DNA samples prepared from human SP3 cells, which had been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3 +/- 0.7) x 10(-3)/Mbp/Gy was deduced for 80 kV X-rays.

Pulsed-field gel electrophoresis was used to separate the chromosomes of the diploid yeast Saccharomyces cerevisiae 211*B after irradiation with X-rays and alpha-particles. After electrophoresis, gels were stained with ethidium bromide, placed on a UV-transilluminator and photographed with a digitizing camera connected to a personal computer. The pictures obtained were processed with the help of specially developed software which allows for the correction of the camera's shading effect and background fluorescence. Linearity between DNA amount and fluorescence was demonstrated. Fluorescence intensity for the band with the lowest electrophoretic mobility was found to decrease exponentially with dose. Based on the known size of the native DNA molecules, double-strand break yields could be calculated. These were found to be (8.2 +/- 0.4) and (14.8 +/- 0.5)10(-12) (g/mol)-1 Gy-1 for 80 keV X-rays and 3.5 MeV 241Am alpha-particles respectively which gives a relative biological effectiveness of 1.8 +/- 0.1.

A new method is presented for measuring DNA double-strand breaks (DSBs) which combines the technique of pulsed-field gel electrophoresis (PFGE) with the idea of velocity sedimentation. Purified DNA samples were treated with restriction enzymes, which results in DNA of sizes which can be separated by PFGE. After electrophoresis, unirradiated DNA shows a size distribution (obtained with the help of a specially developed software program) similar to that obtained with the sedimentation technique; with X irradiation, this distribution is shifted to lower molecular weight with increasing dose. The rate of DSB induction was calculated by comparing the curves obtained experimentally with theoretical distributions (based on the assumption that breaks are formed according to Poisson statistics). The method was tested by measuring X-ray-induced DSBs in P3 (derived from human epithelial teratoma) cells. The induction of DSBs was found to be linear with dose and a rate of 5.4 x 10(-3)/Mbp/Gy was obtained.

Induction of DSBs in the diploid yeast, Saccharomyces cerevisiae, was measured by pulsed-field gel electrophoresis (PFGE) after the cells had been exposed on membrane filters to a variety of energetic heavy ions with values of linear energy transfer (LET) ranging from about 2 to 11,500 keV/microm, (241)Am alpha particles, and 80 keV X rays. After irradiation, the cells were lysed, and the chromosomes were separated by PFGE. The gels were stained with ethidium bromide, placed on a UV transilluminator, and analyzed using a computer-coupled camera. The fluorescence intensities of the larger bands were found to decrease exponentially with dose or particle fluence. The slope of this line corresponds to the cross section for at least one double-strand break (DSB), but closely spaced multiple breaks cannot be discriminated. Based on the known size of the native DNA molecules, breakage cross sections per base pair were calculated. They increased with LET until they reached a transient plateau value of about 6 x 10(-7) microm(2) at about 300-2000 keV/microm; they then rose for the higher LETs, probably reflecting the influence of delta electrons. The relative biological effectiveness for DNA breakage displays a maximum of about 2.5 around 100-200 keV/microm and falls below unity for LET values above 10(3) keV/microm. For these yeast cells, comparison of the derived breakage cross sections with the corresponding cross section for inactivation derived from the terminal slope of the survival curves shows a strong linear relationship between these cross sections, extending over several orders of magnitude.

Cells of diploid yeast, Saccharomyces cerevisiae, were exposed to a variety of energetic heavy ions (provided by the UNILAC facility at the Gesellschaft für Schwerionenforschung, GSI), 241Am alpha-particles and 80-keV x-rays after which they were assessed for DNA double-strand breaks (DSB) using either the neutral sedimentation or the pulsed-field gel electrophoresis (PFGE) technique. Both yielded comparable results. The DSB production cross-sections are compared with inactivation studies performed for the same cells under identical conditions. The measurements show that with lighter ions DSB induction cross-sections increase with linear energy transfer (LET), but the situation is less clear with the heavier ions. A close parallelism was found between DSB induction and cell inactivation in these yeast cells.