End-to-end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens

Two mutant strains harbouring a linear chromosome whose size reached 13 Mb (versus approximately 8 Mb for the wild type) were characterized. This chromosomal structure resulted from the fusion in inverted orientation of two chromosomes partially deleted on the same arm. The fusion occurred by illegitimate recombination between 6 bp repeats. This chromosomal structure was inherited in strict association with a high level of genetic instability (30% of mutants in a single progeny, phenomenon also called hypervariability) and chromosomal instability. In contrast, derivatives, which did not retain the chromosome fusion, showed a wild-type-like instability frequency (c. 1%). Stabilization of the chromosomal structure occurred by chromosome arm replacement or circularization. A high variability of the terminal inverted repeat (TIR) length in the rescued chromosomes (from 5 kb to approximately 1.4 Mb for linear derivatives) was observed. Mutant lineages harbouring the chromosomal fusion are characterized by a highly heterogeneous distribution of DNA in the spores, by the presence of spores without DNA as well as aberrant sporulation figures, and by the production of spores with a low germination rate. The wild-type characteristics were restored in the descendants, which lost the chromosomal fusion. Thus, the fusion of deleted chromosomes initiates a cycle of chromosome instability sharing several levels of analogy with the behaviour of dicentric chromosomes in eukaryotes. We propose that the high instability of the fused chromosomes results from the duplication of a region involved in partitioning of the chromosomes (parAB-oriC ).     

Chromosome cycles of the basidiomycete Cyathus stercoreus (Schw.) De Toni

Summary Chromosome cycles of the basidiomycete Cyathus stercoreus (meiosis and mitosis) are described. The fusion of two nuclei of compatible mating type takes place in the developing basidium at the end of telophase of the presynaptic mitosis. Synapsis follows immediately after nuclear fusion. During synapsis the chromosomes elongate, facilitating pairing. Meiosis and mitosis are essentially similar to those processes in higher organisms. Details of divisional stages are described and illustrated with photomicrographs. The presence of centrioles and spindles is demonstrated. The presence of quadrivalents as well as secondary associations of like chromosomes suggests that Cyathus stercoreus may be a tetraploid species.     

Chromosome damage in mouse-human hybrid cells after BUdR treatment and light irradiation

Mouse cells grown in the presence of bromodeoxyuridine were fused with human cells in the presence of Sendai virus. The cells were then irradiated with blue light. Three days after fusion, the hybrid cell metaphases showed intact human chromosomes and many mouse chromatids with numerous abnormalities, indicating the great potentiality of the technique for genetic analysis and manipulations.     

Chromosomal evolution in the vlei rat, Otomys irroratus (Muridae: Otomyinae): a compound chromosomal rearrangement separates two major cytogenetic groups

G- and C-banding delimits two cytogenetic groups within the vlei rat, Otomys irroratus. One has a diploid number of 2n = 24, resulting from a centric fusion of chromosomes 7 and 12 of the O. irroratus standard coupled with a tandem fusion to chromosome 8. The second has a diploid number of 2n = 28, lacks the compound chromosome, and appears to have a far wider geographic distribution within South Africa. Additionally, the two groups differ through the presence of cytotype-specific heterozygous centric fusions and one to three B chromosomes which appear as floating polymorphisms in the 2n = 28 complex.     

Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells

Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human–mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human–mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg–sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg–sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg–sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

Duplication of a conditionally dispensable chromosome carrying pea pathogenicity (PEP) gene clusters in Nectria haematococca

A supernumerary chromosome called a conditionally dispensable chromosome (CDC) is essential for pathogenicity of Nectria haematococca on pea. Among several CDCs discovered in N. haematococca, the PDA1 CDC that harbors the pisatin demethylation gene PDA1 is one of the best-studied CDCs and serves as a model for plant-pathogenic fungi. Although the presence of multiple copies is usual for supernumerary chromosomes in other eukaryotes, this possibility has not been examined well for any CDCs in N. haematococca. In this study, we produced strains with multiple copies of the PDA1 CDC by protoplast fusion and analyzed dosage effects of this chromosome. Using multiple methods, including cytological chromosome counting and fluorescence in situ hybridization, the fusion products between two transformants derived from the same strain that bears a single PDA1 CDC were shown to contain two PDA1 CDCs from both transformants and estimated to be haploid resulting from the deletion of an extra set or sets of A chromosomes in the fused nuclei. In phenotype assays, dosage effects of PDA1 CDC in the fusion products were evident as increased virulence and homoserine-utilizing ability compared with the parents. In a separate fusion experiment, PDA1 CDC accumulated up to four copies in a haploid genome.     

Chromosome banding homologies in Swamp and Murrah buffalo

Silver staining of Swamp buffalo (2n = 48) metaphase chromosomes revealed telomeric nucleolus organizer regions (NOR's) located on five pairs of autosomes identified by R-banding as numbers 4 p (submetacentric), 8, 20, 22, and 23 (acrocentrics); interphase nuclei also showed no more than five nucleoli. The Murrah buffalo (2n = 50) was previously reported to have telomeric NOR's located on six pairs, -3 p and 4 p (submetacentric), 8, 21, 23, and 24 (acrocentrics). By comparing the two types of buffalo it was concluded that: all of the chromosomes are similar in banding patterns; chromosome 1 of Swamp results from a telomere-centromere tandem fusion between two chromosomes identified as 4 p and 9, respectively, in the Murrah karyotype, thus accounting for the reduced diploid number of Swamp buffalo; the fusion causes the loss of NOR's on the telomeres of chromosome 4, thus accounting for the reduced number of NOR chromosome pairs of Swamp; the presence of a pale C-band are in the region of junction between chromosome 4 and 9 involved in the fusion suggests that the centromeric region of the later is retained and altered.     

Centromere localization at meiosis and the position of chiasmata in the male and female mouse

Techniques for obtaining differential Giemsa staining of the paracentromeric (p.c.) regions of male and female mouse meiotic chromosomes (centromeric heterochromatin) were explored and standard procedures developed for the different meiotic cells in the two sexes. The best result followed the use of heat at controlled pH in Sörensen's phosphate buffer or in Standard Saline Citrate (SSC) solutions. With these techniques, morphological features of the p.c. regions and their variation were studied in normal animals (CFLP strain) and in a strain (AKR) homozygous for a centric fusion [T(11; ?)-1 Ald] between chromosomes No. 6 and No. 15 (Miller et al., 1971). The Y chromosome was often found to show distinct p. c. staining at first and apparently at second meiotic metaphase, and the X and Y chromosomes were found to associate as bivalents by their long arms. Autosomal p.c. regions showed variation in size which might indicate differences between non-homologous chromosomes but a tendency to similarity between homologues. Differences were found between males and females in respect to proportions and variation of bivalents with single and double chiasmata. The relative positions of chiasmata were different in the two sexes. The presence of the centric fusion in the males did not seem to affect the pairing behaviour of the remaining autosomes or of those taking part in the centric fusion. The possibility is discussed that the p.c. regions, to which also other functions would seem to appertain, may be important for chromosome recognition and pairing, possibly on a quantitative basis.     

Chromosomal abnormalities in ascitic fluid from patients with alcoholic cirrhosis.

Cytology and cytogenetics were used to study ascitic fluid obtained from five patients with alcoholic cirrhosis. Cytological examination showed that all fluids contained numerous mesothelial cells and some leucocytes. Cytogenetic analysis showed abnormal karyotypes in cultured cells from all five patients and in uncultured cells from three. A consistent abnormality was the presence of spreads with over 70 chromosomes. Clones of abnormal cells, with marker chromosomes, pseudodiploidy, or aneuploidy, in an effusion are characteristic of malignancy; the abnormal karyotypes fulfilled these criteria. This finding of abnormal karyotypes indicates that transformation of the mesothelium can occur in vivo, and such a reaction may be a reflection of the mutagenic effect of alcohol.     

Assignment of the gene for acid beta-glucosidase to human chromosome 1.

The structural gene for human acid beta-glucosidase (GBA) has been assigned to chromosome 1 using somatic cell hybridization techniques for gene mapping. The human enzyme was detected in mouse RAG cell-human fibroblast cell hybrids by a sensitive double antibody immunoprecipitation assay using a mouse antihuman GBA antibody. No cross-reactivity between mouse beta-glucosidase and human GBA or neutral beta-glucosidase (GBN) was observed. Fifty-two primary, secondary, and tertiary manmouse hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for the human chromosomes. Without exception, the presence of human GBA in these hybrid clones was correlated with the presence of human chromosome 1 or its enzymatic markers, phosphoglucomutase 1 (PGM1), and fumarate hydratase (FH). All other human chromosomes were eliminated by the independent segregation of GBA and their respective enzyme markers and/or chromosomes. Using a RAG X human fibroblast line with a mouse-human rearrangement of human chromosome 1, the locus for GBA was limited to the region 1p11 to 1qter.     

Monochromosomal rodent-human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker

An improved system for the production of a series of rodent-human hybrids selectively retaining single human chromosomes marked in known locations is described. Such hybrids have significant applications in gene mapping and other genetic studies. Human lymphoblastoid lines were infected with the retroviral vector SP-1, which contains the bacterial his-D gene allowing mammalian cells to grow in the presence of histidinol. Microcell fusion of the infected lymphoblastoid cells with CHO cells was used to produce hybrids containing single human chromosomes retained by histidinol selection. Hybrids containing a single human chromosome 9 and a single human chromosome 19 are described. These have been characterized cytogenetically by G-banding, in situ hybridization, and Southern blot analysis.     

Telomeric signals in robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy.

In situ hybridization with synthetic plant telomeric sequences resulted in labeling of all broad bean (Vicia faba) chromosomes at their ends only. Telocentric chromosomes derived by fission of the metacentric satellite chromosome of V. faba also showed signals at both of their ends, whereas the ancestral metacentric did not display signals at its primary constriction, the point of fission. As in V. faba, all acrocentric mouse chromosomes were labeled by in situ hybridization with a vertebrate telomeric probe at both ends of each chromatid exclusively. However, different metacentric Robertsonian chromosomes derived by fusion of defined acrocentrics did not show signals at their primary constrictions. The mechanism of Robertsonian rearrangement leading to a pseudoaneuploid increase or decrease in chromosome number therefore cannot consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material. The additional assumption of a subdetectable deletion of telomeric sequences after fusion and amplification of these sequences following fission is necessary to explain the present observations.     

Chromosome replication in mouse intraspecific hybrids

Hybrid cells (HY SS2 and HY SS6) arising from the fusion of diploid cells of the mouse lymphosarcoma LS/BL and L cells resistant to 8-azaguanine (HGPRT-) showed slower growth and a longer generation time than the parent lines. The inter- and intrachromosomal timing and patterns of early chromosome DNA replication of parent cells was preserved in the hybrid genome and was not influenced by loss of telocentric chromosomes from LS/BL or L (HGPRT-) cells. Thus DNA chromosome replication sequences are not dependent on the presence of a complete set of chromosomes of the parent cells and do not therefore seem to be a result of interaction between chromosomes not segregated in the hybrid genome.     

FRA2B is distinct from inverted telomere repeat arrays at 2q13

Human chromosome 2 was formed by a telomere-to-telomere fusion of two ancestral ape chromosomes. The fusion point is localized in chromosomal band 2q13, which also contains the rare, folate-sensitive fragile site FRA2B. It has been hypothesized that this fragile site may be related to the presence of interstitial telomeric and subtelomeric sequences, which have come to lie in an inverted repeat arrangement as a result of the fusion event. Fluorescence in situ hybridization of a genomic cosmid c8.1, which spans the fusion point, was carried out on metaphase spreads of an individual who expressed the fragile site at 2q13. We show that the fusion point maps distal to this fragile site. Therefore, we conclude that the inverted arrays of telomeric and subtelomeric sequences found at this fusion point are unlikely to correspond to the rare fragile site at 2q13.     

A cis-acting sequence involved in chromosome segregation in Escherichia coli

Eukaryotic chromosomes contain a locus, the centromere, at which force is applied to separate replicated chromosomes. A centromere analogue is also found in some bacterial plasmids and chromosomes, although not yet identified in the well-studied Escherichia coli chromosome. We aimed to identify centromere-like sequences in E. coli with the premise that such sequences would be the first to migrate towards the cell poles, away from the cell centre where DNA replication is believed to occur. We have labelled different loci on the chromosome by integrating arrays of binding sites for LacI-EYFP and phage lambdacI-ECFP and supplying these fusion proteins in trans. Comparison of such pairs of loci suggests the presence of a centromere-like site close to the origin of replication. Polar migration of the site was dependent on migS, a locus recently implicated in chromosome migration, thus providing strong support for migS being the E. coli centromere.     

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere. Edited by: H. F. Willard