Biochemical and molecular biological studies on DNA polymerase of Toxoplasma gondii

对弓形虫RH强毒株速殖子粗提物中的DNA聚合酶活性进行了检测和鉴定。该酶的沉降系数为6.4 S，对应于大约150，000的分子量。与哺乳动物α型DNA聚合酶一样，弓形虫酶对N-乙基马来酰亚胺敏感，并被高离子强度抑制。然而，该酶活性不被作为哺乳动物DNA聚合酶α、δ和γ的抑制剂的阿非迪霉素抑制，并且该活性被作为哺乳动物DNA聚合酶β和γ的抑制剂的ddTTP抑制。Mg^<2+>是酶活性的必需物质，高浓度的K^+明显抑制酶活性。抗人DNA聚合酶α的单克隆抗体不与弓形虫酶结合。因此，弓形虫酶不同于人类酶，可能是设计杀弓形虫药物的有用靶点。RH强毒株的酶活显著高于ME 49弱毒株，说明RH强毒株酶活的提高，使其作为一种生物媒介，比ME 49弱毒株增殖快。为了克隆弓形虫DNA聚合酶α基因，我们合成了一个包含保守碱基的寡核苷酸探针，并将其用于筛选λ GEM中的弓形虫基因组DNA文库，获得了一个阳性克隆。分析插入DNA，发现大小为15 kb，然后亚克隆到pUC 18中进行测序。为克隆弓形虫DNA聚合酶δ基因，采用RT-PCR方法扩增了弓形虫DNA聚合酶δ基因的cDNA片段，并进行了序列测定，证实为弓形虫的真实的基因。用该cDNA片段筛选λ gt 10的弓形虫cDNA文库，鉴定出两个克隆，将其插入片段克隆到pBluescript中进行测序。

A DNA polymerase activity has been detected and characterized in crude extracts from tachyzoites of virulent RH strain of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6.4 S,corresponding to an approximate molecular weight of 150,000. Like mammalian alpha-type DNA polymerases, the T.gondii enzyme was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerase alpha, delta and epsilon, ant the activity was inhibited by ddTTP which is an inhibitor of mammalian DNA polymerase beta and gamma. Mg^<2+> was required for activity and a higher concentration of K^+ markedly inhibited the activity. Monoclonal antibodies against human DNA polymerase alpha did not bind to the T.gondii enzyme. Thus the T.gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs. The activity of virulent RH strain was siginificantly higher than that of avirulent ME49 strain, suggesting that this increased activity of the enzyme of virulent strain contributes to a faster rate of multiplication of the organisms as aompared with that of avirulent strain. For cloning of DNA polymerase alpha gene of T.gondii, an oligonucleotide probe which included the conserved bases was synthesized and used for screening a T.gondii genomic DNA library in lambda GEM.One positive clone was obtained. The insert DNA was analyzed, found to be 15 kb in size, and then subcloned into pUC18 for sequencing. For cloning of DNA polymerase delta gene of T.gondii, a cDNA fragment was produced by RT-PCR and then sequenced and confirmed as the real gene. A T.gondii cDNA library in lambda gt 10 was screened using this cDNA fragment and two slones were identified and their insert DNAs were subcloned into pBluescript for sequencing.