MEASUREMENT OF RADIATION-INDUCED DNA DOUBLE STRAND BREAKS IN YEAST BY PULSED FIELD GEL ELECTROPHORESIS

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Date
1995-05
Authors
SAMUEL, EFFIONG IKPEME
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Abstract
DSB (double strand break) induction in the diploid yeast, Saccharomyces cerevisiae, was measured by the PFGE (Pulsed Field Gel Electrophoresis) technique 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 100 to 11500 KeV/μm, 241Am alpha particles and 80-kV X-rays. After irradiation, the cells were lysed and the DNA purified. The samples were then placed in 1 % agarose gels electrophoresed in a CHEFDR II system. After electrophoresis, the gels were stained with ethidium bromide, placed on a UV-transilluminator and photographed with a computer-coupled digitizing camera. The pictures obtained were then processed with the help of a specially developed computer software which allows for the correction of the camera's shading effect and background fluorescence. The software was used to quantify the ethidium bromide-induced fluorescence after subtracting background fluorescence and the camera's "shading" effect. 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, (DSB) production cross-sections (QDSB) were calculated. Linearity between the induced DSB and fluence was found in all cases. The DSB cross-section increases until it reaches a plateau value of about 1.05 xlO-8 cm2 at about 103 - 4xl03 keV/μm. This could be due to the limited probability of an ion to produce DSB as long as effects by delta electrons in the so-called "penumbra" are negligible so that only interactions in or very close to the track centre contribute significantly. Inside this LET region, the relative biological effectiveness for DSB induction (RBEDSB) (relative to unfiltered 80 kV X-rays) decreases from about 2 down to 0.5. With higher LET values (beginning from about 2xl03 keV/μm), QDSB rises above the plateau. A comparison of the derived DSB production cross-sections with the corresponding cross-section for inactivation shows that a close relationship exists between the two end-points.
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A Thesis submitted to the Post-Graduate School, Ahmadu Bello University, in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy (Physics) Department of Physics Faculty of Science Ahmadu Bello University ZARIA - NIGERIA MAY 1995
Keywords
MEASUREMENT,, RADIATION-INDUCDNA,, DOUBLE STRAND,, GEL ELECTROPHORESIS
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