Nishio Megumi, Seki Yuichi, Ikeda Shogo
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan.
DNA Seq. 2006 Aug;17(4):287-91. doi: 10.1080/10425170600857608.
We studied the genomic structure and biochemical properties of Schizosaccharomyces pombe mitochondrial single-stranded DNA binding protein (mtSSB). We first determined the full-length cDNA sequence of mtSSB and clarified the exon/intron structure of the mtSSB gene (rim1), including the transcription initiation and polyadenylation sites. The cDNA of rim1 gene encoded 150 amino acids and the sequence showed homology to eukaryotic mtSSB and Escherichia coli SSB. We overexpressed mtSSB as a His-tag fusion protein in E. coli and obtained an anti-mtSSB antibody. Gel filtration analysis of S. pombe cell extracts clarified that mtSSB has a tetrameric structure. We also immunochemically detected mtSSB in S. pombe cell extract and showed that 15,000 molecules of mtSSB tetramer are present in a single S. pombe cell. Mature mtSSB lacking the presequence was overexpressed in E. coli in tetrameric soluble form. The recombinant mtSSB bound a single-stranded oligonucleotide and phiX174 virion DNA with almost identical binding activity as E. coli SSB.
我们研究了粟酒裂殖酵母线粒体单链DNA结合蛋白（mtSSB）的基因组结构和生化特性。我们首先确定了mtSSB的全长cDNA序列，并阐明了mtSSB基因（rim1）的外显子/内含子结构，包括转录起始位点和聚腺苷酸化位点。rim1基因的cDNA编码150个氨基酸，其序列与真核生物mtSSB和大肠杆菌SSB具有同源性。我们在大肠杆菌中以His标签融合蛋白的形式过表达mtSSB，并获得了抗mtSSB抗体。对粟酒裂殖酵母细胞提取物的凝胶过滤分析表明，mtSSB具有四聚体结构。我们还通过免疫化学方法在粟酒裂殖酵母细胞提取物中检测到了mtSSB，并表明单个粟酒裂殖酵母细胞中存在15,000个mtSSB四聚体分子。缺少前导序列的成熟mtSSB在大肠杆菌中以四聚体可溶形式过表达。重组mtSSB与单链寡核苷酸和phiX174病毒粒子DNA结合，其结合活性与大肠杆菌SSB几乎相同。
