碩士 / 國立成功大學 / 醫學檢驗生物技術學系 / 96 / Serine hydroxymethyltransferase (SHMT, E.C. 2.1.2.1) is a pyridoxal phosphate-dependent enzyme. It reversibly converts serine and H4PteGlu to glycine and 5, 10-CH2-H4PteGlu, the principal pathway to generate folate-activated one-carbon units required for purine, thymidylate and methionine biosynthesis. In higher organisms more than one SHMT isoform is often present: a cytosolic isoform and an organelle-associated form, usually mitochondria. It is suggested that cytosolic SHMT (cSHMT) regulates one-carbon flow and maintains intracellular one-carbon units homeostasis by alternating the one-carbon flow between dTMP biosynthesis and homocysteine remethylation. Therefore, the intracellular level of cSHMT is believed to be stringently regulated. The aim of this study is to search for the possible regulatory mechanism for zebrafish cSHMT (zcSHMT) expression. We have cloned and characterized the zcSHMT promoter region into promoterless pGL3-basic vector for promoter activating analysis. Serial deletion of the promoter region reveals that the zcSHMT 5’ flanking sequence -72/+25 fragment (zcSHMTp-72/+25) encompassing partial exon 1 sequence is crucial for the transactivation activity. Protein-nucleotide complexes are observed in Electrophoresis Mobility Shift Assay (EMSA) using digoxigenin (DIG)-labeled zcSHMTp-72/+25 probe and incubating with zebrafish liver nuclear extract. Sequence analysis of the zcSHMTp-72/+25 reveals consensus sequences for several transcription factors including Sp1. Significant reduction in transactivation activity is observed when the prospective Sp1 binding sites are abolished by site-directed mutagenesis. However, co-transfection of zebrafish Sp1 coding sequence to ZLE cell results in decreased promoter transaction activity of this promoter region. In addition, the zcSHMTp-72/+25 fragment with abolished Sp1 sites is still capable of forming protein-nucleotide complex with zebrafish liver extract, suggesting components or mechanisms other than that simple-direct binding of Sp1 might be involved in zcSHMT promoter activation. The methods for homocysteine and folate detection were also established and successfully applied to determine folate and homocysteine concentration in bio-samples.
| Identifer | oai:union.ndltd.org:TW/096NCKU5108002 |
| Date | January 2008 |
| Creators | Li-Ting Chen, 陳莉婷 |
| Contributors | Tzu-Fun Fu, 傅子芳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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