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Originally published In Press as doi:10.1074/jbc.M101685200 on May 1, 2001

J. Biol. Chem., Vol. 276, Issue 39, 36557-36565, September 28, 2001
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Xenopus Rhodopsin Promoter
IDENTIFICATION OF IMMEDIATE UPSTREAM SEQUENCES NECESSARY FOR HIGH LEVEL, ROD-SPECIFIC TRANSCRIPTION*

Shobana S. ManiDagger §, Suchitra BatniDagger §, Leigh WhitakerDagger , Shiming Chen||, Gustav Engbretson**Dagger Dagger , and Barry E. KnoxDagger **§§

From the Dagger  Departments of Biochemistry and Molecular Biology and ** Ophthalmology, State University of New York Upstate Medical University, Syracuse, New York 13210, the || Department of Ophthalmology, Washington University School of Medicine, St. Louis, Missouri 63110, and the Dagger Dagger  Department of Bioengineering and Neuroscience, Syracuse University, Syracuse, New York 13244

To understand the mechanisms that control the cell-specific visual pigment gene transcription, the Xenopus rhodopsin 5' regulatory region has been characterized in vivo using transient transfection of Xenopus embryos and transgenesis. The principal control sequences were located within -233/+41, a region with significant conservation with mammalian rhodopsin genes. DNase footprinting indicated seven distinct regions that contain potential cis-acting elements. Sequences near the initiation site (-45/+41, basal region) were essential, but not sufficient, for rod-specific transcription. Two negative regulatory regions were found, one between -233 to -202, with no apparent similarity to known elements, and a second Ret-1-like CAAT (-136/-122) motif. Deletion of either sequence led to a 2-3-fold increase in expression levels, without a change in rod specificity. Sequences between -170 to -146, which contain an E-box motif, were necessary for high level expression in transgenic tadpoles but not in transient transfections. Sequences between -84 and -58, which contained an NRE-like consensus were found to be necessary for high level expression in both assays. Although expression levels were modulated by various proximal sequences in the rhodopsin promoter, none of the tested sequences were found to be necessary for rod specificity. Promoter constructs with a consensus BAT-1 sequence in conjunction with an NRE-like element upstream of the basal promoter directed low level green fluorescent protein expression in the central nervous system in transgenic tadpoles. These results suggest that rod cell-specific expression of rhodopsin is controlled by redundant elements in the proximal promoter.

* This work was supported by National Institutes of Health Grants EY09409, EY11256, and EY12975 (all to B. E. K.), National Institutes of Health Grant EY00667 (to R. B.), and a grant from the Research to Prevent Blindness Foundation and the Lion's Club of Central New York.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Present address: CLONTECH Laboratories, Palo Alto, CA, 94303-4230.

§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750. E. Adams St., Syracuse, NY 13210. Tel.: 315-464-8719; Fax: 315-464-8750; E-mail: knoxb@mail.upstate.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.