(Received for publication, September 8, 1995; and in revised form, October 30, 1995)
From the
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.
In vertebrate retinas, the photoreceptor layer is made up of two
morphologically and functionally distinct cell types, rods and cones
(Dowling, 1987). In most vertebrates, there is a single class of rods
and two to four different classes of cones. However, in amphibians
there are two classes of rods: an abundant (principal) rod with
spectral characteristics similar to rods from other species and a minor
rod with blue-shifted absorption properties (Witkovsky et al.,
1981). In all these cells, phototransduction is mediated by a group of
proteins that control cGMP metabolism, e.g. opsin, transducin
subunit, arrestin, cGMP phosphodiesterase subunits, and cGMP
channel (Hargrave and McDowell, 1992). There are isoforms of each of
these proteins expressed in rods and others expressed in cones
(Dowling, 1987; Lerea et al., 1986; Nir and Ransom, 1992;
Hurwitz et al., 1985; Bognik et al., 1993). Studies
on the developmental regulation of several of these genes demonstrate
that the major control of expression occurs at the level of
transcription initiation (Treisman et al., 1988; Timmers et al., 1993). However, the mechanism(s) regulating the
photoreceptor cell-specific transcription in the vertebrate retina
remain as yet unknown.
A number of studies have focused on the identification of cis-acting DNA elements in the promoters of photoreceptor genes using transgenic mice. Retina-specific reporter gene expression has been found using the 5` upstream regions of the bovine and mouse rhodopsin (Zack et al., 1991; Lem et al., 1991), human red and green opsin (Wang et al., 1992), human and mouse blue opsin (Chen et al., 1994; Chiu and Nathans, 1994a, 1994b) human IRBP (Liou et al., 1991), and mouse rod arrestin (Kikuchi et al., 1993) genes. Biochemical studies have identified and partially characterized nuclear proteins that bind to the 5` upstream regions of rhodopsin (Morabito et al., 1991; Yu et al., 1993; Sheshberadaran and Takahashi, 1994), transducin (Ahmad et al., 1994), and arrestin (Kikuchi et al., 1993). Three sites, Ret 1/PCE1, Ret 2/3, and glass-like, have been found in both mammalian and chicken rhodopsins. Additional transcription factors expressed in the retina have been identified by molecular cloning (Swaroop et al., 1992; Akazawa et al., 1992), and some bind to sequences in the rhodopsin gene (reviewed in Kumar and Zack, 1995). These studies suggest that control of photoreceptor gene transcription may involve a number of different transcription factors that act in concert to produce the unique developmental and cell-specific expression pattern.
Xenopus offers a number of specific advantages as a model system in which to study molecular mechanisms that regulate retinal development and gene expression. First, Xenopus embryology and development has been described in great detail (Nieuwkoop and Faber, 1967). Embryos can be produced in vitro and develop completely outside the female, thus allowing precise manipulation at any time after fertilization (Hamburger, 1960). Second, retinal development proceeds quickly; precursor cells develop to produce the layers of the adult retina by about 3 days post-fertilization (Holt et al., 1988). Third, foreign genes can be introduced into the retina and brain of Xenopus embryos by microinjection techniques or transfection with Lipofectin (Holt et al., 1990; Harris et al. 1995). To study the mechanisms of rod-specific transcription of phototransduction genes, we report here the cloning and characterization of the Xenopus rhodopsin gene, encoding the most abundant phototransduction protein. Furthermore, we show that sequences upstream of the Xenopus rhodopsin gene drive the head-specific expression of a reporter gene in transient transfections of developing Xenopus embryos using Lipofectin (Holt et al., 1990) and by microinjecting early stage blastomeres (Huang and Moody, 1993). This approach will allow a comprehensive study of the cis-acting elements in Xenopus phototransduction genes.
Figure 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.
Figure 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 +1) 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.
In order to demonstrate that XOP1 is the rhodopsin found in the abundant (principal) rod cell, the cDNA was expressed in COS1 cells and the UV-visible absorbance properties determined. To facilitate purification of the expressed protein, the C-terminal 7 amino acids were replaced with the last 14 amino acids of bovine rhodopsin, introducing the epitope for the monoclonal antibody ID4 (Molday and MacKenzie, 1983) into XOP1. After transient transfection of COS1 cells and incubation of the cells with 11-cis-retinal, the visual pigment was solubilized with dodecyl maltoside and purified by immunoaffinity chromatography (Oprian et al. 1987). The UV-visible absorbance spectra had a maximal absorbance at 500 nm, identical to bovine rhodopsin (Fig. 3). This value agrees with that obtained from microspectrophotometry of the abundant rod in Xenopus retina following regeneration with 11-cis-retinal (Witkovsky et al. 1981). Thus, XOP1 encodes the abundant rhodopsin in the adult Xenopus retina.
Figure 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.
The size of the mRNA was determined by Northern
analysis, which showed a single band of 1.7 kb found only in retinal
RNA (Fig. 4A). This size is similar to that found to be
expressed in tadpole heads (Saha and Grainger, 1992). In order to
determine the transcription initiation site, primer extension was
performed with two different antisense primers, P9 and P10. A number of
extension products differing in their relative intensities were
obtained using retinal RNA (Fig. 4B). The major
extension products were 42 bp with P9 and 120 bp with P10, and this
nucleotide is designated as +1 (Fig. 2). Although the size
of the extension product agrees with that found by RACE PCR, the gene
contains a T instead of a G as found in the cDNA at +1. The
transcription initiation site was also confirmed using
poly(A) RNA and P9; the largest extension product
mapped to +1, although the major product mapped to +2. The
differing intensities found in the two RNA preparations may reflect
heterogeneity in the frogs used to prepare the different samples, or
slight differences in primer specificity in the two preparations. There
were additional minor products, reproducibly found in primer
extensions, that occurred at +5 and +6 (Fig. 3B). The existence of multiple extension products
has been reported for a number of genes and is consistent with the lack
of a consensus TATA box in the rhodopsin gene (see below).
Figure 4: Rhodopsin transcript analysis. A, Northern blot. 2 µg 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 µg 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.
Rhodopsin
genes characterized thus far are present in a single copy in the
genome. However, Xenopus contains a pseudotetraploid genome
(Graf and Kobel, 1991), which raises the possibility of multiple copies
for rhodopsin in this species, all of which might be expressed in the
retina. To investigate this possibility, Southern blots were performed
using Xenopus genomic DNA. Using three different enzymes,
multiple bands of similar intensity, including the band expected from
genomic clone gopR1, were observed, even after high stringency washing (Fig. 5A). This suggests that there are four alleles of
rhodopsin in Xenopus. Further evidence for multiple alleles
was found when intron 1 of the rhodopsin gene was amplified from Xenopus genomic DNA by PCR. Four products, of sizes 368, 500,
550, and 650 bp were found (Fig. 5B, lane 1).
Comparisons with the control XOP1 phage (lane 6) identified
the 368-bp product as arising from this gene. The 500- and 550-bp
products amplified to the same level as the 368-bp product, while the
650-bp product was slightly less intense. Comparison of the primer
sequences with the Xenopus violet cone opsin ()and
with other cone opsins from chicken (Okano et al., 1992)
showed little homology and thus would not be expected to amplify under
these PCR conditions. Thus, in Xenopus, there are at least
four genes encoding rhodopsin or a highly homologous opsin perhaps
expressed in the green rod (Witkovsky et al., 1981) Further
work is under way to obtain the sequence of the novel PCR products.
Figure 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
gopRI (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.
Figure 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.
Figure 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 = 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.
The transcriptional activity of the 5.5-kb rhodopsin upstream fragment was also tested in another preparation of embryo heads whose outer epidermal layer had been manually peeled to potentially improve the transfection efficiency in the eye vesicle. This preparation gave an average of a 3-fold enhancement in the luciferase activity driven by pCMVluc compared to that observed in EDTA-treated heads. However, luciferase activity from pXOP(-5500/+41)luc varied widely (Fig. 7, Experiment B). Therefore, although improved access to retinal precursors is achieved by peeled heads, uncontrolled variation makes the study of the opsin promoter difficult.
To specifically target pXOP(-5500/+41)luc to a large population of retinal precursor cells, the reporter DNA was microinjected bilaterally into cleavage stage blastomeres that contribute significant numbers of cells to the stage 42 retina (D1, 8-cell embryo; D111, 32-cell embryo; Kline and Moody(1990) and Huang and Moody(1993)). Following injection of the plasmid DNA, embryos were cultured to stage 42 and assayed for luciferase as before. In injected 32-cell embryos, luciferase activity observed using pXOP(-5500/+41)luc was >200-fold higher than that obtained using pGL2 (Fig. 7, Experiments D and E). Comparable luciferase activity was also observed in embryos injected at the 8-cell stage with pXOP(-5500/+41)luc and this was 2.8% of that obtained using pCMVluc. Therefore, luciferase activity observed upon blastomere injection of retina progenitor blostomeres with pXOP(-5500/+41)luc confirms the transcriptional activity of rhodopsin upstream sequence observed in transient embryo transfections. Further, blastomere injections yielded >3-fold higher luciferase activity using pXOP(-5500/+41)luc than that observed in transient transfection of EDTA-treated heads.
Taken together, the results of these three approaches: transfection of EDTA-treated heads, transfection of peeled heads, and blastomere injection, indicate that the 5.5-kb fragment contains transcriptional control sequences of the rhodopsin gene.
As a first step toward identifying cis-acting
elements controlling the rod cell-specific expression of the Xenopus rhodopsin gene, we have characterized the gene and
expression products, and identified transcriptional activity in
upstream sequences. The Xenopus gene XOPI has an overall
structural organization conserved with other vertebrate rhodopsin
genes. Both sequence comparisons and functional expression of the cDNA
in COS1 cells has identified XOP1 as encoding a rhodopsin. Xenopus has two rod cells expressing distinct rhodopsins (Rohlich et
al., 1989), one absorbing at 520 nm (red rods) and other at 445 nm
(green rods, Witkovsky et al., 1981). The red rod is by far
the more abundant cell, outnumbering green rods by greater than 10-fold
(Rohlich et al., 1989). The abundance of XOP1 in the retinal
cDNA library suggested that it is the rhodopsin in the abundant rod
cell. By expressing the cDNA in COS1 cells, we have identified the
absorbance maximum of XOP1 to be 500 nm, when regenerated with
11-cis-retinal (A). This is the wavelength of the
abundant red rod pigment, when measured in retinas that were bleached
and then regenerated with 11-cis-retinal (Witkovsky et
al., 1981). Normally, Xenopus visual pigments are formed
from 11-cis-dehydroretinal (A
), which leads to a
20-nm red shift in the absorption maximum (Witkovsky et al.,
1981). Thus the COS1 transfection experiments show that XOP1 encodes
the rhodopsin from the red rod.
A Xenopus rhodopsin cDNA (Saha and Grainger, 1992) has been isolated from a tadpole library and has several nucleotide and amino acid differences with XOP1. Although Southern blots and PCR of genomic DNA suggests multiple alleles for rhodopsin (Fig. 5), efforts to identify the unique sequences by PCR using a primer to the tadpole 5`-untranslated region were unsuccessful (data not shown). Based on the unusually high degree of sequence identity, both in the coding and untranslated regions, it is unlikely that the tadpole cDNA and the one reported here arise from different genes since most Xenopus alleles show more than 4% variation in the coding region (Graf and Kobel, 1991). Moreover, the 5` nucleotides (1-222 nucleotides) found in the tadpole cDNA are most similar to an unrelated gene (data not shown), and thus are probably an artifact of library construction. The source of other variants is unclear. Isolation of additional rhodopsin alleles, for example using PCR of intron 1, will permit complete characterization of rhodopsin genes in Xenopus.
To study the Xenopus rhodopsin promoter, we have developed and utilized an assay based
on transient embryo transfections, allowing the analysis of
retina-specific gene xpression in intact Xenopus embryonic
tissue. Using this approach, we have seen high levels of reporter gene
expression in heads transfected with 5.5 kb of upstream sequence. We
have further shown that as little as 600 bp also efficiently drives
luciferase expression in this assay. ()Moreover, we have
extended this approach to other Xenopus genes, including
transducin
-subunit. (
)Studies of mammalian retinal
genes have commonly employed transgenic mice, which require a number of
individual lines and also exhibit position effects of the introduced
transgene. Additionally, retinal cell lines and dissociated primary
cell culture systems for rhodopsin promoter studies are done outside
the normal conditions for retinal development. Thus, the transient
embryo transfection-based promoter assay provides an alternate method
for the detection of transcriptional activity from genomic sequences.
When combined with the use of blastomere injection, it provides a quick
and powerful tool to test the activity and cell specificity of
cis-acting elements controlling retinal genes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L07770 [GenBank]and U23808[GenBank].