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N. Dobri, K. Y. Yasumura, M. V. R. Chavali, B. Chang, R. Ayyagari; Localization and Functional Characterization of Mdm1 Retinal Transcript Associated With Age-Related Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3438.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the potential role of Mdm1 in the retina and the mechanism underlying age related retinal degeneration (arrd2) in a mouse model due to a nonsense mutations in the mouse double minute gene 1 (Mdm1).We present the characterization of the mouse Mdm1 retinal protein (mMdm1RT), its localization in cells and in retinal tissue. Since other double minute proteins Mdm2 and Mdm4 have been identified as p53 interacting proteins, we tested the potential interaction of Mdm1 with p53.
Bioinformatic analyses were carried out using COIL for alpha helices and coiled coil structure; PSORT for topology prediction and CLUSTAL W for sequence comparison. Constructs were generated to express V5 tagged-Mdm1 or eGFP-Mdm1 in transiently transfected NIH3T3 cells. The expression and cellular localization were tested by western blot and immunocytochemistry. Antibodies specific to the mMdm1RT were generated and used to determine the cellular and tissue localization by immunohistochemistry. Interaction of Mdm1 with p53 was studied by co-immunoprecipitation (co-IP) and western blot analysis. Interaction of Mdm1 with DNA was tested by chromatin immunoprecipitation (ChIP) and ChIP on Chip assay.
Bioinformatic analysis revealed the presence of coiled coil structure and two sequence repeate motifs with unknown function. The Mdm1RT has no significant sequence homology with Mdm2 and Mdm4 or any other known proteins in the database. In transiently transfected cells, Mdm1RT is localized predominantly to the nucleus. In the mouse retina, Mdm1RT is highly expressed in all the nuclear layers. Co-IP experiments showed no interaction between Mdm1RT and p53 unlike Mdm2 and Mdm4. Chromatin immunoprecipitation and ChIP on chip assays established interaction of Mdm1RT with several regions in the genome. ChIP assay showed no interaction between Mdm1 and retina specific transcription factors Crx and Nrl.
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