Higashibata H, Fujiwara S, Takagi M, Imanaka T
Graduate School of Engineering, Osaka University, 2-1, Suita, 565-0871, Japan.
Biochem Biophys Res Commun. 1999 May 10;258(2):416-24. doi: 10.1006/bbrc.1999.0533.
Two histone genes, hpkA and hpkB, from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain were cloned, sequenced, and expressed in Escherichia coli cells. Both hpkA and hpkB genes encoded a protein of 67 amino acids, however they possessed the different molecular weight (HpkA, 7,378:HpkB, 7,167). Deduced amino acid sequences of HpkA and HpkB were homologous to other archaeal histones and eucaryal core histones (H2A, H4). Gel mobility shift assays by purified proteins demonstrated that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA. HpkB prevented double stranded DNA from thermal denaturation in less amount than HpkA in vitro. In order to investigate intracellular contents of HpkA and HpkB in KOD1 cells, immunoblot analysis was performed by using anti-HpkA antisera obtained from immunized BALB/c mice, showing that HpkA was less abundantly expressed than HpkB in KOD1 cells. These results suggest that HpkB plays a major role to protect double stranded DNA from thermal denaturation in vivo.
从嗜热古菌柯达嗜热栖热菌（Pyrococcus kodakaraensis）KOD1菌株中克隆、测序了两个组蛋白基因hpkA和hpkB，并在大肠杆菌细胞中进行了表达。hpkA和hpkB基因均编码一种由67个氨基酸组成的蛋白质，但它们具有不同的分子量（HpkA为7378，HpkB为7167）。HpkA和HpkB的推导氨基酸序列与其他古菌组蛋白和真核核心组蛋白（H2A、H4）同源。纯化蛋白进行的凝胶迁移率变动分析表明，与HpkA相比，HpkB对DNA具有更高的亲和力和更强的压缩DNA的能力。在体外，HpkB以比HpkA更少的量就能防止双链DNA发生热变性。为了研究KOD1细胞中HpkA和HpkB的细胞内含量，使用从免疫的BALB/c小鼠获得的抗HpkA抗血清进行了免疫印迹分析，结果表明在KOD1细胞中HpkA的表达量低于HpkB。这些结果表明，HpkB在体内保护双链DNA免受热变性方面起主要作用。
