Characterization of the gene for ADP-ribosylation factor (ARF) 2, a developmentally regulated, selectively expressed member of the ARF family of approximately 20-kDa guanine nucleotide-binding proteins.
ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking in vivo. The six cloned mammalian ARFs can be grouped into three classes based on size and sequence identity. ARF 2 is a class I ARF, whose approximately 2.6-kilobase mRNA exhibits species and tissue selective expression and is developmentally regulated in rat brain. Here we report the sequence, structure, and functional promoter region of the bovine ARF 2 gene, which was facilitated by constructing a composite cDNA. The ARF 2 cDNA, constructed from a partial cDNA clone and polymerase chain reaction-amplified fragments from reverse-transcribed poly(A)+ RNA, was approximately 2270 base pairs (bp) (minus the poly(A) tail). In the 3'-untranslated region, there are two potential polyadenylation signals, ATTAAA and AATAAA, at positions 1064 and 2232, respectively, and two ATTTA motifs, believed to signal mRNA degradation, at positions 2115 and 2165. The ARF 2 gene, represented in three overlapping genomic clones, spans approximately 20 kilobase pairs with five exons and four introns. Consensus sequences for guanine nucleotide-binding and GTP hydrolysis are in separate exons, except for the NKXD sequence, which is divided by intron 4. There are multiple transcriptional initiation sites. Transient transfection of embryonic trachea cells with deletion constructs defined the functional promoter region to be within 400 bp upstream of the most 5' site of transcription initiation. This 400-bp region lacks a TATA-like sequence but contains six inverted CCAAT boxes, four potential Sp1-binding sites, and a potential AP-2-binding site. Although the pattern of expression of ARF 2 is unique among the ARFs, the structures of the class I ARF genes are conserved among its members and across species.
Serventi
I M
IM
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Cavanaugh
E
E
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA; D005314:Embryonic and Fetal Development; D019204:GTP-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D014132:Trachea / Q000166:cytology / Q000196:embryology
D004247:DNA; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors