Abstract
Within the retina there is a circadian clock that controls the 24-h timing of processes such as hormone release, cell movement, and gene transcription. In an effort to better understand the molecular nature of this retinal clock, a differential display (DD) screen was performed to isolate a gene with high amplitude circadian rhythmicity in the Xenopus retina. A novel gene expressed in the early evening in photoreceptor cells was isolated and named nocturnin for night factor. This article outlines the steps we took to study a protein of unknown function, particularly highlighting the analyses one can perform when little more than the primary sequence of a gene is known. In addition, we describe the results of sequence analysis that assisted in predicting the function of nocturnin. We have shown that nocturnin acts as a deadenylase in vitro, removing the poly(A) tail from a mature messenger RNA in a process that either leads to degradation or translational silencing of a message. Although the role of nocturnin in the retina is unknown, future studies to identify target mRNAs that are deadenylated by nocturnin will assist in elucidating its physiological role in this tissue.
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Acknowledgments
A portion of the original data presented in this chapter was generated by Xiaorong Liu, and we thank her for her major contributions toward the current understanding of nocturnin. The authors thank Andrea Forbes, Argy Stampas, and Angie Trevino for constructing the GST-nocturnin plasmids and Paul Hargrave for the gift of the B630N antibody. We also thank Dr. Ammasi Periasamy and the staff of the Keck Center for Cellular Imaging for assistance with confocal microscopy.
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Baggs, J.E., Green, C.B. (2006). Functional Analysis of Nocturnin. In: Liang, P., Meade, J.D., Pardee, A.B. (eds) Differential Display Methods and Protocols. Methods in Molecular Biology, vol 317. Humana Press. https://doi.org/10.1385/1-59259-968-0:243
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DOI: https://doi.org/10.1385/1-59259-968-0:243
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