Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized.
RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives.
RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU.2429/860 |
| Date | 11 1900 |
| Creators | Veiga, Mariana Barçante |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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