ADAR is an adenosine deaminase that acts on dsRNA. Once bound to dsRNA, ADAR deaminates specific adenosines into inosines. If this occurs within the coding region of a transcript the inosine will be read as a guanosine. This can lead to a change in the amino acid at this position and increase protein diversity. In mammals there are three ADAR genes: ADAR1, ADAR2 and ADAR3. However, only ADAR1 and ADAR2 have been shown to be enzymatically active. ADAR1 is widely expressed and can edit both coding RNA and non-coding RNA. ADAR2 is restricted to the CNS and the key transcript that it edits encodes the GluR-B subunit of the glutamate-gated ion channel receptor. Editing of the Q/R site in the GluR-B transcript occurs with an efficiency of more than 99.9% and changes the genomically encoded glutamine into an arginine. This results in an ion channel that is impermeable to calcium. The ADAR2 knock-out mice are viable, but suffer from epileptic seizures and die by day 20. This phenotype can be rescued by expressing the edited R isoform of GluR-B, suggesting that this site is the most important target for ADAR2. Drosophila has only one Adar gene and its product has been reported to edit more than one hundred adenosines in different transcripts. Many of these transcripts encode subunits of ion channels, and it has been hypothesised that lack of ion channel editing causes the behavioural defects and age-related neurodegeneration observed in Adar deletion mutants. In this thesis I investigate the function of ADAR in an uncharacterised Adar mutant, Adar5G1. To characterise the Adar5G1mutant I not only used standard histology but a 3D imaging technique, optical projection tomography (OPT), that had not been reported to be used with Drosophila before this work. OPT allows the internal organs to be imaged without any manual sectioning or dissecting. I used OPT to identify neurodegenerative vacuoles from within the intact head and present the data both in 2D and in 3D. In addition to this, I demonstrate that this technique can be used to image global expression patterns in the Drosophila adult and I relate the TAU-β galactosidase expression pattern to the Drosophila anatomy. The neurodegeneration observed by OPT was confirmed by detailed analysis of stained wax sections. Complete loss of Adar, in the Adar5G1 mutant revealed age-dependent vacuolisation of the retina and mushroom body calyces. The vacuolisation observed in the Adar5G1 mutant was rescued by expression of Drosophila Adar and human ADAR1 p110, and ADAR2. However the cytoplasmic form of ADAR1, ADAR1 p150, did not rescue the vacuolisation of the Adar5G1 mutant. ADAR3, a catalytically inactive ADAR, rescued the vacuolisation phenotype of the Adar5G1 mutant, suggesting that ADAR may have an additional function independent of editing activity. The vacuolisation of the Adar5G1mutant was found not to be associated with type I programmed cell death. However, it was associated with swollen nerve fibres and degrading ommatidia containing multilamellar whorls. Neurodegeneration in various Drosophila mutant models and human neuropathies has been associated with similar cellular structures, suggesting that loss of ADAR results in neurodegeneration common to many of the known neuropathies. Finally, I found that expression of edited isoforms of the nicotinic receptor channel 34E subunit (Nic 34E) failed to rescue the locomotion phenotype of the Adar mutant. However, I found preliminary evidence that one of the lines generated for an edited isoform of Rdl, a subunit of the GABA receptor ion channel, gave a partial rescue of both locomotion and neurodegeneration of the Adar1F4 and Adar5G1 mutant.

No description available.

Thesis (Ph.D. 1974) from the Department of Genetics, University of Adelaide.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 112-115).

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Kristi A. Wharton. Includes bibliographical references.

No description available.

Thesis (Ph. D.)--University of Chicago, Committee on Evolutionary Biology, June 2000. / Includes bibliographical references. Also available on the Internet.

Not all transcribed DNA encodes protein, and some of these noncoding RNAs (ncRNAs), such as roX1 and roX2, may play important roles in the cell. The functional roles of the majority of these, however, remain largely unknown. In this thesis, I first used EST and mRNA evidence to define 2,788 lincRNA loci within the Drosophila melanogaster genome. I suggest that up to 1,652 of these are functional, as 1,411 show evidence for significant evolutionary constraint while 241 fast-evolving loci are enriched in short RNA species. A distinct set of 1,119 lincRNA loci were defined by RNA-seq, the vast majority of which show clear primary sequence constraint. Their expression profiles and enrichment in particular chromatin domains indicate that these lincRNAs are likely involved in developmental regulation. I also identified 42 potential analogous lincRNAs with shared genomic locations between Drosophila and mouse. Constrained, non-embryonic lincRNAs defined by ESTs are transcribed preferentially in the vicinity of protein-coding genes encoding transcription factors and I demonstrated that one of these, which I name dEvf-2, positively regulates the expression of its genomically adjacent transcription factor, Dll, in cell culture. Finally, I used a reverse genetics approach to search for lincRNA promoter mutations and examined the effect of these on lincRNA expression. My findings suggest that many, previously unknown, functional lincRNAs exist within the Drosophila genome and are worthy of further in-depth experimental investigation.

In this thesis I investigated the genetic basis of several behaviours to answer questions surrounding the evolution and mechanistic basis of behaviour. Firstly, I took a single-gene approach to investigate the influence of fruitless (fru) on the courtship behaviour of Drosophila. fru is an alternatively-spliced transcription factor that is necessary for the production of male sexual behaviours, and has also been implicated in producing species-specific differences in courtship song. I investigated the patterns of selection acting on fru at the sequence level and found that positive selection was restricted to the alternatively spliced exons of fru. From this I hypothesised that the positively selected changes in fru would contribute to species-specific differences in courtship song. To test this I examined how isoform-specific fru loss-of-function mutants influence courtship song, and generated “species-swapped” flies whereby regions of fru that showed evidence for positive selection were transferred from four species of Drosophila, into D. melanogaster. Contrary to prediction, I found flies that lacked isoforms containing positively selected regions did not show any differences in courtship song. Unfortunately “species-swapped” flies were not generated in time to examine phenotypes and neuroanatomy as intended. Next, I examined the genetic basis of cold acclimation in two species of Drosophila using a transcriptomic approach. I found that the genes differentially expressed in response to cold acclimation were largely different in each of the species; however, the biological processes they were involved in were broadly similar. Finally, I investigated the transcriptomic changes associated with parental care in the burying beetle Nicrophorus vespilloides to determine if males and females alter the genes they express when parenting alone versus with a partner. I found that males greatly reduced their transcriptional response when parenting with a partner, suggesting they reduce the care they provide when present with a female.