Genetics - Theses
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ItemMutagenesis by X-rays and [125I] iododeoxyuridine in mammalian cells(University of Melbourne, 1985)Ionising radiation causes death and gene mutations in mammalian cells by the induction of DNA damage. The range of damage includes DNA double strand breaks, single strand breaks, base modifications and cross-linking to other molecules. Several studies have implicated DNA double strand breaks as the critical lesions leading to cell death, but relatively little is known about the nature of radiation-induced premutagenic lesions. This is primarily because of the difficulty of observing any one class of DNA damage in isolation. One way to expose cells to radiation is to incorporate radionuclides into DNA during S-phase using nucleotide analogues. When the radionuclide is iodine-125, incorporated as the thymidine analogue 5-iodo-2'-deoxyuridine (I-dUrd), there is on average, one DNA double strand break produced for each radioactive decay event. Thus, when compared with external sources of radiation, 125I decay allows the study of a high yield of DNA double strand breaks, relative to other classes of DNA damage. In this study the mutagenic and cytotoxic effects of X-rays and 125I decays have been compared in cultured mammalian cells. Asynchronous, exponentially growing Chinese hamster ovary (CHO) cells were either irradiated with 250 kVp X-rays at 37�C, or labelled for 1.2 cell population doublings with [125I] I-dUrd and stored at 4�C for 48 hours to accumulate 125I decays. X-ray survival was fitted to a linear-quadratic expression: -InS = ?.D + ?.D2, which yielded ? and ? values of 0.107 (� 0.042) Gy-1 and 2.321 (� 0.543) X 10-2 Gy-2, respectively. The l25I survival was of the simple exponential type with a Do = 34.1 � 0.95 decays. Both agents induced 6-thioguanine resistant mutants which can arise either by point mutations or by rearrangements or deletions leading to inactivation of the hypoxanthine phosphoribosyl transferase (HPRT) gene. For X-rays, there were 8.66 (� 0.0154) X 10-6 mutants Gy-1 and for 125I decays 6.22(� 0.89) X 10-7 mutants per decay. When compared on a direct plot of the logarithm of survival against induced mutation frequency, the mutagenic efficiencies of the two agents were indistinguishable. Neither X-rays nor 125I decays induced mutations at the Na+/K+ ATPase locus, suggesting that the radiation treatments did not induce point mutations. To further analyse the molecular nature of the radiation-induced HPRT mutations, ten X-ray-induced, six 125I-induced and four spontaneous independent HPRT mutant clones were isolated and examined at the enzyme, chromosome and DNA levels. None of the mutants exhibited residual HPRT enzyme activity. The arm, of the X chromosome bearing the HPRT did not show consistent chromosomal G-banding changes. Southern blotting analysis using a cloned mouse HPRT cDNA probe showed that 5/10 X-ray and 2/6 125I-induced HPRT mutants had lost all HPRT coding DNA sequences. A further 2/10 X-ray and 4/6 125I-induced mutants had altered Southern blotting patterns, but retained some HPRT coding sequences. Only one of the four spontaneous HPRT mutants had an altered Southern blotting pattern. The identification of mutants with complete loss of HPRT coding sequences indicates that both the X-rays and 125I decays can cause mutational inactivation by gene deletions involving more than 25 kilobases, which is the approximate size of the hamster HPRT gene. The identification of mutants which had altered Southern blotting patterns, but retained some HPRT coding sequences, indicated that the radiation- induced DNA deletions were probably not much larger than the size of the HPRT gene.