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PubMed1963

LIFE CYCLE ANALYSIS OF MAMMALIAN CELLS. I. A METHOD FOR LOCALIZING METABOLIC EVENTS WITHIN THE LIFE CYCLE, AND ITS APPLICATION TO THE ACTION OF COLCEMIDE AND SUBLETHAL DOSES OF X-IRRADIATION.

Equations are presented describing the accumulation of cells at any part of the life cycle as a result of addition of specific blocking agents. An experimental methodology using these relationships is described which makes possible analysis with relatively high resolution of the distribution of cells throughout the life cycle in normal cultures or those treated with various agents. The action of colcemide on S3 HeLa cells studied by this method revealed that colcemide has no effect on the G1, S, or G2 stages; it blocks cells quantitatively at the metaphase-anaphase region; but it accumulates mitotic figures only from the cells which have not yet entered mitosis at the time of its addition. The technique was also applied to study the efficiency of x-irradiation in delaying the entrance of G2 cells into mitosis. A definite lag was found at the lowest dose studied which was 9 rads. Only the cells confined to a central region of G2 at the time of irradiation are affected by this dose.

Animals↗
PubMed1986

The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells.

A brief Introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light (u.v.) and ionizing radiation (i.r.) and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The section on the Molecular distribution of DNA damage (section 2) focuses on the use of defined DNA molecules to assess the nature, sites and frequency of radiation damage. Recombinant DNA techniques have also been used in the study of enzyme-DNA interactions, to comment upon the rôle of specific types and sites of damage in producing cellular responses. The use of DNA-mediated gene transfer to assess damage and repair (section 3) indicates that recombinant DNA molecules can be used to implicate (or reject) specific types of DNA damage in gene inactivation. Some gene-transfer assays may also be able to confirm the presence of specific repair functions in mammalian cells. Restriction endonucleases are essential for the construction of recombinant DNA molecules, but their ability to cut DNA at specific sequences is also being exploited to implicate the double-strand break as an important type of damage leading to the well-characterized responses of irradiated cells. The DNA double strand break: use of restriction endonucleases to model radiation damage (section 4) documents experiments showing that blunt-ended cuts introduced into cellular DNA are able to produce chromosome aberrations and cell death. Assays based upon the introduction of restriction endonuclease-cut plasmids into radiosensitive and normal cells suggest that sensitivity is in some instances, e.g. the radiosensitive disorder ataxia-telangiectasia, a result of excessive degradation of DNA around broken ends. Identification and cloning of DNA repair genes (section 5) reviews the successful cloning of one human repair gene and the putative identification of others, as well as the lack of success in identifying genes complementing radiosensitive human disorders. Analysis of radiation-induced genetic change (section 6) links the types of DNA damage observed in defined DNA molecules with the types of mutations occurring in irradiated prokaryotes. In mammalian cells recombinant DNA techniques have allowed the nature of mutational changes to be determined for the first time: to date it seems that u.v. produces mainly small (point) mutations while i.r. produces mainly large changes (deletions/rearrangements).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗