Genetics - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 9 of 9
  • Item
    No Preview Available
    Use of gene transfer to study amdS regulation in Aspergillus nidulans
    Littlejohn, Timothy Graham. (University of Melbourne, 1989)
    Expression of the amdS gene of Aspergillus nidulans is regulated by a number of different regulatory genes and coeffectors. In vivo generated cis mutants permitted initial identification of regions 5' to the amdS gene involved in regulation by some of these regulatory genes. In this study, an amdS-lacZ fusion gene was used to follow the regulatory consequences of in vitro generated mutants of the amdS controlling region. Numerous deletion, inversion, insertion and oligonucleotide based mutants were constructed and introduced into A. nidulans using a gene transfer (transformation) technique. Three approaches for the production of transformants suitable for regulatory analysis were assessed; cotransformation, single copy integrations at the argB locus, and gene replacements. A single region of the amdS controlling region was found to be responsible for amdR mediated regulation of amdS, The sequence of the 5' regions of three coregulated genes, gatA, lamA and lamB, revealed that these genes shared this sequence in common. A mutant amdR allele, amdR104c, regulated amdS expression from the same location as the wildtype product. Three regions 5' to amdS were found to be involved in facB mediated regulation of amdS; their action were seen to be synergistic under some circumstances. No homology was found between them, or with the 5' regions of other genes under facB control. A mutant facB allele, facB88, resulted in altered regulation of amdS. Insertion mutants indicated that the wild type products of the amdR and facB genes could regulate amdS when their site(s) of action were moved 5' by several hundred base pairs, and that the products of the mutant alleles showed different responses. In addition, a region 5' to amdS with homology to eukaryotic CCAAT boxes was shown to be required for establishing basal amdS expression. Titration analysis, an in vivo DNA-regulatory product binding assay, was used to show that the same sequences required for amdR mediated expression titrated the amdR product. Individual sites of action of the facB gene product were not seen to titrate the facB gene product, however.
  • Item
    Thumbnail Image
    Genetic and molecular analysis of a positive regulatory gene in Aspergillus nidulans
    Andrianopoulos, Alex. (University of Melbourne, 1989)
    In the ascomycete fungus Aspergillus nidulans, utilization of certain amides, omega-amino acids and cyclic amides (lactams) as carbon and/or nitrogen sources requires the structural genes of amdS, gatA, gabA, lamA and lamB, whose expression is coordinately controlled by the positively-acting regulatory gene amdR. Transcriptional activation by amdR is dependent on omega-amino acid inducers (ligands) such as GABA and ?-alanine. To understand the mechanisms by which amdR exerts its regulatory control over structural gene expression, a program was initiated to clone and characterize the amdR gene. Using DNA- mediated transformation of A.nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Transcript analysis showed that the 2.7kb amdR mRNA is constitutively transcribed at a very low level under all tested conditions. Sequence and transcriptional analysis of amdR showed that it contains three small introns, heterogeneous 5' and 3' transcription sites and multiple codons prior to the major AUG initiator. In addition, the semidominant amdR6c allele was cloned and its lesion identified. The predicted amdR protein sequence has a cysteine-rich "zinc-finger" DNA binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl- terminal half of the product and two sequences homologous to the SV40 large T antigen nuclear localization motif. A series of 5', 3� and internal deletions of amdR were examined in vivo for transcription activator function, showing that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wildtype levels of transcription activation. A number of the amdR deletion products were also shown to compete with the wildtype amdR product in vivo. Dosage phenomena studies using amdR yielded transformants which exhibited stronger growth than the wildtype, indicating increased expression of the relevant structural genes. This suggests that the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Further increases in amdR product concentrations in vivo, through overexpression of the amdR gene, yielded transformants with phenotypic abnormalities. From the molecular and genetic studies presented, a model for amdR-mediated regulation of structural gene expression was formulated.
  • Item
    Thumbnail Image
    Ecological genetics of anthelmintic resistance in nematode parasites of sheep
    Martin, Paul John. (University of Melbourne, 1989)
    Ecological and genetic studies were conducted on anthelmintic resistance in Ostertagia spp. and Trichostrongylus spp., two economically important nematode parasites of sheep. Benzimidazole resistance developed rapidly under field conditions. The selection pressure for resistance related primarily to the efficiency of the anthelmintic and to stock management. These factors determined the relative genetic contribution made, by the survivors of the anthelmintic, to future generations of worms, compared to that made by the free-living sub-population on pasture at the time of treatment. Benzimidazole resistance in Trichostrongylus spp. and Ostertagia spp. was found to be under polygenic control and inherited as an incompletely recessive character with strong maternal influence. Levamisole resistance in the strain of Trichostrongylus spp. studied, was mainly inherited as a sex-linked recessive gene or gene complex although there was some evidence of polygenic influences. The recessive nature of resistance supported the concept of using high dose rates of anthelmintics to delay the onset of resistance. High dose rates increase recessiveness with respect to fitness in the presence of the anthelmintic and remove resistance alleles which are present in heterozygous worms. Reductions in the degree of benzimidazole resistance in Ostertagia spp. occurred too slowly to re-introduce benzimidazoles for parasite control. This occurred under natural selection or counter-selection with levamisole in both field and laboratory studies. Therefore, from a practical perspective, resistance is a problem to avoid rather than manage. As an alternative means of using anthelmintics with a view to preventing resistance, selection studies were conducted using anthelmintics individually or in combination (mixture). Resistance in Trichostrongylus spp. and Ostertagia spp. developed rapidly where a benzimidazole or levamisole anthelmintic was used alone but no resistance developed where the recommended dose rate of both anthelmintics was administered in combination. Furthermore, a combination of a benzimidazole and levamisole, or either with naphthalophos, was found to offer high efficiency (>97%) against field strains of Ostertagia spp. showing resistance to either compound when used alone. The results provide an ecological and genetic understanding of anthelmintic resistance necessary for the strategic implementation of anthelmintics in sustainable worm control programs. The genetic analysis provides the background necessary for simulation models of parasite population dynamics incorporating the development of resistance.
  • Item
    Thumbnail Image
    The molecular characterization of genes involved in acetate metabolism in Aspergillus nidulans
    Sandeman, Ruth Ann. (University of Melbourne, 1988)
    Two acetate-induced genes contained within lambda clones were identified by transformation into Aspergillus nidulans to complement existing mutations. In this way one clone was found to contain the acuE gene, encoding malate synthase, and the other clone contained the facA gene, encoding acetyl CoA synthase. These enzymes are involved in acetate metabolism in A.nidulans and have been shown to be coregulated, together with the acuD gene, isocitrate lyase, and amdS gene, acetamidase, by the facB gene product. The facA+ and acuE+ transformants were studied by Southern analysis, which helped to establish the extent of these genes within the lambda clones and provided a basis for further characterization of these genes. The identity of the facA gene was confirmed by Southern analysis of a facA translocation strain, FAD1. The transcripts of the facA and acuE genes were identified by Northern analysis, and found to be induced by acetate. Transcriptional mapping of both genes established the 5' startpoints of these mRNAs and localized two introns in this region of the facA transcript. The two genes were sequenced and their structures were compared with the gene structure of other sequenced fungal genes. The facA and acuE genes both contain introns, which conform to the expected size of fungal introns and contain recognisable splice site and signal sequences. Both genes also contain promoter and translation initiation termination sequences that conform to the consensus sequences established for other fungal genes. Preliminary Northern analysis of the facA and acuE genes established that the facB gene product is necessary for the induction of transcription by acetate and the 5' regions of these genes were examined for sequences that may be involved in binding the facB gene product. A comparison of the 5' regions of the facA and acuE genes, together with the acuD and amdS genes, failed to reveal sequences of strong homology. However, one sequence repeated in the 5' regions of the facA, acuE and acuD genes did show some homology to the amdl9 region of amdS, which has been shown to be necessary for facB-mediated induction of amdS. Finally, the facA gene sequence was compared to the acu5 gene sequence, which encodes acetyl CoA synthase in Neurospora crassa, and the acuE gene was compared to the aceB gene of Escherichia coli, encoding malate synthase. The facA and acu5 genes were found to be very similar, . although a number of structural differences were apparent at the 5' and N-terminal ends of these genes. These comparisons are discussed in relation to the molecular evolution of these genes.
  • Item
    No Preview Available
    Metallothionein gene regulation in inherited disorders of copper metabolism
    Stevenson, Trevor Wallace. (University of Melbourne, 1986)
    Metallothionein is an unusual, low molecular weight, cysteine rich, metal binding protein found in most vertebrate tissues. Synthesis of metallothionein can be induced by heavy metals, glucocorticoid hormones and also appears to be developmentally regulated. This thesis examines the hypothesis that altered metallothionein gene regulation is the basic defect in inherited disorders of copper metabolism, Menkes' disease in humans, and the Mottled mutants in mice. In both of these disorders copper accumulates in the gut and kidney while there is an apparent copper deficiency in other organs such as liver and brain. To examine whether elevated or unrestrained metallothionein synthesis is the basic defect in these disorders, the level of MT mRNA in liver, kidney and brain of neonatal normal and two of the Mottled mouse mutants were determined. The more severely affected Mottled mutant, brindled, which dies between 14 and 17 days after birth, showed several clear differences in tissue MT mRNA levels from normal mice, and also from the less severely affected blotchy mutants. The brindled mutants did not experience the rise in liver metallothionein mRNA levels shortly after birth observed in both normal and blotchy neonates. A model describing neonatal hepatic copper metabolism and MT mRNA synthesis in the normal, brindled and blotchy mutants is proposed. In brindled mutants, however, inconsistent increases in brain and liver metallothionein mRNA levels were observed In 13-15 day old mice. In order to determine whether these Inconsistent rises were stress related; the levels of mRNA coding for a glucocorticoial responsive gene, Tyrosine Amino Transferase, were measured in individual 15 day old brindled, blotchy and normal mice. Those brindled mice which showed an elevation of liver metallothionein mRNA also showed an elevation of brain metallothionein mRNA, as well an elevation in hepatic Tyrosine Amino Transferase mRNA. These results indicate that the inconsistent rises In liver and brain metallothionein mRNA in the brindled mutants are most probably caused by stress associated with the severity of the phenotype. To further examine metallothionein synthesis without "external" developmental or hormonal influences, the metallothionein mRNA levels, synthesis rates and protein levels were examined in normal and Menkes' disease cultured Continous Lymphoid Cells(CLCs). Menkes' disease cultured cells, during both normal culture growth conditions and in medium with elevated extracellular copper, have higher metallothioneim mRNA levels and than do the normal cells. These elevated metallothionein mRNA levels and synthesis accompany the high levels of metallothionein protein and accumulated copper which have previously been observed in these cells. Upon incubation in low copper serum free medium, however, Menkes' disease cultured cells showed a transient increase followed by a decrease in the metallothionein mRNA levels. The Menkes disease cell metallothionein mRNA levels, when compared to intracellular copper levels, appeared to be responding directly to intracellular copper levels. Incubation in low copper medium which lowered intracellular copper concentration showed that the metallothionein gene was in fact regulated normally in the Menkes disease cells and during normal growth conditions was responding to elevated intracellular copper caused by some other block in intracellular copper transport. Thus the high metallothionein, metallothionein mRNA, and metallothionein synthesis rates observed in Menkes' disease cells are most probably a secondary consequence of some other block in intracellular copper transport.
  • Item
    Thumbnail Image
    Mutagenesis by X-rays and [125I] iododeoxyuridine in mammalian cells
    Gibbs, Richard (Richard A.) (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.
  • Item
    Thumbnail Image
    Copper dependent enzymes in mottled mouse mutants
    Phillips, Alison Marie. (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.
  • Item
    Thumbnail Image
    A sex-influenced protein in Chironomus: detection and characterization
    Cheong, Mei Foong. (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.
  • Item
    Thumbnail Image
    Molecular studies of gene regulation in Aspergillus nidulans
    Atkinson, Peter William ( 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.