Genetics - Theses

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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.
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.
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.
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.
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.
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.
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.
Patterns of protein synthesis in larval and imaginal tissues of Calliphora : an analysis of gene activity during differentiation and development in a holometabolous insect

Martin, Marjorie-Dore (University of Melbourne, 1975)

This study attempts to develop a logically consistent approach to the analysis of the genetic basis of complete metamorphosis in holometabolous insects,using the Australian brown blowfly, Calliphora stygia (Diptera), as the experimental organism. The following aspects of the problem have been examined: 1. Definition of the protein and enzyme spectra of selected larval and imaginal tissues during development. 2. Identification of the homologous gene products amongst various tissues with the view to establishing the uniqueness or overlap of gene readout in the series of tissues. 3. Identification of the times of synthesis of gene products as distinct from the duration of their occurrence leading to a definition of overall patterns of gene activities in the series of tissues. 4. Investigation of a specific model system for hormone action in vitro, namely with fat-body tissue and moulting hormone (MH). Current genetic models of insect metamorphosis have been examined in the light of the findings in this study of C. stygia.
Parthenogenesis, chromosomal polymorphism and morphological variation in Chironomids

Porter, David Laurence. (University of Melbourne, 1973)

Parthenogenesis, in the forms of arrhenotoky, deuterotoky, or thelytoky, is a quite common phenomenon in the animal kingdom. Thelytoky, in which females produce exclusively female progeny in the absence of genetic fertilization, is the most widespread and most mechanistically diverse form of parthenogenesis. It does not follow that thelytoky is the most common ; arrhenotoky, which only occurs in one rotifer, one arachnid and four insect orders (Hartl 1971), is probably the most successful form of parthenogenesis in terms of numbers, due mainly to the presence of over 100,000 species of hymenopterans, the vast majority of which are arrhenotokous. Thelytoky itself is present in a wide variety of forms. The mechanism for the maintenance of thelytoky may be automictic, in which at least the first meiotic division is normal, the chromosomes pairing at prophase and forming bivalents. The zygoid phase is restored by the restitution of anaphase I or metaphase II chromosome plates, fusion of second division products or endomitosis in cleavage nuclei. Alternatively the mechanism may be apomictic, in which meiotic features may be partly or wholly absent, the one or two maturation divisions being equational. Thelytoky may be complete, it being the only manner of reproduction; or it may be cyclical, where it alternates either regularly, or under the influence of environmental factors, with amphimixis or arrhenotoky. Thelytoky may also be either facultative or obligatory. Facultative thelytoky is the situation whereby reproduction is normally bisexual, however a percentage of eggs may develop without fertilization. Obligatory thelytokous forms produce all their offspring without genetic fertilization, reproduction can therefore never be bisexual. There are many thelytokous forms in which the eggs require penetration by the sperm of the same or related species before they develop. In this case, gynogenesis, the sperm makes no chromosomal contribution to the embryo, although there is a cytoplasmic contribution which may have some effect. In this chapter there are two main areas to be examined. The first is to investigate the cytology of two members of the chironomid subfamily Chironominae, Lundstroemia parthenogenetica and Lauterbornia sp. , and to compare them to other thelytokous forms with analogous maturation mechanisms, especially the members of the subfamily Orthocladiinae described by Scholl (1956, 1960). The second area is the discussion of the evolution and properties of thelytokous organisms. Most review articles tend to be limited to a broad presentation of the magnitude of the phenomenon (Oliver 1971), but also order the paper from the point of view of mechanism or animal groupings. Here the discussion is ordered from the point of view of the importance of various phenomena in the evolution and maintenance of thelytokous forms, e.g. hybridization, genetic and ecological considerations. This may not be a more systematic mode of presentation, but it seems to me to be more logical.
Cytogenetic and evolutionary studies on macropathinae (gryllacridoidea : orthoptera)

Mesa, Alejo. (University of Melbourne, 1970)

The Gryllacridoidea are a very ancient and diverse assemblage. One of the four families included in it, the Sohizodactylidae, is confined to the Old World, while the other three, Stenopelmatidae, Gryllacrididae and Rhaphidophorinae, have a world-wide distribution. The latter family Is divided into the subfamilies Ceuthophilinae from North and Central America, Rhaphidophorinae mainly from Europe and Asia, and Macropathinae with a circum-antarctic distribution. Up to date, less than one hundred species of Macropathinae have been described, the majority of them from New Zealand and the surrounding islands. Fourteen species were described from the East and South of Australia including Tasmania and Flinders Island. A few species are from the Southern cone of South America while only one species has been described from Africa, in Cape Town. The majority of these species are forest inhabitants with nocturnal habits. During the day they hide in dark and humid places like hollow logo and or crevices. Caves and tunnels make a suitable place for then to hide in and reproduce. Their density in those places is sometimes remarkable and hence their fame of being mainly cave inhabitants. To collect these insects in forest is more difficult due to the fact that their populations are more scattered. The Macropathinae, like the remaining rhaphidophorids, are wingless. Their body length range from less than 1 cm. to nearly 5 cm. The length from the tip of the antennae to the hind tarsi reaches 45 cm in Gymnoplectron giganteum (Richards 1962). Information on chromosome numbers and chromosomal sex-determining mechanisms in gryllacridids other than Macropathinae is summarised in Table I. According to these data, the chromosome number varies widely from family to family and even within families. The majority of these papers deal with chromosomes at metaphase, information about the fine structure of chromosomes at prophase being very scarce. In the present study a survey on the chromosomes and terminal abdominal segments within Macropathinae was undertaken in order to find possible phylogenetic relationships within the subfamily. Sometimes it was unavoidable to enter the taxonomic field. In this respect, only the genera Miotopus, Pleioplectron and Weta wore fully treated. When new genera end species were involved, a brief, preliminary description was included. Nearly seventy species were investigated, about half of them being new species. About a dozen New Zealand new genera were discovered as well. Approximately fifteen hundred specimens were handled from which nearly four hundred were cytologically investigated. The description of this amount of species will undoubtedly take a long time and falls outside the scope of the present study, which must be only considered a preliminary report.

Genetics - Theses

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