsaI yielded a band specific to the centromeric region only in chromosomes 3 and 3 + X. Furthermore, HinfI digestion yielded only a band at the centromeric region of chromosome 3, whereas DA-DAPI staining revealed a single band limited to the extreme end of the C-band heterochromatin of the short arm of 3 + X. These results suggest that centromeres of Indian muntjac chromosomes contain at least four different types of repetitive DNA. Such diversity in heterochromatin was also confirmed by in situ hybridization using specific DNA probes isolated and cloned from highly repetitive DNA families. Heterozygosity between chromosome homologs was revealed by restriction enzyme banding. Evidence is presented for the presence of nucleolus organizer regions (NORs) on the long arm of chromosome 1 as well as on the secondary constrictions of 3 and 3 + X.Abbreviations DA distamycin A - DAPI 4-6-diamidino-2-phenylindole - NOR(s) nucleolus organizer region(s) - PBS phosphate-buffered saline - PI propidium iodide     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Premeiotic and early meiotic stages in the pollen mother cells of Eremurus and in human embryonic oocytes

Summary The premeiotic and meiotic stages are described in the pollen mother cells of the liliaceous plant Eremurus. In human oocytes from embryonic ovaries, the premeiotic and early meiotic stages up to dictyotene have been identified on morphological grounds. In Eremurus, in which each stage can be independently verified by the sequence of buds situated in a spiral, there is no indication of somatic pairing of homologous chromosomes, nor is there any sign of a premeiotic contraction of the chromosomes. The interphase following this mitosis is, in turn, succeeded by leptotene in which the DNA synthesis occurs, as determined by using autoradiography. This stage is followed by a distance pairing stage in which the homologous chromosomes lie parallel to each other at a distance. In typical zygotene, segments of chromosomes are paired intimately, others are unpaired, and the points at which pairing begins and ends are clearly visible. Each bivalent shows several pairing blocks. Pairing is completed at pachytene; diplotene which is characterized by the separation of the chromosomes follows. In middiplotene the chromosomes collect together in the so-called second contraction stage.The same meiotic prophase stages that occur in Eremurus, including the distance pairing stage, are found in the embryonic human oocytes. In the last premeiotic interphase, the chromosomes appear as condensed prochromosomes which unravel directly to form the leptotene chromosomes. In the oocytes, too, DNA synthesis seems to take place in leptotene.This is publication No. 1947 from the Genetics Laboratory, University of Wisconsin. It has been aided by the following grants from the National Institutes of Health (Washington): GM 15 422 and HD 03 084-03. For excellent photographic help we are grateful to Mr. Walter Kugler, Jr.     

Centromere sizes,positions, and movements in the interphase nucleus

Each microspore of the onion Allium fistulosum (n=8) has 8 chromosomes. It is shown that in the microspore the 8 centromeres aggregate to form 2 or 3 centromeric structures. Subsequently, at early mitotic prophase, these aggregates are resolved into 8 separate centromeres and each becomes structurally double. After mitosis the pollen grain contains 2 nuclei, each with 8 separate and distinct centromeres, clustered at the nuclear envelope. As interphase progresses the centromeres of the vegetative nucleus are no longer at the nuclear envelope and they aggregate into 3 or 4 centromeric masses. In the generative nucleus there is less movement. The interphase centromere movements occur in the absence of microtubules. The centromeres range in size from about 0.10 to 0.17 m³ with an average of 0.14 m³ per centromere.     

Characterization of two moderately repetitive DNA components localized within the β-heterochromatin of Drosophila hydei

Two nuclear DNA fractions from Drosophila hydei were isolated by silver ion and actinomycin D binding and centrifugation in isopycnic salt gradients. Neither fraction is composed of nor does it contain any highly repetitive simple sequence DNA, as shown by melting and reassociation studies. — One fraction has a CsCl density of 1.702 g/cm³ and hybridizes in situ with the -heterochromatin of the chromocenter in polytene cells. This DNA fraction was found to be replicated during polytenization. In diploid cells this 1.702 g/cm³ component hybridizes to the heterochromatin of all four large autosome pairs, the middle part of the long arm of the Y-chromosome, but not to the X-heterochromatin. — A second fraction has a CsCl density of 1.697 g/cm³ and hybridizes in situ with polytene cells to the chromocenter and the nucleolus, but on metaphase chromosomes only to the nucleolus organizer regions.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

Heteropycnosis of an underreplicating chromosome

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that condensed chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. InD. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from theX chromosome, via3 and2, to chromosome4. In the male, the order isX, 2, 3, Y, and4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. TheX chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where theX chromosomes shorten less than the autosomes, and why each of theX chromosomes is 15% shorter than theX chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome3 condense by shortening, while chromosomes2 and4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation:(1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.     

Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a metaphase chromosome cluster. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll^TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca⁺⁺ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.     

Yeast importin-β is required for nuclear import of the Mig2 repressor

Background

In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade.

Results

This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain.

Conclusions

It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.     

The human leukocyte test system

Summary Leukocyte cultures were set up with X-irradiated whole blood (200 R). Cells starting with their DNA synthesis between 25 and 35 h after culture initiation (early replicating cells) were pulse-labeled with tritiated thymidine ([³H] TdR). Mitoses were collected with colcemid in adjacent intervals from 36 up to 72 h after culture initiation. At fixation times of 50, 56, 62, and 72 h enough mitoses for a determination of the frequencies of chromosomal aberrations (dicentric and ring chromosomes) were found. After that the preparations were processed for autoradiography. All mitoses analyzed for chromosomal aberrations were re-analyzed for labeling, and the frequencies of chromosomal aberrations in labeled (=early replicating cells) and unlabeled (=late replicating cells) mitoses were compared. At all fixation times, higher frequencies of dicentric chromosomes were found in labeled as compared to unlabeled mitoses, indicating a higher sensitivity of early replicating cells to X-irradiation in the G₀ stage of the cell cycle.     

Rearrangements between irradiated chromosomes in three-species radiation hybrid cell lines revealed by two-color in situ hybridization

A human-hamster hybrid cell line containing only the human X chromosome (GM06318B) was exposed to 6,000–7,000 rad of X-rays and fused with a mouse cell line (CL1D,TK^-). Three radiation hybrids, LXKC40, LXKC50, and LXKC56, were selected among 39 independent clones containing human material. Two-color in situ hybridization with total genomic DNA probes (cotl human DNA and hamster total genomic DNA) was used to analyse the irradiated chromosome rearrangements. With this three-species model system (human-hamster-mouse) and the chromosome painting process it was possible to determine the origin of each chromosomal fragment in metaphase and interphase. The results obtained indicate preferential rearrangement between irradiated human and hamster chromosomes. Whole, apparently intact hamster chromosomes were observed in all the mitoses. We suggest that these chromosomes could be neoformated from random fragments after irradiation. Hamster and human minichromosomes were also detected. While the integration of human material into the mouse genome was exceptional, the integration of hamster material into mouse chromosomes was more frequent. During interphase the irradiated chromosome domains were often at the periphery of the nucleus. Irradiated material protruded at the periphery of the nuclei. Micronuclei containing hamster material were detected in the vicinity of these protrusions.     

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G₁/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 10⁶ chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole The authors thank Mrs. Jiina Eliáová for her excellent technical assistance and Dr. Slavomir Ondro for the supply of V. faba seeds. A gift sample of APM from the Mobay Corporation (Agricultural Chemicals Division, Kansas City, Mo., USA) is gratefully acknowledged.     

In situ hybridization of “Nick-translated” 3H-ribosomal DNA to chromosomes from salamanders

A technique is described for preparation of ³H-labelled DNA by nick-translation employing deoxyribonuclease I and DNA polymerase I. The labelled DNA can be obtained in high yield with specific activities of 10⁶ cpm/g or more. Ribosomal DNA, isolated from ovaries of young Xenopus laevis, and whole DNA from Plethodon cinereus were labelled in this way. The rDNA was used for in situ hybridization to meiotic chromosomes from P. cinereus, P. vehiculum and P. dunni. Autoradiographs of in situ hybrids were exposed for 5 to 10 days, by which time nucleolus organizer regions on the chromosomes of all 3 species were clearly and specifically labelled. In all eases, labelling was confined to a short region near the middle of the short arm of both halves of a medium length bivalent. It is concluded that nick-translation is a useful and altogether efficient method of labelling nucleic acids for subsequent use in experiments involving in situ hybridizations.     

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK₁ cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In early restoration cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK₁ cells in g₁ and those in G₂/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1–2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.     

Multiple sex chromosomes in Drosophila miranda: A system to study the degeneration of a chromosome

Drosophila miranda possesses an intriguing sex chromosome constitution. While female metaphase plates have 10 chromosomes (diploid set), in males only 9 chromosomes can be identified. The missing homologue has been translocated to the Y, forming a neo-Y chromosome which is polytenized in the salivary gland cells. This report presents a detailed characterization of DNA, isolated from D. miranda flies. In situ hybridizations, using cRNA transcribed from unfractionated D. miranda DNA, reveal hybridization to the neo-Y with label distributed over the entire chromosome. The original partner of the translocated chromosome, X², is essentially unlabelled. These results suggest that repetitive DNA sequences invade the translocated chromosome. This result is discussed with reference to the hypothesis of degeneration of the Y chromosome, formulated by Muller (1918, 1932a).     

Some effects of 2,4,5-trichlorophenoxyacetic acid on the mitotic cycle of lateral root apical meristems of Vicia faba

Roots of Vicia faba were given a one hour pulse label with. ³H-TdR (1 C/ml), either before or after a three hour treatment with a 10^–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G₂) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G₁), because of rapid changes in the lengths of the G₂ and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri.     

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.     

Evidence that F'' lac replicates asynchronously during the cell cycle of Escherichia coli B/r.

Summary The replication of F lac was studied in exponentially growing cultures of E. coli B/r. The cells were pulse induced for the synthesis of -galactosidase and their DNA pulse labelled with ³H thymidine. The cells were then separated into age classes by centrifugation through a sucrose gradient in a zonal rotor. Plasmid replication was measured in each age fraction by three methods: the rate at which -galactosidase could be induced, the amount of label incorporated into CCC plasmid DNA which had been separated from chromosomal DNA on agarose gels, and the amount of label incorporated into plasmid DNA which had been separated from chromosomal DNA by ultracentrifugation through CsCl-EtBr gradients. All these methods gave the same result, that replication of F lac occurs in cells of all ages and is not confined to a part of the cell cycle.     

Examination of newly synthesized DNA in Escherichia coli.

Summary The short (0-20S) Okazaki fragments seen upon pulse-labeling E. coli (thy ⁺, deo ⁺) with ³H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut ⁺, thyl ⁺, polA ⁺), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.