Use this URL to cite or link to this record in EThOS:
Title: An integrative and systems biology approach to Drosophila melanogaster transcriptomes
Author: Chintapalli, Venkateswara Rao
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2012
Availability of Full Text:
Access through EThOS:
Access through Institution:
The availability of fully sequenced genomes of the model organisms including Drosophila, and their subsequent annotation has afforded seamless opportunities for reverse genetics in a complex model organism. With the advent of DNA microarrays to assay the levels of tens of thousands of genes in a single sample, functional genomics has been significantly aided to understand the functions in systems context. These microarrays have been employed predominantly on the RNA samples that are extracted from the whole animals for example at different developmental stages or in response to external stimuli. However, these approaches relied on the expression patterns that represent the sum of transcription coming from all the organs, which do not estimate the tissue-specificity of transcription. The purpose of this thesis is to provide tissue-specific transcriptomes of Drosophila melanogaster that were generated as part of the large FlyAtlas project using Affymetrix Drosophila GeneChips® (or microarrays). These chips, one at a time interrogate the levels of 18,500 transcripts (that represent all known genes) using 18,880 distinct probe sets in a single, total RNA sample. For each tissue, four biological replicates were analysed using the chips and the normalised signal intensities were obtained that represent the relative levels of mRNA expression. Using the transcriptomes, a general analysis was performed for potential novel insights into tissue-specific functions (Chintapalli et al., 2007) (Chapter 3). Then, a comparative analysis of epithelial tissues was performed to understand how the epithelia are organised in terms of their transcriptomes (Chapter 4). The Malpighian tubules are the Drosophila epithelial counterparts of the human kidney. They show asymmetric organisation in the body cavity. FlyAtlas segment-specific tubule transcriptomes allowed the comparison of their potential functional similarities and differences, thus to understand the asymmetry in function (Chapter 5)(Chintapalli, 2012). This identified a human Best vitelliform macular dystrophy (BVMD) disease homolog, Best2 in only the anterior pair of tubules that have the morphologically and functionally distinct enlarged initial (or distal) segment, a storage organ for Ca2+. Bestrophins were accordingly selected as candidate genes to analyse organismal functions, and thus to validate previous two theories that implicated bestrophins as Ca2+-activated Clˉ channels and/or Ca2+ channel regulators (Chapter 6). The confocal microscopy analysis of bestrophin YFP fusion proteins revealed interesting and novel localisations of bestrophins, in that Best1 was found in the apical plasma membranes, Best2 localised to peroxisomes, Best3 and Best4 were found intracellular. The salt survival analysis showed that Best1 is essential in regulating extra salt levels in the body. Furthermore, the fluid secretion analysis showed Best1’s potential role in Ca2+-dependent Clˉ function. Interestingly, the flies with reduced levels of Best2 expression showed increased ability to survive on extra salt food; the basis for this was investigated further in Chapter 7. Best2 was also found abundant in the eyes than anywhere else in the head. A comparative analysis of anterior tubule- and eye-specific transcriptomes revealed a potential overlap of Ca2+ signaling components, in that the PLCβ signaling was one. A neuropeptide Ca2+ agonist, capa1 evoked secondary cytosolic Ca2+ responses were found high in Best2 knockdowns. A quantitative PCR (qPCR) analysis of candidate Ca2+ signaling and homeostasis genes in Best2 mutants revealed their gene expression upregulation, under control-fed and salt-fed conditions than their wildtype controls, fed on similar diet regimes. The norpA that encodes PLCβ was found significantly enriched in the mutants. Cyp6a23 is another gene that was highly upregulated in Best2 mutants; it is a Drosophila homologue of human Cyp11b, a Ca2+-responsive gene implicated in renal salt wasting. Upon the downregulation of Cyp6a23, flies became sensitive to salt diet feeding. Other genes investigated and found to be upregulated in the mutants include transient-receptor-potential (trp) Ca2+ channel and retinal degeneration C (rdgC). Together, these results strongly suggest Best2 as a potential Ca2+ channel regulator, and provide fascinating insight into bestrophin function. Peroxisomal localisation of Best2 in line with the implication that peroxisomes act as dynamic regulators of cell Ca2+ homeostasis led to another aspect of the project (Chapter 8). This study identified two peroxins that are most abundant in the tubules and play essential roles in the novel cyclic nucleotide-regulated peroxisomal Ca2+ sequestration and transport pathway and that are detrimental for peroxisome biogenesis and proliferation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology