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PubMed2004

Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.

Study of the maize (Zea mays L.) somatic chromosomes (2n = 20) has been difficult because of a lack of distinguishing characteristics. To identify all maize chromosomes, a multicolor fluorescence in situ hybridization procedure was developed. The procedure uses tandemly repeated DNA sequences to generate a distinctive banding pattern for each of the 10 chromosomes. Fluorescence in situ hybridization screening trials of nonsubtracted or subtracted PCR libraries resulted in the isolation of microsatellite 1-26-2, subtelomeric 4-12-1, and 5S rRNA 2-3-3 clones. These three probes, plus centromeric satellite 4 (Cent4), centromeric satellite C (CentC), knob, nucleolus-organizing region (NOR), pMTY9ER telomere-associated sequence, and tandemly repeated DNA sequence 1 (TR-1) were used as a mixture for hybridization to root-tip chromosomes. All 10 chromosomes were identified by the banding and color patterns in the 14 examined lines. There was significant quantitative variation among lines for the knob, microsatellite, TR-1, and CentC signals. The same probe mixture identifies meiotic pachytene, late prophase I, and metaphase I chromosomes. The procedure could facilitate the study of chromosomal structure and behavior and be adapted for other plant species.

Chromosome Painting↗
PubMed1972

The role of X-chromosome inactivation during spermatogenesis (Drosophila-allocycly-chromosome evolution-male sterility-dosage compensation).

Inactivation of the single X chromosome in the primary spermatocytes of species with heterogametic males is postulated as a basic control mechanism on the chromosomal level that is required for normal spermatogenesis. This view is supported by (a) cytological observations of X-chromosome allocycly in the primary spermatocytes of all male-heterogametic organisms that were adequately examined, (b) autoradiographic evidence of early cessation of transcription by the X chromosome in the mouse and three species of grasshopper, and (c) the male sterility of animals with certain X-chromosome rearrangements that cannot be attributed to misfunction of specific genes. X-chromosome inactivation during spermatogenesis is proposed as the ideal system for studies of genetic control at the chromosomal level.

Animals↗
PubMed1988

Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer.

We used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for greater than 20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization, we propose the following order for these loci: pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z 1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UM PH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, we have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.

Animals↗
PubMed1998

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens.

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

Amino Acid Sequence↗
PubMed2000

Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe.

PURPOSE: To discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay. MATERIALS AND METHODS: Human lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes. RESULTS: The combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%. CONCLUSION: The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.

Chromosome Aberrations↗
PubMed2005

Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC).

There is a need for quick dose estimation by a simple method in radiation accidents. This study develops a simple and rapid dose estimation protocol for victims of such accidents, in particular those involving high radiation doses. Human peripheral blood lymphocytes (PBL) were gamma-irradiated in vitro at several dose points up to 60 Gy, and were stimulated with phytohaemagglutinin-P (PHA-P) for 2 days to obtain dividing cells. PBL were then forced to condense prematurely, using 50 nM calyculin A, and the obtained chromosome spreads were Giemsa stained. The G2-PCC (prematurely condensed chromosomes) index and chromosome number for each radiation dose point were scored. G2-PCC were stably induced using calyculin A within 24 h delays in stimulation of PBL with PHA-P. The chromosome number of G2-PCC increased steeply with radiation doses up to 30 Gy at a rate of 0.31 Gy(-1) and then decreased at 0.30 Gy(-1) up to 40 Gy. More than 10% of G2-PCC index remained up to a 15 Gy dose. Even after 40 Gy irradiation, about 2% PCC index was obtained, and this value was enough to score a sufficient number of chromosome spreads for analysis. Therefore, the combined use of chromosome number and G2-PCC index allows biodosimetry to be done easily and rapidly. If PCC are not induced using calyculin A, it is strongly suggested that the radiation dose is over 50 Gy. A rapid and easy dose estimation for large dose exposure whole-body was realized by combined analysis of Giemsa-stained chromosome number of G2-PCC and PCC index using calyculin A. This simple method will be of use for rapid decision making of therapy for radiation accident victims. This method also has potential for use as a biodosimeter for partial-body exposure accidents.

Adult↗
PubMed1996

Detection and evaluation of chromosomal aberrations induced by high doses of gamma-irradiation using immunogold-silver painting of prematurely condensed chromosomes.

We developed a method for detecting chromosomal aberrations after irradiation with high doses of gamma-rays. At high dose levels, few of the irradiated cells are able to enter mitosis so that it is difficult to obtain enough mitotic chromosomes using a conventional colcemid block. Therefore, okadaic acid was used to condense prematurely the chromosomes of interphase cells. Even after irradiation with doses > 40 Gy, okadaic acid was able to force sufficient numbers of cells to condense their chromosomes. Segments of chromosomes were then detected by chromosome painting using a gold-conjugated antibody followed by silver enhancement. This simple method allows highly damaged chromosomes to be detected with great sensitivity.

Cell Cycle↗
PubMed1965

CHROMOSOME VELOCITY DURING MITOSIS AS A FUNCTION OF CHROMOSOME SIZE AND POSITION.

Chromosome velocity has been studied in living Melanoplus differentialis spermatocytes by phase contrast cinemicrography. Melanoplus chromosomes (and bivalents) differ in length by as much as 1:3.5. As expected, no size-dependent velocity differences were detected in anaphase, and this is also shown to be true for the less predictable movements during prometaphase congression. The size of the X chromosome can change during observation following x-irradiation, but this is equally without influence on velocity. However, an effect of position on velocity is found in both prometaphase and in anaphase: the chromosomes furthest from the central interpolar axis move 25 per cent faster than more central chromosomes. A simple mechanical model relating frictional resistance and mitotic forces to chromosome velocity is discussed in detail. Calculations from the model suggest that a significant difference in the force acting on a large, as compared with a small chromosome is necessary to account for the observed similarity in velocity. Therefore, it is concluded that the mitotic forces are so organized or regulated that velocity is, within limits, independent of load. The implications of velocity-load independence in relation to the molecular origin of mitotic forces are discussed.

Anaphase↗
PubMed1983

Architecture of metaphase chromosomes and chromosome scaffolds.

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.

Chromatin↗
PubMed2001

Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively.

Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.

Adolescent↗
PubMed1999

Gain of chromosomes 15 and 19 is frequent in both mouse hepatocellular carcinoma cell lines and primary tumors, but loss of chromosomes 4 and 12 is detected only in the cell lines.

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6JxC3H/HeJ F(1) and C3H/HeJxC57BL/6J F(1) mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9). These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.

Animals↗
PubMed1998

Induction of chromosomal aberrations (unstable and stable) by inhibitors of topoisomerase II, m-AMSA and VP16, using conventional Giemsa staining and chromosome painting techniques.

Frequencies of symmetrical and asymmetrical exchange aberrations induced by two inhibitors of topoisomerase II, namely, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) and etoposide (VP16), were estimated in human peripheral blood lymphocytes. The aberrations were scored using conventional Giemsa staining and fluorescence in situ hybridization (FISH) techniques, using chromosome-specific DNA libraries. Stable aberrations (translocations) were detected using two cocktails of DNA libraries specific for three chromosomes, namely 1, 3 and X and 2, 4 and 8, representing approximately 40% of the whole human genome. The frequencies of dicentrics and translocations increased in a dose-dependent manner, however, m-AMSA was found to be a more potent inducer of chromosomal aberrations in comparison with VP16 (at concentrations at which comparable frequencies of aberrations were induced) by 20- to 30-fold. When corrected for DNA content of chromosomes in each cocktail, a higher frequency of translocations with the cocktail consisting of chromosomes 2, 4 and 8 in comparison with 1, 3 and X was evident. The genomic translocation frequency calculated from chromosome painting analysis for m-AMSA exceeded that estimated for dicentrics by approximately 2-fold. However, for VP16 almost equal frequencies of both types of chromosome exchange were found.

Amsacrine↗
PubMed1989

Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes.

A linking library consists of genomic DNA fragments which contain a specific rare restriction enzyme site; such clones are very useful as probes in pulsed field gel electrophoresis and in mapping and cloning large regions of DNA. However, identifying those linking clones which map to a certain chromosomal region can be laborious. Therefore, we have developed a straightforward procedure for constructing a linking library directly from flow-sorted chromosomes. As a test of the approach, a NotI linking library was constructed from the chromosome 17 fraction of a flow-sort of human chromosomes, using only 70 ng of DNA. Thirteen of sixteen linking clones were mapped to chromosome 17, suggesting that the library is highly enriched for this chromosome. This method should be generally applicable to other chromosomes and enzymes as well.

Chromosome Mapping↗
PubMed2005

A tiling microarray expression analysis of rice chromosome 4 suggests a chromosome-level regulation of transcription.

The complete genome sequence of cultivated rice (Oryza sativa) provides an unprecedented opportunity to understand the biology of this model cereal. An essential and necessary step in this effort is the determination of the coding information and expression patterns of each sequenced chromosome. Here, we report an analysis of the transcriptional activity of rice chromosome 4 using a tiling path microarray based on PCR-generated genomic DNA fragments. Six representative rice organ types were examined using this microarray to catalog the transcribed regions of rice chromosome 4 and to reveal organ- and developmental stage-specific transcription patterns. This analysis provided expression support for 82% of the gene models in the chromosome. Transcriptional activities in 1643 nonannotated regions were also detected. Comparison with cytologically defined chromatin features indicated that in juvenile-stage rice the euchromatic region is more actively transcribed than is the transposon-rich heterochromatic portion of the chromosome. Interestingly, increased transcription of transposon-related gene models in certain heterochromatic regions was observed in mature-stage rice organs and in suspension-cultured cells. These results suggest a close correlation between transcriptional activity and chromosome organization and the developmental regulation of transcription activity at the chromosome level.

Chromosome Mapping↗
PubMed1977

Double minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell line.

Four human neuroblastoma cell lines were studied by chromosome banding techniques. All of the lines contained a marker chromosome with a long nonbanding homogeneously staining region (HSR). The HSR-containing chromosome differed in each line. One line contained two classes of cells: one with an HST marker chromosome and the other with double minute chromosomes. Each cell had one of these abnormalities; no cell had both. The presence of two additional chromosomal markers in all cells of this line indicates a common origin. These observations suggest that the double minute chromosomes are derived from the HSR.

Cell Line↗
PubMed2005

Silencing mitosin induces misaligned chromosomes, premature chromosome decondensation before anaphase onset, and mitotic cell death.

Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.

Anaphase↗
PubMed1991

Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins.

Fluram (Fluorescamine; 4-phenylspiro(furan-2(3H),1'-phthalan)-3,3'-dione) is a fluorogenic reagent, which permits the detection of primary amines by forming highly fluorescent pyrrolinone derivatives. This reagent has been used on methanol-acetic acid fixed metaphase chromosomes of mouse and man and proved to be very effective in differentiating chromosome regions in both genomes. Mouse centromeric heterochromatin is highly reactive, showing intense fluorescence in all centromeric regions, whereas human chromosomes show no fluorescence in such regions. In addition, a G-like banding pattern is also obtained in both types of chromosomes. The differential reactivity of each chromosome region showed by this method demonstrates a heterogeneous distribution of chromosome proteins, resulting in a chromosome banding pattern, which is in this case species dependent.

Animals↗
PubMed1998

ZOO-FISH and R-banding reveal extensive conservation of human chromosome regions in euchromatic regions of river buffalo chromosomes.

Commercially available human chromosome (HSA) painting probes were hybridized on river buffalo (Bubalus bubalis, 2n = 50) chromosomes by using FISH and R-banding techniques. Clear hybridization FITC-signals revealed extensive conservation of human chromosome regions in this species and demonstrated that human chromosome probes primarily paint euchromatic regions (R-bands). The present results are discussed in the light of previous gene mapping data obtained in river buffalo and ZOO-FISH data in cattle, and in relation to the standard bovine chromosome nomenclatures. In particular, HSA 8, HSA 10, HSA 11, and HSA 16+7 paint, respectively, BBU 1p, BBU 4p, BBU 5p, and BBU 24, which are homoeologous, respectively, to cattle chromosomes 25, 28, 29 and 27. Thus, these river buffalo chromosome arms can serve as markers to resolve discrepancies in the nomenclature of cattle and related species.

Animals↗