ABSTRACT
Zeta-Crystallin, a major component of the guinea-pig lens proteins, is distantly related to the enzymes of the zinc-containing alcohol dehydrogenase family (ADH). Analysis of the structural similarities between zeta-crystallin and ADH reveals that while characteristics important in maintaining the tertiary structure of the molecule appear conserved, the amino acids binding the catalytic zinc atom are absent in zeta-crystallin. Significantly, zeta-crystallin does not have ADH activity. Previous studies showed that the zeta-crystallin protein is modified in the lens of guinea-pigs affected with an autosomal dominant hereditary cataract. We have further investigated the molecular origin of the lens defect by examining the steady-state levels of zeta-crystallin transcripts in normal and mutant eyes. Our data indicate that no normal zeta-crystallin mRNA is present in the lens of the homozygous animals; instead, a cross-hybridizing lower molecular weight mRNA is detected at significantly reduced concentrations. Heterozygous lenses exhibit both mRNA species.
Subject(s)
Cataract/genetics , Crystallins/genetics , Transcription, Genetic , Amino Acid Sequence , Animals , Cataract/metabolism , Guinea Pigs , Molecular Sequence Data , Protein Conformation , RNA/analysisABSTRACT
zeta-Crystallin is a major component of the water-soluble proteins of the guinea pig lens. We have constructed a lens cDNA library from one- to seven-day-old guinea pigs in the plasmid Bluescript KS+ and used the 16 amino acid (aa) sequence of a CNBr peptide to design an oligodeoxyribonucleotide probe. Analysis of two positive clones and direct sequence of the 5' end of the RNA resulted in the completion of a most probably full-length mRNA comprising 1842 nucleotides (nt). The ATG start codon occurs 83 nt downstream from the 5' end. The open reading frame, ending with a stop codon at nt position 1070, predicts a protein of 328 aa with a calculated Mr of 35,071. Comparison of the amino acid sequence with the National Biomedical Research Foundation protein data base reveals a significant similarity of zeta-crystallin with the enzyme of the alcohol dehydrogenase family.
Subject(s)
Alcohol Dehydrogenase/genetics , Crystallins/genetics , Lens, Crystalline/metabolism , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Codon , Crystallins/biosynthesis , DNA/genetics , Electronic Data Processing , Gene Expression Regulation , Guinea Pigs , Molecular Sequence Data , RNA, Messenger/biosynthesisABSTRACT
The primary structure of zeta-crystallin, a guinea pig lens specific protein, was obtained by cloning the copy of its mRNA from a 0-1 week old cDNA lens library. The protein is 328 amino acids long and 34% of its secondary structure appears in alpha-helical conformation. Comparison of the zeta-crystallin sequence with the sequences of the protein data base bank, revealed the similarity of this lens protein to the enzymes of the long-chain zinc-containing alcohol dehydrogenase family.
