酵母tRNA结合蛋白(43kD蛋白)羧端cDNA的克隆及其序列测定

为了研究酵母分子量为 4 3kD的tRNA结合蛋白的基因来源 ,通过溴化氰部分化学裂解此蛋白 ,产生的 18kD肽段经过蛋白质氨端序列测定 ,测得 18kD肽段氨端部分序列为AFTFKK .针对序列AFTFKK设计简并引物 ,利用简并PCR方法和cDNA的 3′末端的快速扩增方法 (3′RACE) ,成功克隆 4 3kD蛋白mRNA的 3′端序列 .DNA序列测定结果表明 ,该序列位于 3 磷酸甘油酸激酶 (PGK1)基因 10 0 3位— 170 0位 ,长度为 6 98bp ,编码 3 磷酸甘油酸激酶羧基端的 180氨基酸残基以及 3′端非翻译区 .结果证实 ,4 3kD蛋白的基因就是PGK1酶的基因

Cloning and Sequencing of C-terminal cDNA of a Yeast tRNA Binding Protein(43 kD protein)

To further determine the gene of a tRNA binding protein with molecular mass 43 kD, an 18 kD peptide was obtained by partially cleavage of 43 kD protein with bromide cyanogen (CNBr). N terminal sequencing of the 18 kD peptide yielded the sequence AFTFKK. Degenerative primer corresponding to AFTFKK peptide was used to relative assays of degenerate PCR and rapid amplification of 3′ terminal end of cDNA. The final results of the cDNA sequencing verified that the DNA fragment overlapped with 3 phosphoglycerate kinase(PGK1) gene, spaned from position 1003 of PGK1 gene to position 1700 with a length of 698 bp. The 698 bp fragment coded 180 amino acids at the C terminus of PGK1, also including the 3′ untranslated region of PGK1 gene. The results indicated that the gene coded 43 kD protein was identical to the gene of 3 phosphoglycerate kinase.