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• W2077534857 abstract "Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region. Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region. INTRODUCTIONThe neural retina is a specialized part of the central nervous system which both transduces light energy into neurochemical signals and begins initial information processing. It has a complex laminar structure in which there is segregation of form and function. Morphological and thymidine labeling studies have demonstrated that the different types of neuronal and glial cells that make up the retina are born and differentiate in a defined temporal and spatial sequence(1.Carter-Dawson L. LaVail M.M. J. Comp. Neurol. 1979; 188: 263-272Crossref PubMed Scopus (344) Google Scholar). Cell lineage studies, utilizing both retroviral (2.Turner D.L. Cepko C.L. Nature. 1987; 328: 131-136Crossref PubMed Scopus (1117) Google Scholar, 3.Turner D.L. Snyder E.Y. Cepko C.L. Neuron. 1990; 4: 833-845Abstract Full Text PDF PubMed Scopus (537) Google Scholar) and fluorescent dextran (4.Wetts R. Fraser S.E. Science. 1988; 239: 1142-1145Crossref PubMed Scopus (534) Google Scholar) markers, indicate that most, if not all, of these cells arise from common progenitors. However, despite these and other important advances in the cell biology of retinal development(5.Adler R. Friedlander M. International Review of Cytology. 146. Academic Press, San Diego1993: 145-190Google Scholar), the actual molecular mechanisms which regulate cell fate determination and the development of committed progenitors into mature retina cells remain poorly understood.Since development and differentiation of the retina are thought to involve a cascade of events in which different genes are turned on and off in a precisely regulated manner, one approach to studying retinal development is to analyze the mechanisms that control gene expression within the retina. Identification of transcription factors which regulate cell type and lineage-specific gene expression could, for example, lead to the discovery of master regulatory factors analogous to those controlling other lineages, such as the MyoD/myogenin/myf-5 family involved in muscle development(6.Weintraub H. Cell. 1993; 75: 1-20Abstract Full Text PDF PubMed Scopus (924) Google Scholar).Efforts to define the cis-acting DNA elements and trans-acting factors which regulate retina-specific gene expression have so far focused primarily on photoreceptor-specific gene products such as rhodopsin(7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 8.Lem J. Applebury M.L. Falk J.D. Flannery J.G. Simon M.I. Neuron. 1991; 6: 201-210Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 9.Morabito M.A. Yu X. Barnstable C.J. J. Biol. Chem. 1991; 266: 9667-9672Abstract Full Text PDF PubMed Google Scholar, 10.Yu X. Chung M. Morabito M.A. Barnstable C.J. Biochem. Biophys. Res. Commun. 1993; 191: 76-82Crossref PubMed Scopus (26) Google Scholar, 11.Sheshberadaran H. Takahashi J.S. Mol. Cell. Neurosci. 1994; 5: 309-318Crossref PubMed Scopus (25) Google Scholar, 12.Rehemtulla A. Warwar R. Kumar R. Ji X. Zack D.J. Swaroop A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 191-195Crossref PubMed Scopus (183) Google Scholar, 13.Kumar R. Zack D.J. Wiggs J. Molecular Genetics of Ocular Disease. Wiley-Liss, New York1994: 139-160Google Scholar), red and green opsins(14.Nathans J. Davenport C.M. Maumenee I.H. Lewis R.A. Hejtmancik J.F. Litt M. Lovrien E. Weleber R. Bachynski B. Zwas F. Klingaman R. Fishman G. Science. 1989; 245: 831-838Crossref PubMed Scopus (247) Google Scholar, 15.Wang Y. Macke J.P. Merbs S.L. Zack D.J. Klaunberg B. Bennett J. Gearhart J. Nathans J. Neuron. 1992; 9: 429-440Abstract Full Text PDF PubMed Scopus (350) Google Scholar), blue opsin(16.Chiu M.I. Nathans J. J. Neurosci. 1994; 14: 3426-3436Crossref PubMed Google Scholar, 17.Chiu M.I. Nathans J. Vis. Neurosci. 1994; 11: 773-780Crossref PubMed Scopus (55) Google Scholar, 18.Chen J. Tucker C.L. Woodford B. Szel A. Lem J. Gianellaborradori A. Simon M.I. Bogenmann E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2611-2615Crossref PubMed Scopus (46) Google Scholar), interphotoreceptor retinoid-binding protein(19.Borst D.E. Si J.-S. Nickerson J.M. J. Cell Biol. 1991; 115: 312Google Scholar, 20.Liou G.I. Matragoon S. Yang J. Geng L. Overbeek P.A. Ma D.P. Biochem. Biophys. Res. Commun. 1991; 181: 159-165Crossref PubMed Scopus (35) Google Scholar), S-antigen(21.Yamaki K. Tsuda M. Kikuchi T. Chen K.H. Huang K.P. Shinohara T. J. Biol. Chem. 1990; 265: 20757-20762Abstract Full Text PDF PubMed Google Scholar, 22.Breitman M.L. Tsuda M. Usukura J. Kikuchi T. Zucconi A. Khoo W. Shinohara T. J. Biol. Chem. 1991; 266: 15505-15510Abstract Full Text PDF PubMed Google Scholar), arrestin(23.Kikuchi T. Raju K. Breitman M.L. Shinohara T. Mol. Cell. Biol. 1993; 13: 4400-4408Crossref PubMed Scopus (104) Google Scholar), and α-transducin(24.Ahmad I. Yu X. Barnstable C.J. J. Neurochem. 1994; 62: 396-399Crossref PubMed Scopus (16) Google Scholar). Rhodopsin provides a particularly attractive model system for these studies because: 1) both the gene and the protein are well characterized(25.Nathans J. Annu Rev Neurosci. 1987; 10: 163-194Crossref PubMed Google Scholar); 2) its expression is tightly regulated both in terms of cell-type specificity and developmental timing, and it shows diurnal modulation(26.Farber D.B. Danciger J.S. Organisciak D.T. Exp. Eye Res. 1991; 53: 781-786Crossref PubMed Scopus (52) Google Scholar); 3) it is expressed at high levels; and 4) its similarity with the color opsins allows useful homology comparisons (27.Nathans J. Thomas D. Hogness D.S. Science. 1986; 232: 193-202Crossref PubMed Scopus (1168) Google Scholar). Moreover, approximately 30% of cases of autosomal dominant retinitis pigmentosa, a currently untreatable disease in which photoreceptor degeneration leads to blindness, are due to mutations in the rhodopsin gene(28.Dryja T.P. McGee T.L. Reichel E. Hahn L.B. Cowley G.S. Yandell D.W. Sandberg M.A. Berson E.L. Nature. 1990; 343: 364-366Crossref PubMed Scopus (794) Google Scholar, 29.Sung C.-H. Davenport C.M. Hennesey J.C. Maumenee I.H. Jacobson S.G. Heckenlively J.R. Nowakowski R. Fishman G. Gouras P. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6481-6485Crossref PubMed Scopus (337) Google Scholar, 30.Nathans J. Cell. 1994; 78: 357-360Abstract Full Text PDF PubMed Scopus (46) Google Scholar). Development of effective gene therapy for autosomal dominant retinitis pigmentosa will require thorough understanding of rhodopsin regulation(31.Zack D.J. Arch. Ophthalmol. 1993; 111: 1477-1479Crossref PubMed Scopus (21) Google Scholar), particularly since even wild type rhodopsin can lead to retinal degeneration when abnormally expressed(32.Olsson J.E. Gordon J.W. Pawlyk B.S. Roof D. Hayes A. Molday R.S. Mukai S. Cowley G.S. Berson E.L. Dryja T.P. Neuron. 1992; 9: 815-830Abstract Full Text PDF PubMed Scopus (400) Google Scholar, 33.Sung C.H. Makino C. Baylor D. Nathans J. J. Neurosci. 1994; 14: 5818-5833Crossref PubMed Google Scholar).The induction and regulated increase in rhodopsin expression seen during rod development is largely controlled at the transcriptional level(34.Treisman J.E. Morabito M.A. Barnstable C.J. Mol. Cell. Biol. 1988; 8: 1570-1579Crossref PubMed Scopus (93) Google Scholar, 35.Timmers A.M. Newton B.R. Hauswirth W.W. Exp. Eye Res. 1993; 56: 257-265Crossref PubMed Scopus (32) Google Scholar, 36.DesJardin L.E. Timmers A.M. Hauswirth W.W. J. Biol. Chem. 1993; 268: 6953-6960Abstract Full Text PDF PubMed Google Scholar). Transgenic mouse studies utilizing overlapping sets of promoter-lacZ fusion constructs have identified some of the DNA elements that regulate photoreceptor-specific expression of rhodopsin(37.Zack D.J. Methods Neurosci. 1993; 15: 331-341Crossref Scopus (6) Google Scholar). Bovine upstream fragments from −2174 to +70 bp, ( 1The abbreviations used are: bpbase pair(s)kbkilobase pair(s)PCRpolymerase chain reactionRERrhodopsin enhancer regionMOPS4-morpholinepropanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonic acidLCRlocus control regionX-gal5-bromo-4-chloro-3-indoyl β-D-galactosideEMSAelectrophoretic mobility shift assays.) from −735 to +70 bp, from −222 to + 70 bp, and from −176 to +70 bp (relative to the mRNA start site) (7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar) ( 2S. Chen and D. Zack, unpublished results.) as well as murine 4.4 kb and 0.5 kb 5′ fragments (8.Lem J. Applebury M.L. Falk J.D. Flannery J.G. Simon M.I. Neuron. 1991; 6: 201-210Abstract Full Text PDF PubMed Scopus (156) Google Scholar) all direct photoreceptor-specific expression. There are, however, important differences between the various constructs. Although position effects can cause considerable variation, the level of transgene expression is generally higher with the larger constructs than with the smaller ones. In addition, a superior-temporal to inferior-nasal transgene expression gradient is seen with the longer but not with the shorter constructs, which show either a spotty or diffuse pattern of expression.These results suggested that there may be at least two classes of elements regulating rhodopsin expression: a “proximal region” in the vicinity of the mRNA start site (within −176 to +70 bp in the bovine gene) that serves as a minimal promoter capable of directing photoreceptor-specific expression, and a “distal region,” located further upstream, which serves as an enhancer. In addition, the finding of a gradient of expression which is unique to mice with the longer constructs raised the possibility that either the putative enhancer or a different distal sequence might function as a topological element controlling spatial expression across the retina.In this paper we directly address the identity of the putative rhodopsin enhancer. Although the deletion series employed in the initial bovine transgenic experiments suggested that the enhancer and topological regulatory elements were located between −2174 and −734 bp, the mapping was not detailed enough to define a specific location. Based on sequence comparison of the mouse, cow, and human rhodopsin upstream regions, we hypothesized that the enhancer activity might be contained within a highly conserved 100-bp region and have generated transgenic mice that contain promoter-reporter fusion constructs that differ only by the presence or absence of this candidate region. Characterization of these mice indicates that the 100-bp candidate region displays many of the properties of an enhancer. It is not, however, required to establish an expression gradient across the retina. We also present biochemical evidence that bovine nuclear extracts contain both retina-specific and ubiquitously expressed proteins which bind to areas within the enhancer region in a sequence specific manner.EXPERIMENTAL PROCEDURESGeneration of Transgenic MiceTo generate the rhodopsin promoter/lacZ fusion construct that contained the rhodopsin enhancer region (rho-2145), PCR was carried out using a plasmid which contains the bovine rhodopsin upstream sequence from −2174 to +70 bp as template and the primers 5′-ACGAATGGTACCTGGCCACCAGGGGCGTGT-3′ (which contains 6 bp of 5′ spacer DNA, a KpnI site, and spans the sequence from −2145 to −2128) and 5′-CAGGAAGGCCTCTCTGAG-3′ (which spans the sequence from −1599 to −1616). The PCR product was then digested with KpnI and XhoI (which cuts at −1623 bp), gel purified, and directionally cloned into the previously described plasmid Rho −2174/placF (7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar) which had been cut with KpnI and XhoI. The region generated by PCR in the resulting plasmid was resequenced to rule out any PCR-induced mutations. The promoter/lacZ construct lacking the RER (rho-1923) was generated similarly except that the 5′ promoter used in the PCR was 5′-ACGAATGGTACCGCCACACCTGCCTGCCCC-3′ (which contains 6 bp of 5′ spacer DNA, a KpnI site, and spans the sequence from −1923 to −1906). Each construct was then cut with HindIII and KpnI and the purified fragment was used for microinjection.The bovine RER/heterologous promoter fusion construct was generated using the 0.3-kb BamHI/NcoI fragment from plasmid pRed2 (kindly provided by Jeremy Nathans and Yanshu Wang, Johns Hopkins University, Baltimore, MD) which contains the region from −88 to +230 bp from the hsp70 A1 gene(38.Perry M.D. Aujame L. Shtang S. Moran L.A. Gene (Amst.). 1994; 146: 273-278Crossref PubMed Scopus (34) Google Scholar). The fragment, which was gel purified after filling-in the BamHI site with Klenow, was directionally cloned into the plasmid Rho −2174/placF which had been previously digested with XhoI, filled-in with Klenow, cut with NcoI, and then gel purified. The resulting plasmid was cut with HindIII and KpnI to generate the approximately 4.4-kb fragment that was used for microinjection.Transgenic mice were generated at the Johns Hopkins University School of Medicine Transgenic Mouse Facility by pronuclear microinjection of B6AF1 (female) × C57BL/6J (male) embryos using established techniques (39.Hogan B. Constantini F. Lacy E. Manipulating the Mouse Genome: A Laboratory Approach. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar), as described previously(7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar). Animals were screened by PCR as described previously (7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 37.Zack D.J. Methods Neurosci. 1993; 15: 331-341Crossref Scopus (6) Google Scholar) except that 30-bp primers (5′-GATGTGGCGAGATGCTCTTGAAGTCTGGTA3′, 5′-CAAGGCAACTCCTGATGCCAAAGCCCTGCCC-3′, and 5′-AGCTGAGCGCCGGTCGCTACCATTACCAGT-3′) were used instead of the previously described 21-bp primers and the digestion buffer contained 2% Triton X-100 instead of 2% Nonidet P-40.Histology, lacZ Staining, and in Situ HybridizationMice were euthanized and eyes were sectioned or prepared for whole mount analysis as described previously(7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar), except that fixation was performed for 10-15 min at room temperature in MEMFA buffer (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde). Whole mount in situ hybridization was performed using methods that have been described for chick embryos(40.Li H.S. Yang J.M. Jacobson R.D. Pasko D. Sundin O. Dev. Biol. 1994; 162: 181-194Crossref PubMed Scopus (250) Google Scholar). Digoxigenin labeled antisense (T3) and sense lacZ riboprobes (T7) were prepared from appropriately restricted pBluescript II SK+ (Stratagene, La Jolla, CA) containing the 438-bp BamHI/HincII lacZ fragment. Genius reagents were used for labeling and staining according to the manufacturer's directions (Boehringer Mannheim). The probe concentration used for hybridization was 0.2 μg/ml. After hybridization, the eyes were rinsed twice with FSCG buffer (50% formamide, 2 × SSC, and 0.1% CHAPS, and 50 mM glycine) at room temperature, washed twice with the same buffer for 30 min at 60°C, and rinsed four times at room temperature with SCG buffer (2 × SSC, 0.1% CHAPS, and 50 mM glycine) and then digested with RNase A (4 μg/ml) and RNase T1 (20 units/ml) for 30 min at 37°C. Alkaline phosphatase color reactions were performed from 15 min to 18 h, depending on the intensity of the reaction. Eyes were then sectioned as described(40.Li H.S. Yang J.M. Jacobson R.D. Pasko D. Sundin O. Dev. Biol. 1994; 162: 181-194Crossref PubMed Scopus (250) Google Scholar).β-Galactosidase Solution Assayβ-Galactosidase solution assay was performed as described previously(7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar).Preparation of Bovine Nuclear ExtractsNuclear extracts from bovine retina, cerebral cortex, cerebellum, skeletal muscle, liver, heart, and kidney were prepared by the method of Gorski et al.(41.Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar), with minor modifications. Tissues were dissected on ice and, except for retina, were minced prior to homogenization. For each 2-3 g wet weight, tissue was homogenized in 10 ml of buffer A (10 mM HEPES, pH 7.6, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 10% of glycerol) using a motor-driven 30-ml Teflon-glass homogenizer for 10 strokes. The rest of the procedure was as described (41.Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar) except that the ammonium sulfate protein pellet was dissolved at 1 ml/200 A260 units of nuclear lysate and the final dialysis buffer contained 60 mM KCl instead of 40 mM. Protein concentration was determined using the Bio-Rad protein assay system. Aliquots of extract were stored in liquid nitrogen.Electrophoretic Mobility Shift AssaysBand shift assays were performed using standard procedures(42.Chodosh L.A. Albright L.M. Coen D.M. Janssen K. Varki A. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1988: 12.2.1-12.2.10Google Scholar). Probes were labeled either with [γ-32P]ATP and polynucleotide kinase or by filling-in appropriately annealed oligonucleotides with Klenow fragment and [α-32P]dCTP. Approximately 20,000 cpm of labeled probe in a 10-μl reaction volume was incubated on ice for 30 min with 3-10 μg of nuclear extract in mobility shift buffer (60 mM KCl, 25 mM HEPES, pH 7.6, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol) in the presence of the indicated amounts of poly(dI-dC) and then analyzed on a 5% polyacrylamide, 0.5 × TBE gel, at 115 V for 2 h at room temperature. Gels were dried and autoradiographed overnight without enhancement. For the cold oligomer competition experiments, the indicated competitor was added prior to the addition of nuclear extract.DNase I FootprintFor method 2, kinased oligomer primers were used to generate 32P-labeled PCR fragments corresponding to the region −2143 to −1895 bp upstream of the bovine rhodopsin gene(43.Krummel B. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego1990: 184-188Google Scholar). The 5′ primer used in the reaction, 5′-AGCTCACTCGAGCAAGGCCATGAGTTTGAG-3′, contained 6 bp of 5′ spacer DNA, a XhoI site, and spanned the region from −2143 to −2124 bp. The 3′ primer, 5′-AGCTCAGTCGACTGGTAAGTGCTCTGGGGG-3′, contained 6 bp of 5′ spacer DNA, a SalI site, and spanned the region from −1894 to −1911 bp. Amplifications were carried out with one labeled primer. The resulting end-labeled PCR products were gel purified using 2% GTG Nusieve-agarose (FMC, Rockland, ME).For method 1, restriction fragment probes corresponding to the region −2143 to −1895 bp were generated using plasmid p(−2143/-1895)/rho, which contains the PCR product amplified with the above primers cloned into the SalI restriction site of pBluescript II KS+. To prepare probe labeled at its upstream end, p(−2143/-1895)/rho was digested with HindIII, phosphatased with calf intestine alkaline phosphatase, kinased with [γ-32P]ATP and T4 polynucleotide kinase, and digested with Asp718. The desired end-labeled fragment was then gel purified. Probe labeled at its downstream end was prepared similarly except that it was first digested with Asp718 and cut with HindIII after the kinase step.DNase I footprint reactions were carried out using standard procedures (44.Garabedian M.J. LaBaer J. Liu W.-H. Thomas J.R. Hames B.D. Higgins S.J. Gene Transcription: A Practical Approach. IRL Press, Oxford1993: 243-293Google Scholar). Binding reactions contained approximately 10 fmol of probe and 50 μg of bovine nuclear extract in a 50-μl reaction volume for method 1 and 25 μl for method 2. For method 1, binding buffer consisted of 12.5 mM HEPES, pH 7.6, 100 mM KCl, 5 mM ZnSO4, 0.5 mM dithiothreitol, 2% (w/v) polyvinyl alcohol, 10% glycerol, and 1 μg of poly(dI-dC). For method 2, binding buffer consisted of 12.5 mM HEPES, pH 7.6, 60 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 10% glycerol, and 1 μg of poly(dI-dC). After 15 min incubation on ice, the reaction tubes were transferred to room temperature, incubated for 1 min, MgCl2 and CaCl2 were added to give final concentrations of 5 and 2.5 mM, respectively, and then DNase I (Worthington) at the appropriate concentration (see Figure 7:, Figure 8:) was added. Digestion was carried out for the times indicated and then terminated by the addition of 90 μl of stop solution (20 mM EDTA, pH 8.0, 1% (w/v) SDS, 0.2 M NaCl, and 250 μg/ml glycogen) and 10 μl of 2.5 mg/ml proteinase K (Sigma). After incubation at room temperature for 5 min, samples were extracted with phenol/chloroform, precipitated with ethanol, and washed with 75% ethanol. Samples were resolved on standard 6% sequencing gels.Figure 7:DNase I footprint of the RER with retina nuclear extract. A and B, footprint pattern of the RER using method I (see “Experimental Procedures”). C, footprint pattern using method II. In lanes 1-3 and 7-9 the top template strand was labeled; in lanes 4-6 the bottom strand was labeled. The major protected regions on the top strand are labeled I, II, III and IV; the major protected regions on the bottom strand are labeled I′, II′, and III′. Hypersensitive sites are indicated with an “*”. In panel C regions I and IV are indicated for comparative purposes but are shown in parentheses because significant protection is not evident. The sequences corresponding to each of the protected regions are indicated in Fig. 1. The reactions in lanes 1, 4, and 7 did not contain nuclear extract. The reactions in lanes 2, 3, 5, 6, 8, and 9 each contained 50 μg of bovine retina nuclear extract (R). The amounts of DNase I used per 50-μl reaction in lanes 1-9 were 3.3, 80, 40, 3.3, 80, 40, 5, 50, and 50 ng, respectively, and the digestion times were 1 min for lanes 1-6 and 5, 2, and 5 min for lanes 7-9, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSEvolutionary Conservation of the Putative Rhodopsin Enhancer Region and Homology to the Red and Green Opsin Locus Control RegionThe 102-bp distal sequence corresponding to −2044 to −1943 bp in the bovine rhodopsin upstream sequence shows 64% sequence identity when compared with the homologous regions upstream of the human, mouse, and rat rhodopsin genes (Fig. 1). The identity is 77% in the central, more conserved region (−2024 to −1963 bp in the bovine sequence), but decreases at both ends of the sequence. Note that despite the high degree of sequence conservation, the actual position of the distal region relative to the mRNA start site varies significantly between the different species, from 1.5 to 2 kb upstream (human, −1906 to −1805; mouse, −1575 to −1477 bp; and rat, −1537 to −1434 bp). For ease of reference, and based on the data presented below, the distal region will henceforth be referred to as the “rhodopsin enhancer region” (RER).Figure 1:Sequence comparison of distal homology regions from cow, human, mouse, and rat rhodopsin genes. RER sequences from cow, human, mouse, and rat rhodopsin genes were aligned to show maximal homology using GeneWorks 2.3 (Intelligenetics, Mountain View, CA). The 37-bp conserved sequence in the red/green LCR (15.Wang Y. Macke J.P. Merbs S.L. Zack D.J. Klaunberg B. Bennett J. Gearhart J. Nathans J. Neuron. 1992; 9: 429-440Abstract Full Text PDF PubMed Scopus (350) Google Scholar) is also shown and aligned for maximal homology. Bases that are identical in all four rhodopsin genes are enclosed with black lines. Bases that are identical in at least three of the rhodopsin genes are shaded. The base position relative to the mRNA start site of the 3′-most base of each sequence is shown on the right of the figure. The numbers on the bottom of the figure refer to base position for the bovine sequence. The RER, which extends from −2044 to −1943 bp in the bovine gene, is indicated by a bracket. The positions of the four DNase I protected regions (FPI, FPII, FPIII, and FPIV) are shown. The top arrows refer to protection seen with the top strand labeled and the bottom arrow refers to protection seen with the bottom strand labeled. The CGATGG core sequence is underlined. The positions of oligomer pair A (EMSA-A), oligomer pair B (EMSA-B), the putative rhodopsin homeodomain binding site-1 (RHBS-1), and the ret-3 binding site (10.Yu X. Chung M. Morabito M.A. Barnstable C.J. Biochem. Biophys. Res. Commun. 1993; 191: 76-82Crossref PubMed Scopus (26) Google Scholar) are also shown. (Note: the bovine sequence shown contains a correction from the previously published sequence (7.Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (180) Google Scholar) indicating that at residues −2031 to −2030 there should be two Cs rather than one.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)The RER also shows homology to the highly conserved 37-bp sequence in the color opsin locus control region (LCR), an element involved in regulation of the red and green visual pigment gene cluster (15.Wang Y. Macke J.P. Merbs S.L. Zack D.J. Klaunberg B. Bennett J. Gearhart J. Nathans J. Neuron. 1992; 9: 429-440Abstract Full Text PDF PubMed Scopus (350) Google Scholar) (Fig. 1). This area of homology contains a sequence, CTAAT (−1985 to −1981 bp in the bovine sequence), that is similar to the homeodomain consensus binding sequence(45.Catron K.M. Iler N. Abate C. Mol. Cell. Biol. 1993; 13: 2354-2365Crossref PubMed Scopus (177) Google Scholar), and henceforth will be referred to as “rhodopsin homeodomain binding site-1” (RHBS-1). The LCR sequence has a 6-bp deletion, relative to the RER, that is located just upstream of the putative homeodomain binding site. This 6-bp sequence and the putative homeodomain site both appear to be involved in sequence-specific DNA-protein interactions (see below).RER Contains Enhancer ActivityIn order to explore the function of the RER in vivo, transgenic mice were generated with two similar constructs that both contained bovine rhodopsin upstream DNA fused to a lacZ reporter gene, but differed by the presence or absence of the RER (Fig. 2). The construct containing the RER (rho-2045) extended from −2045 to +70 bp, while the construct without the RER (rho-1923) extended from −1923 to +70. The constructs were designed so that rho-2045 woul" @default.
• W2077534857 created "2016-06-24" @default.
• W2077534857 creator A5040841311 @default.
• W2077534857 creator A5041504707 @default.
• W2077534857 creator A5062088552 @default.
• W2077534857 creator A5073242437 @default.
• W2077534857 date "1996-02-01" @default.
• W2077534857 modified "2023-10-17" @default.
• W2077534857 title "RER, an Evolutionarily Conserved Sequence Upstream of the Rhodopsin Gene, Has Enhancer Activity" @default.
• W2077534857 cites W1015136610 @default.
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