Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.Abbreviations DHFR dihydrofolate reductase - MCA 3-methylcholanthrene - NS nickel sulfide     

Jumping translocations in spontaneous abortions

Chromosome translocations involving one donor chromosome and multiple recipient chromosomes have been referred to as jumping translocations (JTs). Acquired JTs are commonly observed in cancer patients, mainly involving chromosome 1. Constitutional forms of JTs mostly involve the acrocentric chromosomes and their satellites and have been reported in patients with clinical abnormalities. Recognizable phenotypes resulting from these events have included Down, Prader-Willi, and DiGeorge syndromes. The presence of JTs in spontaneous abortions has not been previously described. The breakpoints of all JTs occur in areas rich in repetitive DNA (telomeric, centromeric, and nucleolus organizing regions). We report two different unstable chromosome rearrangements in samples derived from spontaneous abortions. The first case involved a chromosome 15 donor. The recipient chromosomes were 1, 9, 15, and 21, and the respective breakpoints were in either the heterochromatic regions or the centromeres. FISH studies confirmed that the breakpoints of the jumping 15 rearrangement did not involve the Prader-Willi region but originated at the centromere or in the proximal short arm. A second case of instability was observed with a rearrangement resulting from a presumed de novo 8;21 translocation. Three JT cell lines were observed. They consisted of a deleted 8p chromosome, a dicentric 8;21 translocation, and an 8q isochromosome. The instability regions appeared to be at the pericentromeric region of chromosome 8 and the satellite region of chromosome 21. Both cases proved to be de novo events. The unstable nature of the JT resulting in chromosomal imbalance most likely contributed to the fetal loss. It appears that JT events may predispose to chromosomal imbalance via nondisjunction and chromosomal rearrangement and, therefore, may be an unrecognized cause of fetal loss.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Selective digestion of mouse metaphase chromosomes

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000xg pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.     

Replication of yeast DNA and novel chromosome formation in mouse cells.

To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

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     

Chromosome banding in Amphibia. XXIV. The B chromosomes of Gastrotheca espeletia (Anura,Hylidae)

The mitotic chromosomes of an Ecuadorian population of the marsupial frog Gastrotheca espeletia were analyzed by means of banding techniques and fluorescence in situ hybridization. This species is characterized by unusual supernumerary (B) chromosomes. The maximum number of B chromosomes is 9 and they occur in three different morphological types. Banding analyses show that the B chromosomes are completely heterochromatic, consist of AT base pair-rich repeated DNA sequences, replicate their DNA in very late S-phase of the cell cycle, and are probably derived from a centromeric or paracentromeric region of a standard (A) chromosome. Exceptionally, the B chromosomes carry 18S + 28S ribosomal RNA genes and the conserved vertebrate telomeric DNA sequence appears to be underrepresented. Flow cytometric measurements of the nuclear DNA content differentiate between individuals with different numbers of B chromosomes. Significantly more B chromosomes are present in female than in male animals.     

Artificial chromosome formation in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15–30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Evgueni V. Ananiev, deceased Evgueni V. Ananiev and Chengcang Wu contributed equally to this work. Novel materials described in this publication may be available for noncommercial research purposes on acceptance and signing of a material transfer agreement. In some cases, such materials may contain or be derived from materials obtained from a third party. In such cases, the distribution of material will be subject to the requisite permission from any third-party owners, licensors, or controllers of all or parts of the material. Obtaining any permission will be the sole responsibility of the requestor.     

Molecular cytogenetics of the equidae. II. purification and cytological localization of a (G + C)-rich satellite DNA from Equus hemionus onager and cross-species hybridization to E. asinus chromosomes

A (G + C)-rich satellite DNA component (p = 1.716 g/ml) has been fractionated from the total DNA of the Iranian subspecies of the Asiatic wild ass, Equus hemionus onager, by successive dactinomycin-CsCl and netropsin sulfate-CsCl isopycnic gradients. Complementary 3H-RNA (cRNA) transcribed from the satellite DNA hybridized predominantly to the centromeric and telomeric constitutive heterochromatic regions of onager chromosomes. These studies have suggested that satellite DNA's with similar sequences are present in the centromeric, as well as telomeric, heterochromatic regions of some onager chromosomes. The centromeric region of the fusion metacentric t(23;24) of the onager is deficient in sequences homologous to the onager 1.716 g/ml satellite DNA, indicating a loss of satellite DNA during fusion or an amplification of the satellite DNA in the centromeric regions of the acrocentric chromosomes 23 and 24 subsequent to fission. Sequences complementary to onager 1.716 g/ml satellite DNA show extensive hybridization to the constitutive heterochromatin of the feral donkey (E. asinus) karyotype, consistent with a view of conservation and amplification of similar or identical sequences in the two species.     

Centromere mitotic recombination in mammalian cells

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.     

Centromere organization in chromosomes of the mouse

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.     

First prenatally detected small supernumerary neocentromeric derivative chromosome 13 resulting in a non-mosaic partial tetrasomy 13q

Neocentromeres are functional centromeres located in non-centromeric euchromatic regions of chromosomes. The formation of neocentromeres results in conferring mitotic stability to chromosome fragments that do not contain centromeric alpha satellite DNA. We present a report of a prenatal diagnosis referred to cytogenetic studies due to ultrasound malformations such as large cisterna magna, no renal differentiation, hypotelorism and ventriculomegaly. Cytogenetic analysis of GTG-banded chromosomes from amniotic fluid cells and fetal blood cells revealed a de novo small supernumerary marker chromosome. Molecular cytogenetic studies using fluorescence in situ hybridization and comparative genomic hybridization showed this marker to be an inverted duplication of the distal portion of chromosome 13q which did not contain detectable alpha satellite DNA. The neocentromeric constriction was located at band 13q31. The presence of a functional neocentromere on this marker chromosome was confirmed by immunofluorescence with antibodies to centromere protein-C. The anatomopathologic study revealed a female fetus with facial dysmorphisms, low set ears and renal dysplasia. Ten small supernumerary neocentromeric chromosomes originating from the distal region of chromosome 13q have been reported to date. There are only three additional cases described with the location of the neocentromere in band 13q31. This is the first reported case detected prenatally.     

The mouse Y* chromosome involves a complex rearrangement, including interstitial positioning of the pseudoautosomal region.

Cytological analysis of the mouse Y* chromosome revealed a complex rearrangement involving acquisition of a functional centromere and centromeric heterochromatin and attachment of this chromosomal segment to the distal end of a normal Y* chromosome. This rearrangement positioned the Y* short-arm region at the distal end of the Y* chromosome and the pseudoautosomal region interstitially, just distal to the newly acquired centromere. In addition, the majority of the pseudoautosomal region was inverted. Recombination between the X and the Y* chromosomes generates two new sex chromosomes: (1) a large chromosome comprised of the X chromosome attached at its distal end to all of the Y* chromosome but missing the centromeric region (XY*) and (2) a small chromosome containing the centromeric portion of the Y* chromosome attached to G-band-negative material from the X chromosome (YX). Mice that inherit the XY* chromosome develop as sterile males, whereas mice that inherit the Y*X chromosome develop as fertile females. Recovery of equal numbers of recombinant and nonrecombinant offspring from XY* males supports the hypothesis that recombination between the mammalian X and Y chromosomes is necessary for primary spermatocytes to successfully complete spermatogenesis and form functional sperm.     

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation

Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability. By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences. Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time. In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes. The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process. The involvement of telomere-related sequences in centromere function is discussed. Received: 15 September 1999; in revised form: 21 November 1999 / Accepted: 24 December 1999     

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae)

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.     

Analysis of mitotic and expression properties of human neocentromere-based transchromosomes in mice

Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model.     

Origin of an apparent B chromosome by mutation,chromosome fragmentation and specific DNA sequence amplification

The present study documents the de novo origin of an apparent B chromosome in Plantago lagopus. The origin was associated with mutation (aneuploidy), chromosome fragmentation, specific DNA sequence amplification, addition of telomeric repeats, and centromeric misdivision. It originated in the progeny of trisome 2, from the excision of 5S rDNA and 18S, 5.8S, 25S rDNA sequences located on chromosome 2, and within a few generations acquired many characteristics of an apparent B chromosome. The B chromosome has preferential transmission through the male (41%, P<0.025) and female gametes (42%, P<0.01) but does not affect plant phenotype. The B chromosome is completely heterochromatic, has a functional centromere and does not pair at meiosis with any A chromosomes of the standard complement. Fluorescence in situ hybridization analysis showed that it arose from massive amplification of 5S rDNA sequences, has 18S, 5.8S, 25S rDNA sequences at the ends of both arms and telomeric repeats at both termini. Ag-NOR-banding and determination of the maximum number of nucleoli in interphase cells indicate that the nucleolar organizer regions at the ends of both arms of the B chromosome are active in organizing nucleoli. RNA blot analysis showed that the 5S rDNA sequences are not transcribed. To our knowledge, this is the first report that fully documents one of the mechanisms by which B chromosomes may arise in nature.     

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.     

Interspecies differences of co-orientation of primary polytene chromosomes in ovarian nurse cells in Drosophila melanogaster, D. simulans and D. mauritiana]

Essential differences in the architecture of primary polytene chromosomes in ovarian nurse cells between Drosophila melanogaster, D. simulans and D. mauritiana were discovered. The individual chromosomes of D. melanogaster (as well as their arms) are separated significantly from each other and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites. D. simulans has the combination of the centromeric parts of the chromosomes X, 2, 3 into the "elongated" chromocentre, and in addition, the arms of chromosomes are also separated and attached to the nuclear envelope. Unlike the rest of the chromosomes, D. mauritiana has the chromosome 3 with firm combinated arms and the large heterochromatic block in the centromere, without being attached to the nuclear envelope.