Genetics - Theses

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Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
The isolation and analysis of the hap genes of Aspergillus nidulans

Papagiannopoulos, Peter. (University of Melbourne, 1996)

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence required for setting the basal level of transcription. Mobility shift assays have identified a factor in A. nidulans nuclear extracts that binds specifically to this CCAAT sequence. In Saccharomyces cerevisiae, the HAP3 and HAP5 genes encode components of a highly conserved multi subunit complex which is able to bind CCAAT sequences. The identification, cloning and sequencing of genes from A. nidulans with homology to HAP3 and HAP5, known as hapC and hapE respectively, is described here. The predicted amino acid sequences of the proteins encoded by the hapC and hapE genes share extensive sequence identity to conserved regions in HAP3 and HAP5 respectively. Furthermore, they both show identity to the histone-fold motif, a motif used widely as a means for protein-protein and DNA- protein interactions. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially poorly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, the hapC deletion results in reduced levels of amdS expression, particularly under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT specific binding to the amdS or gatA promoter, indicating that hapC encodes a component of the complex binding at this sequence. In the presence of the hapC deletion growth on acetamide and amdS
The inheritance of anthelmintic resistance in nematode parasites of sheep

Turney, Kerrin Linda. (University of Melbourne, 1993)

Studies on the inheritance of anthelmintic resistance were conducted. Three anthelmintics were considered; levamisole, ivermectin and closantel, and selection processes involved in conferring resistance were compared and contrasted. This study showed that sex-linked resistance was present in a field derived strain of Trichostrongylus colubriformis. Laboratory selection on another strain of T. colubriformis showing only a low level of levamisole resistance, led to the strain becoming highly resistant and the genetic basis of this resistance was sex-linked. The results indicated that high level levamisole resistance is inherited as a sex-linked recessive character and is probably monogenic. This confirms earlier work by Martin and McKenzie (1990b). The inheritance of resistance to ivermectin was found to be partially dominant in Haemonchus contortus and T. colubriformis and results were consistent with resistance in both species being controlled by a polygenic system. At the recommended dose rate of ivermectin the H. contortus heterozygotes showed a high level of survival and this has important consequences for the farmer as resistance will increase rapidly under selection (Georghiou and Taylor 1977). ' Results on the inheritance of resistance to closantel on resident worm burdens were consistent with resistance being conferred by many genes with a degree of recessiveness. Fitness estimates were made on susceptible, resistant and hybrid phenotypes. There were no significant differences in fitness between the F, and susceptible phenotypes at any time during the decay of closantel. However, both these phenotypes showed significantly reduced fitness compared with the resistant phenotype during the first 30 days after treatment with closantel. This suggests that there is no selection between the hybrids and the susceptibles at the concentrations of closantel used in this study, and implies that resistance is polygenic. The results provide information on the genetic basis of resistance and how selection for resistance may operate in the field. This information can be used to develop and modify worm control programs so that the onset of resistance is delayed or avoided.
Structural and functional analysis of the amdR positively-acting regulatory gene in Aspergillus oryzae

Xiao, Wenwang. (University of Melbourne, 1992)

The mechanism of the amdR regulation has been well characterized in Aspergillus nidulans. The corresponding A.oryzae gene was cloned and characterized in this study, which provides an insight into the conservation of the regulatory proteins during evolution. The A.oryzae homologue was isolated by cross hybridization with the A.nidulans amdR gene, and shown to complement the A.nidulans amdR44- mutant. Induction studies indicated that the A.oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A.nidulans protein. Inactivation of the A.oryzae amdR gene further confirmed its regulatory role in GABA utilization. Transformation of the A.nidulans amdR gene restored the amdR activity in the gene inactivation mutant of A.oryzae, indicating again that these two amdR genes are functionally interchangeable. Sequence comparison of the two amdR genes showed that the overall homology of DNA sequence throughout the coding region was 64%; and that the conservation of the two polypeptides was 85%, with 76% identity. The conserved amino acid sequences are concentrated in the putative functional domains, such as DNA binding, nuclear localization, transcription activation, and inducer interaction. Two stretches of amino acid sequences which are outside the known functional domains are significantly divergent between two species, suggesting that these represent regions of little functional importance. The A.nidulans amdR gene contains three introns, and the first and third introns have been found at identical positions within the A.oryzae gene. Intron II region is probably a coding region in A.oryzae due to its lack of consensus 5' splice site and a high degree of homology with the corresponding A.nidulans sequence. The transcriptional initiation, termination and splice signals are similar between these two amdR genes, although the overall DNA sequences in these regions have diverged. The extent of sequence divergence between these two amdR genes indicates that the species have evolved separately for a period of time. However, the sequence divergence has been restrained to those changes which have little effect on the functional activity of the amdR gene products.
Investigations of Drosophila melanogaster as a model for insecticide resistance studies

Adcock, Gregory John. (University of Melbourne, 1991)

Standard techniques were defined for using Drosophila melanogaster as a model organism for insecticide-resistance studies. The dosage mortality curves for sensitve (wild-type (WT)) strains (CS and attached-X) challenged with dieldrin, diazinon and cyromazine were defined to choose a concentration to screen for monogenically resistant survivors. Ethylmethanosulphonate mutagenesis was used to create diversity within the sensitive populations prior to screening. From a screen of 600,000 embryos, and the rescreening of 160 survivors, two stabley cyromazine resistant strains were isolated. By standard mapping techniques the resistance in these two strains was found to be conferred by different genes (or gene complexes), designated Cyr-1 and Cyr-2. These mapped respectively within 1 m.u. of ri on chromosome III, and in the region of vg on chromosome II. The dosage mortality for various combinations of the resistance alleles showed that Cyr-1 confers 3xWT tolerance when heterozygous and homozygous, while Cyr-2 confers 2xWT when heterozygous and 4xWT when homozygous. Fitness tests on the resistance alleles showed that Cyr-1 causes a significant decrease in developmental time and survival compared with CS. Neither of the resistance alleles, however, appear to affect developmental homeostasis since the asymmetry values of resistant strains are similar to that of WT. It is argued on the basis of these results, with some modification to the techniques, that Drosophila melanogaster can be a useful model for studying the inheritance of resistance.
Biological monitoring of the Yarra River macroinvertebrates, (December 1983 - December 1986)

Pettigrove, Vincent. (University of Melbourne, 1990)

This thesis details the first phase of a monitoring programme of the aquatic macroinvertebrates of the Yarra River which aims to detect and assess long term trends in the condition of the River and its tributaries. Spatial and temporal trends identified in the fauna over the first phase of this study are identified and related to various physico-chemical parameters and catchment activities. The project design of this study is assessed in terms of whether it appears to be able to meet the project objectives and whether it can be operated in a more efficient and effective manner. In addition, areas that require further investigation in lieu of the findings of this study are discussed. The condition of the macroinvertebrates was assessed by investigating faunal composition and structure through the use of diversity indices, multivariate analyses (direct gradient analysis, ordination and cluster analysis), and dietary analysis of selected taxa. Observed spatial changes in the communities were associated to physico-chemical data by describing any patterns observed and by using a step-wise discriminant analysis. The frequency of structural abnormalities in two species of chironomids was assessed as a means of determining whether they were stressed by toxic contaminants, and the value of this technique in biological assessment is also evaluated. The macroinvertebrate fauna was quantitatively sampled at nine locations on the Yarra River (Upper Yarra, Upper Woori Yallock, Lower Woori Yallock, Healesville, Coldstream, Warrandyte, Alphington and Dights Falls) and one location on Woori Yallock Creek (Yarra!ock) on seven occasions between December 1983 and December 1986. The analyses indicated that a considerable deterioration exists in the condition of the fauna progressively downstream and was associated with water quality (nutrients, turbidity, organic enrichment, and toxicants at Alphington) and physical characteristics of individuals sites (water depth, the type of substratum and surrounding riparian vegetation). Specific findings included: . A large faunal change was apparent between riffle sites at Upper Yarra and Upper Woori Yallock, possibly due to higher levels of nutrients and the more open riparian vegetation present at the Upper Woori Yallock site. . A considerable change in the fauna occurred between pool sites at Healesvilie and Coldstream: a more depauperate Coldstream fauna was attributed to increased organic enrichment and turbidity levels, which probably originated from Olinda Creek. . Considerable differences were found between the faunal composition of the Warrandyte pool site and that of other pool communities upstream. Although nutrient and turbidity levels were higher at Warrandyte, the faunal changes were primarily attributed to physical differences, in particular the study area at Warrandyte had greater water depth and less organic material than upstream pool sites. . The macroinvertebrates communities at Alphington appeared to be markedly more stressed than those of the pool communities investigated. Some changes can be attributed to the physical condition of this site, but the very low numbers of taxa recorded and the high frequency of abnormal mandibles and menta detected in populations of the chironomid Polypedilum tonnoiri from this site, strongly suggest that one or more toxic substances may be also stressing the fauna. - The high flows that prevailed in the Yarra from July to December 1986 appeared to have a large impact on the number of taxa and individuals present at most of the lowland sites. The way in which the fauna responded to these high flows varied between sites as a result of their different physical attributes and whether or not the high flows had a large impact on water quality. - The fauna at Yarra!ock on the Woori Yallock Creek appeared to be primarily affected by high turbidities and nutrient levels and it was not possible to elucidate the impact of toxic pollution, although biocides were widely used in the catchment for various types of intensive agriculture. It is likely that future monitoring of the Yarra River macroinvertebrates will be successful if: . Eight replicates continue to be collected from each site sampled. . The fauna from both the pool and riffle habitats continue to be monitored (as they provide a broader insight into the various factors influencing the condition of the fauna than assessing only one type of habitat). . The fauna is also collected during February/March (when the fauna may be most stressed by various water quality parameters), in addition to the fauna being sampled between April to June and late November to January. . The frequency of structural abnormalities in selected species continue to be monitored (as it is a unique means of determining whether or not the fauna appears to stressed by toxicants). . That the macroinvertebrates of the major tributaries are surveyed to determine what impact they these tributaries may have on the condition of the Yarra River. Highest priority should be given to those streams that appear to be strongly influencing the condition of the Yarra River. . that additional sites be located in areas where there appeared to be a large change in the condition of the fauna. - Several areas for future investigation have been identified that will help substantiate whether the factors that appeared to be influencing species abundance and distribution are in some way responsible for the changes observed in the fauna. These areas are: . An assessment of the benthic flora. . An investigation of the microdistribution of the fauna, particularly in relation to the importance of near-bed flows. . Research into the taxonomy of groups that are currently not well known, particularly those groups that appear to sensitive to changes in the condition of the stream (eg Oligochaeta). . An investigation of the relationship between the amount of sediment and the abundance of individuals in an airlift sample. . An assessment of the importance of drift at certain sites where it could help explain variation in species diversity and abundance. . An investigation of the impact of turbidity on macroinvertebrate composition and structure. Furthermore, there is a need to determine the importance of rare taxa in interpreting the invertebrate data as this may influence whether subsampling and some techniques used to assess the data can be used in future monitoring of the Yarra macroinvertebrates.
Molecular investigations of calcium-binding proteins

Delbridge, Margaret Lorraine. (University of Melbourne, 1990)

Using polyclonal antibodies raised against a Drosophila Ca2+-binding protein (DCABP-23), a Drosophila head cDNA library constructed in the expression vector ?gt11 was screened in order to isolate the gene encoding DCABP-23. Three clones were isolated and subjected to Southern, Northern and sequence analyses. These analyses showed two of the clones to be chimeric. The overlapping regions of all three clones, though probably not encoding DCABP-23, appear to encode a Drosophila cystatin-like protein. The Drosophila cystatin-like protein has homology to cystatins isolated from other organisms, and has been mapped, by in situ hybridization to polytene chromosomes to region 88 B/C on chromosome 3. The reasons why these clones were isolated in the library screen for DCABP-23 will be discussed.

Genetics - Theses

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