Tsai S C, Haun R S, Tsuchiya M, 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 Dec 5;266(34):23053-9.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. Five different human ARFs have been identified by cDNA cloning. Northern analysis using ARF 3-specific oligonucleotides identified two mRNAs of 3.7 and 1.2 kilobases (kb). We report here the complete nucleotide sequence of the 3.7-kb ARF 3 mRNA derived from three overlapping cDNAs isolated from human hippocampus and fetal brain cDNA libraries, as well as the structure of human ARF 3 gene. Sequences of two overlapping genomic clones indicated that the ARF 3 gene spans approximately 18.3 kb and contains five exons and four introns. The conserved amino acid sequences involved in guanine nucleotide binding by ARF 3 are distributed among separate exons, as found in other GTP-binding protein genes. Translation initiates in exon 2 which includes the sequence GXXXXGK that probably participates in phosphate binding and GTP hydrolysis. The sequence DVGG in exon 3 coordinates binding of Mg2+ and the beta-phosphate of GDP. In the ARF 3 gene in contrast to those of other GTP-binding proteins, the sequence NKXD (which is thought to contribute to the specificity of interaction with the guanine ring) is divided between exons 4 and 5. The latter encodes the COOH-terminal 53 amino acids of ARF 3 and contains greater than 2500 base pairs of untranslated DNA. The sequence AATTAA is 19 bases 5' to the polyadenylation addition site of the 3.7-kb mRNA. Multiple transcription start sites were identified by primer extension and S1 and mung bean nuclease analyses. The 5'-flanking region of exon 1 contains neither a TATA nor a CAAT box, but is high in GC content (greater than 70%) and includes three potential Sp1-binding sites (GC box), consistent with the promoters described for several housekeeping genes. The 1.2-kb ARF 3 mRNA is shown to arise by use of an alternative polyadenylation signal (AACAAA) at nucleotide 1091 within the ARF 3 cDNA.
ADP核糖基化因子（ARFs）是一类分子量约为20 kDa的鸟嘌呤核苷酸结合蛋白，在体外可刺激霍乱毒素的ADP核糖基转移酶活性。通过cDNA克隆已鉴定出五种不同的人类ARFs。使用ARF 3特异性寡核苷酸进行的Northern分析鉴定出两条分别为3.7和1.2千碱基（kb）的mRNA。我们在此报告从人海马体和胎儿脑cDNA文库中分离出的三个重叠cDNA所得到的3.7 kb ARF 3 mRNA的完整核苷酸序列，以及人类ARF 3基因的结构。两个重叠基因组克隆的序列表明，ARF 3基因跨度约为18.3 kb，包含五个外显子和四个内含子。与其他GTP结合蛋白基因一样，ARF 3中参与鸟嘌呤核苷酸结合的保守氨基酸序列分布在不同的外显子中。翻译起始于外显子2，其中包含可能参与磷酸结合和GTP水解的序列GXXXXGK。外显子3中的序列DVGG协调Mg2+与GDP的β-磷酸的结合。与其他GTP结合蛋白的基因不同，在ARF 3基因中，序列NKXD（被认为有助于与鸟嘌呤环相互作用的特异性）在外显子4和5之间分开。后者编码ARF 3的COOH末端53个氨基酸，并包含超过2500个碱基对的非翻译DNA。序列AATTAA位于3.7 kb mRNA的多聚腺苷酸化添加位点上游19个碱基处。通过引物延伸以及S1和绿豆核酸酶分析鉴定出多个转录起始位点。外显子1的5'侧翼区域既没有TATA盒也没有CAAT盒，但GC含量很高（大于70%），并包括三个潜在的Sp1结合位点（GC盒），这与几个管家基因的启动子情况一致。1.2 kb的ARF 3 mRNA显示是通过使用ARF 3 cDNA内第1091位核苷酸处的一个替代多聚腺苷酸化信号（AACAAA）产生的。
