Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster.

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.

Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa.

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.

[Mutations induced by X-rays and some chemical reagents changing Drosophila melanogaster life span].

It has been shown that most of Drosophila melanogaster mutant lines obtained as a result of X-rays irradiation (XI) as well as of the combined action of XI and some chemical agents are characterized by decreased indexes of average (7-40 %) and maximal (1-35 %) life span. Insertion-excision processes at the instable genes white and cut are among the reasons of decreased vitality and shortened life span in induced mutants. Collection of neurodegenerative mutants has been obtained under the influence of ENU. Fast dying of flies and decreased vitality correlated with time point of neurodegenerations in brain structure.

Modulation of chromosome damage localization by DNA replication timing.

PURPOSE: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xpe) and a late replicating long heterochromatic arm (Xqh). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model. MATERIALS AND METHODS: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2'-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xpe labeled) or late S-phase (Xqh labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies. RESULTS: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xpe while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xqh. CONCLUSIONS: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.

Inhibition of Atm and/or Atr disrupts gene silencing on the inactive X chromosome.

ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

[Induction of mutations in unirradiated X-chromosome of Drosophila melanogaster hybrids of repair deficient females mei-41 [D5] and mus209 [B1] and irradiated males M5]. / Induktsiia mutatsii v neobluchennoi X-khromosome Drosophila melanogaster u gibridov samok, mutantnykh po reparatsii linii mus209 i mei-41, i obluchennykh samtsov M5.

It has been analyzed the frequency of the recessive lethal mutations in the unirradiated X-chromosome of Drosophila. Females of wild type (CS) as well as of error-prone (mei-41) and error-free (mus209) mutant strains were used. In CS hybrids the increasing of the mutation rate (p < 0.05) was found. In muc209 hybrids the mutation rate was not affected. In mei-41 hybrids the tendency to decreasing of the mutation rate was found. The obtained results demonstrate the possible role of error-prone repair in the inducing of mutations in the unirradiated X-chromosome in the presence of irradiated homologue.

Exceptional conservation of horse-human gene order on X chromosome revealed by high-resolution radiation hybrid mapping.

Development of a dense map of the horse genome is key to efforts aimed at identifying genes controlling health, reproduction, and performance. We herein report a high-resolution gene map of the horse (Equus caballus) X chromosome (ECAX) generated by developing and typing 116 gene-specific and 12 short tandem repeat markers on the 5,000-rad horse x hamster whole-genome radiation hybrid panel and mapping 29 gene loci by fluorescence in situ hybridization. The human X chromosome sequence was used as a template to select genes at 1-Mb intervals to develop equine orthologs. Coupled with our previous data, the new map comprises a total of 175 markers (139 genes and 36 short tandem repeats, of which 53 are fluorescence in situ hybridization mapped) distributed on average at approximately 880-kb intervals along the chromosome. This is the densest and most uniformly distributed chromosomal map presently available in any mammalian species other than humans and rodents. Comparison of the horse and human X chromosome maps shows remarkable conservation of gene order along the entire span of the chromosomes, including the location of the centromere. An overview of the status of the horse map in relation to mouse, livestock, and companion animal species is also provided. The map will be instrumental for analysis of X linked health and fertility traits in horses by facilitating identification of targeted chromosomal regions for isolation of polymorphic markers, building bacterial artificial chromosome contigs, or sequencing.

[The "image" of the regulatory gene in experiments with Drosophila]. / "Obraz" reguliatornogo gena v opytakh na drozofile.

The mutants referred to as facultative dominant lethals were selected in the progeny of gamma-irradiated Drosophila males. The mutant males were viable and fertile, though their crosses with females of the yellow line yielded no daughters. The mutations obtained differed from the common mutations by (1) extremely varying penetrance of F1 hybrids from crosses with various lines; (2) the uncertain relationships between the mutant and normal alleles; (3) the different expression in somatic and germ cells; (4) the dependence of the expression on the sex of the parent carrying the donor mutations; (5) the mass morphosis formation and (6) the frequent reversal to the norm. These mutations are assigned to the regulatory group and their specific expression (see above) can be helpful in identifying regulatory gene mutations. We assume that the specific expression of the mutations studied is related to specific properties of the regulatory genes. These properties are as follows: (1) only one out of two homologous regulatory genes located on one homolog is in an active state, (2) in the haploid chromosome set the regulatory gene is represented by several alleles (cys-alleles); (3) only one allele ensures the regulatory gene activity.

Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus.

PURPOSE: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. MATERIALS AND METHODS: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain. RESULTS: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain. CONCLUSIONS: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.

A biophysical model for estimating the frequency of radiation-induced mutations resulting from chromosomal translocations.

Gene mutations can be induced by radiation as a result of chromosomal translocations. A biophysical model is developed to estimate the frequency of this type of mutation induced by low-LET radiation. Mutations resulting from translocations are assumed to be formed by misrejoining of two DNA double strand breaks (DSB), one within the gene and one on a different chromosome. The chromosome containing the gene is assumed to occupy a spherical territory and does not overlap spatially with other chromosomes. Misrejoining between two DSB can occur only if the two DSB are closer than an interaction distance at the time of their induction. Applying the model to mutations of the hprt gene induced in G0 human lymphocyte cells by low-LET radiation, it is calculated that mutations resulting from translocations account for about 14% of the total mutations. The value of the interaction distance is determined to be 0.6 micrometers by comparing with the observed frequency of translocations in the X-chromosome.

Induction, processing and persistence of radiation-induced chromosomal aberrations involving hamster euchromatin and heterochromatin.

Euchromatic and heterochromatic regions are easily distinguished in Chinese hamster sex chromosomes, hence offering the possibility of studying the role of chromatin structure in the induction, processing and persistence of radiation-induced chromosome damage. X-ray (4 Gy)-induced breaks in the euchromatic Xp and in the heterochromatic Xq were analysed immediately and 4h after irradiation by premature chromosome condensation (PCC) in combination with either FISH using chromosome arm-specific probes or Giemsa staining. The study, performed with female Chinese hamster splenocytes, was extended to a 34 h recovery followed by arm-specific FISH in metaphase. A significant over-involvement of the heterochromatic Xq in radiation-induced breakage was observed at all sampling times (p<0.001). However, the heterochromatic state had little effect on the processing of the induced lesions. In a second experiment, the persistence of radiation-induced chromosome aberrations (CAs) involving Xp, Xq and Y chromosome was studied with cultured Chinese hamster male splenocytes sampled 30, 56 and 96 h after irradiation (4 Gy). A higher involvement of the heterochromatic regions (Xq and Y) in radiation-induced CAs was again observed in the first sampling time (p<0.001), suggesting that Chinese hamster heterochromatin could be more radiosensitive than euchromatin. Cells with CAs involving heterochromatin were apparently less persistent than those with lesions involving euchromatin. This observation could be attributable to either the distribution of CA per cell or to the fraction of potentially stable exchanges.

Cytogenetic monitoring of hospital workers occupationally exposed to ionizing radiation using the micronucleus centromere assay.

A cytogenetic study was performed in lymphocytes of hospital workers occupationally exposed to X- and gamma-rays using the micronucleus centromere assay. A comparison of the data for the exposed group and an age-matched group of non-exposed hospital workers showed a significant (P < 0.05) increase in centromere-positive micronuclei for the radiation workers, while no effect on centromere-negative micronuclei was present. The observed systematic increase in micronucleus frequency with age was mainly due to increased chromosome loss, reflected in the centromere-positivity of the micronuclei. The micronucleus frequencies were 40% higher in females than in males, which can again be attributed to higher chromosome loss. Two exposed individuals showed exceptionally high micronucleus yields, 90% of which were centromere-positive. In situ hybridization with a centromeric probe for chromosome X shows that X chromosome loss is responsible for these high micronucleus yields. In the studied population, smoking had no significant effect on the micronucleus yields. The results obtained indicate that in contrast to the predominantly clastogenic action of acute exposure to ionizing radiation, the aneugenic properties of radiation may be important after long-term chronic low dose exposure.

Radiation-associated sarcomas are characterized by complex karyotypes with frequent rearrangements of chromosome arm 3p.

Ionizing radiation is a well-known risk factor for sarcoma development. To investigate whether radiation-associated sarcomas are characterized by chromosome aberrations that distinguish them from de novo sarcomas, we identified those patients in our series of more than 500 cytogenetically abnormal sarcomas that fulfilled the following criteria: (1) each patient should have been irradiated for another malignancy at least 3 years prior to the sarcoma diagnosis, and (2) the sarcoma should have developed within the field of radiation. Ten patients fulfilling these criteria could be retrieved (median age at sarcoma diagnosis was 55 years, range 17-79; median latency period between primary tumor and radiation-associated sarcoma was 9 years, range 4-30). The diagnoses were typical for radiation-associated sarcomas: 2 each of malignant fibrous histiocytoma, leiomyosarcoma, and pleomorphic sarcoma, and 1 each of osteosarcoma, fibrosarcoma, myxofibrosarcoma, and spindle cell sarcoma. All 10 cases had relatively complex karyotypes with multiple, mostly unbalanced, structural rearrangements, similar to what has been reported in de novo sarcomas of the corresponding histologic subtypes. The only cytogenetic features that were unusually frequent among the radiation-associated sarcomas were the finding of unrelated clones in 3 cases, and loss of material from chromosome arm 3p, in particular 3p21-3pter, in 8 cases. Loss of the same chromosome segment has been described in 4 of the 8 previously published cases of radiation-associated sarcomas that have been analyzed after short-term culturing, which makes this imbalance significantly (P < 0.001) more frequent among radiation-associated sarcomas (12 of 18 cases) than among unselected cases of the corresponding histologic subtypes (74 of 282 cases). In contrast to the cytogenetic results, no 3p deletions were detected among the 6 cases of the present series that could be analyzed by comparative genomic hybridization (CGH). The most frequent imbalance detected by CGH was gain of 15cen-q15 (3 cases), followed by loss of chromosome 13 and gain of 5p, and 7cen-q22, each detected in 2 cases.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells.

PURPOSE: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD: Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS: Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS: It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.

Heterogeneity of Chinese hamster X chromosomes in X-ray-induced chromosomal aberrations.

PURPOSE: To study the frequencies and distribution of X-ray-induced chromosomal aberrations in different arms as well as different regions of the long arms of Chinese hamster X chromosomes by fluorescence in situ hybridization (FISH). MATERIAL AND METHODS: Female embryonic primary cells of Chinese hamster were exposed to 1 and 4 Gy X-rays during the G1 stage and metaphases were collected 20 h later with colcemid-blocking. Induced aberrations involving different arms as well as different regions of the long arm of the X chromosomes were analysed by three-colour FISH using arm-specific painting probes. Distributions of aberrations in the arms were statistically analysed by chi2-test, with the hypothesis that aberrations are proportionally distributed on the basis of their relative lengths of the arms. RESULTS: The long arms of the X chromosomes were frequently involved in breaks than was the short arm. The result of the chi2-test indicates a non-proportional distribution of breaks between the arms, while exchanges (dicentrics and translocations) involving the arms were proportionally distributed. Differential involvement of regions of the long arm, i.e. Xq1 and Xq2, in breaks was also observed. Xq21, a known common fragile site in the X chromosome, was often involved in terminal deletions. CONCLUSION: Arm-specific probes of Chinese hamster chromosomes are useful for the detailed study of X-ray-induced aberrations in the X chromosome. The heterogeneity of the Chinese hamster X chromosome in response to X-ray-induced aberrations exists not only between the short (euchromatin) and the long (heterochromatin) arms, but also between different heterochromatic regions of the long arm of the X chromosome.

Increased chromosome exchange frequencies in iodo-deoxyuridine-sensitized human SW-1573 cells after gamma-irradiation.

The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. The yield of chromosome exchanges involving chromosome 2 was higher than those involving chromosome-X. On the basis of the DNA content the relative involvement of the X-chromosome in exchange frequencies after 2 Gy was much higher than of chromosome 2. After 4 Gy the relative involvement of both chromosomes in exchanges is approximately equal. After radiosensitization, increased chromosome exchange frequencies are observed in both studied chromosomes. For the total chromosome exchange frequencies the sensitizer enhancement ratio (SER) at 2 Gy is 1.8 and 1.3 for chromosome 2 and X respectively. The SER at 4 Gy for total exchange frequencies is 1.6 and 1.9 chromosome 2 and X respectively. For reciprocal exchanges at 2 Gy higher SER values and at 4 Gy lower SER values were observed for both chromosomes.

Mutatect: a mouse tumour model for detecting radiation-induced mutations in vivo.

A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.

Study on genotoxic effects of the space environment: a comparison between experienced cosmonauts and unexposed Russian twins.

The molecular analysis of T-lymphocytes from experienced cosmonauts and seven pairs of unexposed twins was performed [M. Khaidakov, D. Young, H. Erfle, A. Mortimer, Y. Voronkov, B.W. Glickman, Molecular analysis of mutations in T-lymphocytes from experienced soviet cosmonauts, Environ. Mol. Mutagen, 30 (1997) 21-30; J. Curry, G. Bebb, J. Moffat, D. Young, M. Khaidakov, A. Mortimer, B.W. Glickman, Similar mutant frequencies observed between monozygotic twins, Human Mutation, 9 (1997) 445-451]. Hprt mutant frequencies (MF) in both datasets were considerably higher (38.0+/-14.6x10(-6) in cosmonauts, and 18.5+/-8.9x10(-6) in twins) than in the background Western control (8-12x10(-6)), [A.D. Tates, F.J. van Dam, H. van Mossel, H. Shoemaker, J.C.P. Thijssen, V.M. Woldring, A.H. Zwinderman, A.T. Natarajan, Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations, Mutation Res., 253 (1991) 199-213; R.F. Branda, L.M. Sullivan, J.P. O'Neill, M.T. Falta, J.A. Nicklas, B. Hirsch, P.M. Vacek, R.J. Albertini, Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations, Mutation Res., 285 (1993) 267-279]. The distribution of mutations by class in the twin dataset was essentially similar to the background Western control, whereas cosmonaut samples demonstrated a significant excess of splice errors and complex mutations. The distribution of base substitutions showed similar trends in both the cosmonaut and twin samples, which are quite distinct compared to those seen in the Western control. The differences observed between cosmonaut and twin samples (a 2-fold higher MF and an excess of complex mutations in cosmonaut mutational spectra) could be an indication of possible effects of the space environment. However, these changes could also be age-related because the twin group was, on average, 17 years younger. Moreover, very similar patterns of base substitution distribution in both datasets suggest the involvement of certain region-specific factors reflected in mutational spectra. In order to discriminate between occupation and region-specific factors contributing to mutagenesis, an additional study involving trainees and cosmonauts with recent long-term flight experience is required.

Radiosensitivity and repair of the inactive X-chromosome. Insights from FISH and immunocytogenetics.

The inactive X-chromosome provides a unique opportunity to study the role of transcriptional activity and chromatin condensation in the repair of chromosome damage. We induced chromosome breakage in human lymphocytes with X-rays (1 or 2 Gy) in either G0 and G1 phase of the cell cycle, and in the presence or absence of an inhibitor of double strand break repair, adenine 9-beta-D-arabinofuranoside (Ara-A). Chromosomal aberrations involving the X-chromosome were detected by means of fluorescence in situ hybridization with an X-chromosome specific red painting probe. The activation status of the X-chromosomes involved in the chromosomal aberrations was determined by simultaneous immunocytogenetics with FITC-conjugated antibodies against BrdUrd incorporated at late S-phase to distinguish the late replicating inactive X-chromosome in green-yellow. This multicolor approach allowed us to study and compare breakage and the extent of repair in the active and inactive X-chromosome. Our data indicate that both chromosomes responded with a similar radiosensitivity. This observation was consistent at both X-ray doses and at the two stages of the cell cycle analyzed. However, the number of chromosomal aberrations involving the inactive X-chromosome was increased after repair inhibition with Ara-A. The differential sensitivity to repair inhibition was observed in G0 after 1 Gy and in G1 after 2 Gy. Thus, the activation status of the X-chromosome might be a source of heterogeneity in breakage and repair. These observations suggest that there is heterogeneous repair when the active and the inactive X-chromosomes are compared and that the observed fragility is the result of a compromise between the actual number of breaks induced in each chromosome and their differential processing.