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Displacement of chromosomes in mitosis: a technique for assessing differential chromosome error.

To assess whether displacement--the appearance of individual chromosomes inside of the ring of chromosomes attached to spindle fibers--is a valid method of analyzing chromosome misdivision, displacement data from six healthy female subjects were compared with data for mitotic and meiotic nondisjunction. Statistical analysis indicated that nondisjunction and displacement are equivalent methods of assessing differential chromosome error. Tests for homogeneity of the data sets showed no significant heterogeneity for the mitotic data, but chromosomes 1, 2, 11, 16, 17, 19, and 20 were responsible for significant heterogeneity in the meiotic data.

Adult↗

Structure and chromosomal mapping of a highly polymorphic repetitive DNA sequence from the pseudoautosomal region of the mouse sex chromosomes.

The pseudoautosomal region of the Mov15 mouse strain is marked by a Moloney murine leukemia provirus. The sequences flanking the Mov15 provirus were molecularly cloned and shown to consist of a tandemly repeated sequence of 31 nucleotides. Copy number variation of this repeat most likely accounts for the polymorphism in the mouse pseudoautosomal region detected with a probe from the flanking sequences. In situ hybridization to metaphase chromosomes showed heavy labeling of the pairing region of the X and Y chromosomes. The repetitive sequence was also found at the subtelomeric region of three autosomes. A similar level of amplification as the one seen on the sex chromosomes seems to be present on chromosomes 9 and 13. Lower copy number appear to be present on chromosome 4.

Animals↗

Localization of four human chromosome 21 genes--SOD1, ETS2, IFNAR, and CBR--to two different chromosomes in the marsupial species Macropus eugenii.

We have mapped the chromosomal location of four genes previously assigned to human chromosome 21--Cu/Zn superoxide dismutase (SOD1), the protooncogene ETS2, the interferon alpha/beta receptor gene (IFNAR), and the carbonyl reductase gene (CBR)--in the tammar, Macropus eugenii. The genes are localized on two separate autosomes: SOD1 and CBR map to chromosome 7 and ETS2 and IFNAR map to chromosome 3 or 4. These results provide the first example of asynteny between SOD1/CBR and ETS2/IFNAR in a mammalian species. The results suggest that either this synteny group has been disrupted in the marsupial lineage, or, alternatively, the genes located on human chromosome 21 may have been joined after the marsupials diverged from the eutherian mammals some 130-150 million years ago.

Alcohol Oxidoreductases↗

A new rat repetitive DNA family shows preferential localization on chromosome 3, 12 and Y after fluorescence in situ hybridization and contains a subfamily which is Y chromosome specific.

A DNA segment, containing a so far unknown repetitive DNA sequence has been isolated by reassociation of sheared total rat genomic DNA. FISH with this probe gave a strong hybridization signal on the satellites and in the centromeric region of chromosomes 3 and 12 and on the q-arm of the Y chromosome. A much weaker signal was seen in the centromeric region of chromosomes 11, 19 and X. The repeat unit of this repetitive DNA sequence is a 195-200 bp monomer, which is tandemly repeated. Screening of a rat genomic lambda library resulted in the isolation of variant members of this repeat family. FISH results with these members showed differences in their hybridization pattern especially when posthybridization washings were performed under higher stringency. Under these conditions one phage gave a strong hybridization signal only on the Y chromosome; other phage showed only weak hybridization patterns on different chromosomes. A subclone of the Y-specific phage was sequenced and showed large sequence homology with the 195-200 bp monomer. In Southern blot experiments this Y-specific sequence detects several male specific sequences, although some cross-hybridization with closely related sequences in both male and female DNA can also be observed.

Animals↗

Synteny conservation between parts of human chromosome 4q and bovine and ovine chromosomes 6.

Booroola Merino sheep are characterized by a high ovulation rate attributable to the presence of the FecB allele of the FEC gene. This gene, which has been assigned to sheep chromosome 6, is linked to the gene for EGF, which in man is located on the long arm of chromosome 4 (HSA 4q). To increase the number of known markers on sheep chromosome 6, we used comparative mapping data from sheep, man, and cattle. In our study, we show a synteny between EGF and the genes PDHA2 and FGF5 (from HSA 4q) and microsatellites ILSTS018, ILSTS090, and ILSTS093 (from bovine chromosome 6) in sheep. We also show that the conservation between HSA 4q and sheep chromosome 6 is disrupted between EGF and FGF2.

Animals↗

Mitotic chromosomal bcl-2. I. Stable expression throughout the cell cycle and association with isolated chromosomes.

Bcl-2 is present in a cytoplasmic distribution in cells that express high levels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and others have shown that bcl-2 is present on the surface of mitotic chromosomes. However, monoclonal antibodies reactive with an epitope representing amino acids 41-54 of the bcl-2 sequence did not detect bcl-2 in other phases of the cell cycle. This study extended those earlier findings to determine if bcl-2 was expressed as a cyclin or if this pattern was an artifact of immunocytochemistry. Immunofluorescence studies in several other human cell lines showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A comparison of available commercial antibodies showed that, in spite of reactivity with denatured bcl-2 on Western blots, clear reactivity with bcl-2 in fixed cells was found only with those reactive with the (a.a. 41-54) epitope. With RNase protection and Western blot analyses, cells synchronized at various stages of the cell cycle showed constant levels of bcl-2 mRNA and protein. Analysis of bcl-2 using Western blots showed a band with the same apparent molecular weight as that seen in comparison with authentic bcl-2 overexpressed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, permeabilized preparations was influenced by protease treatment, phosphate, and pH. Studies using isolated chromosomes showed that much of the bcl-2 in these cells was attached to chromosomes in mitosis, had the expected molecular weight, and was phosphorylated in the same manner as that seen in whole-cell extracts. These results show that bcl-2 is not a cyclin and that the bcl-2 localized on chromosomes is the same molecule seen by immunoblotting. These results suggest that the reactive (a.a. 41-54) epitope present in bcl-2 is somehow modified or masked in interphase.

Animals↗

Tiling microarray analysis of rice chromosome 10 to identify the transcriptome and relate its expression to chromosomal architecture.

BACKGROUND: Sequencing and annotation of the genome of rice (Oryza sativa) have generated gene models in numbers that top all other fully sequenced species, with many lacking recognizable sequence homology to known genes. Experimental evaluation of these gene models and identification of new models will facilitate rice genome annotation and the application of this knowledge to other more complex cereal genomes. RESULTS: We report here an analysis of the chromosome 10 transcriptome of the two major rice subspecies, japonica and indica, using oligonucleotide tiling microarrays. This analysis detected expression of approximately three-quarters of the gene models without previous experimental evidence in both subspecies. Cloning and sequence analysis of the previously unsupported models suggests that the predicted gene structure of nearly half of those models needs improvement. Coupled with comparative gene model mapping, the tiling microarray analysis identified 549 new models for the japonica chromosome, representing an 18% increase in the annotated protein-coding capacity. Furthermore, an asymmetric distribution of genome elements along the chromosome was found that coincides with the cytological definition of the heterochromatin and euchromatin domains. The heterochromatin domain appears to associate with distinct chromosome level transcriptional activities under normal and stress conditions. CONCLUSION: These results demonstrated the utility of genome tiling microarray in evaluating annotated rice gene models and in identifying novel transcriptional units. The tiling microarray sanalysis further revealed a chromosome-wide transcription pattern that suggests a role for transposable element-enriched heterochromatin in shaping global transcription in response to environmental changes in rice.

Chromosomes, Plant↗

Developmental abnormalities in mouse embryos tetrasomic for chromosome 11: apparent similarity to embryos functionally disomic for the x chromosome.

Adopting a mating system involving two different Robertsonian translocations with monobrachial homology, we studied the early development of mouse embryos trisomic or tetrasomic for chromosome 11. A developmental delay of 12-24 hours was evident in trisomic embryos at embryonic day (E)7.5, whereas tetrasomic embryos apparently had stopped growth by E6.5 without formation of extraembryonic structures. This extremely severe developmental abnormality found in tetrasomic embryos is similar to that reported in embryos having two active X chromosomes in extraembryonic cell lineages. Autosomal tetrasomy, but not autosomal trisomy, can lead to such early developmental errors. Thus, a reasonable inference would be that the X chromosome is twice as active as the autosome. Probably, the X chromosome became upregulated in response to the evolutionary necessity of minimizing haplo-insufficiency brought about by miniaturization of the Y chromosome.

Aneuploidy↗

[Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei].

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.

Animals↗

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.

Alpha Particles↗

[Detection of numerical chromosomal aberrations in epididymal sperm of mice using three-color FISH with chromosome-specific DNA probes].

To detect malsegregation of chromosomes during meiosis in male mice, three-color FISH using DNA probes specific for mouse chromosomes X, Y and 8 was performed on epididymal sperm decondensed for 30 min each in 10 mmol/L DTT and 4 mmol/L LIS, and conventional chromosome counting was also carried out on C-banded MMII cells. Comparisons between these two methods indicate as the following: (1) three-color FISH is a simple, rapid and sensitive approach; (2) The estimate of the frequency of aneuploidy obtained from three-color FISH analysis is more reliable since extremely large numbers of sperm (more than 10,000 sperm per animal) are scored; (3) The rates of cells arrested at meiosis I and meiosis II (representing diploid sperm) can be detected only by using the three-color FISH approach; (4) Malsegregation of chromosomes occurred during both meiosis I and meiosis II can be detected simultaneously by using this new approach. The probes and scoring criteria used in FISH on interphase sperm nuclei, and the necessity of using three or more chromosome-specific DNA probes to thoroughly evaluate aneugenic effects of factors tested on meiosis in male mice were discussed.

Animals↗

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.

Chromosome Aberrations↗

MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice.

Msh4 (MutS homolog 4) is a member of the mammalian mismatch repair gene family whose members are involved in postreplicative DNA mismatch repair as well as in the control of meiotic recombination. In this report we show that MSH4 has an essential role in the control of male and female meiosis. We demonstrate that MSH4 is present in the nuclei of spermatocytes early in prophase I and that it forms discrete foci along meiotic chromosomes during the zygotene and pachytene stages of meiosis. Disruption of the Msh4 gene in mice results in male and female sterility due to meiotic failure. Although meiosis is initiated in Msh4 mutant male and female mice, as indicated by the chromosomal localization of RAD51 and COR1 during leptonema/zygonema, the chromosomes fail to undergo normal pairing. Our results show that MSH4 localization on chromosomes during the early stages of meiosis is essential for normal chromosome synapsis in prophase I and that it acts in the same pathway as MSH5.

Animals↗

[Cytogenetic maps of lampbrush chromosomes of newts of the genus Pleurodeles: an algorithm of lampbrush chromosome identification in Pleurodeles waltl by immunocytochemical marker-loop staining with polyclonal anti-Ro52 antisera].

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodeles newts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoprotein (RNP) marker loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain newt lampbrush chromosome preparations. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker loops along the chromosome length were constructed.

Algorithms↗

A chromosome 14-specific human satellite III DNA subfamily that shows variable presence on different chromosomes 14.

We describe a new subfamily of satellite III DNA (pTRS-63), which, by a combination of in situ hybridization to human metaphase chromosomes and analysis of a panel of somatic cell hybrids, is shown to be specific for human chromosome 14. This DNA has a basic 5-bp repeating unit of diverged GGAAT which is tandemly repeated and organized into either one of two distinct higher-order structures of 5 kb (designated the "L" form) or 4.8 kb (designated the "S" form). In addition, a third (Z) form, representing no detectable levels of this satellite III subfamily, is found. Results from five somatic cell hybrid lines and from a number of informative human individuals suggest that, on any one chromosome 14, only one of the three forms may exist. Subchromosomally, this sequence has been mapped to the p11 region and is distal to the domain occupied by another previously described satellite III subfamily (pTRS-47) found on chromosome 14. The pTRS-63 sequence described adds to the understanding of the structural organization of the short arm of human chromosome 14 and should be useful for the investigation of the molecular etiology of the frequently occurring t(13q14q) and t(14q21q) Robertsonian translocations.

Base Sequence↗

Heterochromatin and chromosome evolution: a FISH probe of Cebus apella paraguayanus (Primate: Platyrrhini) developed by chromosome microdissection.

Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.

Animals↗

[Study of super-structure of eukaryotic chromosomes. I. The super-structure of metaphase chromosomes in pigs].

The super-structure of fine and close metaphase chromosomes was studied with the optical micro multistage amplifying system designed by author himself, which could show the fine structure about 15nm in diameter of chromosomes. The micrographs of chromosomal super-structure made of DNA-nuclear protein or non-histone protein were shown respectively in the present paper. The chromotids were composed of long fibres about 20-30nm in diameter in a regular helical form directly. The characters and parameters of chromosomal super-structure were described, and a dynamical helical model of the chromosomal structure was suggested.

Animals↗

[Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes].

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.

Chromosome Mapping↗