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N Golestaneh, S Kumar, J Fan, AB Chepelinsky; Transcription Regulatory Elements of the Lens MIP/aquaporin 0 Gene . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4642.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lens Major Intrinsic Protein (MIP)/Aquaporin 0 is specifically expressed in the lens fibers, functions as a water channel and plays an important role in lens transparency. We have cloned the lens MIP gene from human and mouse to characterize the regulatory elements and transcription factors that regulate specific expression of this gene in the lens. Methods: Murine MIP gene 5'-flanking promoter sequences with or without MIP intronic sequences were inserted in a luciferase reporter vector. New born rat lens explants cultured with bFGF, with or without insulin and/or IGF-1 were transfected with various MIP promoter reporter constructs. The promoter activities of these constructs were subsequently compared. Endogenous MIP transcription in cultured lens explants was determined by RT-PCR. Results: Alignment of the human and mouse MIP gene revealed several highly conserved domains in their 5'-flanking, introns one, two, three and 3'-non-coding regions. Based on these observations, we inserted intronic sequences evolutionarily conserved at the 3'-end of the luciferase reporter gene to map regulatory elements present in the MIP gene intronic regions that may modulate MIP expression. We found experimental conditions to achieve MIP transcription in rat explanted lens epithelia cultured under differentiating conditions in the presence of bFGF, IGF-1 and insulin. We were able to transfect and determine MIP promoter activity in differentiating lens epithelia explants. Genomic DNA sequence analysis showed that the timeless gene exon 1 is located proximal to the MIP gene 3'-end. Therefore, the MIP gene 3'-non coding and/or 3'-flanking domains may also contain promoter/regulatory elements of the timeless gene. Conclusion: We have characterized several regulatory elements of the MIP gene promoter in the 5'-flanking region of the gene. The characterization of additional regulatory elements in intronic regions will allow us to understand the tight regulation of the MIP gene. Additionally, the close proximity of the MIP and timeless genes presents the challenge to dissect the regulatory elements of two genes in tandem with completely different spatial and temporal regulation, one lens specific, the other one expressed in many tissues. One involved in lens transparency, the other one involved in circadian rhythms.
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