XB-ART-18548J Biol Chem. February 9, 1996; 271 (6): 3179-86.
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.
PubMed ID: 8621718
Article link: J Biol Chem.
Grant support: EY09409 NEI NIH HHS , EY10096 NEI NIH HHS
Genes referenced: id4 nrl prss1 ret rho sp1
Article Images: [+] show captions
|FIG. 1. The structure of the Xenopus gene. A, restriction map of the genomic clone gopRI. B, the structure of the rhodopsin gene (XOP1) consisting of five exons (numbered boxes) with untranslated (stippled fill) and translated (solid box) regions indicated. C, the mRNA is 1684 bp in length. D, the cDNA clones, XOP71 and RACE PCR, used in DNA sequence determination.|
|FIG. 2. Xenopus rhodopsin gene sequence. The nucleotide sequence of the coding region with deduced amino acids and 1.2 kb of sequence upstream. The major transcription start site (numbered 11) is shown. The position of the introns are indicated (inverted triangle). Primers (described under “Experimental Procedures”) are underlined. Potential transcription control sequences are underlined and lettered in italics (see “Results” for more details). Numbering includes the omitted intron sequences.|
|FIG. 3. UV-visible absorbance spectra of XOP1. XOP1 cDNA containing a replacement of the carboxyl-terminal 7 amino acids with the ID4 monoclonal antibody epitope, was transiently transfected into COS1 cells, incubated with 11-cis-retinal and purified by immunoaffinity chromatography in dodecyl maltoside. Maximal chromophore absorbance for both XOP1 (1) and bovine (2) rhodopsin occurred at 500 nm. The protein absorption peak occurs at 280 nm.|
|FIG. 4. Rhodopsin transcript analysis. A, Northern blot. 2 mg of Xenopus adult total brain (lane 1) and retinal (lane 2) RNA was resolved on a denaturing agarose gel, and rhodopsin transcripts were detected by hybridization with a cDNA probe (nucleotides 315–856). The single 1.7-kb retina-specific message is indicated (arrow). Arrowheads represent molecular weight markers of 9.4, 7.5, 4.4, 2.4, and 1.4 kb from top to bottom. B, primer extension. Primer extension of total RNA with radioactively labeled antisense primer P10 is shown. Reactions were carried out using 5 mg of total retinal RNA (lane 1) and with a 50-fold excess of unlabeled P10 (lane 2) or total brain RNA (lane 3). A sequencing ladder using the same primer is also shown. The underlined nucleotide indicates the major transcription start site and minor start sites are indicated by smaller asterisks.|
|FIG. 5. A, Southern analysis. Southern blot of Xenopus genomic DNA undigested (lane 1) or digested with SacI (lane 2), EcoRI (lane 3), or BamHII (lane 4), hybridized with an exon 1 probe (nucleotides 1–444) and washed at high stringency. B, PCR of intron 1 using genomic DNA. PCR using primers P1 and P12 (see Fig. 2) were used to amplify intron 1 from genomic DNA (lanes 1–3 and 5) or from genomic clone lgopRI (lanes 6–9). Amplifications were carried out with P1 and P12 (lanes 1 and 6), P1 (lanes 2 and 7), P12 (lanes 3 and 8), no primers (lanes 5 and 9), and primers alone, no genomic DNA (lane 4). The sizes of the four products found in lane 1 are 367, 500, 550, and 650 bp.|
|FIG. 6. Homologies with other vertebrate rhodopsin upstream sequences. A, proximal sequence homology. Sequence alignment of the ;450 immediate upstream nucleotides of the Xenopus (XEN), chicken (CHK), human (HUM), bovine (BOV), rat (RAT), and mouse (MUS) rhodopsin genes is shown. Alignments were created using a window size of 6 and a stringency of 67%. Gaps introduced in the sequence for optimal alignment are shown by dots. Regions containing greater than 75% identity across species are shaded. Transcription start sites (boxed nucleotides) and position of the initiator methionine (arrow) are indicated. Potential GC boxes binding SP1 are indicated with dotted underline and pyrimidine tracts with solid underline. B, nucleotide identities of the Xenopus upstream sequence with glass elements, proximal and distal, with core sequences underlined. Nucleotides conserved between Xenopus and chicken are shown in italics, and across all three species are indicated in bold. C, homologies of the Xenopus rhodopsin upstream sequence with the human retinal leucine zipper binding sequence, NRL, and rat Ret2 are shown with nucleotide identities in bold.|
|FIG. 7. Rhodopsin upstream sequences direct transient expression of luciferase in Xenopus embryos. A, the luciferase reporter constructs are diagrammed with the luciferase gene (luc) transcribed from left to right. Solid boxes indicate genomic sequences from XOP1 and GL2 is the (promoterless) control plasmid. B, Luciferase levels obtained from transient expression experiments. Stage 26/27 embryos were dissected and treated with trypsin in the presence of EDTA prior to lipofection (Experiments A and C). Additional embryos were dissected and the head epidermis was manually removed prior to trypsinization and lipofection (Experiment B). Embryonic tissue was incubated to the equivalent of stage 42/43 and assayed for luciferase activity. Activities are presented as RLU/embryo, where 1 pg of luciferase 5 85,000 RLU. Early stage blastomeres (8-cell or 32-cell, Experiment D or E, respectively) were injected with plasmid and cultured to stage 42, when luciferase levels were determined.|