Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Structural analysis and complete physical map of Arabidopsis thaliana chromosome 5 including centromeric and telomeric regions.

Previously, we have reported a fine physical map of Arabidopsis thaliana chromosome 5, except for the centromeric and telomeric regions, by ordering clones from YAC, P1, TAC, and BAC libraries of the genome consisting of the two contigs of upper arm and lower arm, 11.6 M bases and 14.2 M bases, respectively. Here, the remaining centromeric and telomeric regions of chromosome 5 are completely characterized by the ordering of clones and PCR amplifications. Chromosome 5 of Arabidopsis thaliana ecotype Columbia is about 28.4 M bases long. The centromeric region is estimated at about 2 M bases long between two 5S-rDNA clusters. The 180-bp repeat region mainly consists of blocks of 180-bp tandem family and various type retroelements dispersed over a 500-kb region. The telomeric regions of chromosome 5 are characterized by PCR cloning, sequencing and hybridization. The telomere repeats at both ends are about 2.5-kb long and interestingly, telomere-associated repeats (approximately 700 bp) are found near both ends of chromosome 5.     

Two new cases of the Christchurch (Ch1) chromosome 21: evidence for clinical consequences of de novo deletion 21P-

We performed an investigation of two unrelated cases with extremal variants of chromosome 21 without visible materials of the short arms (Christchurch or Ch1 chromosome). In the first case chromosome 21p- was initially detected during routine cytogenetic amniocentesis. Chromosomal variant was inherited from phenotypically normal father to phenotypically normal fetus (phenotypically normal boy after the birth). The second case of chromosome 21p- was detected in 7 years old boy, referred to cytogenetic analysis due to mental retardation and mild congenital malformation, including prenatal hypoplasia, microcephaly, low-set dysplastic ears, short nose, micrognatia, short neck. Molecular characterization of 21p-variant chromosomes was performed by the use of FISH with DNA probes specific to the short arm and centromeric region of chromosome 21 (telomeric, beta-satellite, ribosomal, classical satellite and alphoid DNA probes). Chromosomes 21p-hybridized positively only with telomeric DNA at both chromosomal ends and alphoid DNA probes at centromeric region of the first patient. In second case (de novo deletion of 21p), the Ch1 was associated with clinical phenotype and loss of telomeric and subtelomeric DNA in the p-arm of chromosome 21. Therefore, the complete absent of the short arm of chromosome 21 may be considered as abnormal. We propose that de novo deletion 21p- could have negative consequences due to absence of large portion of chromosomal DNA from the p-arm (telomeric, satellite or ribosomal DNAs) and following imbalance in organization and functioning of genome.     

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retrotransposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed. Received: 30 July 1999 / Accepted: 19 September 1999     

Cytogenetic analysis of some Brazilian marsupials (Didelphidae: Marsupialia)

Three species of marsupials from the Amazon region (Marmosa cinerea, Caluromys lanatus, and Didelphis marsupialis) and two from the region of S?o Paulo (Didelphis marsupialis and Didelphis albiventris) were studied. The G-banding pattern of the species with 2n = 14 (M. cinerea and C. lanatus) was very similar, as well as the pattern of G-bands in the species with 22 chromosomes (Didelphis). All of the autosomes of M. cinerea and D. albiventris have centromeric C-bands and the Y chromosome is totally C-band positive. The long arm of the M. cinerea X chromosome is completely C-band positive except for a negative band close to the centromeric region. In D. albiventris the long arm of the X chromosome is C-band positive except for a negative band close to the telomeric region. In M. cinerea the silver-stained nucleolar organizer regions (Ag-NORs) are found in the acrocentric chromosomes, being located in the telomeric region of one pair and in the centromeric region of the other pair. Caluromys lanatus has centromeric Ag-NORs in one acrocentric and in one submetacentric chromosome pairs. Didelphis marsupialis has three chromosome pairs with telomeric Ag-NORs. In D. albiventris the Ag-NORs are terminal and located in both arms of one pair and in the long arm of two pairs of chromosomes.     

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.     

New telomere formation during the process of chromatin diminution in Ascaris suum

Chromatin diminution in the parasitic nematode Ascaris suum represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. At the molecular level, it is a rather complex event including chromosome breakage, new telomere formation and DNA degradation. Analysis of a cloned somatic telomere (pTel1) revealed that it has been newly created during the process of chromatin diminution by the addition of telomeric repeats (TTAGGC)n to a chromosomal breakage site (Müller et al., 1991). However, telomere addition does not occur at a single chromosomal locus, but at many different sites within a short chromosomal region, termed CBR1 (chromosomal breakage region 1). Here we present the cloning and the analysis of 83 different PCR amplified telomere addition sites from the region of CBR1. The lack of any obvious sequence homology shared among them argues for a telomerase-mediated healing process, rather than for a recombinational event. This hypothesis is strongly supported by the existence of 1-6 nucleotides corresponding to and being in frame with the newly added telomeric repeats at almost all of the telomere addition sites. Furthermore, we show that telomeres are not only added to the ends of the retained chromosomal portions, but also to the eliminated part of the chromosomes, which later on become degraded in the cytoplasm. This result suggests that de novo telomere formation during the process of chromatin diminution represents a non-specific process which can heal any broken DNA end.     

Evolutionarily plastic regions at human 3p21.3 coincide with tumor breakpoints identified by the "elimination test"

We have previously found with the microcell hybrid-based "elimination test" that human chromosome 3 transferred into murine or human tumor cells regularly lost certain 3p regions during tumor growth in SCID mice. The most common eliminated region, CER1, is approximately 2.4 Mb at 3p21.3. CER1 breakpoints were clustered in approximately 200-kb regions at both telomeric and centromeric borders. We have also shown, earlier, that tumor-related deletions often coincide with human/mouse synteny breakpoints on 3p12-p22. Here we describe the results of a comparative genomic analysis on the CER1 region in Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, Gallus gallus, Mus musculus, Rattus norvegicus, and Canis familiaris. First, four independent synteny breaks were found within the CER1 telomeric breakpoint cluster region, comparing human, dog, and chicken genomes, and two independent synteny breaks within the CER1 centromeric breakpoint cluster region, comparing human, mouse, and chicken genomes, suggesting a nonrandom involvement of tumor breakpoint regions in chromosome evolution. Second, both CER1 breakpoint cluster regions show recent tandem duplications (seven Zn finger protein family genes at the telomeric and eight chemokine receptor genes at the centromeric side). Finally, all genes from these regions underwent horizontal evolution in mammals, with formation of new genes and expansion of gene families, which were displayed in the human genome as tandem gene duplications and pseudogene insertions. In contrast the CER1 middle region contained evolutionarily well-conserved solitary genes and a minimal amount of retroposed genes. The coincidence of evolutionary plasticity with CER1 breakpoints may suggest that regional structural instability is expressed in both evolutionary and cancer-associated chromosome rearrangements.     

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.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

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.     

The controlling sequence for site-specific chromosome breakage in Tetrahymena

Site-specific chromosome breakage occurs in many ciliated protozoa during nuclear differentiation. We have determined the cis-acting sequence that controls this process in Tetrahymena thermophila. The Tetrahymena ribosomal RNA gene is bounded by two breakage sites. Injection of this gene into developing macronuclei leads to breakage at these sites. Deletion analysis has localized the sequences essential for breakage to a 28 bp region that includes a 15 bp sequence (Cbs) known to be present in other breakage sites. Insertions of Cbs allow breakage to occur at new sites, which is accompanied by elimination of surrounding DNAs and formation of telomeric sequences, as it is at natural sites. Thus, Cbs is the necessary and sufficient sequence signal for chromosome breakage in Tetrahymena.     

Duplication of the bcr and gamma-glutamyl transpeptidase genes.

The Philadelphia (Ph') translocation involves rearrangement of the bcr gene located on chromosome 22. Hybridization experiments revealed the presence of multiple bcr gene-related loci within the human genome. Two of these were molecularly cloned and characterized. Both loci contain exons and introns corresponding to the 3' region of the bcr gene. Restriction enzyme and DNA sequence analysis indicate a very high degree of conservation between bcr and the two related genomic sequences. Both bcr-related loci are located on chromosome 22, one centromeric, the other telomeric, of the bcr gene. Within the two bcr related genomic sequences, fragments or the complete coding sequences of an unrelated gene were found to be present. This gene was identified; it encodes gamma-glutamyl transferase, an enzyme involved in the glutathione metabolism.     

Origin and molecular structure of a midget chromosome in a common wheat carrying rye cytoplasm

The origin and molecular structure of the midget chromosome that is retained in a common wheat with rye cytoplasm, were studied by using fluorescent in situ hybridization (FISH). FISH with biotinylated rye genomic DNA as a probe clearly showed that the midget chromosome had originated from certain part(s) of rye chromosome(s). The midget chromosome did not possess sequences similar to wheat rDNA nor to a rye telomeric sequence with a 350 bp repeat unit. However, another repetitive sequence (120 bp family) of rye was found to occur at one end of the midget chromosome. The telomeric repeat sequences from Arabidopsis thaliana cross-hybridized to both ends of the midget chromosome as well as to wheat chromosomes. From the results obtained in this and previous studies, it is assumed that the midget chromosome originated from part of a rye chromosome, most likely the centromeric region of chromosome 1R, and that the telomeric sequences were synthesized de novo.by R. Appels     

Mechanisms of chromosome number evolution in yeast

The whole-genome duplication (WGD) that occurred during yeast evolution changed the basal number of chromosomes from 8 to 16. However, the number of chromosomes in post-WGD species now ranges between 10 and 16, and the number in non-WGD species (Zygosaccharomyces, Kluyveromyces, Lachancea, and Ashbya) ranges between 6 and 8. To study the mechanism by which chromosome number changes, we traced the ancestry of centromeres and telomeres in each species. We observe only two mechanisms by which the number of chromosomes has decreased, as indicated by the loss of a centromere. The most frequent mechanism, seen 8 times, is telomere-to-telomere fusion between two chromosomes with the concomitant death of one centromere. The other mechanism, seen once, involves the breakage of a chromosome at its centromere, followed by the fusion of the two arms to the telomeres of two other chromosomes. The only mechanism by which chromosome number has increased in these species is WGD. Translocations and inversions have cycled telomere locations, internalizing some previously telomeric genes and creating novel telomeric locations. Comparison of centromere structures shows that the length of the CDEII region is variable between species but uniform within species. We trace the complete rearrangement history of the Lachancea kluyveri genome since its common ancestor with Saccharomyces and propose that its exceptionally low level of rearrangement is a consequence of the loss of the non-homologous end joining (NHEJ) DNA repair pathway in this species.     

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.     

Mapping the distribution of the telomeric sequence (T(2)AG(3))(n) in rock wallabies,Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization. ii. the lateralis complex

The distribution of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) was examined by fluorescence in situ hybridization in the six Petrogale (rock wallabies) taxa of the lateralis complex. As expected, the (T(2)AG(3))(n) sequence was located at the termini of all chromosomes in all taxa. However, the sequence was also present at several nontelomeric (viz., interstitial and centromeric) sites. The signals identified were associated with either ancient rearrangements involved with the formation of the 2n = 22 plesiomorphic macropodine karyotype or more recent rearrangements associated with karyotypes derived from the 2n = 22 karyotype. Interstitial (T(2)AG(3))(n) signals identified on chromosomes 3 and 4 in all six species of the lateralis complex and a large centromeric signal identified on chromosome 7 in the five subspecies/races of P. lateralis appear to be related to the more ancient rearrangements. Subsequent chromosome evolution has seen these signals retained, lost, or amplified in different Petrogale lineages. Within the lateralis complex, in two submetacentric chromosome derived by recent centric fusions, the telomeric sequence was identified at or near the centromere, indicating its retention during the fusion process. In the two taxa where chromosome 3 was rearranged via a recent centromeric transposition to become an acrocentric chromosome, the telomeric signal was located interstitially.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

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.     

Microsatellite-centromere mapping in Cynoglossus semilaevis using gynogenetic diploid families produced by the use of homologous and non-homologous sperm

Twenty-one microsatellite markers were studied in three meiogynogenetic families of Cynoglossus semilaevis gunther for centromere mapping using half-tetrad analysis. Among the 13 mapped loci, 10 were estimated to be located in the telomeric region, one in the centromeric region, and two in the intermediate region of the chromosome. This study provides a basis for constructing a linkage map of C. semilaevis .