ISSN:
1573-4943
Keywords:
γ-Crystallin
;
amphibian lens
;
polymerase chain reaction (PCR)
;
sequence comparison
;
multigene family
;
phylogenetic tree
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract γ-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 ofγ-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 encodingγ-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 knownγ-crystallins from teleostean fishes. PCR-amplified product corresponding toγ-crystallin isoforms was obtained, which was then subcloned in pUC18 vector and transformed intoEscherichia coli strain JM109. Plasmids containing amplifiedγ-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 twoγ-crystallins each with a deduced protein sequence of 177 amino acids including the translation-initiating methionine. Theseγ-crystallins of pI 6.364 and 6.366 contain a low-methionine content of 2.81%, in contrast to 11–16% obtained for thoseγ-crystallins with high-methionine content from most teleostean lenses. Pairwise sequence comparison of bullfrogγ-crystallins with those published sequences ofγ-crystallins from carp, shark,Xenopus and anotherRana 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 ofγ-crystallins even from closely related species ofRana frogs. The sequence analysis and comparison of various isoforms of the frogγ-crystallin family provide a firm basis for identifying these lens proteins as members of a multigene family more complex than that reported for mammalianγ-crystallins.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01886816
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