CLONING AND SEQUENCING OF A DEOXYRIBONUCLEIC-ACID COPY OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE MESSENGER RIBONUCLEIC-ACID ISOLATED FROM CHICKEN MUSCLE
CLONING AND SEQUENCING OF A DEOXYRIBONUCLEIC-ACID COPY OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE MESSENGER RIBONUCLEIC-ACID ISOLATED FROM CHICKEN MUSCLE
复制标题
DOI：
10.1021/bi00276a013
复制
发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
SCHWARTZ, RJ
中科院分区：
文献类型：
--
作者：
DUGAICZYK, A;HARON, JA;SCHWARTZ, RJ
从 3 周龄鸡的胸肌中纯化出 3-磷酸甘油醛脱氢酶 (GAPDH)，并用于在兔子中制备特异性抗血清。将该抗血清与体外翻译测定结合以监测 GAPDH mRNA 的纯化。从相同的胸肌中分离 RNA，并通过多种技术连续分级，产生纯度至少为 50% 的 GADPH mRNA 制剂。针对该纯化的 RNA 制备双链 c[互补]DNA，将其插入 pBR322，并用于转化大肠杆菌。通过与针对纯化的RNA制备的cDNA探针进行集落过滤杂交来筛选重组体。然后通过使用 pGAD-28-纤维素从总 Poly(A) RNA 中选择 cRNA，然后在体外翻译杂交选择的 RNA，对具有最大插入片段 pGAD-28 的杂交阳性克隆进行表征。通过在十二烷基硫酸钠-聚丙烯酰胺凝胶上与纯GAPDH共迁移、用特异性抗GAPDH抗血清沉淀、氰基化指纹分析和AMP-琼脂糖亲和层析，表明单一翻译产物是GAPDH。 pGAD-28 用几种限制性内切酶进行定位，然后通过 Maxam 和 Gilbert 的方法进行测序。发现 1261 个核苷酸的插入片段在 5'' 端、整个编码区包含 29 个核苷酸的非编码序列，以及 3'' 非编码区的 230 个核苷酸，包括聚腺苷酸添加信号 (AATAAA) 和聚腺苷酸尾部的前 5 个残基。
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-wk-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GADPH mRNA which was at least 50% pure. Double-stranded c[complementary]DNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select cRNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, precipitation with specific anti-GAPDH antiserum, cyanylation fingerprinting and AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5'' end, the entire coding region, and 230 nucleotides of the 3''-noncoding region including a poly(A) addition signal (AATAAA) and the first 5 residues of the poly(A) tail.