Using three-color chromosome painting to test chromosome aberration models.

Abstract

Ionizing radiation induces DNA doublestrand breaks (DSB), which interact pairwise to produce chromosome aberrations. There have long been two main competing theories of such pairwise DSB-DSB interactions. The "classical" theory asserts that an unrepaired DSB makes two ends that separate within the cell nucleus, with each end subsequently able to join any similar (nontelomeric) end. The "exchange" theory asserts that at a DSB the chromatin does not separate completely; rather the DSB ends remain associated until repair, or an illegitimate recombination involving another DSB, occurs. The DSB-DSB interaction mechanism was tested by using three-color fluorescence in situ hybridization to paint chromosomes and observe "three-color triplets": three broken and misrejoined chromosomes having cyclically permuted colors. We observed 18 "three-color triplets" in 2000 cells after 2.25 Gy of y-irradiation. On the exchange model in its standard form such three-color triplets cannot occur, so this model is inconsistent with the observations. On the classical model, formalized as a discrete time Markov chain embedded at the transitions of a continuous time Markov chain, the frequency ofoccurrence of three-color triplets can be computed by Monte Carlo simulations. The number of threecolor triplets predicted mathematically by the classical model was found to be slightly larger than the observed number. Thus our data, together with our computer simulations, exclude the standard form of the exchange model but are compatible with the classical model. The results are also compatible with other, more complicated models.

A proposed system for scoring structural aberrations detected by chromosome painting

“…6h and j have been observed in irradiated cells by using whole chromosome paint ing techniques (Natarajan et al. 1992;Lucas and Sachs. 1993;Brown and Kovacs.…”

“…This is clearly a problem for high doses of low-LET radiations such as X-and y-rays (Brown and Kovacs. 1993;Lucas and Sachs. 1993;Marder and Morgan.…”

DNA Cleavage/Repair and Signal Transduction Pathways in Irradiated Breast Tumor Cells

“…This result implies that, in the parental GHO-D422 cells, either parental sequence 1 or parental sequence 2 (or both) was initially on a different chromosome than aprt, and that the exchange produced at least one chromosomal translocation. In irradiated cells, cyclically permuted threeway exchanges were detected previously at the chromosomal level using three-colour FISH (Lucas and Sachs 1993), and these events were taken as evidence that exchanges in general probably result from independent binary interactions between two DNA ends, rather than any concerted interactions among pairs of ends.…”

“…In the simplest model, free DNA double-strand break ends are assumed to have a significant but limited diffusion radius in the nucleus, resulting in a finite possibility of being misjoined to the ends of other double-strand breaks in the DNA of different chromosomes in the same nucleus (Bender et al 1974, Savage 1993). However, in the absence of molecular data, more complex models involving, for example, specific cooperative interactions among pairs of ends, extensive exonucleolytic single-strand exposure, aberrant homologous recombination among closely related sequences on different chromosomes, or other forms of homologous pairing, cannot be excluded (Savage 1989, Thacker et al 1992, Lucas and Sachs 1993, Nicolas et al 1995.…”

Novel Approaches in Genetic Toxicology of Pesticides by Applying Fluorescent in Situ Hybridization Technique

“…More thorough evaluation of translocation frequency by applying chromosome painting FISH in regards to duration of pesticide exposure considering various confounders has been conducted on a group of pesticide plant workers exposed to mix of carbofuran, chlorpyrifos, metalaxyl and dodine (Zeljezic et al, 2009). In this study approach used in ionizing radiation biodosimetry has been applied, painting chromosomes 1, 2, and 4 by FISH in red, Lucas & Sachs (1993) is used to extrapolate observed translocations frequencies for painted chromosomes to total genomic translocation frequency. In lymphocytes of pesticide plant workers genomic translocation frequency was significantly higher than in matching controls (P ScheffePostHoc =0.000004), being higher in females (0.0062±0.0027 per cell) than in males (0.0043±0.0016) regardless of adjustment for the age difference.…”