Mitochondrial Gene Expression in Early Sea Urchin Development

Elliott, David James (1990) Mitochondrial Gene Expression in Early Sea Urchin Development. PhD thesis, University of Glasgow.

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Mitochondrial transcripts are differentially expressed, particularly the rRNAs which are expressed at a higher level than the mRNAs and tRNAs. Most work on animal mitochondrial gene expression has been done in vertebrates, in which differential expression of the rRNA genes relative to the mRNA/tRNA genes seems to be achieved at the level of transcriptional initiation/termination. Transcription of the contiguous rRNA genes initiates in an intergenic sequence located immediately upstream of them, but is subsequently attenuated to limit RNA polymerase readthrough into the mRNA/tRNA genes. The mRNA and rRNA genes are thought to have distinct transcriptional initiation sites located immediately downstream of the rRNA initiation site, but RNA polymerase initiating at this downstream site reads through the attenuator into the mRNA/tRNA genes. Sequence analysis has shown that the organisation of the sea urchin mitochondrial genome differs from that found in vertebrates. Of particular importance for the control of gene expression is the fact that the rRNA genes are separated by the genes encoding 2 mRNAs and several tRNAs, and also an extended intergenic sequence. These differences mean that alternative mechanism from that in vertebrates must be operating to give differential expression of the rRNA and mRNA/tRNA genes. There is also a further level of complexity in sea urchin mitochondrial gene expression, in that the ratio of rRNA:mRNA is developmentally modulated. Using RNA filter hybridisations, I have shown that the ratio of rRNA:mRNA is developmentally modulated. mRNA levels increase about tenfold between the egg and blastula stages of development, whereas rRNA levels decrease slightly. The levels of high molecular weight transcripts containing the information for more than one gene change between different developmental stages, suggesting that pathways of RNA synthesis operate in a developmentally regulated manner. In order to get an insight into how the rRNA:mRNA ratio is established and developmentally modulated in sea urchins, I mapped mitochondrial transcripts at the egg and blastula stages of development. These experiments showed that the 5' ends of both rRNA genes are directly adjacent to their upstream genes, so neither rRNA gene has an intergenic sequence directly upstream. However, the 3' ends of both rRNAs overlap with the transcripts encoded by the adjacent downstream genes. These overlapping transcripts must be synthesised by mutually exclusive pathways, most likely at the level of RNA processing. Transcript termini seemed to be the same at both developmental stages. I have used two complementary techniques in order to map transcriptional start sites, and transcription units, on the sea urchin mitochondrial genome. Firstly I have used in vitro capping with the enzyme guanylyl transferase, which specifically labels the 5' triphosphate groups on RNA (which result from transcriptional initiation rather than processing). This approach has not given entirely consistent results, but has identified two regions of the sea urchin mitochondrial genome which are complementary to transcripts with 5' triphosphate termini. Secondly I have 3H-labelled sea urchin RNA in vivo. in pronase-permeabilised sea urchin eggs, and have identified the major labelled mitochondrial transcripts by gel and hybridisational analyses. I have used this method along with ultra-violet (u.v.) mapping, in which transcription is terminated in a dose-dependent fashion depending on distance from the transcriptional initiation site. This can be used to order genes within transcription units, and has shown that whereas the synthesis of 16S rRNA is relatively u.v. resistant, the synthesis of both 12S rRNA and COI mRNA are sensitive to even the lowest doses of u.v. irradiation. This suggests that 16S rRNA is synthesised from a transcriptional initiation site located closely upstream, and that COI mRNA and 12S rRNA are both synthesised from transcriptional initiation sites located far upstream of their genes. The genes encoding COI and 16S rRNA are adjacent, demonstrating that at least in some cases adjacent genes are not necessarily co-transcribed from the same promoter. 16S rRNA and COI mRNA may have distinct primary transcripts, and the choice of which mature transcript is selected by the mutually exclusive synthetic pathways may be determined by their repective sites of transcriptional initiation, perhaps by affecting secondary structure at the 16S rRNA/COI gene boundary. The u.v.-mapping data is thus consistent with a transcriptional initiation site located just upstream of the 16S rRNA gene.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics, Developmental biology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78089
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:41
Last Modified: 30 Jan 2020 15:41

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