(一)鯉魚r-crystallin 基因之研究 (二)牛蛙POMC基因之表現

Abstract

（一）： 已知鯉魚水晶蛋白中含有高甲硫胺酸（highme thionin）的γ水晶蛋白質，從其c-DNA基因庫中已?選到兩個高甲硫胺酸（high methionin）的γ水晶蛋白質的基因，分別命名為γ-m1，及γ-m2 。 利用γ-m1，當做探針（probe) ，從鯉魚眼睛genomic基因庫中篩選到一個genomic菌株，此genomic菌株含有約3.5kb的genomic DNA，經由Enzyme mapping sou thern blotting的方法，知道其真正含有C-DNA部分集中於約0.7kb的區域中，此長度與γ-m1 C-DNA長度差不多，一個genomic-DNA與其C-DNA長度一樣可能性很小，大部分genomic DNA均含有intron將此約0.7kb區域定序後，發現其為γ-m1 C-DNA。 （二）： 已從牛蛙腦下腺poly A mRNA所建構的C-DNA基因庫中篩選到牛蛙POMC基因，現將此POMC基因clone至表現載體（Expression vector）於E.Coli中表現，所選用的表現載體PRIT2T具λ PR promoter，λ PR promoter可大量而有效率的生產我們所需，PRIT2T表現載體所產生之產物為protein A’-fusion protein , protein A’部分可有助於產物之分離，建構完成的POMC表現質體pRIT2T-POMC送入N4830-1 host後產生之protein A’- Fusion protein，分子量大約35 KD左右此產物比所預期的70KD小，經由核酸定序分析，得知2bp reading frame shift，因而造成POMC基因只translate 37個胺基酸即遇到TAA Stop codon.

(一) Carp lens contain unusually high methionine(12-15%) γ- crysallins We have got two γ- crystallins clones of such high methionine contents from the c - DNA library of carp, that is , γ-m1 and γ- m2. In order to understand the structure of high-methionine γ-crystallines genomic DNA, a positive high-methionine γ-crystallin genomic clone had been screened out from the genomic DNA library of carp lens by using r – m1 acting as screening probe. This high-methionine γ- crystalline genomic clone is about 3.5 Kb in length. By methods of enzyme mapping and Southern blotting , we know that only about 0.7 Kb region of this genomic clone really belong to high-methionine γ-crystallin genomic DNA. This genomic DNA is about the same length as γ-m1 cDNA. It is almost impossible for a genomic DNA to be the same length as cDNA. Introns are necessary element of most genomic DNA. By sequencing this 0.7Kb region. We know that this genomic clone is not γ-crystallin genomic DNA. (二) Bullfrog POMC gene has been screened out from bullfrog pituitary cDNA library. Now we clone POMC gene into pRIT2T fusion expression vector to express POMC gene in E. Coli. pRIT2T fusion expression vector carrying . λPr promoter can efficiently express POMC gene. pRIT2T fusion expression vector produce proteinA'-fusion protein. Product can easily separate by lgG affinity column. POMC expression plasmid (pRIT2T-POMC) is transformed into N4830-1 E. Coli, and produces proteinA'-POMC fusion protein. Molecular weight of this product is about 35 KD that is smaller than the molecular weight we expect. By DNA sequencing method, we know that because of 2 b.p. deletion we get this product.