Murtagh J J, Mowatt M R, Lee C M, Lee F J, Mishima K, Nash T E, Moss J, Vaughan M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 May 15;267(14):9654-62.
Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.
蓝氏贾第鞭毛虫是一种具有真核生物和原核生物特征的肠道原生寄生虫。为了确定蓝氏贾第鞭毛虫中是否存在鸟嘌呤核苷酸结合蛋白基因，通过聚合酶链反应以及与编码保守GTP结合结构域序列互补的混合寡核苷酸探针杂交，对基因组DNA文库和cDNA文库进行了筛选。鉴定出一个与哺乳动物ADP核糖基化因子（ARFs）具有高度序列同一性的基因，据信该基因在囊泡运输中起重要作用。蓝氏贾第鞭毛虫ARF基因有一个573个碱基的开放阅读框，编码191个氨基酸，与已知的哺乳动物和酵母ARFs有63%-70%的同一性。ARFs之间的序列保守性在假定的GTP结合结构域中最为显著。在两种不同的蓝氏贾第鞭毛虫分离株中发现了一种约750个碱基的单一ARF mRNA种类。对蓝氏贾第鞭毛虫ARF转录本进行引物延伸和RNA测序，发现其5'非翻译区较短（6个碱基），大小与其他蓝氏贾第鞭毛虫转录本中的相似。蓝氏贾第鞭毛虫提取物含有ARF活性，如霍乱毒素催化的ADP核糖基化受到刺激所示，在大肠杆菌中作为融合蛋白表达的蓝氏贾第鞭毛虫ARF同样表现出生化活性。它在蓝氏贾第鞭毛虫中的存在与以下观点一致：ARF在这种原生动物与其他真核生物分化之前（约15亿年前）就已出现，并且一种类似ARF的蛋白质可能是其他几类信号转导鸟嘌呤核苷酸结合蛋白的祖先，包括异源三聚体G蛋白的α亚基。
