Kitayama S, Kohoroku M, Takagi A, Itoh H
Institute of Physical and Chemical Research, Saitama, Japan.
Mutat Res. 1997 Nov;385(2):151-7. doi: 10.1016/s0921-8777(97)00048-7.
Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D. radiodurans was isolated. Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained. After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar). The putative protein has a conserved ATP binding domain characteristic of DNA helicases. However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E. coli homologue. The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents.
用一种限制性内切酶消化耐辐射球菌的染色体DNA后，以λ噬菌体为载体构建了一个部分基因组文库。分离出一个噬菌体克隆，其DNA能够弥补耐辐射球菌辐射敏感突变体的缺陷。使用噬菌粒载体进行亚克隆后，获得了一个含有1.2 kb插入DNA的杂交质粒。测定核苷酸序列后，推导的氨基酸序列与大肠杆菌的RuvB蛋白显示出密切的同源性；约81%的氨基酸（总共310个残基）是同源的（152个相同，100个氨基酸相似）。推测的蛋白质具有DNA解旋酶特有的保守ATP结合结构域。然而，与大肠杆菌同源物相比，在ruvB上游序列中我们未发现RuvA蛋白的SOS启动子和开放阅读框。该突变体用外源DNA转化的速率与野生型细胞相同，但它对紫外线、γ射线和链间交联试剂中度敏感。
