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Biomedical subjects

J Meyne

Publications and source records attributed to J Meyne.

At least 19 recordsLinked to original sources
PubMed1999

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Animals↗
PubMed1999

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.

Animals↗
PubMed1996

Ordered tandem arrangement of chromosomes in the sperm heads of monotreme mammals.

A very old unanswered question in classical cytology is whether chromosomes are arranged randomly in sperm or whether they occupy specific positions. Even with modern methods of chromosome painting, it is difficult to resolve this question for the very condensed and almost spherical sperm head of most mammals. We have taken advantage of the unusual fibrillar sperm head of monotreme mammals (echidna and platypus) to examine the position of chromosome landmarks in a two-dimensional array. We used fluorescence and radioactive in situ hybridization to telomeric, rDNA, and unique sequences to show that chromosomes are arranged tandemly and in a defined order in the sperm nucleus.

Animals↗
PubMed1996

On the origin of lateral asymmetry.

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2'-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.

Animals↗
PubMed1996

CO-FISH reveals inversions associated with isochromosome formation.

Despite the likely prevalence and documented biological impact of inverted DNA sequences in humans and other species, our ability to detect them on a routine basis is limited. The technique of chromosome orientation fluorescence in situ hybridization (CO-FISH) was used to detect obligate chromosome inversions associated with isochromosome formation in two human cell lines. Simultaneous hybridization of a strand-specific telomeric probe allowed us to deduce the absolute orientation of repetitive DNA sequences associated with the inverted region. These results show that, in principle, CO-FISH could be used to detect virtually any type of inversion, including those likely to escape detection by other methods. Prospective applications of the technique are discussed in relation to its principal limitation, the present availability of suitable single-stranded DNA probes.

Base Sequence↗
PubMed1996

A new method for detecting pericentric inversions using COD-FISH.

A new approach for detecting chromosomal inversions, based on the recently developed technique of chromosome orientation and direction fluorescence in situ hybridization (COD-FISH), is presented. COD-FISH is a strand-specific modification of standard FISH technology which allows the hybridization of single-stranded probes to one, and only one, chromatid of a metaphase chromosome. It can be used to determine the absolute 5'-to-3' direction of DNA target sequences with respect to the short-to-long arm direction of a given chromosome. Since an inversion reverses the orientation of DNA sequences within the inverted region, an inversion becomes detectable as a "switch" in probe signal from one chromatid to the other, when compared to a reference probe outside of the inverted region. Pericentric inversions in chromosomes 1, 8, 10, and X, which had previously been identified by chromosome banding, were analyzed by the COD-FISH technique. The results presented here demonstrate that COD-FISH can be used for the detection of pericentric inversions and that, in some instances, it provides additional information not obtainable by more conventional methods of cytogenetic analysis. Practical limitations of the COD-FISH technique are also discussed.

Centromere↗
PubMed1995

FISH analysis of the telomere sequences of bulldog ants (Myrmecia: formicidae).

Chromosomes from several species of ants from the genus Myrmecia were hybridized with deoxyoligomer probes of either (T2AG2)7, the putative insect telomere repeat sequence, or (T2AG3)7, the vertebrate telomere repeat sequence. While both sequences hybridized over a range of stringency conditions, (T2AG2)n was clearly the predominant sequence at the termini of the Myrmecia chromosomes. No interstitial sites of either sequence were detected. The genus Myrmecia has a wide range of karyotypes, with chromosome numbers ranging from 2n=2-84. It has been hypothesized that the ancestral karyotype was 2n=4 and karyotype evolution proceeded with an increase in chromosome number. In the absence of detectable interstitial sites of telomere sequence, it is interesting to speculate on the origin of the new telomeres as the chromosome numbers increased.

Animals↗
PubMed1995

Direction of DNA sequences within chromatids determined using strand-specific FISH.

The 5' to 3' direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5' to 3' direction of three repetitive DNA sequences, each for a different human repeat family.

Base Sequence↗
PubMed1994

Identification of trisomy in Macaca fascicularis by fluorescence in situ hybridization with a human chromosome 13 DNA library.

A juvenile macaque monkey with abnormal phenotypic and behavioral features was studied cytogenetically. An additional autosome was found in over 90% of the animal's cultured cells. This chromosome, subsequently identified as number 16 in the macaque karyotype by G-banding, was shown to be mostly homologous with human chromosome 13 using fluorescence in situ hybridization of a human chromosome specific cosmid library. Although the monkey, now deceased, exhibited some abnormal physical and behavioral features, none of the severe clinical characteristics associated with human chromosome 13 trisomy were apparent. We suggest that the incomplete expression of 13-trisomy observed could result if the macaque chromosome were deficient in some of the region(s) of chromosome 13 common to humans affected with the disorder.

Animals↗
PubMed1994

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA.

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the -A-B-A-B-A- form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant -B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashion along the DNA strand.

Base Sequence↗
PubMed1993

Strand-specific FISH reveals orientation of chromosome 18 alphoid DNA.

A method is described for making in situ hybridization strand specific. Through the use of a synthetic DNA probe of a repetitive sequence in the centromeric region of chromosome 18, it is shown that the repeats exist in a head-to-tail tandem array. The method should be useful for studies of the molecular organization and mapping of chromosomes.

Base Sequence↗
PubMed1992

SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastic progression of human fibroblasts.

In human fibroblasts, the expression of SV40 large T antigen is known to cause a variety of chromosomal aberrations and especially dicentric chromosomes. In some cases, the later aberrations have been reported to be reversible telomeric associations. We report here aberration and chromosome number studies of twenty-nine T antigen positive lineages, studied from their initiation by transfection of T antigen sequences into human diploid fibroblasts, until crisis or immortalization occurred or, in some cases until the lines became tumorigenic in nude mice. The data show that T antigen consistently produced chromosomal instability of both number and structure by an active process that began before transformation indicators were positive and continued throughout neoplastic progression. The most frequently observed aberrations were dicentric chromosomes, which were shown to be true dicentrics by examination by in situ hybridization with telomeric sequences. These data are consistent with the hypothesis that T antigen causes human fibroblasts to become neoplastically transformed by successive rounds of chromosomal mutation and lineage evolution.

Antigens, Polyomavirus Transforming↗
PubMed1992

Highly conserved repetitive DNA sequences are present at human centromeres.

Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.

Animals↗
PubMed1992

Interstitial hybridization sites of the (TTAGGG)n telomeric sequence on the chromosomes of some North American hylid frogs.

Interstitial hybridization sites for the (TTAGGG)n telomeric repeat sequence were present in all seven species of hylid frogs examined and in a triploid hybrid between two of the species. Intra- and interspecific differences and similarities in hybridization sites agreed with what is known about the systematics of these species. Chromosome fusions, fissions, and inversions do not appear to have played a role in the evolution of the interstitial sites for the telomeric repeat in the species examined.

Animals↗
PubMed1991

The centromere-kinetochore complex: a repeat subunit model.

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

Animals↗
PubMed1991

Ribosomal RNA gene site polymorphism in Bufo terrestris.

Individual specimens of Bufo terrestris were discovered that possessed ribosomal gene locations in addition to those normally found. Every specimen from an island population that was examined had extra sites, whereas fewer individuals from coastal mainland populations and none from inland populations had them. Although the extra ribosomal gene locations probably did not arise through gross structural chromosome rearrangements, their origin remains unclear.

Animals↗