Analysis of the induction of chromosome aberrations at the molecular level using restriction endonucleases

Author

Richard Alan Winegar

Date of Award

8-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

R. Julian Preston

Committee Members

Frank Larimer, Ray Popp, Larry Waters

Abstract

A confounding factor in the study of radiation-induced chromosome aberration induction is the wide variety of DNA lesions that radiation produces. Restriction endonucleases (REs), which produce double strand breaks (DSBs) at specific DNA sequences, were used to study the induction of chromosome aberration induction by specific DNA lesions. Osmolytic shock of pinocytic vesicles reliably and effectively introduced REs into viable CHO cells. REs, which act as a radiomimetic agent, displayed a linear dose response for aberration induction. Cut-site frequency rather than cut-end structure appeared to determine a particular enzymes's efficiency at aberration induction.

DNA repair inhibition studies indicated RE-induced double-strand break (DSB) repair is complete within 1 hour after treatment. G₁ studies using cytosine arabinoside (araC) and aphidicolin indicated there is a synthesis step in the repair of RE-induced DSB. 3-aminobenzamide (3-AB), a poly(ADP-ribosyl)ation inhibitor, enhanced RE-induced aberrations during G₁; its effect appears to be determined by the amount or distribution of RE-induced DSB. During G₂ RE treatments, araC enhanced chromatid deletion and achromatic lesion yields, but not exchange yields. 3-AB had little effect on G₂ induced aberrations. The DNA repair inhibition experiments suggest the presence of at least two repair mechanisms for RE-induced DSB: recombinational repair involving a synthesis step, and ligational repair regulated by poly(ADP-ribosyl)ation.

Interaction experiments involving different pairs of REs gave additional evidence for ligational repair. It appears, however, that cut-end structure affects the ability of different RE-induced DSBs to interact.

Preliminary data were obtained on the development of two different systems to study nonrandom chromosomal alterations. Studies with different mouse-hamster hybrid cell lines indicated that X rays induced aberrations throughout the hybrid genome. Mitomycin C, however, preferentially induced aberrations in the mouse centromeric heterochromatin, which is rich in repetitive satellite DNA sequences. Not I, an enzyme with a rare recognition sequence, appeared to induce aberrations in only a few locations in the CHO karyotype. These two systems may provide a valuable tool for studying the role nonrandom chromosomal changes play in cell transformation.

Recommended Citation

Winegar, Richard Alan, "Analysis of the induction of chromosome aberrations at the molecular level using restriction endonucleases. " PhD diss., University of Tennessee, 1987.
https://trace.tennessee.edu/utk_graddiss/12190