虎皮蛙α2巨球蛋白之基因選殖The cDNA Clone Specific for the Rana tigrina Frog Protein Homologue to α2-macroglobulin

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虎皮蛙 α2 巨球蛋白之基因選殖

The cDNA Clone Specific for the Rana tigrina Frog Protein Homolo gue to α2-macroglobulin

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低等脊椎動物的 α2M 分子之基本單元 , 是由兩條蛋白鏈所組成的單體 ; 分 化至兩棲類時 ,α2M 失去其 β-α 結合位之修飾訊號 , 因為蠑螈 , 母雞 , 哺乳 類之 α2M 基本單元僅有一條蛋白鏈。青蛙的 αM 就是存在於此重要的分化 時期之蛋白 , 可以提供我們進一步演化上的訊息 , 分析兩棲類青蛙的 α 2M 基本單元結構 , 有助於更精確的釐清 α2M 分子的分化情形。本實驗將虎皮 蛙肝臟之 mRNA 用 PCR 增幅其中之硫酯位區段 , 很成功的得到六種不同的次 選殖純株 , 並將其中三個純株的序列和插入長度加以分析。得知 FPCR-5, FPCR-11,FPCR-16 的插入長度分別是 222 對鹼基 ,222 對鹼基與 219 對鹼基。

其中 FPCR-16 與人類 α2M,C3,C4 之相對應核酸序列依序有 68.5 ％ ,53.9

％ ,56.2 ％的類似性 , 而 FPCR-5 及 FPCR-11 所推求之氨基酸序列則較類似人 類的 C3,C4 。故將 FPCR-16 定為疑似青蛙 α2M 之硫酯位區段序列。使用 硫酯位合成探針 , 對青蛙的 cDNA 基因庫進行純株篩選 , 得到具有硫酯位的 cDNA, 並使用假設為 α2M 之 FPCR-16 作第二種探針。從北方點墨實驗得知 青蛙 α2M 顯現 4.6kb 及 2.1kb 兩條主線 , 可解釋為青蛙 α2M 及 α1M 分子量之 不同。本論文也討論 α2M 硫酯位核酸序列下游 165 對鹼基處 , 有一段長為 六個鹼基對 , 暫時定名為 "AG-GG I.S." 的序列 , 可能與演化的插入性序列 有關。

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The molecular structure of the vertebrate α2M acquired The molecular structure of the vertebrate α2M acquired a two- subunit chain structure prior to the emergence of pri- mitive vertebrates. By the time of divergence of amphibian, α 2M lost the β-α processing signal, since α2M of salaman- dra, hen and mammalian species have a single chain-subunit. Frog α M appeared to represent this critical divergent stage, and will provide us with further evolutionary informations.To analysis of the subunit structure of α2M from amphibian frog will help to define more exactly its molecular divergency. This work decribes the PCR strategy used to amplify the thioester region from Rana tigrina pantherina frog liver mRNA. It has been successful to isolate six independent subclones, and the nucleotide sequences of the three of their inserts were

determined. The insert size of FPCR-5, FPCR-11, FPCR-16 clone is 222bp, 222bp, and 219bp respectively. The nucleotide

sequence of FPCR-16 insert showed 68.5 ％ ,53.9 ％ , and 56.2 ％ identity with corresponding regions of human α2M,C3, and C4.

The other two clones,FPCR-5 and FPCR-11, revealed the deduced amino acid sequence more homologous to human C3 and C4.There- fore, FPCR-16 was tentatively identified as frog α2M. CDNA

clones concerning with the partially sequence of α 2M were isolated from frog liver cDNA libraries by using the synthetic probe containing thioester sequence and the

虎皮蛙α2巨球蛋白之基因選殖The cDNA Clone Specific for the Rana tigrina Frog Protein Homologue to α2-macroglobulin

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determined. The insert size of FPCR-5, FPCR-11, FPCR-16 clone is 222bp, 222bp, and 219bp respectively. The nucleotide

sequence of FPCR-16 insert showed 68.5 ％ ,53.9 ％ , and 56.2 ％ identity with corresponding regions of human α2M,C3, and C4.

The other two clones,FPCR-5 and FPCR-11, revealed the deduced amino acid sequence more homologous to human C3 and C4.There- fore, FPCR-16 was tentatively identified as frog α2M. CDNA

clones concerning with the partially sequence of α 2M were isolated from frog liver cDNA libraries by using the synthetic probe containing thioester sequence and the

insert fregment of the putative frog α2M, FPCR-16, as second probe. By northern blot analysis of frog liver RNA, frog α2

M showed two major band at 4.6Kb and 2.1Kb. The estimated size ofα2M was probably reflecting differences of α1M and α2M.

The evolutionary relationship of 6bp insert sequence, named

AG-GG I.S., 165bp far down stream region of thioester site is

discussed.