Genetics - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemThe mammalian stonin proteins : from translation to degradation(University of Melbourne, 2009)Recent work has centred around the role of stonin 2 in clathrin-mediated endocytosis (CME) of the integral synaptic vesicle protein synaptotagmin I. Without a mode of internalisation of this protein, synaptic vesicles that are formed from the plasma membrane are fusion-incompetent and therefore no neurotransmitter can be released (Mohrmann, 2008). Stonin 2 was first identified on the basis of homology with the Stoned B gene of Drosophila melanogaster. The stoned B protein is encoded in the second open reading frame (ORF) of a dicistronic transcript that encodes another protein involved at the synapse, named stoned A. It was in 1973 that the locus was first identified in a screen for temperature sensitive paralytic mutants. These mutants were shown to have defective nervous systems and had aberrant synaptic vesicle retrieval at the plasma membrane of the neuromuscular junction (NMJ). Like stonin 2, stoned B interacts with synaptotagmin I through its C-terminal domain that shows significant homology to the ?2 subunit of the clathrin adaptor protein complex AP-2. Stonin 2 is not the only stoned B homologue in mammalian genomes, with another protein named stonin 1 showing equal protein similarity. In this report, the stonin 1 antibodies that had previously been utilised (Arnott 2004) were re-characterised and it was concluded that they were only useful to detect expressed recombinant protein. Affinity purified stonin 2 antibodies, created in this project, were successfully utilised to detect both endogenous stonin 2 and recombinant expressed-tagged stonin 2 for both Western blot and immunocytochemistry, which required a novel antibody purification method to be created to achieve a greater resolution. After analysis of expressed tagged stonin proteins in the neuroendocrine PC12 cell line, it was suspected that both proteins were being rapidly degraded. This led to the discovery that both stonin 1 and 2 were being targeted for degradation by the ubiquitin proteasome system that appears to be neuroendocrine specific. Stonin 2 is additionally cleaved by an unknown protease to produce C-terminal fragments that are the predominant protein species in PC12 cells. The analysis of the stonin 2 protein fragmentation pattern compared to the endogenous banding pattern led to the discovery that protein variation is also achieved through initiation of translation at internal start sites. Although the specific methionine residues were not characterised, at least two protein species were identified that were the result of internal initiation. These protein species may be regulated through the use of a short upstream open reading frame that overlaps the predicted stonin 2 ORF start codon. The internal start sites for translation initiation would give rise to N-terminally truncated protein species that would lack one or two critical AP-2 binding motifs that may modulate the function of stonin 2. The final series of experiments presented in this thesis analyse the localisation and protein-protein interactions of the stonin proteins through immunocytochemical analysis of exogenously expressed-tagged stonin proteins during inhibition of the ubiquitin proteasome system. This lead to the finding that when stonin 1 is bound to internal membranes it is predominantly bound to internal structures that can be motile. Under these conditions, it was also shown that stonin 1 could bind synaptotagmin I, although it is unlikely to occur at the plasma membrane. Membrane-bound Stonin 2 is found predominantly sub-cortically or at the plasma membrane. The sub-cortical localisation may be at sites that vesicles are, or will be, attached to the plasma membrane. Stonin 2 can be part of a protein complex that involves AP-2, synaptotagmin I and the fission molecule dynamin I. These results provide new insight into the role that both stonin 1 and 2 play in the cellular trafficking network. The protein variation identified in Stonin 2 adds further complexity to the myriad of regulatory events that can occur to modulate synaptic plasticity.
-
ItemMammalian homologues of the stoned gene(University of Melbourne, 2004)Genetic and biochemical studies have shown that the stoned gene of Drosophila melanogaster is essential for normal recycling of synaptic vesicles. The dicistronic stoned transcript encodes two products; STNA, a novel protein, and STNB which has similarity with the ? subunits of adaptor complexes. However it is not yet known specifically how these proteins function during synaptic transmission. This investigation sought to identify murine homologues of the stoned proteins, in order to further our understanding of these essential neural components. During this study, two STNB homologues were identified, mSTNB1 and mSTNB2, which also have similarity to the ? adaptins. Although no sequence homologues of STNA appear to be present in mammalian genomes, this does not exclude the possible existance of a functional STNA homologue. The characterisation of mSTNB1 revealed that the mRNA transcript is widely expressed. However unlike STNB, neither the mSTNB1 mRNA or protein is enriched in the brain. The mSTNB1 protein has also been found in a range of cell lines, including CHO cells. An analysis of heart cross-sections revealed that mSTNB1 is abundant in a subset of non-neuronal cells. Comparison of mSTNB1 to subcellular markers in CHO cells indicated that mSTNB1 is associated with punctate structures, where it partially co-localises with the Golgi-associated 58kDa protein. This may represent the ER-to-Golgi intermediate compartment (ERGIC), which is involved in the early secretory pathway. Consistent with this, mSTNB1 is not found associated with the endocytic trafficking compartments. These observations have led to the hypothesis that the STNB family of proteins may be involved in the trafficking of large vesicular carriers, such as synaptic vesicle precursors or the ERGIC, rather than the biogenesis of small vesicles.
-
ItemCytochrome P450 gene expression in Drosophila melanogaster( 2008)Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.