Immediate upstream sequence of arrestin directs rod-specific expression in Xenopus.
Arrestins are a family of proteins that modulate G protein-coupled receptor responses with distinct arrestin genes expressed in rods and cones. To understand the regulatory mechanisms controlling rod-specific expression, the abundant Xenopus rod arrestin cDNA and a partial genomic clone, containing the immediate upstream region and amino terminus of the polypeptide, have been characterized. The deduced polypeptide has approximately 69% identity to other vertebrate rod arrestins. Southern blot analysis and polymerase chain reaction of intronic sequences demonstrated multiple alleles for rod arrestin. DNase I footprinting with retinal proteins revealed four major DNA binding sites in the proximal promoter, coinciding with consensus sequences reported in mammalian promoters. Purified bovine Crx homeodomain and mouse Nrl proteins protected a number of these sites. A dual approach of transient embryo transfections and transgenesis was used to locate transcriptional control sequences essential for rod-specific expression in Xenopus. Constructs containing -1287/+113 of 5' upstream sequence with or without intron 1 directed high level expression, specifically in rods. A construct containing only -287/+113 directed expression of green fluorescent protein solely in rod cells. These results suggest that the Crx and Nrl binding sites in the proximal promoter are the primary cis-acting sequences regulating arrestin gene expression in rods.
Mani
S S
SS
Department of Biochemistry and Molecular Biology, Department of Ophthalmology, SUNY Health Science Center at Syracuse, New York 13210, USA.
Besharse
J C
JC
Knox
B E
BE
D000818:Animals; D030801:Animals, Genetically Modified; D019393:Arrestin / Q000235:genetics*; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D002417:Cattle; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000235:genetics; D017403:In Situ Hybridization; D008164:Luminescent Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection; D014981:Xenopus / Q000196:embryology / Q000378:metabolism*
D019393:Arrestin; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; C084884:Nrl protein, mouse; D012333:RNA, Messenger; D015534:Trans-Activators; C109288:cone rod homeobox protein; D049452:Green Fluorescent Proteins