絲胺酸羥基甲基轉移酶（serine hydroxymethyltransferase，SHMT，E.C. 2.1.2.1）須與pyridoxal phosphate（PLP）結合才具酵素活性，它可催化四氫葉酸（THF）與5, 10-甲烯基四氫葉酸（5,10-methylene-THF）及絲胺酸（serine）與甘胺酸（glycine）的可逆式反應。此外它可與大部分葉酸結合，調控葉酸循環中單碳官能基的流向，影響核苷酸、胺基酸的生合成及甲基化，與基因表現、染色體穩定有關。在大多數的多細胞生物中，發現有一種形式以上的SHMT存在：細胞質型和另一種胞器相關型，通常為粒線體型。在人與斑馬魚個體中，細胞質型SHMT（cSHMT）的mRNA具有組織特異性。因此想研究斑馬魚cSHMT（zcSHMT）調控機轉。我們從斑馬魚染色體選殖zcSHMT 基因5端上游promoter序列至pGL3-basic載體，轉染至斑馬魚肝臟上皮細胞（zebrafish liver epithelial cell，ZLE）測定啟動子活性。並從RLM-RACE cDNA Library構築zcSHMT 5’-UTR決定轉錄起始點。經過縮減啟動子序列並測定轉錄活性後，發現含轉錄活性的最小啟動子序列為zcSHMT 5端序列-72/+25（zcSHMTp-72/+25）。zcSHMTp-68/+1也具有啟動子活性，但有明顯下降。將zcSHMTp-72/+25與zcSHMTp-68/+1作探針與斑馬魚肝臟核蛋白進行Electrophoresis Mobility Shift Assay （EMSA）結合反應，發現有蛋白與探針形成具專一性複合物。去除-72/-25序列，-25/+25不具有啟動子活性。推論有轉錄因子結合-72/-25序列引響起動子活性。運用網路軟體transcription element search software（TESS）分析可能結合至-72/-25序列的轉錄因子，結果顯示有多種轉錄因子結合序列。其中包含兩個Sp1結合序列。利用點突變將Sp1結合位置改變，其轉錄活性呈現有意義下降。但共同轉染斑馬魚Sp1表現質體與zcSHMTp-72/+25/pGL3-basic質體至ZLE，其轉錄活性未上升反而有意義下降。將zcSHMTp-72/+25中的Sp1結合位置改變後，發現仍可與斑馬魚肝臟核蛋白結合，推論Sp1不是直接結合在zcSHMT啟動子序列，可能需透過其他轉錄因子協助。另外我們建立測定葉酸及同半胱胺酸方法，初步結果顯示大量表現斑馬魚10-甲醛四氫葉酸去氫酶（zebrafish 10-formyl-THF dehydrogenase，zFDH）的293T細胞其培養基中，所含同半胱胺酸濃度有明顯升高的現象。

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.

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