Mode of ATM-dependent suppression of chromosome translocation

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.     

Clinical consequences of a human non-fluorescent Y chromosome (Ynf)

A new case of ambiguous genitalia and immature tissue in the left gonad is presented. Cytogenetic findings with various techniques demonstrated that the distal two-thirds of the long arm of the Y chromosome is deleted. Q-banding showed a non-fluorescent Y; three positive bands were however noted when the DA/DAPI technique was applied. After a review of the literature, it was concluded that the non-fluorescent Y chromosome (Ynf) when inherited from generation to generation is a heteromorphism in normal males. However, in our case, where the proband's Y is lacking the fluorescent segment, a simple deletion does not appear to adequately explain the DA/DAPI positive bands. Possibly, a deletion followed by a structural rearrangement of the non-fluorescent segment had occurred de novo. The highly Y-specific DNA sequences present in the fluorescent segment are absent in these patients. The abnormal development in these cases is due to the presence of the 45,X cell line. The gene responsible for spermatogenesis has been localized to the non-fluorescent region in the long arm of the Y chromosome. Furthermore, it is concluded that two types of non-fluorescent Y chromosomes can be found in the population; one is a normal inherent heteromorphic variant, while the other appears to be an abnormality, especially in cases with azoospermia. Such distinctions should clearly be established prior to genetic counseling for patients with so called Ynf or del (Yd).     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Dicentric chromosome frequency analysis using slit-scan flow cytometry

Slit-scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following gamma irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV gamma-rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit-scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear-quadratic manner according to the relation f(D) = 4.52 x 10(-5) + 5.72 x 10(-5) D + 1.19 x 10(-4) D2.     

A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints.

Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.     

Lack of synergistic effect between X-ray and UV irradiation on the frequency of chromosome aberrations in PHA-stimulated human lymphocytes in the G1 stage.

PHA-stimulated human lymphocytes in the G1 stage were irradiated with UV radiation and X-rays, and the cells were analyzed for chromosomal aberrations in the first mitotic division. The frequency of dicentric chromosomes after single X-irradiation in the G1 stage was about twice the yield in the G0 stage. No increase in the yield of dicentrics was observed after combined irradiation with UV and X-rays. This is contrary to the finding for G0 lymphocytes, where a 2-fold increase of chromosome aberrations was observed. UV irradiation of G1 lymphocytes induced chromatid-type aberrations whereas no significant yield of dicentric chromosomes was observed. This is in agreement with previous findings in Chinese hamster cells in the G1 stage [7]. Irradiation of G0 lymphocytes with UV radiation induce a low frequency of dicentric chromosomes. Thus, the present data indicate that the ratio between chromosome-type and chromatid-type aberrations is different in the G1 and G0 stages in human lymphocytes irradiated with UV radiation.     

Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

A stable dicentric chromosome: Both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric configuration

A stable, dicentric human chromosome, which is known from light microscopy to show a 50:50 distribution between monocentric/dicentric appearance, was examined by conventional electron microscopy and after labelling the centromere with anticentromere antibodies from CREST serum. Both centromeres of the chromosome developed kinetochores whether in monocentric or dicentric configuration. The eight monocentrics observed had all developed kinetochores at the centromere outside the constriction; at least six of them also had kinetochores at the centromere in the constriction. The dicentrics from glutaraldehyde fixed cells had spindle microtubules attached to both kinetochore sets irrespective of monocentric/dicentric configuration. The chromosome thus appeared to use both centromeres, either equally or with one serving a chromatid adhesion function while the second was used for transport along the spindle.     

Postmating reproductive isolation and modification of the ‘sex ratio’ trait inDrosophila subobscura induced by the sex chromosome gene arrangement A2+3+5+7

Drosophila subobscura males were trapped in Tunis, and mated to different lab strains. The offspring from 15% of these wild Tunisian males consisted of more than 90% females. Chromosome analysis showed that these males had carried the A2+3+5+7 which was described as 'sex ratio' chromosome, endemic in North Africa and the Canary Islands. The mean female frequency in the total offspring of all trapped males was 61%. This percentage was stable for more than ten years. F1 females from the mating of wild Tunisian males to Küsnacht standard females were backcrossed to Küsnacht standard males. In the offspring of this back cross, A2+3+5+7-males were sterile. The fertility of A2+3+5+7-males could be restored in two ways: 1) When the Küsnacht standard autosomes were replaced by Tunisian autosomes, most of the A2+3+5+7-males were again fertile. The A2+3+5+7-chromosome seems to be incompatible with autosomes from a geographically distant region. 2) After exchanging autosomes between lines, in which A2+3+5+7-males were 100% sterile, fertility could be restored in 30% of the A2+3+5+7-males. All males carrying one specific A2+3+5+7 stayed sterile as well in combination with autosomes from different lines as with Tunisian autosomes. The Y-chromosome and the cytoplasm was the same in sterile and in fertile A2+3+5+7-males. Therefore the origin of the Y-chromosome and the cytoplasm could not play a major role in sterility. The percentage of fertile males varied for different Y-chromosomes. Thus the Y-chromosomes may have some influence on fertility in this study. The restored fertility of A2+3+5+7-males can be explained assuming complementation. Defects of autosomes, and perhaps of the Y-chromosomes, could differ from line to line. Genomic changes may have happened when the A2+3+5+7 was in the genome together with autosomes and Y-chromosomes from Swiss populations. The A-chromosome which prevented fertility in all combinations, is thought to be itself defective. In one cross the 'sex ratio' trait was modified. In the offspring of some males the male to female ratio was 1:1. The variable sex ratio in the offspring from different males may have been an effect of the autosomes. In short, the intraspecific hybrid sterility and modification of the 'sex ratio' trait in D. subobscura indicate that: a) an incompatibility possibly existed between the gene arrangement A2+3+5+7 from one population and autosomes respectively Y-chromosomes from a population isolated from the former. b) In addition unidentified genomic changes occurred, c) induced by the A2+3+5+7-chromosome. d) The sex chromosomes A and Y, and the autosomes were involved.     

Identification of several chromosome aberrations in man by the fluorescent method using quinacrine yprite]

The applications of the fluorescent staining of chromosomes with quinacrine mustard allowed to identify a dicentric Y-chromosome in two patients with defected external gynaetalies: a boy of 15 years old and a girl of 2 years old. Both the patients had mosaicism of sex chromosomes: 45, x/46, x dic (Y). The dicentric Y-chromosome, resembling chromosome, 16, had bright luminescence of the thelomeric regions characteristic of the normal Y-chromosome. Besides, a balanced autosomic translocation t (1, 14) (q 31, q 3) was found in the girl identified also with quinacrine mustard fluorescent staining.     

A dicentric iso (Y) chromosome in an infertile male

A dicentric Y chromosome was detected in a 30-year-old azoospermic male patient who was found to be mosaic for 45,X/46,X,dic Y(qter----p11::p11----qter). The dicentric iso (Y) chromosome was identified conclusively with C-banding, G-banding and Q-banding techniques. The relationship of structural abnormalities of the Y chromosome and azoospermia is discussed.     

Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast

We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.     

Induction of complete and incomplete chromosome aberrations by bleomycin in human lymphocytes

Bleomycin (BLM) is a clastogenic compound, which due to the overdispersion in the cell distribution of induced dicentrics has been compared to the effect of high-LET radiation. Recently, it has been described that in fibroblast derived cell lines BLM induces incomplete chromosome elements more efficiently than any type of ionizing radiation. The objective of the present study was to evaluate in human lymphocytes the induction of dicentrics and incomplete chromosome elements by BLM. Peripheral blood samples have been treated with different concentrations of BLM. Two cytogenetic techniques were applied, fluorescence plus Giemsa (FPG) and FISH using pan-centromeric and pan-telomeric probes. The observed frequency of dicentric equivalents increases linearly with the BLM concentration, and for all BLM concentrations the distribution of dicentric equivalents was overdispersed. In the FISH study the ratio between total incomplete elements and multicentrics was 0.27. The overdispersion in the dicentric cell distribution, and the linear BLM-concentration dependence of dicentrics can be compared to the effect of high-LET radiation, on the contrary the ratio of incomplete elements and multicentrics is similar to the one induced by low-LET radiation (~0.40). The elevated proportion of interstitial deletions in relation to total acentric fragments, higher than any type of ionizing radiation could be a characteristic signature of the clastogenic effect of BLM.     

Sex-specific differences in meiotic chromosome segregation revealed by dicentric bridge resolution in mice

The meiotic properties of paracentric inversion heterozygotes have been well studied in insects and plants, but not in mammalian species. In essence, a single meiotic recombination event within the inverted region results in the formation of a dicentric chromatid, which usually breaks or is stretched between the two daughter nuclei during the first meiotic anaphase. Here, we provide evidence that this is not the predominant mode of exchange resolution in female mice. In sharp contrast to previous observations in other organisms, we find that attempts to segregate the dicentric chromatid frequently result not in breakage, stretching, or loss, but instead in precocious separation of the sister centromeres of at least one homolog. This often further results in intact segregation of the dicentric into one of the meiotic products, where it can persist into the first few embryonic divisions. These novel observations point to an unusual mechanism for the processing of dicentric chromosomes in mammalian oogenesis. Furthermore, this mechanism is rare or nonexistent in mammalian spermatogenesis. Thus, our results provide additional evidence of sexual dimorphism in mammalian meiotic chromosome behavior; in "stressful" situations, meiotic sister chromatid cohesion is apparently handled differently in males than in females.     

The effect of oxygen concentration on the X-ray induction of chromosome aberrations in human lymphocytes.

In vitro dose--response curves, for unstable chromosome aberration induction in human lymphocytes under conditions of full oxygenation or of anoxia, have been obtained using 250 kVp X-rays. Dicentric yields have been fitted to the quadratic function Y = alpha D + beta D2. An Oxygen Enhancement Ratio (OER) ranging from 7.2 to 2.7 was calculated from the coefficients of these curves possibly indicating that the data do not fit to the dose-modifying model of the oxygen effect although the differences are not statistically significant. A similar analysis of total aberration data yields an OER of 3.6 to 2.7 fitting much more satisfactorily to the dose-modifying model. Variations in dicentric yield induced by 3.0 Gy and 0.75 Gy of X-rays with increasing oxygen concentration were plotted and for each dose a constant dicentric yield was observed at oxygen levels of 2 and 250 ppm. Above 250 ppm yields increased steeply up to about 1% oxygen and then more gradually to a maximum at 100% oxygen.     

Identification and characterization of a new type of asymmetrical dicentric chromosome derived from a single maternal chromosome 18

Molecular cytogenetic analysis identified a new type of dicentric chromosome involving different breakpoints at 18q in a female fetus. The chromosome anomaly was designated as an asymmetrical pseudoisodicentric chromosome 18, 46,XX,psu dic(18)(pter-->q11.2::q21.3-->pter)mat. A series of BAC clones for 18q11.2 and q21.3 regions were used to identify one breakpoint within the region q11.2 between 19.8 and 21.6 Mb from the telomere of 18p and another breakpoint within q21.3 between 55.4 and 56.9 Mb from the telomere of 18p by FISH analysis. Real-time quantitative PCR and microsatellite analysis further verified that the dicentric chromosome was maternal in origin and resulted from a break-reunion between sister chromatids of a single maternal chromosome. We propose that a loop-type configuration of sister chromatids took place and that the break-reunion occurred at cross sites of the loop to form an asymmetrical isodicentric chromosome during either mitosis or meiosis. In this case, the asymmetrical pseudoisodicentric resulted in an 18pter--> q11.2 duplication and an 18q21.3-->qter deletion, which could have led to certain dysmorphic features of 18q- syndrome in this fetus.     

The effect of hyper- and hypothermia on induced telomeric chromosome fusion]

After administration of colcemid and 5-BrdU in the cell culture, the cells pass through the first interphase to delay in mitosis. Then the cells overcome the colcemid blockade, and polykaryocytes with micronuclei are formed. The second interphase in followed by the second mitosis, during which dicentric chromosomes are observed. These dicentrics are the result of telomeric chromosome fusion. The action of hyperthermia (40 degrees C) during the whole period of colcemid and 5-BrdU treatment or that of the hyperthermia (40 degrees C) only during the first 17 hours (the first interphase and the first mitosis) lead to the increased frequency of dicentrics. Under condition of hypothermia (34 degrees C) the frequency of dicentric formation decreases. Changes in cultivation temperature during the latest 25 hr of colcemid and 5-BrdU action (the second interphase and the second mitosis) exert no influence on the dicentric formation frequency. Because there are no dicentrics in cells during the metaphase of the first mitosis, it is supposed that the temperature--sensitive period may be the latest steps of colcemid blockade, i.e. the period of formation of micronuclei.     

Time-effect relationship for unstable chromosome exchange levels in Chernobyl clean-up workers

The post-irradiation changes of dicentric and centric ring levels were studied in Chernobyl liquidators using the data of 507 individual chromosomal surveys of persons sampled at different time after their activities at Chernobyl NPP accident zone. The time-effect relationship within 0-10.5 years after exposure was displayed as exponential decline of the mean chromosome exchange frequency with average decay half-time 2.2 y. During 10.5-13 years after exposure the increasing and stabilization of chromosome exchange yield on the level 2-3-times higher than control was observed. In the first few months after irradiation the dicentric and centric ring frequency in liquidators had the clear reverse correlation with the duration of person's duties at the Chernobyl zone. The parameters of unstable chromosome exchange elimination were independent on the initially induced aberration yield, that resulted in earlier reaching the subcontrol level in persons who had more protracted duration of duties at the Chernobyl accident zone.     

Regularities in forming chromosome aberrations in cattle lymphocytes from irradiation in vitro

Aberrations in lymphocytes of cattle blood exposed to gamma-radiation with doses from 1 to 7 Gy were studied. The rate of variable cells depended linearly on the irradiation dose, whereas the total frequency of aberrations, as well as that of dicentric and annular chromosomes followed a linear-quadratic dependence.