生長休止基因7蛋白產物功能及其mRNA表現調節之研究

Abstract

gas7由處於細胞靜止期的小鼠纖維母細胞株－NIH3T3中以基因捕捉法被選殖出來．用DNA strider軟體分析gas7的cDNA序列找到一421個胺基酸的ORF其被預測可轉譯出一48kDa的蛋白．將gas7 cDNA序列及預測的胺基酸序列以GCG軟體與Genembl，Swissport等DNA，蛋白質銀行內已知序列比較，發現gas7為一新的基因，與任何已知基因無顯著相似性．在本篇論文中我報告了對gas7蛋白質產物功能的探討及其RNA表現的分析． 為了研究gas7蛋白產物的功能，我由定位其蛋白產物開始著手．由in vitro translation，co-translation及membrane strippig實驗發現，Gas7在被轉譯時並不會進入內質網膜（ER），故可推測其應是一細胞質中或細胞核內的蛋白．要研究一蛋白質的功能，抗體是最有用的工具之一．因此我著手製備抗Gas7蛋白的抗體．我構築了一GST-Gas7融合蛋白，讓其在細菌中大量表現．然後將得到的融合蛋白注射到兔子體內使其產生抗血清．此抗血清可專一的認識由細菌表現的融合蛋白及在細胞暫時表現的Gas7蛋白．用此抗血清去作有gas7基因暫時表現的小鼠纖維母細胞之免疫螢光染色來定位Gas7蛋白，確定其是一細胞質中的蛋白．同時發現有暫時表現gas7的細胞會有外形的改變．我們推測Gas7蛋白對細胞的cytoskeleton有影響． 由小鼠多組織北方轉漬法發現gas7的RNA產物在小腦的表現最多．為了確定其蛋白是否亦在小腦表現最多．用上述抗血清以西方轉漬法（Western Blot）偵測小腦蛋白萃取物，結果偵測到－與在NIH3T3細胞中大小不同的蛋白（38kDa）．用小腦的RNA作5'RACE，發現小腦表現的RNA與gas7在5端的序列不同．此RNA isoform可找到－ORF其被預測可轉譯出－38kDa的蛋白．我們推測alternative splicing對gas7在不同組織的表現扮演重要的角色，此機制可能造成不同大小的蛋白被產生．而在不同的組織有不同的功能．

Growth-arrest-specific (gas) gene 7 was identified using a promoter trapping vector from serum-starved NIH3T3 cells. Its cDNA (2875 bp) was cloned and sequenced. GCG and DNA strider analyses showed that gas7 is a novel gene and contains an open reading frame (ORF) of 421 amino acids with a predicted molecular weight of 48 kDa. This putative protein has no significant homology with any known protein and contains no transmembrane domain. I report here the characterization of the functions of Gas7 protein. Using an in vitro translation assay, I demonstrated that a 48 kDa protein can be translated from gas7 mRNA, and that the protein does not translocate into canine microsomal membranes. These results indicate that Gas7 isn't a membrane or secretory protein. Using a membrane stripping assay, I also demonstrated that Gas7 is not an integral membrane protein. To analyze the Gas 7 protein, a GST-Gas7 fusion protein was expressed in E. coli, and subsequently was used for the production of polyclonal antibodies. By using the polyclonal antibodies and the Western blot assay, a 48 kDa protein was detected from gas7 transiently transfected NIH3T3 cells. Furthermore, by using confocal microscopy and immunofluorescence staining of gas7 transiently transfected NIH3T3 cells, I demonstrated that Gas7 is a cytosolic protein. This transient expression of gas7 resulted in a striking morphology change of NIH3T3 cells. This result suggests that the Gas7 protein has an effect on the cytoskeleton of the transfected NIH3T3 cells. To investigate the mRNA expression of gas7, using mouse Multiple-Tissue Northern Blot, we found that the transcripts of gas7 are most abundant in the cerebellum and they exist in two different sizes. To investigate whether Gas7 protein is preferentially expressed in the mouse cerebellum, we performed a Western blot and detected a 38 kDa protein, whose size is different from that of the protein produced in NIH3T3 cells, in the extract of mouse cerebella. Using 5' rapid amplification of cDNA ends (RACE) from the total RNA of mouse cerebella, I found an alternatively spliced transcript which is different from gas7 cDNA in the 5' end. This transcript contains an ORF that specifies a putative protein of MW 38 kDa. These results suggest that alternative splicing is an important mechanism for gas7 gene expression in brain tissues.