Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells.
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.
Tsuchiya
M
M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Price
S R
SR
Tsai
S C
SC
Moss
J
J
Vaughan
M
M
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002645:Chickens; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D008322:Mammals; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011544:Pseudogenes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D000089982:ADP-Ribosylation Factor 6; D015342:DNA Probes; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat