Studies On The Messenger-rnas For Gamma Crystallins In The Rat Lens

The purpose of this study was to gain an understanding of the expression of the gamma crystallin proteins in the rat lens during the course of lens differentiation and development.;In vitro translation results showed that at least 20% of the mRNA extracted from the neonatal lens and that at least two transcripts were coding for gamma crystallin proteins.;Several gamma crystallin cDNA clones were isolated. Nucleotide sequencing information was obtained from two of the cDNA clones (A12 and G5). The clones A12 and G5 correspond exactly in amino acid sequence with the genes gamma (4-1) and gamma (1-1) (genomic clone designation Shoenmakers et al., 1984). Previously only gamma (2-1) and gamma (3-1) had been shown to be expressed in the lens. Thus expression of four of the six gamma crystallin genes of the rat has now been demonstrated conclusively by this study and that of Moorman et al. (1982).;Two other cDNA clones (D11 and D12) prepared in this study seem to be derived from transcript(s) distinct from either G5 or A12 and as yet have not been assigned.;Plasmid clones A12, G5, D11 and D12 were also hybridized to dot-blots of rat lens RNA, and were then melted in a controlled manner. Clone A12 appeared to be the most abundant (35%) of the gamma crystallins when measured by this method. Clone G5 was abundant (20%) as well. Clones D11 and D12 had similar melting profiles, but little of the gamma crystallin mRNA pool was made up of the transcript(s) from which these clones were derived.;In situ hybridizations using A12 and G5 as probes were carried out on 10 um sections of 17 day embryonic and 2 day newborn rat eyes. Specific hybridization could be observed in the lens fibre cells using both clones. Only background levels of hybridization were observed in lens epithelial cells and surrounding tissues and humours. A gradient of hybridization occurred in embryonic lenses increasing from the equator inward and following the progression of fibre cell elongation. There was some indication in newborns that a zone of cortical fibre cells contained the highest levels of gamma crystallin mRNA. (Abstract shortened with permission of author.