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Molecular Cloning of Ovine Connexin44 and Temporal Expression of Gap Junction Proteins in a Lens Cell Culture

From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis.

PURPOSE. The lens plasma membranes of several mammalian species have been shown to contain three different connexin proteins. The goal of this study was to clone the sheep homologue of rat connexin46 identified as sheep connexin44 and to determine the temporal changes in the expression of the three sheep connexin proteins in a lens primary cell culture system.

METHODS. A sheep genomic library was screened with a rat lens connexin46 cDNA probe. Lens junctional protein and mRNA levels were determined in a sheep primary cell culture system by Western and Northern blot analyses, respectively.

RESULTS. Sheep connexin44, the homologue of rat lens connexin46, was identified as a single-copy gene with a predicted molecular weight of 43,989 Daltons that is contained within a single exon. Northern blot analysis detected a 2.2-kb connexin44 transcript in RNA isolated from lens but not that isolated from heart, kidney, liver, or lung. During the in vitro differentiation of lens epithelial cells from 5 to 20 days in culture, connexin43 mRNA levels declined approximately 75%, whereas connexin49 RNA levels increased approximately 24 fold. The 40% decrease in the level of connexin43 protein and the 21-fold increase in the level of connexin49 protein did not directly correlate with the changes in mRNA levels encoding these proteins during this same period. Although detectable, the amount of connexin44 mRNA and protein remained low throughout the 20-day period during which lens cells were grown in culture. Neither mRNA nor protein encoding MP20 or MP26 transcripts could be detected in even the oldest 20-day lens cultures.

CONCLUSIONS. Steady state mRNA levels of sheep connexin43 and connexin49 do not appear to be the only factor regulating the expression of these genes during in vitro differentiation of lens cells in culture. Although a decreased level of expression of connexin43 was accompanied by an increased level of expression of connexin49 over the 20-day period in culture, connexin44 mRNA and protein levels remained low throughout this 20-day period. Overall, these results suggest that these junctional proteins have a unique temporal pattern of expression during differentiation, and this lens primary cell culture system provides a valuable tool to better understand this process.

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