Hoffman D C, Prescott D M
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Campus Box 347, Boulder, CO 80309-0347, USA.
J Mol Evol. 1997 Sep;45(3):301-10. doi: 10.1007/pl00006234.
The complete macronuclear DNA polymerase alpha gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase alpha encoding approximately 1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase alpha amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase alpha amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase alpha sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol alpha biochemistry at the molecular level.
完整的大核DNA聚合酶α基因，此前已在新奥克特里查中测序，现从基因组大核文库中克隆出来，并对低等毛滴虫三裂奥克特里查进行了测序。通过PCR获得了编码约1000个氨基酸（约为开放阅读框的三分之二）的DNA聚合酶α的大核DNA克隆，并对大弹跳虫、全列虫属、绿色副侧毛虫、披针侧毛虫、泰勒豆形伪角毛虫、贻贝伪角毛虫、鸡尿壶菌和大尾柱虫进行了测序。从DNA聚合酶α氨基酸序列推断出的系统发育关系已被用于阐明先前由细胞皮层形态确定的分类关系。基于DNA聚合酶α氨基酸序列的低等毛滴虫系统发育与形态学和其他分子系统发育不一致。基于这些数据，我们断言，与形态学数据相反，新奥克特里查和三裂奥克特里查是不同的物种，并且我们建议将寡毛类的大弹跳虫重新分类为低等毛滴虫。这项工作还扩展了真核生物DNA聚合酶α序列的可用数据库，并为诱变实验提出了新的氨基酸序列靶点，以便在分子水平上继续对DNA pol α生物化学进行功能剖析。
