Characterization of the Xenopus rhodopsin gene.
The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.
Batni
S
S
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA.
Scalzetti
L
L
Moody
S A
SA
Knox
B E
BE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D004330:Drosophila; D004625:Embryo, Nonmammalian; D005091:Exons; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics*; D017299:Rod Opsins / Q000096:biosynthesis / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection; D014714:Vertebrates; D014981:Xenopus / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D008156:Luciferases; D012172:Retinaldehyde