噬菌体T5稳定RNA基因的克隆

Cloning of genes for bacteriophage T5 stable RNAs.

作者信息

Ksenzenko V N, Kamynina T P, Kazantsev S I, Shlyapnikov M G, Kryukov V M, Bayev A A

出版信息

Biochim Biophys Acta. 1982 May 31;697(2):235-42. doi: 10.1016/0167-4781(82)90082-3.

Abstract

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage lambda XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4-5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI 440 fragment contains the gene for tRNA 10 (tRNAAsp), the EcoRI/HindIII 220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNAHis), and the EcoRI/HindIII 960 fragment contains only a part of the gene for tRNA 9 (tRNAGln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp.

摘要

来自T5噬菌体缺失区域的一个EcoRI酶切片段（440个碱基对）和两个EcoRI/HindIII酶切片段（220和960个碱基对）已作为载体插入噬菌体λ XIII和质粒pBR322中。通过在硝酸纤维素滤膜上与体内32P标记的T5 4 - 5 S RNA杂交来研究重组DNA分子。对从滤膜上洗脱的RNA进行二维聚丙烯酰胺凝胶电泳分离和指纹分析，以鉴定由克隆片段编码的RNA。为了准确确定这些RNA的基因定位，用T1和胰RNA酶处理RNA - DNA杂交体，并对抵抗RNA酶作用而稳定的洗脱RNA片段进行电泳。结果表明，EcoRI 440片段包含tRNA 10（tRNAAsp）的基因，EcoRI/HindIII 220片段包含RNA III（107个碱基）的基因以及RNA I（107个碱基）和tRNA 12（tRNAHis）部分基因，EcoRI/HindIII 960片段仅包含tRNA 9（tRNAGln）部分基因。这些基因在T5噬菌体物理图谱上的排列如下：-tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp。