Sequence analysis of frog alpha-crystallin cDNA and its deduced primary structure: comparison of alpha A subunit chains among different vertebrate species.
alpha-Crystallin which is present in the lenses of all vertebrate species is a major lens protein with blocked amino terminus. To facilitate the cloning and sequence determination of amphibian alpha-crystallin, total cDNA mixture was constructed from the poly(A)+mRNA of eye lenses from the bullfrog. cDNAs encoding alpha A and alpha B chains of alpha-crystallin were then amplified by polymerase chain reaction (PCR) using primers based on the N- and C-terminal amino-acid sequences of conserved mammalian alpha-crystallin subunit chains. PCR-amplified product corresponding to alpha A crystallin subunit was obtained, which was then subcloned into pUC18 vector and then transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing DNA inserts coding for alpha A-crystallin constructed a complete full-length reading frame of 522 base pairs covering a deduced protein sequence of 173 amino acids including the universal translation-initiating methionine. The frog alpha A shows 95, 82 and 81% sequence identity to alpha A crystallin chains of another species of Rana frog, bovine and human, respectively, revealing the close relationship among alpha A crystallins from remotely related species. The present report on the determination of primary structure for frog alpha A-crystalline chain through cDNA cloning and sequencing has provided the missing N-terminal segment of alpha A sequence reported for another amphibian species and would represent the first complete frog alpha A-crystallin sequence solved by PCR technique.
Lu
S F
SF
Laboratory of Crystallin Research, National Taiwan University, Taipei.
Pan
F M
FM
Chiou
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002645:Chickens; D003459:Crystallins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D011892:Rana catesbeiana / Q000235:genetics*; D011898:Ranidae; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014714:Vertebrates / Q000235:genetics*
D003459:Crystallins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins