Investigation of the molecular mechanisms of hereditary cataracts using as a model the nuclear hereditary cataract of strain 13/N guinea pigs continues. This year, the focus was on the further characterization of the zeta-crystallin-cDNA present in the lens of the cataractous animal. We developed a new method to construct cDNA libraries from small amounts of tissue and obtained a cDNA library from four heterozygous guinea pig lenses. Screening of this library produced normal and defective zeta-cDNAs. Sequencing of the cataractous zeta-cDNA revealed a 102 bp deletion corresponding exactly to 34 amino acids. The deletion, which does not interfere with the reading frame, is located toward the carboxy end of the protein. Analysis of the genomic DNA from normal and affected animals indicated that the deleted mRNA region is present in the genome of the cataractous animals. This result suggests that a mutation affecting the splicing mechanism is possibly the cause of the mRNA deletion. In studies of the role of the zeta mRNA in the liver of guinea pigs, we isolated four clones from an adult liver cDNA library. Upon sequencing, two of the clones showed the poly A tail at different positions in the mRNA, confirming different processing of the zeta-crystallin gene in the liver versus the lens. Sequencing the coding region revealed that the amino acids of the liver protein are identical to those of the lens protein. Having successfully cloned the complete zeta-crystallin gene, we are now analyzing it. Thus far, we have characterized six introns and a very long exon comprising the untranslated 3'end of the mRNA. Sequencing one of the clones has revealed the presence of one exon that corresponds exactly to the deleted region of the zeta mRNA of the cataractous animals.
NIH ExPorter
NIH ExPorter