鯉魚視網膜紫質基因之分子結構

Abstract

為了研究視覺基因的分子結構與有關魚類生理學視覺訊息傳導的生化起始反應，首先得定出視覺基因的核酸序列．從鯉魚視網膜cDNA基因庫，第一種視紫質的cDNA (Tsai et al., 1994)與第二種視紫質部分的cDNA (Chong, 1994)已被選殖．本論文的實驗定出完整的第二種視紫質的cDNA序列並分析之．第二種視紫質的胺基酸序列顯示兩種視紫質之間有五個胺基酸相異，即(i)第一種的第十九個胺基酸是Val而第二種的是Ile (Val-19-Ile), (ii)Ile-54-Val, (iii)Ile-108-Val, (iv) Val-169-Glu，與(v)His-315-Asn．除此，第二種視紫質基因的3’端非轉譯區比第一種的長99鹼基，並且含四個polyadeny1ation signal而不是一個．再者，本人建立了一個genomic基因庫並從裡面分離出第二種視紫質的genomic DNA．此genomic DNA被定序及其轉錄之起始位置(transcription start site)也經引子延長化驗(primer extention assay)而被確定．實驗結果顯示，(1)此基因序列沒有內子(intron),(2)其轉錄起始位置其他脊椎動物相同，及(3)在其上游序列含有保守區域(conserved region)和視網膜專一性核蛋白結合位置(retina-specific nuclear protein binding sites)．而南方分析(Southern analysis)與反轉錄聚合酵素鍊鎖反應(reverse transcriptase-polymerase chain reaction)表示兩種視紫質乃存在於同一個鯉魚群但是不同的個體裡．另一方面，利用綠光視覺接受器(green opsin)的cDNA當做探針，本人從genomic基因庫篩選出一個類似retinal epithelium-specific protein的克隆(clone)．結果其中一片段的序列與人類衛星去氧核醣核酸(human satellite DNA)有66％相似而另一段則與thromboxane synthase有95％相似．但此克隆的真正身份卻有待進一步的研究．

To investigate the molecular structures of the visual pigment genes and the initial biochemical reactions involved in the visual signal transduction relevant to fish physiology, the sequence of the visual pigment gene has to be determined. A putative cDNA encoding carp type I rhodopsin (Tsai et al., 1994) and a partial cDNA encoding type II rhodopsin (Chong, 1994) had been cloned from a retinal cDNA library. In the present work, the cDNA of the type II rhodopsin was completely sequenced and analyzed. The deduced amino acid sequence of type II rhodopsin reveals that there are only five amino acid residues differences between type I and type II, i.e.. (i) the 19th residue in type I is Val, instead of Tie in type II (Val-19-Ile), (ii) Ile-54-Val, (iii) Ile-108-Val, (iv) Val-169-Glu, and (v) His-315-Asn. Besides, there are 4 polyadenylation signals in type II rhodopsin instead of I in type I, and the 3’ untranslated region of type II is 99 bases longer. Furthermore, a genomic library of the common carp was constructed and the genomic DNA of type II rhodopsin was isolated. The genomic DNA was sequenced and the primer extension assay was performed to determine the transcription start site. The results reveal that (1) the gene is not interrupted by any intron, (2) the initiation of the transcripts of rhodopsin II is similar to that of the other vertebrates, and (3) there are conserved regions and retina-specific nuclear protein binding sites found in the upstream region. The Southern analysis of genomic DNA and the reverse transcriptase-polymerase chain reaction imply that the two types of rhodopsins are from different individuals of the same population. On the other hand, a clone (3aV1) which resembles the retinal epithelium-specific protein gene was screened from the genomic DNA library using the partial cDNA of green opsin as a probe. The results indicate that a fragment of the deletion clones of 3aV1 is 66% identical to the human satellite DNA and another fragment is 95% identical to the thromboxane synthase. However, the real identity of the clone remains to be investigated.