Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.     

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

Summary In spite of Carl Rabl's (1885) and Theodor Boveri's (1909) early hypothesis that chromosomes occupy discrete territories or domains within the interphase nucleus, evidence in favor pf this hypothesis has been limited and indirect so far in higher plants and animals. The alternative possibility that the chromatin fiber of single chromosomes might be extended throughout the major part of even the whole interphase nucleus has been considered for many years. In the latter case, chromosomes would only exist as discrete chromatin bodies during mitosis but not during interphase. Both possibilities are compatible with Boveri's well established paradigm of chromosome individuality. Here we show that an active human X chromosome contained as the only human chromosome in a Chinese hamster x man hybrid cell line can be visualized both in metaphse plates and in interphase nuclei after in situ hybridization with either ³H- or biotin-labeled human genomic DNA. We demonstrate that this chromosome is organized as a distinct chromatin body throughout interphase. In addition, evidence for the territorial organization of human chromosomes is also presented for another hybrid cell line containing several autosomes and the human X chromosome. These findings are discussed in the context of our present knowledge of the organization and topography of interphase chromosomes. General applications of a strategy aimed at specific staining of individual chromosomes in experimental and clinical cytogenetics are briefly considered.     

Uniparental chromosome elimination in the early embryogenesis of the inviable salmonid hybrids between masu salmon female and rainbow trout male

Chromosome elimination through chromosome loss and partial deletion is known to be one of the causes of embryonic inviability in some salmonid interspecific hybrids. Using fluorescence in situ hybridization and related techniques, including whole chromosome painting and comparative genomic hybridization, parental origin of eliminated chromosomes was identified in the inviable hybrids between masu salmon (Ms, Oncorhynchus masou) female and rainbow trout (Rb, O. mykiss) male at the early embryonic stage prior to death. In these hybrids, the haploid Rb chromosome number decreased to nearly half, whereas the Ms chromosomes were retained as one or occasionally two full haploid complements. The Rb chromosomes were also involved in the frequently observed fragments and micronuclei. Whereas the occurrence of fragments was constant throughout the observed period, chromosome loss occurred mainly from just after fertilization to the blastulae stage. In tissue sections and cell spreads of late blastula, some Rb chromosomes were trapped in the midzone from ana- to telophase, resulting in micronuclei at the subsequent interphase. Micronuclei and mitotic abnormalities were also observed in the androgenetic haploid hybrids. However, such abnormalities were seldom or never observed in the viable reciprocal hybrids. The present findings suggest that the paternal Rb chromosomes in the inviable hybrids are preferentially eliminated through mitotic abnormalities during early embryogenesis, owing to a possible incompatibility between the maternal Ms cytoplasm and paternal Rb genome. Received: 22 August 1996; in revised form: 14 November 1996 / Accepted: 20 November 1996     

Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential

Foxtail millet (Setaria italica), a member of the Poaceae grass family, is an important food and fodder crop in arid regions and has potential for use as a C(4) biofuel. It is a model system for other biofuel grasses, including switchgrass and pearl millet. We produced a draft genome (～423 Mb) anchored onto nine chromosomes and annotated 38,801 genes. Key chromosome reshuffling events were detected through collinearity identification between foxtail millet, rice and sorghum including two reshuffling events fusing rice chromosomes 7 and 9, 3 and 10 to foxtail millet chromosomes 2 and 9, respectively, that occurred after the divergence of foxtail millet and rice, and a single reshuffling event fusing rice chromosome 5 and 12 to foxtail millet chromosome 3 that occurred after the divergence of millet and sorghum. Rearrangements in the C(4) photosynthesis pathway were also identified.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

Comparative mapping reveals a complex relationship between the pearl millet genome and those of foxtail millet and rice

Comparative genetic maps were constructed of the pearl millet genome with foxtail millet and used to describe the homoeology between the genomes of pearl millet, foxtail millet and rice. Despite the close taxonomic relationship of pearl and foxtail millet, their genomes were highly, rearranged. A comparison of the millet and rice genomes indicated that most of these rearrangements were likely to have taken place in pearl millet. Two duplications were identified in pearl millet. A duplication between the distal segments of linkage groups 1 and 4 corresponds to the ancient duplication previously identified between rice chromosome arms 11S and 12S and foxtail millet chromosomes VII and VIII. The other putative duplication, also between regions of linkage groups 1 and 4, is likely to be species-specific. The exploitation of the comparative maps in pearl millet research is discussed. Received: 10 February 1999 / Accepted: 6 July 1999     

Chromosome elimination in sciarid flies

The programmed elimination of part of the genome through chromosome loss or chromatin diminution constitutes an exceptional biological process found to be present in several diverse groups of organisms. The occurrence of this phenomenon during early embryogenesis is generally correlated to somatic versus germ-line differentiation. A most outstanding example of chromosome elimination and genomic imprinting is found in sciarid flies, where whole chromosomes of exclusive parental origin are selectively eliminated at different developmental stages. Three types of tissue-specific chromosome elimination events occur in sciarids. During early cleavages, one or two X paternal chromosomes is/are discarded from somatic cells of embryos which then develop as females or males respectively. Thus, the sex of the embryo is determined by the number of eliminated paternal X chromosomes. In germ cells, instead, a single paternal X chromosome is eliminated in embryos of both sexes. In addition, while female meiosis is orthodox, male meiosis is highly unusual as the whole paternal chromosome set is discarded from spermatocytes. As a consequence, only maternally derived chromosomes are included in the functional sperm. This paper reviews current cytological and molecular knowledge on the tissue-specific cell mechanisms evolved to achieve chromosome elimination in sciarids.     

The presence of amplified regions affects the stability of chromosomes in drug-resistant Chinese hamster cells

The stability of chromosomes carrying amplified CAD (carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) or DHFR (dihydrofolate reductase) genes was studied in V79 Chinese hamster cell derivatives resistant to PALA (N-phosphonacetyl-L-aspartate) and MTX (methotrexate), respectively. Cells were maintained in the presence of the selective drugs during the study. In both metaphase chromosomes and interphase nuclei, amplified regions were localized by in situ hybridization. In MTX-resistant cells, the amplification-bearing chromosome moved sluggishly at anaphase and gave rise to bud-shaped formations in interphase nuclei. It is suggested that these buds could eventually separate as micronuclei. In both MTX- and PALA-resistant cells, amplified DNA was observed in micronuclei in interphase and in displaced chromosomes in metaphase. Finally, amplification-bearing dicentric chromosomes were found in both drug-resistant cell lines. Cumulatively, these observations indicate that the presence of the amplified region in a chromosome renders it unstable: chromosomes bearing an amplified region tended to be excluded from cells, and rearrangements were more frequent than in normal chromosomes.     

Emergence of Micronuclei and Their Effects on the Fate of Cells under Replication Stress

The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor cells.     

Induction of paternal genome loss by the paternal-sex-ratio chromosome and cytoplasmic incompatibility bacteria (Wolbachia): A comparative study of early embryonic events

Paternal genome loss (PGL) during early embryogenesis is caused by two different genetic elements in the parasitoid wasp, Nasonia vitripennis. Paternal sex ratio (PSR) is a paternally inherited supernumerary chromosome that disrupts condensation of the paternal chromosomes by the first mitotic division of fertilized eggs. Bacteria belonging to the genus Wolbachia are present in Nasonia eggs and also disrupt paternal chromosome condensation in crosses between cytoplasmically incompatible strains. Cytoplasmic incompatibility Wolbachia are widespread in insects, whereas PSR is specific to this wasp. PGL results in production of male progeny in Nasonia due to haplodiploid sex determination. The cytological events associated with PGL induced by the PSR chromosome and by Wolbachia were compared by fluorescent light microscopy using the fluorochrome Hoescht 33258. Cytological examination of eggs fertilized with PSR-bearing sperm revealed that a dense paternal chromatin mass forms prior to the first metaphase. Quantification of chromatin by epifluorescence indicates that this mass does undergo replication along with the maternal chromatin prior to the first mitotic division but does not replicate during later mitotic cycles. Contrary to previous reports using other staining methods, the paternal chromatin mass remains condensed during interphase and persists over subsequent mitotic cycles, at least until formation of the syncytial blastoderm and cellularization, at which time it remains near the center of the egg with the yolk nuclei. Wolbachia-induced PGL shows several marked differences. Most notable is that the paternal chromatin mass is more diffuse and tends to be fragmented during the first mitotic division, with portions becoming associated with the daughter nuclei. Nuclei containing portions of the paternal chromatin mass appear to be delayed in subsequent mitotic divisions relative to nuclei free of paternal chromatin. Crosses combining incompatibility with PSR were cytologically similar to Wolbachia-induced PGL, although shearing of the paternal chromatin mass was reduced. Wolbachia may, therefore, block an earlier stage of paternal chromatin processing in the fertilized eggs than does PSR. © 1995 Wiley-Liss, Inc.     

An apomictic polyhaploid obtained from a pearl millet x Pennisetum squamulatum apomictic interspecific hybrid

Summary In a research program to transfer apomixis from Pennisetum squamulatum Fresen to pearl millet, P. americanum L. Leeke, a polyhaploid plant (2n=21) was discovered in the uniform open-pollinated progeny of an apomictic interspecific hybrid (2n = 41) between pearl millet and P. squamulatum. The polyhaploid was shorter, less vigorous and was smaller morphologically than its maternal parent. It probably originated by parthenogenetic development of a reduced gametophyte in the apomictic interspecific hybrid. The most common metaphase I chromosome association in the polyhaploid was 4 bivalents plus 13 univalents. Irregular chromosome distribution, tripolar spindles, bridges and fragments were observed at anaphase I and telophase I. The polyhaploid was male-sterile and partially female- fertile having multiple aposporous embryo sacs in 95% of the ovules. Seed set was 3% when open-pollinated and 33% when pollinated with pearl millet pollen. Low seed set was due to competition among multiple embryos developing in the same ovule. Seventeen progeny from seed produced under open-pollination on the polyhaploid each had 2n=21 chromosomes and were morphologically uniform and identical to the female parent. The expression of obligate apomixis in the polyhaploid conditioned by the P. squamulatum genome between the simplex and duplex condition indicates that apomictic reproduction is possible in nonpolyploid plants.     

Aneuploidy and ageing: sex chromosome exclusion into micronuclei

In lymphocyte cultures, the number of aneuploid cell nuclei increases with proband age mainly because of the loss of sex chromosomes. Since one possible cause of aneuploidy in cell nuclei is chromosomal lag at anaphase, with subsequent chromosome loss via micronucleus formation, we scored 5000 interphase nuclei from ten female and ten male probands for associated micronuclei. Whereas, in young (< 10 years) probands, an average of 0.15% interphase nuclei exhibited micronuclei, the frequency rose to 0.46% in older probands (> 70 years). In situ hybridizations with X-specific and Y-specific DNA probes were carried out, and the signal distribution in ten nuclei with associated micronuclei was documented for each donor. Our results indicate that the exclusion of sex chromosomes into micronuclei doubles during a human life, from 11% in young probands to 20% in old donors.     

Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells

We previously reported that exogenous histone H1, when injected into mitotic cells, disrupts the synchronous progression of mitotic events by delaying chromosome decondensation. This strategy was utilized to determine whether any other interphase proteins are also able to disrupt normal mitotic processes, when introduced into the mitotic phase. We found that a chromatin subfraction from bovine liver nuclei induced postmitotic micronuclei formation in a dose-dependent manner when injected into the prometaphase of rat kangaroo kidney epithelial (PtK(2)) cells. Close observation showed that, in the case of injected mitotic cells, the mitotic spindles were disrupted, chromosomes became scattered throughout the cytoplasm, and actin filaments were organized ectopically. In addition, when the fraction was injected into interphase cells, extra actin filaments were formed and microtubule organization was affected. In order to determine whether the micronuclei formation resulted from the ectopic formation of actin filaments, we examined the effect of the actin polymerization inhibitor, cytochalasin D. The results showed that the drug inhibited micronuclei formation. From these findings, we concluded that this chromatin subfraction contains actin polymerization activity, thus causing the disruption of mitotic spindles.     

A comparison between short-term evolution of micronuclei induced by X-rays and colchicine in root tips of Vicia faba

The short-term evolution of micronuclei derived from acentric fragments and whole chromosomes was studied in root tips of Vicia faba. Micronuclei were induced by X-rays (30 cGy and 120 cGy) and colchicine (10(-5) M and 3 X 10(-4) M). Frequencies of chromosome breakage or loss of micronuclei in interphase and mitotic cells were studied. The DNA content of micronuclei in interphase cells was also measured. Micronuclei derived from whole chromosome showed a higher probability to survive and to undergo mitotic condensation in synchrony with main nuclei than micronuclei derived from an acentric fragment. PCC (Premature Chromosome Condensation) was not observed for both types of micronuclei in Vicia faba, in contrast to the ones reported in mammalian cells in culture.     

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.     

Association of BAF53 with mitotic chromosomes

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.     

Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise

The in vitro micronucleus test with Syrian hamster embryo (SHE) cells assays the induction of micronuclei by chemical agents. Both chromosome fragments and lagging chromosomes can give rise to micronuclei. Nevertheless, only limited information is available on the ultrastructure of micronuclei and the mechanisms of their formation. Diethylstilbestrol (DES), a non-mutagenic carcinogen, as well as its analogue 3.3'-DES induce micronuclei in SHE cells. A comparison of the dose response of DES-induced micronucleus formation with the previously published ones for aneuploidy and transformation shows that all 3 run in parallel. Thus, a functional relationship between these endpoints, in the SHE system, may be implied. The present study is designed to address the formation of micronuclei using supravital UV microscopy, to test for the presence of defined chromosome domains within micronuclei using immunocytochemistry, and to define aspects of their ultrastructure by electron microscopy. Supravital UV microscopy showed that 3.3'-DES induces displacement of chromosomes/chromatids during prophase/anaphase and formation of micronuclei during cytokinesis. Immunocytochemistry revealed that micronuclei contain, at high frequencies, CREST antibody-reactive kinetochores, indicating the presence of whole chromosomes or centric fragments in these structures. Moreover, transmission electron microscopy showed that micronuclei exhibit ultrastructural details typical of interphase nuclei. Specifically, micronuclei exhibited morphological evidence of a nuclear lamina and segregation of karyoplasm into euchromatic and heterochromatic regions. All micronuclei examined were enclosed by a nuclear envelope of normal morphology and showed nuclear pore complexes. Together the findings provide evidence that DES interferes with the mitotic apparatus as early as prophase, resulting in the formation of micronuclei and, as a consequence, in the loss of chromatids or chromosomes.     

Cytogenetics of pearl millet

Summary The somatic karyotype of pearl millet Pennisetum americanum (L.) Leeke. (2n = 14) has been studied in several cultivars, but few cytological markers have been discovered which could help in the easy identification of the chromosomes. Analysis of pachytene bivalents permits such identification but is feasible only in a few cultivars. Recently, several lines having telocentric chromosomes have been produced and classified but their potentialities as cytogenetic tools have yet to be explored. Some African populations of pearl millet carry B-chromosomes in their karyotype. Cytogenetics of B-chromosomes has been reported in great detail. Bs undergo spontaneous changes to produce deficient- and iso-chromosomes. The main effect of B-chromosomes is on chiasma frequency which is exerted by the relative amounts of chiasma promoting euchromatin and the chiasma depressing heterochromatin in the Bs. Haploid plants occur occasionally and sometimes show a low degree of seed set, offering a possibility of establishing homozygous inbred lines. Cytogenetics of several spontaneous and induced autotetraploids have been reported. In general quadrivalent formation between the seven sets of four homologues was random. Seed set of the autotetraploids could be improved by selection; improved seed fertility was found to be associated with increased chiasma frequency, increased quadrivalent frequency and regular distribution of chromosomes at anaphase I. Genes controlling morphological characters of plant phenotype segregate independent of those controlling fertility and in pearl millet polyploidy per se is not limiting to plant vigour. Primary trisomics represent the best studied among the aneuploids of pearl millet. All the seven primary trisomics have been identified and described. Some were used in assigning genes to specific chromosomes but in general trisomies have poor vigour and fertility, and show low frequency of transmission. Apart from B-chromosomes, cytogenetics of interchanges has been the best studied aspect of pearl millet. The frequency of co-orientation of an interchange complex at metaphase I, which determines the fertility or sterility of the interchange heterozygote, is influenced by the genetic background and thus is theoretically amenable for selection leading to improved fertility of the heterozygote. Interchange tester-stocks have been assembled which can be used to identify the chromosomes involved in any newly obtained interchange. A complex interchange line involving all the chromosomes of the complement has also been produced, but the ring-of-fourteen produces total male and female sterility.Genotypic control of mitosis and meiosis has been reported, with reference to chromosome numerical mosaicism, multiploid sporocytes, desynapsis and chromosome fragmentation, and male sterility. Pearl millet being a largely outbreeding species, forced inbreeding was mainly found to result in loss of morphological vigour and reduction in mean chiasma frequency per PMC. Interspecific hybrids between pearl millet and several related species have been cytologically investigated and homology of the seven chromosomes of pearl millet with seven of the fourteen chromosomes of P. purpureum has been demonstrated. Cytogenetic evidence from haploids, autopolyploids and interspecific hybrids has indications to suggest that the haploid number of x = 7 is derived from x = 5, but the evidence is inconclusive and needs critical evaluation.