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Structure and upstream region characterization of the human gene encoding rod photoreceptor cGMP phosphodiesterase α-subunit

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Abstract

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the α-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an ∼500 bp sequence upstream of the mousePde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putativecis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed foot-prints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.

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Abbreviations

PDE6:

photoreceptor-specific family of 3′-5′ cyclic nucleotide phosphodiesterases

PDE6A:

locus encoding human rod photoreceptor cGMP phosphodiesterase α-subunit gene

PDE6B:

locus encoding human rod photoreceptor cGMP phosphodiesterase β-subunit gene

PCR:

polymerase chain reaction

RACE:

rapid amplification of cDNA ends

tsp:

transcription start point

RET1/PCE1:

cis-element found in the upstream region of many photoreceptor-specific genes

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, University of South Alabama College of Medicine, 36688-0002, Mobile, AL

    Mohamed K. Mohamed, Russell E. Taylor & Steven J. Pittler

  2. Department of Ophthalmology, University of South Alabama College of Medicine, 36688-0002, Mobile, AL

    Steven J. Pittler

  3. Center for Eye Research, University of South Alabama College of Medicine, 36688-0002, Mobile, AL

    Mohamed K. Mohamed, Douglas S. Feinstein, Xizhong Huang & Steven J. Pittler

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  1. Mohamed K. Mohamed
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  3. Douglas S. Feinstein
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Mohamed, M.K., Taylor, R.E., Feinstein, D.S. et al. Structure and upstream region characterization of the human gene encoding rod photoreceptor cGMP phosphodiesterase α-subunit. J Mol Neurosci 10, 235–250 (1998). https://doi.org/10.1007/BF02761777

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