Facile cloning and sequence analysis of goose delta-crystallin gene based on polymerase chain reaction.
To facilitate the cloning of delta-crystallin gene, the product of which is a major lens protein present in the avian and reptilian lenses, polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of goose lenses. The PCR product was then subcloned into pUC19 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing 1.4 kb DNA inserts encoding delta-crystallin constructed a complete and unambiguous full-length reading frame of 1401 base pairs covering a deduced protein sequence of 465 amino acids excluding the universal translation-initiating methionine. The goose delta-crystallin shows 88, 94, 88 and 69% sequence identity to pigeon delta, duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, similar to duck delta 2 and in contrast to pigeon delta crystallin, goose delta-crystallin appears to possess high argininosuccinate lyase activity despite the fact that a highly homologous structure is shared among these homologous proteins. Structural analysis and comparison of these closely related delta-crystallin homologues with or without enzymatic activity should be of value in unraveling the intriguing evolutionary process leading to the development and evolution of active enzymatic crystallins in the lenses of certain avian species.
Yu
C W
CW
Laboratory of Crystallin Research, National Taiwan University and Institute of Biological Chemistry, Taipei.
Chiou
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001123:Argininosuccinate Lyase / Q000737:chemistry; D001483:Base Sequence; D001717:Birds; D003001:Cloning, Molecular / Q000379:methods*; D003459:Crystallins / Q000737:chemistry / Q000235:genetics*; D004247:DNA; D005777:Geese; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D003459:Crystallins; D004247:DNA; D001123:Argininosuccinate Lyase