Tsuchiya M, Price S R, Tsai S C, Moss J, Vaughan M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Feb 15;266(5):2772-7.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.
ADP核糖基化因子（ARFs）是分子量约为20 kDa的鸟嘌呤核苷酸结合蛋白，作为霍乱毒素ADP核糖基转移酶活性的GTP依赖性别构激活剂。通过克隆已鉴定出四种哺乳动物ARF，分别称为ARF 1 - 4。在低严谨条件下，用牛ARF 2 cDNA与哺乳动物多聚腺苷酸加尾（poly(A)+）RNA杂交产生多条带，随后使用ARF特异性寡核苷酸探针将这些带分配到已知的ARF基因。然而，一些与cDNA杂交的条带（如3.8和1.3千碱基（kb）的mRNA）的相对信号强度与用单个ARF特异性寡核苷酸探针观察到的强度不一致。这些不一致表明其他ARF样mRNA与已知的ARF mRNA迁移在一起。为了探究这种可能性，使用牛ARF 2 cDNA筛选了环磷酸腺苷（cAMP）分化的HL - 60 λZAP文库。通过将阳性克隆与每种ARF物种特异性的寡核苷酸探针杂交来鉴定与已知ARF基因（1、3和4）对应的克隆；对ARF 2 cDNA阳性、寡核苷酸阴性的克隆进行测序。鉴定出两个新的ARF样基因，ARF 5和ARF 6，它们分别编码180和175个氨基酸的蛋白质。两种蛋白质都含有被认为参与鸟嘌呤核苷酸结合和GTP水解的共有序列。ARF 5在推导的氨基酸序列上与同样有180个氨基酸的ARF 4最相似。ARF 6推导的氨基酸序列除了丝氨酸/苏氨酸替换外与推定的鸡假基因（CPS1）相同，在大小和推导的氨基酸序列上与其他ARF物种不同。对于来自各种组织和培养细胞的哺乳动物poly(A)+ RNA，ARF 5优先与1.3 kb的mRNA杂交，而ARF 6与1.8和4.2 kb的mRNA杂交。这些mRNA的大小与其他ARF（ARF 1，1.9 kb；ARF 2，2.6 kb；ARF 3，约3.8和1.3 kb；ARF 4，l.8 kb）相似这一事实解释了先前观察到的cDNA和ARF特异性寡核苷酸杂交模式之间的不一致。所有六个ARF cDNA彼此之间的相似性高于与其他约20 kDa的鸟嘌呤核苷酸结合蛋白的相似性。
