The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays. R olling circle amplification (RCA) is a molecular cytogenetic technique used with a padlock oligonucleotide probe to detect single base changes in isolated nucleic acids (1-5). Padlock probes are composed of Ϸ100 nucleotides that hybridize to targets of Ϸ30 bases. The 30-base target-binding region of the probe is split into two 15-base segments placed in opposite orientation at each end of the linear probe so that a circle must be formed for hybridization to occur (6, 7). At 10 bases per helical turn, the hybridized probe wraps around its target three times, and the remaining 70 bases form an unhybridized singlestranded loop. Posthybridization DNA ligation connects the two ends of the probe in the middle of the 30-base binding region. The unbound 70-base loop facilitates probe circularization and permits Ϸ20 bases to serve as a primer recognition site for DNA polymerase to replicate the circle. RCA is an isothermal process in which the polymerase progresses continuously around the loop until the 100 bases have been replicated hundreds or thousands of times. Incorporating a labeled nucleotide during the RCA reaction produces sufficient signal for easy visualization of the target.Application of RCA to in situ targets in fixed or permeabilized cells has not been uniformly successful to date. Whereas recent work has demonstrated that the concept is viable (8), DNA detection efficiencies of 20-30% lessen the utility of RCA as an assay. Lack of success has been attributed to possible blocking of the polymerase by the target strand, and it was suggested that this problem might be overcome by cutting the target DNA strand near the RCA probe's hybridization site (5). Under these conditions, DNA polymerase could free the probe from the target, in effect spinning the probe away from the target, keeping the polymerase from being blocked during the amplification process. Here, we report that in addition to restriction enzyme digestion of DNA, additional steps were required to achieve consistent and satisfactory results for RCA in situ. Whereas heat denaturation is typically used to render the target DNA single stranded, we found that complete removal of the nontargeted DNA strand by digestion with exonuclease III significantly increased the efficiency of the process.We also demonstrate the use of RCA to detect mRNA in cytological preparations. Using appropriate image analysis techniques, the RCA assay is sufficiently quantitative to enable transcriptionally mediated dose-response curves to be generated. Inc...

Low-dose ionizing radiation alters the gene expression profiles of mammalian cells, yet there is little understanding of the underlying cellular mechanisms responsible for these changes or of their consequences for genomic stability. We investigated the cytogenetic adaptive response of human lymphoblastoid cell lines exposed to 5 cGy (priming dose) followed by 2 Gy (challenge dose) compared to cells that received a single 2-Gy dose to (a) determine how the priming dose influences subsequent gene transcript expression in reproducibly adapting and non-adapting cell lines, and (b) identify gene transcripts that are associated with reductions in the magnitude of chromosomal damage after the challenge dose. The transcript profiles were evaluated using oligonucleotide arrays and RNA obtained 4 h after the challenge dose. A set of 145 genes (false discovery rate = 5%) with transcripts that were affected by the 5-cGy priming dose fell into two categories: (a) a set of common genes that were similarly modulated by the 5-cGy priming dose irrespective of whether the cells subsequently adapted or not and (b) genes with differential transcription in accordance with the cell lines that showed either adaptive or non-adaptive outcomes. The common priming-dose response genes showed up-regulation for protein synthesis genes and down-regulation of metabolic and signal transduction genes (>10-fold differences). The genes associated with subsequent adaptive and non-adaptive outcomes involved DNA repair, stress response, cell cycle control and apoptosis. Our findings support the importance of TP53-related functions in the control of the low-dose cytogenetic radioadaptive response and suggest that certain low-dose-induced alterations in cellular functions are predictive for the risk of subsequent genomic damage.

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Ethylenebis(dithiocarbamate) (EBDC) fungicides are used heavily in the United States. EBDCs (e.g., mancozeb, maneb) are metabolized to ethylene thiourea (ETU). The EPA classifies ETU as a carcinogen, based on thyroid and other cancers in rodents, and has restricted the use of EBDCs, while requiring workers to use protective equipment. There are no data on the potential carcinogenicity of EBDCs in humans, and there is only one study on human genotoxicity. ETU is known to cause decreases of thyroxine (T4) and increases in thyroid-stimulating hormone (TSH) in rodents. We have studied cytogenetic outcomes and serum thyroid hormone levels among 49 heavily exposed workers without protective equipment spraying EBDC on tomatoes in Mexico. We also studied 14 lightly exposed landowners and 31 nonexposed controls. Urinary ETU was used to compare exposure between groups. We found an increase in TSH (p = 0.05) among applicators compared to controls, but no decrease in thyroid hormone (T4). We found increases in sister chromatid exchange (p = 0.03) and in chromosome translocations (chromosome aberrations that persist through cell division) for applicators compared to controls (p = 0.05). However, the subset of reciprocal translocations showed a lesser increase (p = 0.24). Our data suggest that EBDCs affect the thyroid gland and the lymphocyte genome among heavily exposed workers. However, our data are limited to subclinical outcomes, are of borderline statistical significance, and should be interpreted with caution.ImagesFigure 1.