Sequence Analysis of Frog α-Crystallin cDNA and Its Deduced Primary Structure: Comparison of αA Subunit Chains Among Different Vertebrate Species
1995
Abstract α-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 α-crystallin, total cDNA mixture was constructed from the poly(A) + mRNA of eye lenses from the bullfrog. cDNAs encoding αA and αB chains of α-crystallin were then amplified by polymerase chain reaction (PCR) using primers based on the N- and C-terminal amino-acid sequences of conserved mammalian α-crystallin subunit chains. PCR-amplified product corresponding to αA 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 αA-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 αA shows 95, 82 and 81% sequence identity to αA crystallin chains of another species of Rana frog, bovine and human,respectively, revealing the close relationship among αA crystallins from remotely related species. The present report on the determination of primary structure for frog αA-crystallin chain through cDNA cloning and sequencing has provided the missing N-terminal segment of αA sequence reported for another amphibian species and would represent the first complete frog αA-crystallin sequence solved by PCR technique.
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