A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

The behavior of allocyclic chromosomes in Bloom's syndrome

The behavior of individual allocyclic chromosomes has been analyzed in lymphocytes of a sister and a brother with Bloom's syndrome. Of 4,633 diploid cells, 115 showed allocyclic chromosomes, and 74 of these had 44, 45 or 46 normal metaphase chromosomes accompanied by one or two allocyclic chromosomes. Of 56 tetraploid cells, 9 contained such chromosomes. The allocyclic chromosomes appeared pulverized or extended corresponding to S or G₂ PCC. We have proposed the hypothesis that individual allocyclic chromosomes do not, as a rule, come from micronuclei, as has often been assumed, but have been left behind in their cycle. This would be caused by a mutation or deletion of a hypothetical coiling center situated near the centromere of each chromosome arm. The following observations agree with our explanation but less well or not at all with the idea of micronuclei: (1) In only 9.6% of the cells does the allocyclic chromosome lie at the edge of the metaphase plate. (2) In 24 cells a part of a chromosome is pulverized while the rest is in metaphase. (3) Both a pulverized and an extended chromosome were present in the same cell. (4) A pulverized acrocentric is often nose-to-nose with a normal D or G chromosome. (5) No allocyclic chromosomes corresponding to G₁ PCC have been found in our material. (6) When a ring is replaced by an allocyclic chromosome, it is usually a member of a 46-chromosome complement. Furthermore, the occurrence of allocyclic chromosomes is correlated with that of other chromosome anomalies which do not follow a Poisson distribution. Allocyclic chromosomes are also more frequent (16%) in tetraploid than in diploid cells (2%).     

Über die Primärwindungen menschlicher Chromosomen

Zusammenfassung In Routinepräparat befinden sich 70–80% der Chromosomensätze im Stadium der Metaphase. Die Zahl der Coils pro Chromosom ist konstant. Nach Zählung der Windungen kann danach karyotypiert werden. Die Zahl der Windungen bei Prophasechromosomen ist größer, die der Anaphasechromosomen kleiner. Die Ganghöhe ist in diesen drei untersuchten Stadien der Mitose konstant=0,55 (±0,15 ).

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The primary coils of human chromosomes

Summary In a routine chromosome preparation 70–80% of chromosome sets are at the stage of metaphase. The number of coils per chromosome is constant. Karyotyping is possible on the basis of the number of primary coils. There are more primary coils in chromosomes at the stage of prophase and less in chromosomes in anaphase. The pitch is constant in all three stages of mitosis examined=0.55 (±0.15 ).

     

Characterization of human chromosomal unit fibers

A structural component of mitotic chromosomes that partially explains the compaction of DNA within mitotic chromosomes is suggested on the basis of the occurrence of long, regular cylindrical structures in preparations of isolated human chromosomes. These structures, unit fibers, of a rather constant diameter of about 4,000 Å have been postulated to be formed by coiling of the 250T2–300 Å solenoid chromatin fiber that itself is formed by coiling of the 100 Å string of nucleosome fiber. The human chromatid would thus be composed by a hierarchy of helices with contraction ratios for DNA at each level of coiling of 7 (string of nucleosomes), 5 (solenoid) and 40 (4,000 Å unit fiber or super-solenoid) which results in an overall contraction ratio for DNA in the unit fiber structures of about 1,400, which is approximately 5-fold less than the final contraction of DNA in intact chromatids of condensed metaphase chromosomes. The present report concerns more detailed studies with respect to the dimensions and cytochemical properties of the unit fiber structures observed in preparations of isolated human mitotic chromosomes that provide direct and indirect evidence in support of their super-solenoid structure and relate to known properties of human mitotic chromosomes.     

The kinetochores of Caenorhabditis elegans

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 m in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18–0.2 m in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 m, and an electron lucent middle layer of 0.03 m. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.     

Chromosome recombination and defective genome segregation induced in Chinese hamster cells by the topoisomerase II inhibitor VM-26

We found that 4-demethylepipodophyllotoxinthenylidene--d-glucoside (VM-26; Teniposide), which specifically inhibits the enzyme DNA topoisomerase II, induces the formation of quadriradial chromosomes in Chinese hamster ovary cells. VM-26 traps topoisomerase II molecules when they are covalently integrated into DNA during their reaction. Quadriradial chromosomes are formed by reciprocal exchange of double-stranded DNA between single chromatids of two different chromosomes. Using synchronised cells, we found that they were formed after a single replication cycle in the presence of VM-26 at a low concentration (0.008M), which does not affect DNA replication, and occurred in 50% of the mitotic cells at a concentration of 0.16 M. They were also formed when VM-26 was present for only 1.5 h before mitosis, after the completion of S-phase DNA replication. Chromatids bearing a translocated segment of another chromatid, which were derived from recombined chromosomes, were observed in late metaphase cells. Segregation of the daughter genomes was defective in many mitotic cells, probably because chromatids with two or no centromeres and kinetochores, formed from chromosomes recombined between their centromeres, could not be segregated. In the light of evidence that topoisomerase II molecules covalently integrated in DNA are trapped and therefore more abundant in the presence of VM-26, and that this enzyme can effect recombination of double-stranded DNA in vitro, we interpret these observations as evidence that topoisomerase II can mediate chromosome recombination in vivo.by M. Trendelenburg     

Synaptonemal complex formation in hybrids of Lolium temulentum x Lolium perenne (L.)

Comparisons were made between two kinds of tetraploids derived from the hybrid Lolium temulentum x L. perenne. One hybrid behaves like an autotetraploid with multivalents at first metaphase of meiosis in pollen mother cells. The other behaves like an allotetraploid, in which pairing at first metaphase is restricted to bivalents comprised of strictly homologous chromosomes. The diploidisation of the latter form is controlled by determinants located on both the normal, A chromosomes and on supernumary B chromosomes. Reconstruction of synaptonemal complexes and their elements, from serial sections through pollen mother cell nuclei examined under the electron microscope, reveals that at zygotene pairing in both forms results in multivalent formation involving non-homologous as well as homologous chromosomes. The mechanism responsible for the diploidisation is, therefore, not based on a restriction of pairing at early meiosis to homologous chromosomes but on a correction or transformation of the multivalent chromosome associations to bivalents subsequent to zygotene. The transformation is not completed until late pachytene. In the multivalent-forming tetraploid a maximum of four chromosomes are associated at first metaphase. Yet configurations of a higher valency are found at zygotene. There is, therefore, a partial transformation of multivalents even in this autotetraploid form which restricts configurations at metaphase I to homologous and homoeologous chromosomes only. In both hybrids some homologous bivalents are not the product of resolution of multivalents but result from two-by-two pairing from the beginning of zygotene.     

Complete characterization of a large marker chromosome by reverse and forward chromosome painting

Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the degenerate oligonucleotide primed — polymerase chain reaction, we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (reverse chromosome painting) and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome (forward chromosome painting) and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.     

Synaptonemal complex formation in hybrids of Lolium temulentum × Lolium perenne (L.)

The chromosomes of Lolium temulentum are longer and contain on average 50% more nuclear DNA than the chromosomes of L. perenne. In the hybrid, despite the difference in length and DNA content, pairing between the homoeologous chromosomes at pachytene is effective and the chiasma frequency at first metaphase in pollen mother cells is high, about 1.6 per bivalent, comparable to that in the L. perenne parent. Electron microscopic observations from reconstructed nuclei at pachytene show that synaptonemal complex (SC) formation in 40% of bivalents is perfect, complete and continuous from telomere to telomere. In others, SCs extend from telomere to telomere but incorporate lateral component loops in interstitial chromosome segments. Even in these bivalents, however, pairing is effective in the sense of chiasma formation. The capacity to form perfect SCs is achieved by an adjustment of chromosome length differences both before and during synapsis. Perfect pairing and SC formation is commoner within the larger bivalents of the complement. At zygotene, in contrast to pachytene, pairing is not confined to homoeologous chromosomes. On the contrary there is illegitimate pairing between non-homologous chromsomes resulting in multivalent formation. There must, therefore, be a mechanism operative between zygotene and pachytene that corrects and modifies associations in such a way as to restrict the pairing to bivalents comprised of strictly homoeologous chromosomes. Such a correction bears comparison with that known to apply in allopolyploids. In the hybrid and in the L. perenne parent also, certain specific nucleolar organisers are inactivated at meiosis.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

Preleptotene chromosome contraction in Lilium longiflorum “Croft”

A period of chromosome contraction between premeiotic interphase and leptotene was regularly observed in three samples of Lilium longiflorum Croft. Extensive preleptotene chromosome contraction was also observed in L. longiflorum Ace and in the Lilium hybrid Enchantment. Although the stage resembles late mitotic prophase, microsporocytes never develop to metaphase, but despiralize to leptotene, and regular alignment, pairing and chiasma formation follow. As preleptotene chromosome contraction is discovered in an increasing number of organisms it becomes less likely that it represents a true reversion to mitosis. However its absence in many organisms and its extreme variability in others do not support the concept of preleptotene chromosome contraction as a regular meiotic stage. It is suggested that the line of demarcation between mitosis and meiosis is often imprecise, and meiocytes may fluctuate to some extent between these states before a final transition to meiosis is made. The occurrence and extent of this fluctuation may possibly be related to some externally produced substances required for the orderly development of meiotic prophase.     

Chromosomal passengers: Toward an integrated view of mitosis

Conclusions The immunocytochemical taxonomy experiments cited above have identified a class of chromosomal antigens whose properties were not predicted by earlier models of mitosis. Our theory describing one possible explanation for the transfer of these antigens from the chromosomes to the spindle midzone at the metaphase: anaphase transition must now be subjected to further experimental tests. The phenotypes of cells microinjected with antibodies to passenger proteins should enable us to identify mitotic processes dependent on these proteins, as in the example of CHO1 antibody blocking mitotic progression (Nislow et al. 1990). In addition, the availability of cDNA clones and high titer antibodies may enable homologues of these components to be identified in organisms in which they can be subjected to genetic analysis.For the time being, we suggest that current views of the relative roles of chromosomes and cytoskeletal components in mitosis may require revision. Our hypothesis takes the current model for the role of the kinetochores in organizing the bipolar mitotic spindle (Kirschner and Mitchison 1986) a step further. The process of assembling a functional spindle and positioning the cleavage furrow may entail a degree of functional cooperation between chromosomes and cytoskeletal components far beyond that envisioned before now.     

Human high-affinity FcγRI (CD64) gene mapped to chromosome 1q21.2-q21.3 by fluorescence in situ hybridization

The human FcRI gene encodes for a highaffinity Fc receptor that plays pivotal roles in the immune response. We have used fluorescence in situ hybridization analysis to localize the FcRI gene to human chromosome 1. The human FcRI (CD64) gene has been assigned to human chromosome 1q21.2-q21.3 using R-banded human (pro)metaphase chromosomes.     

Silver staining the chromosome scaffold

Cytological silver-staining procedures reveal the presence of a core running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this core and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic core staining was unaffected. Control experiments suggested that the core does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific core staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.     

Genetic transformation and “graft-hybridization” in flowering plants

Summary Recent pollination experiments with highly irradiated (100,000 r) pollen in Nicotiana have shown that radiation-pulverized pollen chromatin can cause genetic transformation of the egg. A new model is proposed here for integration of chromatin fragments into host chromosomes. It is also proposed that heterochromatin may be involved in the process of gene transfer, and in the phenomena of meiotic drive associated with gene transfer.It is suggested that this discovery throws new light on the phenomenon of graft-hybridization. In spite of many reports to the contrary, graft-hybrids have so far been explained only on the basis of their being chimaeras. A mechanism is suggested here by which they may result from genetic transformation.     

Linear differentation of cereal chromosomes

Summary The BSG test was used in an investigation of the linear differentiation in rye variety Zhitkinskaya, common wheat variety Aurora and two secondary Triticale namely AD-196 and F-1239.Chromosomes of Aurora variety and wheat chromosomes within Triticale may be easily divided into constant and variable chromosomes as described previously (lordansky et al. 1977; Zurabishvili et al 1977).It is necessary to emphasize that the diversity of variable chromosomes underlies karyotypical polymorphism within wheat and Triticale species. The polymorphism observed exists in parallel with strict homomorphism of homologous chromosomes.In IB chromosomes of Aurora variety, the short arm is substituted by the rye chromosome arm. The karyotype of Triticale AD-196 consists of six pairs of rye chromosomes and fifteen pairs of wheat chromosomes.     

Unequal X Chromosomes in Bradysia Hygida (Diptera:Sciaridae) Females: Karyotype Assembly and Morphometric Analysis

The mitotic chromosomes of Bradysia hygida(Diptera:Sciaridae) neuroblast cells are described together with their morphometric data. Giemsa-stained neuroblast chromosomes from female and male larvae confirm the chromosome number of this species, 2n=8 (XX) and 2n=7 (XO), respectively. The karyotype assembly reveals two metacentric autosomic pairs, the A and B chromosome; a subtelocentric, the C chromosome, the smallest one; and a sexual unequal metacentric pair, X chromosome, in female karyotype and a one sexual metacentric X chromosome in male. The implications of the unequal X chromosome pair are discussed.     

Preparation and karyotype analysis of mitotic chromosomes of the freshwater spongeSpongilla lacustris

The present study documents for the first time the karyotype and mitotic chromosomes of a sponge. For the studies the freshwater spongeSpongilla lacustris (Lin. 1758) was used. Its karyotype comprises nine different chromosome pairs ranging in size from 2.1 to 0.7 m. Changes in size and shape of the chromosomes during the progression of mitosis are documented both light and electron microscopically. The data reveal that the lowest multicellular eukaryotes, the sponges, have already reached a high level of evolution of the mitotic mechanism.This work is dedicated to Prof. N. Weissenfels, Bonn     

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis

Summary. Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3±4.1µm) than those developing 2 PPBs (49.8±4.1µm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42µm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.Correspondence and reprints: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.