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J.S. Friedman, H. Cheng, R. De Nicola, K.P. Mitton, Y. Jiang, D. Hicks, A. Swaroop; Elucidation of the Minimal Transactivation Domain of NRL, the bZIP Transcription Factor Essential for Rod Differentiation and Function . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3533.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To determine the minimal peptide domain required for transactivation of promoters by the rod-specific bZIP transcription factor NRL. Methods:A series of five progressive N-terminal deletions and six internal fragments of NRL were cloned into the yeast two-hybrid vector pHybLexZeo (Invitrogen) in frame with the LexA-DNA binding domain. Full length NRL and DD10, a naturally occurring dominant negative deletion of NRL, were used as positive and negative controls respectively. Identical NRL fragments were also cloned into mammalian expression vector pCDNA4c (Invitrogen) in frame with the His and Xpress epitope-tags. Yeast strain L40 were transformed with the pHybLexZeo constructs and the transformants were grown under selective conditions (minus-His, +Zeo, +50mM 3-AT, +X-gal). NRL constructs in pCDNA4c are being used in transfection experiments of mammalian 293 cells and examined through luciferase assays. Results:Experiments with NRL deletion constructs demonstrated that the deletion of NRL residues 1 to 30 did not affect the transactivation of His and lacZ reporter genes in yeast assays. Subsequent deletions after NRL residue 75 showed no transactivation ability when grown under selection on X-gal containing media. The smallest internal NRL fragment containing transactivation function comprised of amino acids 30 to 93. NRL residues 1 to 57 were unable to transactivate in the yeast assay. Conclusions:These results suggest that NRL’s minimal transactivation domain is likely to encompass amino acids 30 to 93. The smallest minimal domain is potentially from residues 58 to 75. Determination of NRL’s minimal domain can provide a better understanding of how NRL regulates its downstream target genes in rod photoreceptors and in designing novel transcription factors with specific properties.
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