Molecular cloning of porcine soluble angiotensin-binding protein.
Soluble angiotensin-binding protein (sABP) is a 75-kDa cytosolic protein that binds angiotensins and its analogues with high affinity. In this study, the primary structure of porcine sABP is determined by cDNA cloning. Based on the partial amino acid sequences of sABP tryptic fragments, fully degenerate oligonucleotides were synthesized, and used as primers for polymerase chain reactions to amplify the corresponding sABP cDNA fragment from porcine liver first-strand cDNA. By using initially the polymerase chain reaction product and later partial cDNA clones as probes, porcine heart and liver cDNA libraries were screened, and several positive clones were obtained including one covering the entire coding region. From the cDNA sequence, an open reading frame that encodes sABP as a 704-amino acid protein with molecular mass of 80,800 daltons is predicted. No significant homology was seen between sABP and other proteins in GenBank and NBRF data bases, including the angiotensin-related proteins such as angiotensin converting enzyme, renin, and AT1 angiotensin II receptor. Northern blot analysis of poly(A)+ RNA revealed that the mRNA for sABP is expressed as 5.3- and 2.8-3.2-kilobase transcripts. These transcripts are generated by the use of alternative polyadenylation signals. Within the 3'-untranslated region of the cDNA sequence downstream from the polyadenylation signals for smaller transcripts, a porcine short interspersed repetitive element (SINE) was found; only the longer 5.3-kilobase transcript had the SINE sequence.
Sugiura
N
N
Department of Biological Sciences, Tokyo Institute of Technology, Japan.
Hagiwara
H
H
Hirose
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D013552:Swine
D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C076530:ABP1 protein, Sus scrofa; D004247:DNA