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ItemNo Preview AvailableUse of gene transfer to study amdS regulation in Aspergillus nidulans(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.
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ItemGenetic and molecular analysis of a positive regulatory gene in Aspergillus nidulans(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.
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ItemEcological genetics of anthelmintic resistance in nematode parasites of sheep(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.
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ItemThe molecular characterization of genes involved in acetate metabolism in Aspergillus nidulans(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.