PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.
The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.
Ploskonosova
I I
II
Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushkino, Russian Federation.
Baranov
V I
VI
Gaziev
A I
AI
D000199:Actins / Q000235:genetics; D000375:Aging / Q000235:genetics*; D000818:Animals; D001921:Brain / Q000528:radiation effects*; D001923:Brain Chemistry / Q000528:radiation effects*; D004247:DNA / Q000528:radiation effects*; D018736:DNA Adducts; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005720:Gamma Rays / Q000009:adverse effects*; D005796:Genes / Q000528:radiation effects*; D005803:Genes, Immunoglobulin / Q000528:radiation effects; D007073:Immunoglobulin E / Q000235:genetics; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011836:Radiation Tolerance / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D013047:Specific Pathogen-Free Organisms; D013154:Spleen / Q000737:chemistry / Q000528:radiation effects*
D000199:Actins; D018736:DNA Adducts; D007073:Immunoglobulin E; D004247:DNA; C093010:DNA polymerase, Thermus thermophilus; D004259:DNA-Directed DNA Polymerase