Cataracts of varied etiologis, including heriditary cataracts, are associated with altered levels of gamma-crystallins. We now have succeeded in cloning different gamma-crystallin cDNA sequences and we have determined the complete nucleotide sequence of a bovine gamma-crystallin mRNA. Gamma-crystallins are characteristically synthesised in the terminally differentiating lens fibre cells and therefore, can be used as convenient markers of differential gene expression. The present study will attempt to elucidate the molecular genetic mechanisms involved in the development and regulation of the spatially and temporally controlled heterogeneity of gamma-crystallins in the calf and the human lens, during normal differentiation and diseased states. Toward this goal, using the cloned probes already available in our laboratory, we will isolate the major human gamma-crystallin gene(s) and characterize them by complete sequence analyses. Using recombinant DNA techniques, DNA sequences will be deleted from or added to the isolated human and/or bovine gene(s). The expression of these genes will be monitored in cell-free systems and in vivo, by microinjecting them into the nuclei of the lesn epithelial cells, maintained in primary cultures (gamma-crystallins are not synthesised in these cells). Transcriptional regulation in the differentiated lens fibres and epithelial cells will be investigated both in vitro and in vivo by studying the processing of the nuclear transcripts of the gamma-crystallin mRNA. Utilizing restriction enzymes and specific human and bovine cloned DNA probes, we will attempt to map sequence polymorphisms, mutations or defects in the gamma-crystallin gene sequences of the individuals suspected of having cataracts of genetic origin. This study will investigate the possible involvement of the regulation of gene expression in the maintenance of ocular transparency and in the development of cataractous lenses.
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