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