Characterization of gamma-crystallin from the eye lens of bullfrog: Complexity of gamma-crystallin multigene family as revealed by sequence comparison among different amphibian species

gamma-Crystallin is the major and most abundant lens protein present in the eye lens of lower vertebrates such as amphibian and piscine species. To facilitate structural characterization of gamma-crystallins isolated from the lens of the bullfrog (Rana catesbeiana), a cDNA mixture was synthesized from the poly(A)(+) mRNA isolated from fresh eye lenses. cDNA encoding gamma-crystallin was then amplified using polymerase chain reaction (PCR) based on two primers designed according to the relatively conserved N- and C-terminal sequences of known gamma-crystallins from teleostean fishes, PCR-amplified product corresponding to gamma-crystallin isoforms was obtained, which was then subcloned in pUC18 vector and transformed into Escherichia coli strain JM109, Plasmids containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method, Sequencing several clones containing DNA inserts of about 0.54 kb revealed the presence of two isoforms with an open reading frame of 534 base pairs, covering two gamma-crystallins each with a deduced protein sequence of 177 amino acids including the translation-initiating methionine, These gamma-crystallins of pI 6.364 and 6.366 contain a low-methionine content of 2.81%, in contrast to 11-16% obtained for those gamma-crystallins with high-methionine content from most teleostean lenses, Pairwise sequence comparison of bullfrog gamma-crystallins with those published sequences of gamma-crystallins from carp, shark, Xenopus and another Rana frog, bovine, and human lenses indicates that there is only 46-63% sequence similarity among these species, revealing that amphibians possess a very complex and heterogeneous group of gamma-crystallins even from closely related species of Rana frogs, The sequence analysis and comparison of various isoforms of the frog gamma-crystallin family provide a firm basis for identifying these lens proteins as members of a multigene family more complex than that reported for mammalian gamma-crystallins.