Sequence-Analysis of Frog αB-Crystallin cDNA: Sequence Homology and Evolutionary Comparison of αA, αB and Heat Shock Proteins

Abstract

α-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine α-crystallins possess genuine chaperone activity similar to small heat-shock proteins. In order to facilitate the determination of the primary sequence of amphibian αB-crystallin, cDNA encoding αB subunit chain was amplified using a new "Rapid Amplification of cDNA Ends" (RACE) protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to αB subunit was then subcloned into pUC18 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the automatic fluorescence-based dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts coding for αB-crystallin subunit constructed only one complete full-length reading frame of 522 base pairs similar to that of αA subunit, covering a deduced protein sequence of 173 amino acids including the universal translation-initiating methionine. The frog αB crystallin shows 69, 66 and 56% whereas αA crystallin shows 83, 81 and 69% sequence similarity to the homologous chains of bovine, chicken and dogfish, respectively, revealing a more divergent structural relationship among these αB subunits as compared to αA subunits. Structural analysis and comparison of αA- and αB-crystallin subunits from eye lenses of different classes of vertebrates also shed some light on the evolutionary relatedness between αB/αA crystallins and the small heat-shock proteins.