Isolation and initial characterization of the 5' flanking region of the human and murine cyclic guanosine monophosphate-phosphodiesterase beta-subunit genes.
OBJECTIVE
As an initial approach to study the mechanisms that direct photoreceptor-specific expression of the rod cyclic guanosine monophosphate-phosphodiesterase beta-subunit (beta-PDE) gene, the 5' flanking regions of the human and mouse genes were cloned and analyzed.


METHODS
Genomic libraries were screened and clones containing the 5' upstream region of the beta-PDE gene were isolated and sequenced. Primer extension and ribonuclease protection assays were used to determine the transcription initiation sites. Sequences were compared using dot-matrix analysis and nucleotide alignment to determine potential regulatory elements that have been conserved through evolution. DNA-protein interactions were examined using DNAse I footprinting.


RESULTS
The beta-PDE gene 5' sequence contains two distinct transcription start sites and lacks a TATA box. A stretch of approximately 30 nucleotides just upstream of the first transcribed nucleotide is strongly conserved in both species. This sequence contains a TATA-like element and a -CTAATC- motif previously described in other photoreceptor-specific genes. A highly-conserved AP-1 element, the recognition site for members of the jun and the fos oncoproteins family, is also present in this proximal region. DNAse I footprinting revealed an array of retinal proteins binding to these elements.


CONCLUSIONS
The beta-PDE 5' region features match those of a highly tissue-specific gene in which factors restricted to the retina might play a role in gene activation. Elements conserved through evolution in the human and mouse sequences were found and analyzed as potential cis-acting elements. The availability of the human beta-PDE 5' upstream sequence will allow patients with retinal degeneration to be screened for possible mutations in these control sequences.
Di Polo
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A
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA.
Bowes Rickman
C
C
Farber
D B
DB
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D015723:Gene Library; D005809:Genes, Regulator / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012160:Retina / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human; C517438:Pde6b protein, mouse