©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Single Cis-acting Element in a Short Promoter Segment of the Gene Encoding the Interphotoreceptor Retinoid-binding Protein Confers Tissue-specific Expression (*)

(Received for publication, July 21, 1994)

Nicoletta Bobola (1) Emilio Hirsch (2) Adriana Albini (3) Fiorella Altruda (2) Douglas Noonan (3) Roberto Ravazzolo (1)(§)

From the  (1)Institute of Biology and Genetics, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy, the (2)Department of Genetics, Biology and Medical Chemistry, University of Torino, Via Santena 5 bis, 10126 Torino, Italy, and the (3)National Institute for Research on Cancer, Viale Benedetto XV 10, 16132 Genova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from -123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between -45 and -58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between -86 and -106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the -45 to -58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5`-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.


INTRODUCTION

Development in mammals is programed to define patterns of differentiated cells in specialized tissues. During this developmental process, tissue-specific genes become coordinately expressed due to mechanisms of regulation, largely at the transcriptional level. The interphotoreceptor retinoid-binding protein (IRBP) (^1)gene represents a good model for investigating regulation of expression of neural retina-specific genes. IRBP is a large lipoglycoprotein that constitutes approximately 70% of the protein component of the interphotoreceptor matrix(1, 2) . Although widely distributed among the vertebrates, it has a highly restricted tissue-specific expression and is found in the interphotoreceptor matrix of the retina(3) . IRBP mRNA is present in photoreceptor cells of the retina, prevalently in rod cells, and, at very low levels, in a subgroup of pinealocytes(4) . IRBP is also expressed by retinoblastoma-derived cell lines in vitro(5) , and the level of IRBP expression can be altered by agents that affect retinoblastoma cell differentiation(6) .

IRBP can bind a variety of retinoids(7) , and it has been suggested that it acts by actively transporting retinoids between photoreceptor cells and the retinal pigmented epithelium (8) and/or that it acts as a ``buffer'' protein for retinoids(9) . All of the bovine and human IRBP genes have been cloned(10, 11) ; the sequences of the human and murine 5`-flanking regions that are upstream from the transcription start site have been described(12) . The human IRBP gene has been mapped to the centromeric region of chromosome 10(13) .

The exquisite tissue-specific expression of IRBP indicates that there must be specific factors that control the expression of this gene. Initial investigations on the mechanism of tissue-specific expression of the IRBP gene have mainly been directed to the elucidation of transcriptional regulation. Hypomethylation of CpG islands in the fragment between -1578 and -1108 of the IRBP promoter and at the beginning of the first exon correlates with gene expression(14) . Mice that are transgenic for a reporter gene fused to a 1.3-kilobase pair human IRBP promoter segment show tissue-specific expression of the reporter gene(15) . A shorter 212-bp promoter fragment linked to a reporter gene showed tissue-specific expression(12) .

Here we report the results of a detailed analysis of a short IRBP promoter segment (-123 to +18). The ability to confer retina-specific expression of reporter gene has been studied in transgenic mice. By comparing the wild type 124-bp promoter fragment with a mutated version of the same fragment, we demonstrated that a cis-acting element that contains a repeated GATTAA motif is essential for tissue-specific expression. An analysis of binding sites for trans-acting factors contained in nuclear extracts of retinoblastoma IRBP-expressing cell lines compared with nuclear extracts of nonexpressing cell lines has been performed. We demonstrate that the same cis-acting element that was able to confer tissue-specific expression of the IRBP gene binds a factor present only in IRBP-expressing cells.


MATERIALS AND METHODS

DNA Constructions

Plasmid pNB20 contains a 1.3-kilobase pair fragment of the human IRBP gene (from -1398 to +18 relative to the transcription start site) inserted upstream of the lacZ reporter gene in the HindIII site of plasmid pbetaGal(16) . The same HindIII site of pbetaGal was used to generate plasmid pNB21, which contains a promoter fragment from -123 to +18, and plasmid pNB22. Plasmid pNB22 is identical to pNB21 except for two substitution mutations in the promoter sequence between -48 and -52 and between -61 and -64, as indicated in Fig. 3. All recombinant plasmids were analyzed by restriction mapping and by DNA sequencing.


Figure 3: Nucleotide sequences reporting cis-acting elements, oligonucleotides used as labeled probes or competitors in DNA/protein binding assays, and substitutions introduced by mutagenesis. Lowercase letters indicate the nucleotide substitutions introduced by mutagenesis. Solid lines indicate DNase I protections in the promoter fragment and the GATTAA elements in the oligonucleotides. Dotted lines indicate mutated GATTAA elements in oligonucleotides.



Mutagenesis Procedure

Site-directed mutagenesis for pNB22 was first performed according to the method of Higuchi et al.(17) using a two-step polymerase chain reaction procedure to prepare a plasmid containing the -48 to -52 substitution. A second mutagenesis was performed with the same procedure to obtain the -61 to -64 substitution. The mutated fragment was reinserted in the original vector substituting the wild type fragment.

Transgenic Mice

Gene constructs were excised from plasmid backbone with XhoI-BamHI digestion and purified from agarose gel by Geneclean (BIO 101, Inc., Vista, CA).

Transgenic mice were produced by pronuclear injection into B6D2 F2 fertilized eggs (18) of 500 copies/ml DNA diluted in 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. Founder mice were identified by Southern blot analysis of EcoRI-digested tail DNA(19) .

Transgene expression was detected on frozen sections of fixed organs. Specimens were fixed for 2 h in 4% paraformaldehyde in phosphate-buffered saline (10 mM phosphate buffer, pH 7.4, 150 mM NaCl), incubated for 2 h in 30% sucrose in phosphate-buffered saline for cryopreservation, embedded in OCT compound (Miles), and frozen in liquid nitrogen. 10-µm-thick sections were collected on gelatin-coated slides and stained for anywhere from 2 h to overnight (depending on the transgene) in 1 mg/ml X-gal (Sigma), 5 mM K(3)Fe(CN)(6), 5 mM K(4)Fe(CN)(6), and 2 mM MgCl(2) in phosphate-buffered saline with 0.001% Triton X-100. Nuclear fast red was used for counterstaining.

Cell Culture and Preparation of Nuclear Extracts

Two retinoblastoma cell lines, Y-79 and Weri RB-1, both of which express the IRBP gene, and HeLa-S3 and H-9 lymphoblasts, which do not express IRBP, were used for nuclear extract preparation. Nuclear extracts were prepared according to the method of Dignam et al.(20) . All steps were carried out at 4 °C, with phenylmethylsulfonyl fluoride (0.5 mM) added to each buffer as a protease inhibitor. The nuclear extracts were dialyzed against a buffer containing 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol, aliquoted, and stored at -80 °C.

DNase I Footprinting

A 3` end-labeled 147-bp fragment of the human IRBP promoter containing the promoter sequence from -123 to +18 was prepared as follows. The recombinant plasmid pNB20 was cut with HindIII and labeled at the 3` end with Klenow enzyme and a dideoxynucleotide triphosphate mixture containing [P]dCTP. RsaI was used for the second digestion, and the end-labeled fragment was purified on a polyacrylamide gel. The DNA binding reaction was performed in a final volume of 50 µl containing 15 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 mM NaCl, 5% glycerol, 2.5 µg of poly(dI-dC), 25 fmol of the end-labeled DNA probe, and nuclear extract (20-30 µg of protein) or buffer used for dialysis of nuclear extracts. The binding reaction was allowed to proceed at room temperature for 15 min. DNase I (Boehringer Mannheim, grade I, stored at -20° C at a concentration of 1 mg/ml in 50% glycerol) was appropriately diluted (1:60 for samples containing nuclear extract and 1:800 for samples without extract) immediately before use in an ice-cold buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 62.5 mM MgCl(2), 10 mM CaCl(2), and 1 mg/ml bovine serum albumin, and 2 µl of diluted DNase I were added to the binding mixture and incubated for 1 min (samples with extract) or 15 s (samples without extract) at room temperature. The reaction was stopped with 15 µl of 5% SDS, 125 mM EDTA, and 0.7 mg/ml tRNA and subjected to phenolchloroform extraction and ethanol precipitation. The digestion products were then analyzed by electrophoresis on denaturing 8% polyacrylamide gel with 7 M urea. An aliquot of the same end-labeled DNA fragment was also subjected to the G + A sequencing reaction (21) and loaded on the same gel for identification of protected sequences.

Gel Retardation Assay

The assay was performed by incubating 2-3 µg of nuclear extract with 5 fmol of an end-labeled oligonucleotide (approximately 10,000 cpm) in a 10-µl incubation mixture containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.35 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 1 µg of poly(dI-dC) as a nonspecific competitor for 15 min at room temperature or, alternatively, with the same buffer lacking NaCl. Following electrophoresis in a 5% polyacrylamide gel in 0.25 times TBE (22 mM Tris borate, and 0.5 mM EDTA), gels were dried prior to autoradiography at -80 °C. When the assay was performed to compare nuclear extracts from different sources, we used the same amount of nuclear protein (3 µg of total protein/10 µl of assay mixture).


RESULTS

Tissue-specific Expression of the Human IRBP Promoter

In order to elucidate the transcriptional properties of the human IRBP promoter, chimeric plasmids containing IRBP promoter sequences fused to the lacZ reporter gene were analyzed by expression in transgenic mice. We show in Fig. 1that a promoter segment containing the first 123 bp upstream of the transcription start site (pNB21) was sufficient to confer full expression of the reporter gene in the retinal photoreceptors as well as in pinealocytes. Expression was highly regulated, because it was detectable only in the eye and in the pineal gland. X-gal staining was particularly evident in the outer plexiform layer and in the inner segment of the photoreceptors; staining in the outer nuclear layer appeared after a longer exposure to the beta-galactosidase substrate. Remarkably, only some pinealocytes were shown to express the lacZ reporter gene.


Figure 1: Sections of retina and epiphysis of transgenic mice carrying the pNB21 construct. A section (10-µm thick) from an adult eye stained for 2 h with X-gal is shown. A, beta-galactosidase activity is present only in the photoreceptor cell layer. g, ganglion cell layer; i, inner nuclear layer; p, photoreceptor cells nuclei (outer nuclear layer); r, retinal pigment epithelium. Bar corresponds to 50 µm. B, lower magnification of the same histological section shown in A. n, optic nerve. Bar corresponds to 50 µm. C, coronal section (15-µm thick) at the epiphysis level of an adult brain stained with X-gal. beta-Galactosidase activity is present in some pinealocytes located in the wall of the pineal stalk (arrowheads). e, lumen of the pineal peduncle. Bar corresponds to 100 µm.



The pNB20 plasmid, containing a 1.3-kilobase pair promoter segment, showed the same tissue-specific expression, although beta-galactosidase activity was detected only in a few photoreceptor cells and not in pinealocytes, a pattern similar to that reported by others for the same fragment(22) .

The specificity and strength of the 124-bp promoter addressed the search for binding sites of tissue-specific trans-acting factors inside its sequence.

Identification and Analysis of Nuclear Factor Binding Sites

To identify DNA/protein binding sites in the 124-bp promoter region immediately upstream of the transcription start site, we prepared nuclear extracts from either IRBP-expressing (retinoblastoma Y79 and Weri Rb-1) or nonexpressing (epithelioid HeLa and lymphoblastoid H9) cell lines and performed DNase I footprinting analysis of a HindIII-RsaI fragment that contained the promoter fragment from -123 to +18. We observed that an area of DNase I protection, between -45 and -58, which we termed ``A,'' could be detected only after incubation with nuclear extract from retinoblastoma cell lines (Fig. 2, lanes 3 and 12). Another area of DNase I protection, between -86 and -106, which we termed ``B,'' was detectable after incubation with extracts from all cell lines, although with different intensities (Fig. 2, lanes 3, 8, and 12-14). In order to test the sequence specificity of the A and B protein binding sites, we synthesized double-stranded oligonucleotides containing the protected sequences (shown in Fig. 3, together with the sequence of the promoter fragment). Unlabeled A and B oligonucleotides in molar excess with respect to the labeled probe in the footprinting assay were both able to compete the binding of nuclear factors to the respective A and B elements (Fig. 2, lanes 4 and 5 and lanes 9 and 10, respectively). We also observed in several experiments that the competition of binding to A in the presence of retinoblastoma cell nuclear extract gave rise to a more efficient protection in the B site, the size and intensity of which became comparable with that observed with HeLa nuclear extract.


Figure 2: DNase I footprinting of a segment of the IRBP promoter with nuclear extracts of different cell lines. A segment of plasmid pNB1 containing the sequence between -123 and +18 was labeled at its 3` end, incubated without or with nuclear extract (NE) from different cell lines, and treated with DNase I. Lanes 1 and 6, Maxam-Gilbert G + A sequencing reaction; lanes 2, 7, and 11, DNase I digestion pattern of the naked DNA incubated without cell nuclear extract; lanes 3-5, incubated with 28 µg of nuclear extract from Weri-RB1 cells without competitor (lane 3) or with competition by a 250-fold molar excess of a double-stranded A oligonucleotide (lane 4) or a double-stranded B oligonucleotide (lane 5); lanes 8-10, incubation with 30 µg of nuclear extract from HeLa cells, without (lane 8) or with competition by a 250-fold molar excess of either a double-stranded A oligonucleotide (lane 9) or a double-stranded B oligonucleotide (lane 10); lanes 12-14, incubation with 20 µg of nuclear extract from Y79, HeLa, and H9 cells, respectively, without competitor.



Taken together, these results suggest that nuclear factors present in cell lines that express IRBP bind two different elements in this short promoter sequence in a sequence-specific manner, and that one of these factors is contained only in nuclear extracts from cells that express IRBP. Moreover, the factors interacting with the A and B sites seem to interfere with each other for their respective binding.

Characterization of DNA-binding Proteins in Different Cell Nuclear Extracts

The binding properties of the sequence-specific factors binding to the A and B elements were further studied by gel retardation assay (GRA). In GRA with a labeled double-stranded oligonucleotide containing a GATTAA motif inside the -45 to -58 protected sequence, a retarded complex was detected with nuclear extract from the Weri-Rb1 retinoblastoma cell line and absent with HeLa cell nuclear extract (Fig. 4, lanes 1 and 2). This same complex, also detectable with Y79 retinoblastoma cell nuclear extract (data not shown), could be competed by the specific oligonucleotide (Fig. 4, lanes 3-7). The complex was more evident with low salt concentration in the binding buffer of GRA. Among already characterized transcription factors, members of the GATA family have been shown to bind sequences similar to GATTAA(23, 24) . The results of competition experiments in GRA with specific GATA oligonucleotide and of GRAs in the presence of anti-GATA1, anti-GATA2, and anti-GATA3 antibodies clearly excluded that the trans-acting factor binding to the A element is a member of the GATA family (data not shown).


Figure 4: Gel retardation assay with the A oligonucleotide and nuclear extracts from Weri-Rb1 and HeLa cell lines. A 5` end-labeled double-stranded A oligonucleotide was incubated with crude nuclear extract (NE) of Weri-RB1 (lanes 1 and 3-7) or HeLa cells (lane 2). The same unlabeled double-stranded oligonucleotide was included as a competitor (Comp.) at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 200-fold molar excess in lanes 4-7, respectively).



With a labeled double-stranded oligonucleotide containing the B element, we observed a major retarded complex with nuclear extracts from both Weri-RB1 and HeLa cells (Fig. 5, square bracket). This complex could be specifically competed by the same unlabeled oligonucleotide (Fig. 5, lanes 4-7). A computer analysis of the B sequence, which is relatively GC-rich, indicated sequence similarity with binding sites of two already characterized transcription factors, SP1 and AP2. A double-stranded oligonucleotide containing the consensus SP1 recognition sequence (Promega, Madison, WI) competed the formation of the complex as efficiently as the oligonucleotide containing the B element (Fig. 5, lanes 8-11), whereas no competition was observed with an oligonucleotide containing the consensus AP2 recognition sequence (Promega) (data not shown). Some bands that migrate more quickly and that are present in low abundance appeared to be also due to sequence-specific binding, being competed by the B oligonucleotide but not by the SP1 or the AP2 oligonucleotides.


Figure 5: Gel retardation assay with the B oligonucleotide and nuclear extracts from Weri-Rb1 and HeLa cell lines. A 5` end-labeled double-stranded B oligonucleotide was incubated with crude nuclear extract (NE) of Weri-RB1 (lanes 1 and 3-11) or HeLa cells (lane 2). The same unlabeled double-stranded oligonucleotide was included as a competitor (Comp.) at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 250-fold molar excess in lanes 4-7, respectively). A consensus SP1 oligonucleotide (Promega) was included as a competitor at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 200-fold molar excess in lanes 8-11, respectively).



From these observations we conclude that a factor, present in IRBP-expressing cells, binds a double-stranded oligonucleotide containing a GATTAA sequence. Furthermore, an ubiquitous factor, possibly SP1 according to the competition experiment, binds the B element.

Effect of Mutations in the 123-bp Promoter Fragment

The results of DNase I footprinting and GRA prompted us to prepare chimeric plasmids containing promoter segments in which the element that was able to bind a tissue-specific factor was mutated. The 124-bp IRBP promoter contains a GATTAA motif centered around -50 that is included within the A protected element. A second GATTAA motif is also present as an inverted repeat shortly upstream with an interval of 4 bp, although it is outside of the DNase I A protected element. To verify whether this second upstream motif might be recognized by the same factor that binds in the footprinting and GRA, once the protected one was mutagenized, we performed further competitions in GRA. Fig. 6shows that the specific complex formed using the A oligonucleotide as a labeled probe and Weri-Rb1 nuclear extract could be competed by the same specific oligonucleotide (Fig. 6, lanes 2-4) as well as by a longer oligonucleotide that included both GATTAA elements (AL oligonucleotide, Fig. 6, lanes 5-7). Binding ability was still shown, although more weakly, by an oligonucleotide containing the mutated downstream motif and the wild type upstream motif (1M oligonucleotide, Fig. 6, lanes 8-10), whereas when a single motif in a mutagenized form was present in the oligonucleotide (AM oligonucleotide), virtually no competition was observed (Fig. 6, lanes 11-13). For these reasons, to perform an efficient site-directed mutagenesis that excluded the possibility of any binding of the specific trans-acting factor, we prepared a plasmid (pNB22) derived from pNB21 containing mutations in both GATTAA motifs. Plasmid pNB22 was used to generate transgenic mice. Of the three pNB22 transgenic lines analyzed, none showed X-gal staining in photoreceptor cells or in other tissues.


Figure 6: Effect of different competitor oligonucleotides on binding of a tissue-specific factor to the A oligonucleotide. A 5` end-labeled double-stranded A oligonucleotide was incubated with crude nuclear extract of Weri-RB1 cells without competitor (Comp., lane 1) or with increasing molar excess (50-, 100-, and 200-fold molar excess with respect to the labeled probe for all competitor oligonucleotides) of the A oligonucleotide (lanes 2-4), AL oligonucleotide (lanes 5-7), 1M oligonucleotide (lanes 8-10), and AM oligonucleotide (lanes 11-13).




DISCUSSION

In this paper we show that a 124-bp fragment derived from the IRBP promoter fused to the lacZ reporter gene was able to drive expression of the reporter gene in transgenic mice. Moreover, we identify a cis-acting element located inside this fragment that is responsible for tissue-specific expression.

Comparison of 5`-flanking regions of the human and murine IRBP genes shows a high degree of sequence homology (76%) in the first 277 bp upstream of the transcription start site and with 79% homology in a second region between -1277 and -1526(12) , confirming the importance of the most proximal region in controlling IRBP gene. Our results demonstrate that the first 124 bp contain the elements that are necessary and sufficient for correct tissue localization of the gene product in the adult mouse. IRBP is a protein expressed early in embryonic development(25) , and our work is in progress to assess whether the 124-bp minimal promoter is also sufficient for correct temporal expression of the reporter gene during embryonic development.

Because it is known that IRBP gene transcription can be modulated by light (26) and by agents such as cAMP(6) , it is also interesting to evaluate whether this minimal promoter fragment might be able to modulate gene expression in response to different factors. Our preliminary sequence analysis in search of potential responsive cis-acting elements indicated that an AP1-like element and an ATF-like element overlap in a region centered around -115.

We have characterized the 124-bp promoter region sufficient for tissue-specific expression by DNase I footprinting and GRA using nuclear extracts prepared from IRBP-expressing cells (retinoblastoma cell lines) and nonexpressing cell lines. A previous report(32) , in which a characterization of DNA/protein interactions in the first 300 bp of the IRBP promoter was performed by GRA using retina extracts and nuclear extracts from Y79, shows that these extracts give rise to comparable retarded complexes, different from complexes formed when other tissue extracts were used.

Our results showed that the 124-bp promoter contains two protein-binding sites that are recognized by different factors. One, named B, binds apparently ubiquitous factors, among which is SP1 or SP1-related factor, and our data (^2)suggested that this B element represses transcription. The second, called A, that contains a GATTAA motif in its central part, binds a factor that is present only in nuclear extracts of IRBP-expressing cells and seems to be the most probable candidate for tissue-specific regulation of the gene. We demonstrated, by analysis of transgenic mice carrying the reporter gene fused to the 124-bp IRBP promoter, which had been modified in the A element by site-directed mutagenesis, that this element is essential for tissue-specific expression of the gene. In transgenic mice bearing the mutated version of the promoter, the expression of the reporter gene was completely abolished both in the retina and in the pineal gland.

The GATTAA element, besides playing an essential role in the IRBP promoter, probably by binding a tissue-specific factor, appears to be highly conserved in the 5`-flanking region of several photoreceptor-specific genes in humans and in other species of vertebrates(27, 28, 29) . Moreover, in different species of Drosophila this same GATTAA element is also contained in the 5`-flanking region of a variety of photoreceptor-specific genes. Site-directed mutagenesis of a sequence containing the GATTAA element in rhodopsin genes of Drosophila melanogaster has been shown to cause the loss of reporter gene expression(30) , similar to what we have demonstrated for the IRBP promoter. Thus, the importance of the GATTAA element is indicated both by the high degree of conservation of this sequence in the 5`-flanking regions of different photoreceptor-specific genes even in very different species (e.g. humans and species of Drosophila) and by its localization inside regions that have been demonstrated to be involved in gene regulation(27, 30) .

The IRBP promoter lacks a consensus TATA box. It is questionable whether the GATTAA motif centered around -50 functions as a site of interaction of TFIID components necessary for the assembly of the basal transcription machinery. Some photoreceptor-specific genes contain both the GATTAA motif and the TATA box. On the other hand, the sequence surrounding the transcription start site in IRBP matches the consensus PyPyA^1(T/A)PyPy, which is indicated as initiator element for TATA-less promoters(31) .

The identification of the tissue-specific factor(s) interacting with the cis-acting element that we have described will contribute substantially toward understanding the mechanism of regulation of the IRBP gene and, quite likely, the regulation of many retinal/visual transduction genes.


FOOTNOTES

*
This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro to (R. R. and A. A.), a grant from Consiglio Nazionale delle Ricerche ``Ingegneria Genetica'' (to D. N.), and by a Telethon grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: IBiG-University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy. Tel.: 39-10-353-8981; Fax: 39-10-353-8978.

(^1)
The abbreviations used are: IRBP, interphotoreceptor retinoid-binding protein; bp, base pair(s); X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; lacZ, gene encoding beta-galactosidase; GRA, gel retardation assay.

(^2)
N. Bobola, A. Albini, D. Noonan, and R. Ravazzolo, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Gianni Bruzzone for preparation of the photographs.


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