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.
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ItemCopper dependent enzymes in mottled mouse mutants(University of Melbourne, 1984)The activities of two copper dependent enzymes, cytochrome c oxidase and superoxide dismutase, were determined, and related to intracellular copper concentrations, in six tissues of mice carrying the x-linked defects in copper homeostasis, brindled and blotchy, at the mottled locus. The mutants were assayed throughout post natal development in the presence and absence of a sub-cutaneous copper load. Copper replete and copper deficient genetically normal mice were used as experimental controls. Mutant tissues could be divided into enzyme deficient, that is brain, heart and skeletal muscle, and tissues with normal enzyme activity, liver, kidney and lung. Copper concentration was highly correlated with enzyme activity in deficient tissues. In mutant tissues with normal activity intracellular copper could be reduced, (liver) greatly increased (kidney) or close to normal (lung). Comparisons with genetically normal copper deprived mice suggest that copper availability to the cell, rather than within the cell, is responsible for the enzyme deficiency in brain, heart and skeletal muscle and that mutant gene expression and the accompanying copper accumulation phenotype is confined to epithelial rich tissues, at least in the brindled mouse. However, the possibility of intracellular expression of the basic defect in these enzyme deficient tissues has not been excluded. The response of copper dependent enzymes to subcutaneous copper injection (50 ?g copper) differed in skeletal muscle in the two mutants and studies on the distribution of radioactive isotopes of copper showed small but consistent tissue specific differences between the brindled and blotchy hemizygote. These results, together with previously published data on lysyl oxidase activity suggest that different polypeptides are defective in the two mutants. In early post natal development the activity of both copper dependent enzymes studied increased significantly in a number of tissues in copper replete mice. An inability to meet the increased copper requirements that accompany these developmental changes, particularly in the maturing brain, is believed to be responsible for the death of the brindled mouse at the mid-suckling stage. In the more viable blotchy mutant the compensatory effects of stunted growth may decrease the magnitude of the developmentally related enzyme deficiency in some tissues but cannot account for the phenotypic differences between the mutants. A number of hypotheses are proposed to explain the experimental results, and the objectives and directions of future research projects are indicated.
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ItemA sex-influenced protein in Chironomus: detection and characterization(University of Melbourne, 1984)The synthesis of a protein in Chironomus was indicated to be sex-influenced by its female-specific occurrence detected electrophoretically in a range of geographically distinct species. These species included Ch. tentans derived from Canada, Ch. duplex from Australia and Ch. species a from New Zealand. The sex-influenced protein was found to contain two subunits of similar molecular weight at approximately 17,800 dal tons. By virtue of its positive staining with benzidine, this protein was indicated to be one of the haemochironomins, a class of predominant haemolymph proteins synthesized during the larval stage. The presence of this sex- influenced protein in the female haemolymph was correlated to the stages of development. It was found to be absent in haemolymph of the female fourth instar larvae younger than phase 3 in Ch. tentans and phase 5 in Ch. duplex. It was present throughout the later phases and persisted in the haemolymph of the female pupa. Tissue distribution studies indicated that the sex-influenced protein was found solely in the haemolymph in the female larva. However, pupal ovaries containing yolky oocytes as well as adult eggs were indicated electrophoretically to contain the sex-influenced protein. An additional and reliable method for identifying the sex-influenced protein in female larval haemolymph and in eggs of the various species was based on the criterion of antigenic specificity to an antiserum raised against the protein isolated in the present study. The results suggested that the female-specific synthesis of the sex-influenced protein may be correlated to. female-specific requirement for it in egg formation.
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ItemMolecular studies of gene regulation in Aspergillus nidulans( 1985)The amdA regulatory gene of Aspergillus nidulans controls the expression of both the acetamidase (amdS) gene and a gene, designated aciA, which encodes a 42 000 dalton polypeptide. The cloning and analysis of the aciA gene is described in this thesis. The aciA gene is a single copy gene approximately 2kb in size and encodes two messenger RNA species of 1.45 and 1.55kb•in size. Both of these aciA transcripts are regulated in an identical fashion. The aciA gene is demonstrated to be induced in the presence of the carbon source acetate and evidence is presented which indicates that it may also be subject to carbon catabolite repression. None of the other regulatory circuits which are known to control amdS expression affect the expression of the aciA gene. The 5' non-coding region of the aciA gene was sequenced and both the primary and secondary structures within this region are compared with the corresponding regions of the amdS gene and a cis-acting mutant of the amdS gene which increases the amdA-mediated regulation of this gene. The aciA gene possesses a canonical TATA box and also contains a number of other sequences which are similar to sequences found in the 5' non-coding region of the amdS gene. The most interesting of these is a 23bp purine-rich region which is similar to a purine-rich region occurring in approximately the same position in the amdS gene. This sequence is found to be duplicated in the amdS mutant which is subject to increased regulation by the amdA gene. Experiments in which A. nidulans was transformed with multicopy plasmids containing DNA fragments from the aciA gene have indicated that the aciA sequence responsible for the titration of the amdA gene product is located in the 5' non-coding region of this gene. A model for the regulation of the aciA gene by the amdA gene, acetate and carbon catabolite repression is presented. The possible significance of small repeated sequences within the purine-rich regions of aciA and amdS is also discussed.