Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes.

We have studied the induction of chromosomal aberrations in human lymphocytes exposed in G0 to X rays or carbon ions. Aberrations were analyzed in G0, G1, G2 or M phase. Analysis during the interphase was performed by chemically induced premature chromosome condensation, which allows scoring of aberrations in G1, G2 and M phase; fusion-induced premature chromosome condensation was used to analyze the damage in G0 cells after incubation for repair; M-phase cells were obtained by conventional Colcemid block. Aberrations were scored by Giemsa staining or fluorescence in situ hybridization (chromosomes 2 and 4). Similar yields of fragments were observed in G1 and G2 phase, but lower yields were scored in metaphase. The frequency of chromosomal exchanges was similar in G0 (after repair), G2 and M phase for cells exposed to X rays, while a lower frequency of exchanges was observed in M phase when lymphocytes were irradiated with high-LET carbon ions. The results suggest that radiation-induced G2-phase block is associated with unrejoined chromosome fragments induced by radiation exposure during G0.     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

New Application of the Comet Assay: Chromosome�CComet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.     

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

Role of Mammalian Rad9 in Genomic Stability and Ionizing Radiation Response

Eukaryotic cells have evolved DNA damage response mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. The Schizosaccharomyces pombe Rad9 gene was initially identified as encoding a cell cycle checkpoint protein. When the mammalian homologue of S. pombe Rad9 was inactivated, however, chromosomal instability was observed even in the absence of DNA damaging agents. Both an increase in chromosome end-to-end associations and telomere loss were observed in cells with inactivated mammalian Rad9. This telomere instability correlated with enhanced S- and G2-phase specific cell killing, delayed kinetics of γ-H2AX foci appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase specific DNA damage repair. Inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S- and G2-phase of the cell cycle. These newly defined functions of mammalian Rad9 are discussed in relation to telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.     

DNA breaks promote genomic instability by impeding proper chromosome segregation

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells

Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human–mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human–mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.     

Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

Cytosine arabinoside is a potent clastogen and does not affect the repair of X-ray-induced chromosome fragments in unstimulated human lymphocytes

The metabolic inhibitor of DNA synthesis cytosine arabinoside (ara-C) is known to induce chromosome aberrations in human lymphocytes. It has been recently argued, however, that there is no unequivocal evidence that ara-C can damage chromosomes directly. Therefore, the effect of ara-C on unstimulated human lymphocytes was examined directly by means of the premature chromosome condensation technique. In about 50% of the cells, ara-C effectively induced chromosome fragments, which did not show rejoining even after the chemical was washed out. These results suggest that a possible selection against damaged cells in their progress to mitosis could result in the low yields of ara-C-induced chromosome aberrations reported in the literature. The effect of ara-C on the repair of radiation-induced chromosome aberrations was also examined. Ara-C did not affect the rejoining of the chromosome fragments induced in unstimulated human lymphocytes by 6 Gy of X-rays.