鸡胚DNA聚合酶β信使核糖核酸及其体外翻译产物的特性研究

Characterization of messenger RNA for chick-embryo DNA polymerase beta and its translation product in vitro.

作者信息

Yamaguchi M, Takahashi T, Yasuda K, Shimura Y, Matsukage A

出版信息

Eur J Biochem. 1983 Jun 15;133(2):277-82. doi: 10.1111/j.1432-1033.1983.tb07459.x.

DOI:10.1111/j.1432-1033.1983.tb07459.x

PMID:6852040

Abstract

A specific immunoprecipitation method, using rabbit anti-(chick DNA polymerase beta) IgG was applied to detect the polypeptide of DNA polymerase beta among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr = 40 000 was specifically immunoprecipitated from [35S]methionine-labeled translation products and was competitive with the purified DNA polymerase beta for the antibody. Furthermore, the 40 000-Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase beta was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)-rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.

摘要

采用一种特定的免疫沉淀方法，即使用兔抗（鸡DNA聚合酶β）IgG，来检测从鸡胚中提取的mRNA在体外翻译产物中DNA聚合酶β的多肽。从[35S]甲硫氨酸标记的翻译产物中特异性免疫沉淀出一条分子量为40000的多肽，它与纯化的DNA聚合酶β竞争抗体。此外，体外获得的分子量为40000的翻译产物具有DNA聚合酶活性，这可通过在含DNA的聚丙烯酰胺凝胶中电泳后原位检测来确定。DNA聚合酶β的mRNA是聚腺苷酸化的，根据体外翻译中[35S]甲硫氨酸的掺入情况，其含量估计为富含多聚（A）的总RNA的0.001%。在变性条件下通过区带沉降和琼脂糖凝胶电泳确定该mRNA的大小约为1800个核苷酸。