From the Biochemistry Section, Laboratory of Retinal
Cell and Molecular Biology, and the
Immunology and Virology
Section, Laboratory of Immunology, National Eye Institute, National
Institutes of Health, Bethesda, Maryland 20892-2740 and the
¶ Departments of Cellular Biology and Anatomy and of
Ophthalmology, Medical College of Georgia,
Augusta, Georgia 30912-2000
Received for publication, August 15, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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We have characterized NORPEG, a novel
gene from human retinal pigment epithelial cells (ARPE-19), in which
its expression is induced by all-trans-retinoic acid. Two
transcripts (~3 and ~5 kilobases in size) have been detected
for this gene, which is localized to chromosome band 5p13.2-13.3.
Placenta and testis showed the highest level of expression among
various human tissues tested. Six ankyrin repeats and a long
coiled-coil domain are present in the predicted sequence of the NORPEG
protein, which contains 980 amino acid residues. This ~110-kDa
protein was transiently expressed in COS-7 cells as a FLAG fusion
protein and immunolocalized to the cytoplasm. Confocal microscopic
analysis of the NORPEG protein in ARPE-19 cells showed threadlike
projections in the cytoplasm reminiscent of the cytoskeleton.
Consistent with this localization, the expressed NORPEG protein showed
resistance to solubilization by Triton X-100 and KCl. An ortholog of
NORPEG characterized from mouse encoded a protein
that showed 91% sequence similarity to the human NORPEG protein. The
expression of Norpeg mRNA was detected in mouse embryo
at embryonic day 9.5 by in situ hybridization, and the
expression appears to be developmentally regulated. In adult mouse, the
highest level of expression was detected in the seminiferous tubules of testis.
The retinal pigment epithelium
(RPE)1 is a monolayer of
highly differentiated cells located between the choroid and neural retina in the eye (1, 2). It provides nutrients to photoreceptor cells
and contains melanin pigments that absorb excess light radiation. RPE
cells are polarized: the basolateral membrane side encounters choroidal
circulation, whereas the apical microvillus membrane side faces
photoreceptors. These cells are indispensable for the regeneration of
11-cis-retinal, the visual chromophore, and carry out the
phagocytosis and degradation of photoreceptor outer segment discs
undergoing circadian shedding (1, 3). The RPE also plays a
critical role in infectious and inflammatory diseases affecting the
retina (4), and macular degeneration, a major visual system disorder
associated with aging, has been attributed to RPE dysfunction (5,
6).
Several cell lines derived from human RPE have been recently
established (7-9). ARPE-19, a rapidly growing cell line established by
Dunn et al. (8), is noted for its ability to retain many structural and functional characteristics of intact RPE. ARPE-19 cells
exhibit a distinctly epithelial morphology, noticeable pigmentation, and polarized distribution of cell-surface markers (8, 10). They
express several genes specific for the RPE; assimilate photoreceptor outer segment discs by phagocytosis; and show polarized expression of
reduced folate transporter-1, folate receptor- To study gene expression in the RPE, we have constructed a cDNA
library with poly(A)+ RNA preparations isolated from
ARPE-19 cells. A cDNA clone identified during the initial
characterization of this library was found to represent a novel human
gene. We have further characterized this gene and shown that its
expression is regulated by retinoic acid. We have also characterized an
ortholog of this gene from mouse, and its expression, when analyzed by
in situ hybridization, appears to be developmentally regulated.
Cell Culture and cDNA Cloning--
Human retinal pigment
epithelial cells (ARPE-19), a rapidly growing human RPE cell line, were
grown at 37 °C in a humidified atmosphere of 5% CO2 in
Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin as described previously (12). Cells were grown on 100-mm
culture dishes and treated with all-trans-retinoic acid when
they were 60-80% confluent.
Total RNA was isolated from ARPE-19 cells using RNAzol B (Tel-Test,
Inc., Friendswood, TX). Poly(A)+ RNA fractions were
separated using oligo(dT)-cellulose and used for cDNA synthesis and
library construction using a ZAP Express cDNA synthesis kit and ZAP
Express cDNA Gigapack III Gold cloning kit (Stratagene, La Jolla,
CA). Poly(A)+ RNA preparations from ARPE-19 cells or mouse
testis were used for reverse transcriptase-PCR. The RNA preparations
were reverse-transcribed with an oligo(dT) primer, and the first strand
cDNA preparations were used as template for PCR. Rapid
amplification of cDNA ends (RACE) was performed using 5'-and
3'-RACE systems obtained from Life Technologies, Inc. (14). Sequencing
of cDNA clones and PCR amplification products was performed on an
Applied Biosystems 370A DNA sequencer using the Taq dideoxy
termination sequencing kit (PerkinElmer Life Sciences).
DNA and protein sequences were analyzed using the BLAST similarity
search program supported by NCBI. Multiple sequence alignment was performed using the ClustalW program (15). The Paircoil program was
employed for predicting coiled-coil regions (16). ExPASy proteomic
tools were used for analyzing protein physicochemical properties.
Chromosomal Localization--
A human genomic P1 library was
screened by PCR using primers 5'-TGACCCAGCTCAAACAAC and
5'-ACCATTGCATCAGTCATAGCAGC, designed from the NORPEG
cDNA sequence (Genome Systems, Inc., St. Louis, MO). The identity
of one of the P1 clones thus obtained was verified by PCR using
different sets of primers and by sequencing some of the amplification
products. Purified DNA from this clone was labeled with
digoxigenin-dUTP by nick translation and used as a probe for in
situ hybridization. The labeled probe was combined with sheared
human DNA and hybridized to metaphase chromosomes derived from
phytohemagglutinin-stimulated peripheral blood lymphocytes in a
solution containing 50% formamide, 10% dextran sulfate, and 2× SSC.
Specific hybridization signals were detected by incubating the
hybridized slides in fluorescein-conjugated anti-digoxigenin antibodies, followed by counterstaining with
4,6-diamidino-2-phenylindole.
Northern Blot Analysis--
RNA preparations were subjected to
agarose gel electrophoresis in the presence of formaldehyde,
transferred to a Nytran membrane (Schleicher & Schüll), and
hybridized with a cDNA probe labeled with 32P (17).
Hybridization was carried out using QuickHyb hybridization solution
(Stratagene) containing labeled probe (~2 × 106
cpm/ml) and denatured salmon sperm DNA (0.1 mg/ml) at 68 °C for 1 h. The blots were then washed under stringent conditions and exposed to Kodak XAR film or analyzed using a PhosphorImager (STORM 860, Molecular Dynamics, Inc., Sunnyvale, CA).
Peptide Antibodies--
Antibodies were produced against two
synthetic peptides designed from the predicted sequences of the human
and mouse NORPEG proteins. The first peptide (NORPEG-28,
CENGDAEKVASLLGKKGAS) contained a cysteine residue at the N terminus in
addition to amino acids 28-45 of the human or mouse NORPEG protein.
The second peptide (NORPEG-961, DVQKVLKQILTMCKNQSQKK) contained amino
acids 961-980 of the human or amino acids 960-979 of the mouse NORPEG
protein. The peptides were synthesized, purified to >90% by high
pressure liquid chromatography, conjugated to keyhole limpet
hemocyanin, and used for immunization of rabbits (Bethyl Laboratories,
Inc., Montgomery, TX). The antisera (anti-NORPEG-28 and
anti-NORPEG-961) were purified using immunoaffinity columns prepared
with peptides linked to agarose using cyanogen bromide. The
affinity-purified antibody preparations were passed over keyhole limpet
hemocyanin immunosorbent to remove keyhole limpet hemocyanin immunoreactivity.
Expression of FLAG Fusion Protein in COS-7
Cells--
Poly(A)+ RNA preparations isolated from ARPE-19
cells were reverse-transcribed with 5'-AGAGCTCTCCTGTGCTGA
(corresponding to bp 3465 to 3448 of NORPEG cDNA)
as a primer using Superscript II reverse transcriptase (Life
Technologies, Inc.). The reaction mixture was treated with RNase, and
an aliquot was subjected to PCR with high fidelity Pfu Turbo
DNA polymerase (Stratagene) employing 5'-GTGGAGCAGCCAGCTGGGTC and
5'-CATCAGGACCAGACCTC as sense and antisense primers, respectively. The
amplification product, corresponding to bp 16-3327 of
NORPEG cDNA, was cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). This plasmid DNA was used as a template for
PCR with primers 5'-cttgcggccgcCATGAAGAGCTTGAAAGCG and
5'-agctggatccCTTCTTTTGAGACTGGTTTT. These primers contained
the restriction enzyme sites (underlined) NotI and
BamHI, respectively (extra nucleotides added to create restriction sites are shown in lowercase letters). Pfu Turbo
DNA polymerase was used as the enzyme. The PCR product, containing bp
111-3051 of NORPEG cDNA, was digested with restriction
enzymes and cloned into the NotI and BamHI sites
of the pFLAG-CMV-2 expression vector (Sigma). The fusion protein
containing the FLAG epitope at the N terminus was transiently expressed
in COS-7 cells (CRL-1651, American Type Culture Collection). The cells
were transfected with plasmid DNA using LipofectAMINE reagent (Life
Technologies, Inc.) and allowed to grow for 72 h.
The extracts prepared from transfected cells were analyzed for
expressed FLAG fusion protein by Western blotting using anti-FLAG M2
monoclonal antibody (Sigma) or antibody preparations against NORPEG.
SDS-polyacrylamide gel electrophoresis was carried out using
NUPAGE 4-12% BisTris gels and MOPS/SDS running buffer (Novex, San Diego, CA), and then the protein bands from the gels were electroblotted onto nitrocellulose membranes. The WesternBreeze chromogenic immunodetection system (Novex), which utilizes alkaline phosphatase-conjugated secondary antibody, was used for detecting immunoreactivity.
The expression of FLAG fusion protein was also analyzed by
immunofluorescence microscopy. COS-7 cells were grown to 60-80% confluence on 8-well glass chamber slides (Nalge Nunc, Naperville, IL)
and transfected with the NORPEG construct using LipofectAMINE reagent
as described above. The cells were then allowed to grow for 48 h
before fixing for 5 min in 1:1 methanol/acetone mixture precooled to
Laser Scanning Confocal Microscopic Analysis of the NORPEG
Protein in ARPE-19 Cells--
The ARPE-19 cells were grown on Nunc
8-well chamber slides coated with laminin and were allowed to
differentiate for at least 4 weeks (12). The cells were fixed with
ice-cold methanol and blocked with blocking solution (Novex) for 1 h at room temperature. Cells were incubated with anti-NORPEG-28
antibody preparations for 3 h in a humidified chamber at room
temperature and then were washed in antibody wash solution (Novex).
Incubation with 0.1% pre-bleed serum or with buffer only served as a
negative control. After rinsing, all samples were incubated overnight
at 4 °C with fluorescein-conjugated AffiniPure goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a
dilution of 1:100. Cells were optically sectioned (z series)
using a Nikon Diaphot 200 laser scanning confocal imaging system
(Molecular Dynamics, Inc.). Images were analyzed using the Image
Display 3.2 software package (Silicon Graphics, Mountain View, CA).
Immunohistochemistry--
To examine the expression of the
NORPEG protein, testes and eyes from 5-6-week-old albino (ICR) mice
were enucleated, frozen immediately in Tissue-Tek OCT (Miles
Laboratories, Elkhart, IN), and sectioned at 10-µm thickness.
Immediately before the experiment, sections were fixed in ice-cold
acetone for 5 min. Endogenous peroxidase activity was quenched by
incubating the sections for 15 min with the peroxidase solution.
Sections were washed in phosphate-buffered saline and blocked for
1 h with blocking solution. Sections were incubated with
anti-NORPEG-28 or anti-NORPEG-961 antibody preparations. Immunoreactivity was detected by peroxidase staining using reagents from Dako Corp. (Carpinteria, CA).
In Situ Hybridization--
In situ hybridization was
performed by Phylogeny Inc. following the procedure of Lyons
et al. (18) as described before (19). The cRNA probes were
prepared from linearized cDNA templates (bp 2232-2619 region of
mouse Norpeg cDNA cloned into the pBluescript II
KS+ vector) using an in vitro transcription kit
(Ambion Inc.) and 35S-UTP (Amersham Pharmacia Biotech) and
subjected to alkali hydrolysis to give a mean size of 70 bases.
Hybridization was carried out at 52 °C in a solution containing 50%
deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl
(pH 7.4), 5 mM EDTA, 10 mM NaPO4,
dextran sulfate, 1× Denhardt's solution, 50 µg/ml total yeast RNA,
and ~60,000 cpm/µl 35S-labeled cRNA probes (antisense
or sense). The sections were then subjected to stringent washing at
65 °C in 50% formamide, 2× SSC, and 10 mM
dithiothreitol. Following washing and RNase A treatment, the slides
were dehydrated, dipped in Kodak NTB-2 nuclear track emulsion, and
exposed for 2-3 weeks in light-tight boxes with desiccant at 4 °C.
Photographic development was carried out using Kodak D-19. The slides
were counterstained lightly with toluidine blue and analyzed using both
the light- and dark-field optics of a Ziess Axiophot microscope.
Identification and Characterization of NORPEG cDNA from Human
and Mouse--
A cDNA library was constructed using
poly(A)+ RNA fractions isolated from the human RPE cell
line ARPE-19. During the characterization of this library, we found a
cDNA clone whose sequence did not show any similarity to published
sequences at the time of its isolation. The complete cDNA sequence
for this novel gene was obtained using 5'- and 3'-RACE techniques and
by reverse transcriptase-PCR. The 4925-bp cDNA sequence for the
gene, which we termed NORPEG (novel
retinal pigment epithelial cell
gene; GenBankTM/EBI accession number AF155135),
shows an open reading frame with an initiation codon at position 112 and a stop codon (TAA) at position 3052, thus leaving a long
3'-untranslated region. It should be noted that a cDNA clone
(mRNA for the KIAA1334 protein; GenBankTM/EBI
accession number AB037755) as well as a partial cDNA clone (DKFZp564G013; GenBankTM/EBI accession number AL050011)
matching the sequence reported here recently appeared in the data base.
We have also obtained the cDNA sequence for an ortholog of
NORPEG that is expressed in mouse testis employing reverse
transcriptase-PCR and RACE techniques. The mouse Norpeg
cDNA (GenBankTM/EBI accession number AF274866) is 4879 bp long and contains an open reading frame with an initiation codon at
bp 221 and a stop codon (TAA) at position 3158. The mouse
Norpeg cDNA showed 81% sequence identity to the human
NORPEG cDNA.
The predicted human and mouse NORPEG proteins contain 980 and 979 amino
acid residues, respectively (Fig.
1A). They show 84% identity
and 91% similarity. The human NORPEG protein is predicted to have a
molecular mass of 110,042 Da, a pI of 5.83, and six potential
N-glycosylation sites. In comparison, the mouse homolog is
predicted to have a molecular mass of 108,852 Da, a pI of 5.88, and
four potential N-glycosylation sites. The domain
organization of the NORPEG protein is shown in Fig. 1B.
Using BLAST analysis, the N-terminal regions of both human and mouse
NORPEG protein sequences were found to be similar to several ankyrin
repeat-containing proteins. Six ankyrin repeats spanning amino acids
18-240 were identified by ProfileScan for the human NORPEG protein.
Alignment of the six repeats with the consensus sequence for the
ankyrin motif is shown in Fig. 1C. In mouse, the six ankyrin
repeats spanned amino acids 20-242. BLAST analysis also showed that a
large segment of both the human and mouse NORPEG proteins (amino acids
400-900) shares similarity with coiled-coil helical domains of several proteins, including restin (Reed-Steinberg
cell-expressed intermediate filament-associated protein),
myosin heavy chain, and golgin-245 (20-22). Further analysis indicated
11 coiled-coils between amino acids 341 and 947 for the human NORPEG
protein (Fig. 1D). In comparison, the mouse NORPEG protein
contains 10 coiled-coils between amino acids 341 and 946 (data not
shown).
Chromosomal Localization and Gene Structure of Human
NORPEG--
The chromosomal localization of the human
NORPEG gene was determined by PCR screening of a chromosome
mapping panel (NIGMS Human Genetic Mutant Cell Repository at the
Coriell Institute for Medical Research, Camden, NJ) with
oligonucleotides 5'-TGACCCAGCTCAAACAAC and 5'-ACCATTGCATCAGTCATAGCAGC.
These primers spanned the NORPEG cDNA sequence region of
bp 2249-2462. A 214-bp amplification product, whose identity was
verified by DNA sequencing, was observed when a DNA preparation from a
chromosome 5 monochromosomal somatic cell hybrid was used as the
template. Further confirmation of the localization of the
NORPEG gene to human chromosome 5 was obtained by
fluorescence in situ hybridization. The DNA preparation obtained from a genomic P1 clone containing the NORPEG gene
was used as the probe. The probe hybridized to the short arm of
chromosome 5 (Fig. 2A). The
localization was further verified by co-hybridizing this probe with a
probe specific for the cri-du-chat locus, which has been
previously been mapped to the long arm, 5q22 (Fig. 2B). Measurements of 10 hybridized chromosomes showed that the
NORPEG gene is located 29% of the distance from the
centromere to the telomere of chromosome arm 5p; this is an area
corresponding to band 5p13.2-13.3 (Fig. 2C). Also in
support of this localization, several genomic clones designated as
chromosome 5 clones that appeared recently in the data base (high
throughput genomic sequencing) were found to contain segments of DNA
sequences of NORPEG cDNA. Comparison of the cDNA
sequence of the NORPEG gene with genomic clone sequences
(AC008950, AC016602, AC025269, AC025754, AC025769, AC026801, AC055815,
and AC008690) allowed us to determine the exon/intron boundaries in
this gene. As illustrated in Fig. 2D, the NORPEG
gene contains 18 exons and spans >120 kb.
Expression of NORPEG mRNA in Human Tissues and Cells and Its
Regulation by Retinoic Acid--
RNA preparations obtained from
ARPE-19 cells were analyzed by Northern blotting using a cDNA probe
for the NORPEG gene. As shown in Fig.
3A, a strong signal was
observed at ~5 kb, with a weak signal at ~3 kb. Hybridization of a
human tissue RNA blot with this probe showed that the gene is also
highly expressed in placenta and testis (data not shown). Therefore,
RNA preparations from these two sources were also subjected to Northern
blot analysis (Fig. 3A). Like the ARPE-19 cells, placenta
showed a highly intense signal at ~5 kb, with a weak signal at ~3
kb. Interestingly, a reverse trend was observed in testis; the intense
signal was at ~3 kb, and the weak signal was at ~5 kb. These two
distinct mRNA species were subjected to further analysis. Blots
containing RNA preparations from ARPE-19 cells and testis were analyzed
with probes representing various segments of the NORPEG
cDNA. Several probes generated from bp 1-3000 of the cDNA
hybridized with both the ~5- and ~3-kb messages, whereas those
generated from the bp 3124-4774 region hybridized only with the
~5-kb message. As shown in Fig. 3B, a probe representing
the bp 2559-3000 region hybridized with both the ~5- and ~3-kb
messages, whereas a probe representing the bp 3124-3666 region
hybridized only with the ~5-kb message. Thus, it appears that the
~3-kb message lacks most of the long 3'-untranslated region present
in the ~5-kb message. NORPEG mRNA was also found to be
highly expressed in several human cancer cell lines (Fig.
3C).
NORPEG mRNA expression in ARPE-19 cells was induced by
all-trans-retinoic acid (Fig. 3D). The ~5-kb
transcript showed a concentration-dependent increase when
the cells were treated with retinoic acid for 48 h. A >5-fold
induction of this transcript was detected in the presence of 1 µM all-trans-retinoic acid. The ~3-kb
transcript, due to its low abundance in ARPE-19 cells, is not quite
visible in the autoradiogram presented. This is especially true in the case of untreated cells. However, signal for the shorter transcript is
clearly noticeable in lanes representing RNA samples from cells treated
with Expression of the Human NORPEG Protein in COS-7 Cells as a FLAG
Fusion Protein--
The entire coding region of the human
NORPEG cDNA was cloned into an N-terminal FLAG
expression vector, and the FLAG fusion protein was transiently
expressed in COS-7 cells. Immunofluorescence microscopic analysis of
these cells using an anti-FLAG monoclonal antibody showed intense
immunoreactivity in the cytoplasm (Fig. 4). A similar staining pattern was also
observed when anti-NORPEG-28 antibody was used as the primary
antibody.
The expressed NORPEG protein was also detected on a Western blot as an
~110-kDa band using an anti-FLAG monoclonal antibody preparation
(Fig. 4E). The immunoreactivity was associated with the
pellet fraction when the cell homogenate was centrifuged. Treatment of
the resulting pellet with Triton X-100 and 1.5 M KCl did
not result in the solubilization of the ~110-kDa protein. The
expressed protein was analyzed using antibody preparations against
peptide sequences from the N-terminal (amino acids 28-45) as well as
C-terminal (amino acids 961-980) regions of the NORPEG protein. Both
of these antibody preparations (anti-NORPEG-28 and anti-NORPEG-961)
were found to recognize the ~110-kDa band (Fig. 4, F and
G). The preimmune serum, as expected, did not show any reactivity toward this band (data not shown).
Analysis of the NORPEG Protein in Differentiated Human RPE
Cells--
Differentiated ARPE-19 cells are known to exhibit polarized
distribution of certain proteins (10, 12). Therefore, the distribution
of the NORPEG protein was analyzed in these cells. ARPE-19 cells were
allowed to differentiate and then immunostained using anti-NORPEG-28
antibody and fluorescein-conjugated secondary antibody. The
immunostained cells were subjected to laser scanning confocal
microscopic analysis. The cells were optically sectioned in a
horizontal and vertical plane. The distribution of immunoreactivity was
throughout the cytoplasm of the ARPE-19 cells (Fig.
5). The x-y scans
(horizontal optical sections) revealed a threadlike distribution of the
NORPEG protein throughout the cytoplasm. The x-z
scans (vertical optical sections) showed distribution throughout the
cells, indicating that expression is not limited to the apical or basal
membranes of the cells. Again, the vertical scans of the cells revealed
extensive expression throughout the cytoplasm. In experiments using
both antibodies, the pre-bleed controls showed minimal labeling of the
cells.
Analysis of Norpeg mRNA Expression in Mouse during
Development--
Norpeg mRNA expression was analyzed in
mouse embryo by in situ hybridization at various stages of
development. At 9.5 days, expression (as indicated by a high level of
silver grains) was detected in branchial arch mesenchyme, forebrain,
hindbrain, midbrain, and neural tube (Fig.
6). At 12.5 days, a hybridization signal was detected in the hindbrain, forebrain, lung, genital eminence, spinal ligaments around vertebrae and ribs, and around the cartilage of
ribs and nasal sinuses (Fig. 7). Signal
was also detected in frontonasal mass, mandibular arch, optic sulcus,
spinal ganglia and hind limb bud (data not shown). At 15.5 days,
expression was detected in the ventricular layer of neurons subjacent
to the neocortex, around the nasal sinuses, bronchioles of the lung, kidney, and around the vertebrae of the tail (Fig.
8). Signal was also seen in the olfactory
bulb (data not shown).
Analysis of a mouse tissue RNA blot with a Norpeg cDNA
probe showed that the Norpeg mRNA expression was very
high in testis compared with all other tissues (data not shown).
Therefore, we analyzed the expression of Norpeg mRNA in
adult mouse testis by in situ hybridization (Fig.
9). The mRNA expression was very high in seminiferous tubules that contain differentiating sperm.
Immunohistochemical Analysis of the NORPEG Protein in Mouse Testis
and Retina--
Mouse testis was subjected to immunohistochemical
analysis using anti-NORPEG-28 antibody. Fig.
10A (Testis) shows a
hematoxylin- and eosin-stained cross-section of mouse testis. The
arrow points to one of several seminiferous tubules. Each
tubule is surrounded by a lamina propria; and immediately internal to
this, the tubule is lined with the supporting cells (of Sertoli). Fig.
10B (Testis) shows a similar section of testis subjected to
immunohistochemistry. The supporting cells are intensely positive, as
indicated by the reddish-brown precipitate. Fig.
10C (Testis) shows a section incubated with the pre-bleed
control, where no positive reaction was observed.
Fig. 10 also shows the results from the immunohistochemical
analysis of mouse retina. Fig. 10A (Retina) shows a section
stained with hematoxylin and eosin for comparison with the other panels of retina subjected to immunohistochemistry procedures
(B-D, Retina). As shown in Fig. 10B
(Retina), there is an intense positive reaction for the NORPEG protein
in the ganglion cell layer and in Müller cell fibers spanning the
inner portion of the retina. The outer plexiform layer shows an intense
positive reaction, as does the RPE. Fig. 10C (Retina) is a
high magnification of the inner portion of the retina, showing the
intense positive reaction in ganglion cells and vertically oriented
Müller cell fibers. Fig. 10D (Retina) is a high
magnification demonstrating the positive reaction in the RPE. The
photoreceptor cells of the outer nuclear layer are negative for NORPEG.
There was also expression in the optic nerve (data not shown). The
pre-bleed controls showed no labeling (data not shown).
This study has resulted in the identification and characterization
of NORPEG, a novel gene localized to human chromosome 5, as
well as an ortholog of this gene from mouse. The proteins encoded by
human and mouse genes contain 980 and 979 amino acid residues, respectively, and share 91% sequence similarity (84% identity) between them. These proteins appear to represent a new gene family since they did not show any close similarity to published protein sequences in the data base. The closest match observed was 45% similarity (28% identity) between the N-terminal region of the NORPEG
protein and the protein sequence encoded by an mRNA that is
overexpressed in dog thyroid tissue following thyrotropin stimulation (GenBankTM/EBI accession number X99145).
The human and mouse NORPEG proteins contain identical structural
domains. The C terminus of the NORPEG protein contains a large
coiled-coil domain covering ~60% of the protein. This region shows
similarity to coiled-coil regions of several other proteins such as
restin, myosin heavy chain, and golgin-245 and could be involved in
self-aggregation or interaction with other proteins (20-22). The
second structural domain seen in the NORPEG protein (both human and
mouse) is an ankyrin repeat region. It contains six ankyrin repeats,
each composed of 33 amino acid residues, toward the N terminus. Ankyrin
repeats are known to be present in a large number of proteins and are
involved in protein-protein interactions (23). As many as 24 of these
repeats are present in ankyrin, the protein that anchors the
cytoskeleton to erythrocyte membranes (23, 24). I The open reading frame of the human NORPEG cDNA was
expressed as a FLAG fusion protein. It was recognized by an anti-FLAG monoclonal antibody and found to have the expected molecular mass of
~110 kDa. The fusion protein was identified as that of NORPEG based
on its immunoreactivity toward antibodies raised against peptides
designed from the N- and C-terminal regions of the predicted sequence.
Immunofluorescence microscopic analysis indicated that the fusion
protein is localized to the cytoplasm. The NORPEG protein appears to be
a cytoskeletal protein based on the threadlike staining in the
cytoplasm observed by laser scanning confocal microscopic analysis of
ARPE-19 cells. The resistance of the expressed NORPEG protein to
solubilization by Triton X-100 and 1.5 M KCl is also in
support of this conclusion (27).
The functional role of the NORPEG protein remains to be elucidated.
Nevertheless, the expression pattern of this gene could offer an
insight into its possible function. The RPE, testis, and placenta are
three sites where the highest level of NORPEG expression is
observed. In the eye, RPE cells are joined by tight junctions in the
region of the apical membrane. The RPE tight junctions form a portion
of the blood-retinal barrier (1). In testis, Norpeg
is expressed in seminiferous tubules, and the expression appears to be
localized to Sertoli cells. These cells, joined at their bases by tight
junctions to create a permeability barrier between the extratubular and
intratubular compartments, are part of the blood-testis barrier (28).
The cell type in placenta that expresses NORPEG is not yet
identified. But, syncytiotrophoblast cells in this organ constitute the
blood-placental barrier (29, 30). Thus, the high level of
NORPEG expression in the RPE, testis, and placenta would
indicate that the putative cytoskeletal protein encoded by this gene
could be involved in some type of barrier function. A second feature
that the RPE, testis, and placenta have in common is retinoid
metabolism and transport (3, 31, 32). Therefore, it is possible that
the NORPEG protein may play a key role in this process. An explanation
of the developmental expression pattern of NORPEG in
neuronal tissues as well as in prenatal kidney and lung will require
further functional characterization of the encoded protein.
Although NORPEG is mainly expressed as an ~5-kb transcript
in many tissues and cells analyzed, an ~3-kb transcript, the major form found in testis, was also detected. Both the ~5- and ~3-kb transcripts were also detected for mouse Norpeg (data not
shown). The ~5-kb transcript appears to represent the cDNA
sequence reported here for NORPEG. The ~3-kb transcript failed to
hybridize to the cDNA probes representing the ~2-kb-long
3'-untranslated region of the NORPEG cDNA sequence.
Thus, it appears that the difference in length of the 3'-untranslated
regions in these two transcripts is most likely due to the use of
alternative polyadenylation signals. Sequences similar but not
identical to the canonical AATAAA sequence are found at several
positions within the 3'-untranslated region. A large number of genes
are known to generate two or more transcripts due to the presence of
multiple polyadenylation sites within a 3'-terminal exon (33, 34). This
process results in the formation of multiple transcripts differing in
the length of their 3'-untranslated regions. These transcripts
generally exhibit altered stability, translatability, and
tissue-specific expression. This is due to the fact that the
3'-untranslated region is known to harbor various cis-regulatory elements controlling these parameters. The
3'-untranslated region of the ~5-kb NORPEG transcript
contains five AUUUA repeats, a regulatory element implicated in
mRNA instability (35). Thus, cis-regulatory elements
similar to this present in the 3'-untranslated region could be
responsible for the observed tissue-specific expression of the ~5-kb transcript.
The ~3- and ~5-kb transcripts of NORPEG could also be
generated by a different mechanism. Several genes are known to yield multiple transcripts by alternative 3'-terminal exon usage (33). In the
case of the ~5-kb transcript of NORPEG, the 3'-terminal exon consists of the last 81 bases of the coding region plus the whole
3'-untranslated region. This long exon could be replaced by an
alternative short exon(s) to form the ~3-kb transcript. The results
obtained by Northern blot analysis did not eliminate this type of
alternative splicing. The resulting shorter transcript will encode a
variant of the NORPEG protein with an altered C terminus in comparison
with the normal NORPEG protein encoded by the larger transcript.
Although there is no evidence for alternative splicing according to
expressed sequence tags found in the GenBankTM/EBI Data
Bank, further investigation is needed to rule out this possibility.
The reason for the preferential expression of the ~3-kb transcript of
NORPEG in testis is not known. However, testis-specific expression of transcripts with shortened 3'-untranslated regions has
been reported for several genes containing multiple polyadenylation signals (36-38). The shorter transcripts are comparatively more stable
since the truncation of the 3'-untranslated region effectively eliminates various elements contributing to the mRNA instability. The selection of stable transcripts is thought to be necessary to
compensate for the minimal transcription occurring during later stages
of spermatogenesis (38).
Interestingly, the expression of NORPEG mRNA in ARPE-19
cells is induced by all-trans-retinoic acid. The treatment
resulted in the increased expression of both the ~3- and ~5-kb
transcripts, indicating that the retinoid effect is at the
transcriptional level. A mechanism involving the transcript
stabilization mediated through the 3'-untranslated region should have
resulted in the preferential increase in the longer transcript. The
observed effect is similar to the transcriptional regulation reported
for fibroblast growth factor (int-2) transcripts in F9
cells, in which the induction by retinoic acid is not dependent on the
length of the 3'-untranslated region (39). Retinoic acid is a key
regulator of many biological functions, including cell differentiation,
proliferation, and development and it regulates the transcription of a
number of genes by its ability to bind specific nuclear receptors,
i.e. retinoic acid receptors and retinoid X receptors
(40-43). These receptors mediate their effect by binding to specific
DNA sequences (retinoic acid response elements and retinoid X response
elements) on the target genes. It remains to be elucidated whether the
observed increase in NORPEG mRNA in response to retinoic
acid treatment is a direct effect mediated through the retinoid
receptors interacting with retinoic acid response elements or retinoid
X response elements, possibly present in the regulatory region of the
NORPEG gene. Studies are currently underway to clone and
characterize the promoter region of the human NORPEG gene.
ARPE-19 cells do respond to all-trans-retinoic acid by
increasing the expression of retinoic acid receptor- In situ hybridization analysis of Norpeg mRNA
in developing mouse embryo (9.5-15.5 days gestation) showed that it is
highly expressed in nervous tissue that appears to be less
differentiated, in dividing neurons, in mesenchyme surrounding
developing cartilage, in spinal ligaments, and in prenatal kidney and
lung. The presence of the Norpeg message in the early
stages of embryonic development and its expression pattern during
development suggest that this gene may play an important developmental
role. Also, the fact that Norpeg expression is apparently
regulated by retinoic acid is in support of this role since retinoic
acid is a key player in early vertebrate development (45-47). In adult
mouse, Norpeg mRNA expression remains high in testis.
Interestingly, the expression is localized to seminiferous tubules that
contain differentiating sperm. Retinoic acid is known to play an
important role in spermatogenesis (48, 49), and since Norpeg
mRNA is up-regulated by retinoic acid, this gene could be a
potential target for retinoic acid in testis.
Expression of the NORPEG protein was detected in mouse retina in the
RPE, Müller cells, and ganglion cells by immunocytochemistry. It
will be of interest to determine whether this expression pattern is
related to the regulation of the Norpeg gene by
retinoic acid. Retinoic acid is produced in neural retina and the RPE
in the developing mouse eye, and the RPE is the principal site of
retinoic acid synthesis in postnatal and adult mouse eyes (50).
Cellular retinoic acid-binding protein I has been detected in early
differentiating ganglion cells (51). A retina-specific nuclear
receptor, which is thought to regulate the transcription of the
cellular retinaldehyde-binding protein gene through possible
interaction with the retinoic acid receptor and the retinoid X
receptor, is reported to be highly expressed in the RPE and
Müller glial cells (52).
In summary, we have identified a novel gene, NORPEG,
localized to chromosome 5 and expressed in cultured human RPE cells, where its expression is induced by all-trans-retinoic acid.
This gene is also highly expressed in human placenta, testis, and
several cancer cell lines. Two transcripts were detected for this gene. An ortholog of NORPEG expressed in mouse was also
characterized, and its expression appears to be developmentally
regulated. The NORPEG protein contains an ankyrin repeat domain as well
as a long coiled-coil domain and appears to be associated with the cytoskeleton. The precise role that this retinoic acid-responsive and
developmentally regulated novel gene plays in cell structure and
function remains to be elucidated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and
Na+-K+-ATPase (8, 11, 12). They are also
capable of expressing growth factors and cytokines (10,
13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. The slides were incubated in the presence of anti-FLAG M2
or anti-NORPEG-28 antibody for 1 h at 37 °C. Immunoreactivity was detected using the appropriate fluorescein-conjugated secondary antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Predicted amino acid sequences and structural
characteristics of human and mouse NORPEG proteins. A,
predicted amino acid sequences for human and mouse NORPEG proteins.
Identical amino acid residues are indicated by asterisks,
and evolutionarily conserved amino acid residues are indicated by
colons and periods. B, domain
organization of the NORPEG protein. aa, amino acids.
C, alignment of six ankyrin repeats of the human NORPEG
protein with the ankyrin (ANK) repeat consensus sequence
(the last sequence shown in the alignment; X is any amino
acid residue). D, Paircoil analysis of the human NORPEG
protein sequence shows 11 coiled-coils between amino acids 341 and
947.
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Fig. 2.
Chromosomal localization and gene structure
of human NORPEG. A,
fluorescence in situ hybridization analysis using a
NORPEG probe (indicated by arrows) showed that
the gene is localized to human chromosome 5. B,
co-hybridization of the NORPEG probe (indicated by
arrows) and a probe specific for 5q22 (indicated by
arrowheads) to chromosome 5. C, idiogram of
chromosome 5 showing the subchromosomal localization of
NORPEG to the 5p13.2-13.3 region (indicated by the
arrowhead). D, scale model of the
NORPEG gene structure. Exons are represented by
rectangles; horizontal lines represent intron
sequences. Introns >10 kb are broken by diagonal hatch
marks. The size of intron 3, identified by a question
mark, is unknown.
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Fig. 3.
Expression of human NORPEG
mRNA and its induction by all-trans-retinoic
acid. A, Northern blot analysis showed the presence of
two NORPEG transcripts ~3 and ~5 kb in size. The ~3-kb
message is abundant in testis (lane T), whereas the ~5-kb
message is abundant in placenta (lane P) and ARPE-19 cells
(lane A). Left and right panels are
x-ray films of the same blot exposed for 3 and 16 h, respectively.
B, the ~3-kb message lacks the long 3'-untranslated region
found in the ~5-kb message. A cDNA probe (left panel)
made from the coding region of NORPEG (bp 2559-3000)
hybridized with both the ~5- and ~3-kb mRNA species from the
ARPE-19 cells (lane A) and testis (lane T); but a
cDNA probe (right panel) made from the 3'-untranslated
region (bp 3124-3666) hybridized only with the ~5-kb mRNA.
C, NORPEG mRNA, predominantly the ~5-kb
form, was detected in several of the cancer cell lines analyzed:
promyelocytic leukemia HL-60 (lane 1), HeLa cell S3
(lane 2), chronic myelogenous leukemia K-562 (lane
3), lymphoblastic leukemia MOLT-4 (lane 4), Burkitt's
lymphoma Raji (lane 5), colorectal adenocarcinoma SW480
(lane 6), lung carcinoma A549 (lane 7), and
melanoma G361 (lane 8). D, NORPEG
mRNA expression in ARPE-19 cells was increased following
all-trans-retinoic acid treatment. Total RNA preparations
obtained from ARPE-19 cells treated for 48 h with various
concentrations of all-trans-retinoic acid were analyzed by
Northern blotting. The upper panel shows a blot probed with
a NORPEG cDNA probe, whereas the lower panel
shows 18 S and 28 S rRNA bands on an ethidium bromide-stained gel. The
chart shows the relative amount of radioactivity associated
with bands on the Northern blot as analyzed with a
PhosphorImager.
0.5 µM retinoic acid. Thus, it appears that the ~3-kb transcript is also induced by this treatment.
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Fig. 4.
Expression of the human NORPEG protein in
COS-7 cells as a FLAG fusion protein. The open reading frame of
the human NORPEG cDNA was cloned into the pFLAG-CMV-2
expression vector. COS-7 cells were transfected with the plasmid and
analyzed by immunofluorescence microscopy. The cells were fixed and
treated with the indicated primary antibody and then with a
fluorescein-conjugated secondary antibody. A, transfected
cells treated with anti-FLAG M2 monoclonal antibody (magnification × 100). B, transfected cells treated with anti-FLAG M2
monoclonal antibody (magnification × 400). C,
transfected cells treated with anti-NORPEG-28 antibody
(magnification × 400). D, control cells treated with
anti-FLAG M2 monoclonal antibody (magnification × 100). COS-7
cells transfected with the plasmid were also analyzed by Western
blotting. E, blot probed with anti-FLAG M2 monoclonal
antibody showing that the expressed protein was not solubilized by
Triton X-100 and high concentrations of KCl. Cells were separated from
the culture medium (lane 1) and then homogenized in
Tris-buffered saline. The homogenate was centrifuged, and the
supernatant (lane 2) was separated from the pellet
(lane 3). Supernatants were also obtained by sequential
extraction of this pellet with the following solutions: Tris-buffered
saline (lane 4) and Tris-buffered saline containing 0.5%
Triton X-100 (lane 5), 1% Triton X-100 (lane 6),
and 1.5 M KCl plus 0.5% Triton X-100 (lane 7).
The resulting final pellet was also analyzed (lane 8).
Molecular mass markers are shown in lane M. F,
blot probed with an anti-NORPEG-28 antibody preparation. Lane
M, molecular mass markers; lanes 1 and 2,
pellet and supernatant from detergent extraction, respectively.
G, blot probed with an anti-NORPEG-961 antibody preparation.
Lane M, molecular mass markers; lanes 1 and
2, pellet and supernatant from detergent extraction,
respectively.
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Fig. 5.
Analysis of the NORPEG protein in ARPE-19
cells by laser scanning confocal microscopy. The NORPEG protein in
ARPE-19 cells was detected with anti-NORPEG-28 antibody and
fluorescein-conjugated secondary antibody. A, horizontal
(x-y) scan; B, vertical
(x-z) scan; C, horizontal
(x-y) scan of the pre-bleed control.
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Fig. 6.
In situ hybridization analysis of
Norpeg mRNA expression in 9.5-day mouse
embryo. Dark-field micrographs are shown. Sections probed
with sense cRNA (A and B) showing low background
levels of silver grains served as controls. Antisense cRNA
(C, sagittal section; D, oblique sagital section
of two embryos) detected Norpeg mRNA expression, as
indicated by high levels of silver grains, in branchial arch mesenchyme
(ba), forebrain (fb), hindbrain (hb),
midbrain (mb), and neural tube (nt).
d, decidua; h, heart; ys, yolk
sac.
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Fig. 7.
In situ hybridization analysis of
Norpeg mRNA expression in 12.5-day mouse
embryo. The control sense cRNA probe (A and
B) showed a low background. Antisense cRNA
(C and D) detected Norpeg
mRNA expression in the hindbrain (hb), forebrain
(fb), lung (lu), and genital eminence
(ge). Expression was also detected around the cartilage of
the ribs (r) and nasal sinuses (ns) as well as in
areas that appear to be spinal ligaments around the vertebrae
(v) and ribs (r). h, heart;
li, liver; to, tongue; si, small
intestine; sc, spinal cord.
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Fig. 8.
In situ hybridization analysis of
Norpeg mRNA expression in 15.5-day mouse
embryo. The sense cRNA probe (A-C) showed a low
background. The antisense cRNA probe (D-F) detected
Norpeg mRNA expression in the ventricular layer of
neurons subjacent to the neocortex (cx), around the
nasal sinuses (ns), in bronchioles of the lung
(lu), in kidney (k), in genital eminence
(ge), and around the vertebrae of the tail (t).
hb, hindbrain; mb, midbrain; h, heart;
j, jaw; r, rib; bl, bladder;
oc, occipital cartilage; th, thymus;
sc, spinal cord; to, tongue; li,
liver; v, vertebrae; si, small intestine.
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Fig. 9.
In situ hybridization analysis of
Norpeg mRNA expression in adult mouse testis.
Antisense cRNA (A) showed high level expression of
Norpeg mRNA in seminiferous tubules (st) that
contain differentiating sperm. ta, tunica albuginea;
epi, epididymis. The sense cRNA control probe (B)
showed a low background level of silver grains as expected.
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Fig. 10.
Immunohistochemical analysis of NORPEG
expression in mouse testis and retina. Sections of testis and
retina were immunostained using anti-NORPEG-28 antibody and
peroxidase-conjugated secondary antibody. TESTIS:
A, hematoxylin- and eosin-stained cross-section shows one of
several seminiferous tubules (arrow). Each tubule is
surrounded by a lamina propria; and immediately internal to this, the
tubule is lined with the supporting cells (of Sertoli). B,
section stained with the antibody shows that supporting cells are
intensely positive as indicated by the reddish-brown color.
C, no staining was observed for the section incubated with
the pre-bleed control. RETINA: A, section stained
with hematoxylin and eosin for comparison. rgc, retinal
ganglion cells; ipl, inner plexiform layer; inl,
inner nuclear layer; opl, outer plexiform layer;
onl, outer nuclear layer; is, inner segment;
os, outer segment; rpe, retinal pigment
epithelium. B, section stained with the antibody shows an
intense positive reaction in the ganglion cell layer, in Müller
cell fibers spanning the inner portion of the retina. The outer
plexiform layer shows an intense positive reaction, as does the RPE.
C, a higher magnification of the inner portion of the retina
shows the intense positive reaction in ganglion cells and Müller
cell fibers. D, a high magnification shows the positive
reaction in the RPE. The photoreceptor cells of the outer nuclear layer
are negative.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, the inhibitor of
nuclear factor-
B, and the GA-binding protein
(GABP
), a
transcription factor, contain five and four ankyrin repeats,
respectively (25, 26). The presence of ankyrin repeats and the
coiled-coil domain in the NORPEG protein indicates a potential for
interaction with other cellular proteins.
and retinoid X
receptor-
(data not shown). Also, it has been reported that retinoic
acid delays the expression of RPE-specific genes in ARPE-19 cells
(44).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Cynthia Jaworski for analyzing the gene structure and valuable suggestions and to Dr. Paul Russell for valuable suggestions and critical reading of the manuscript.
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Note Added in Proof |
---|
While this article was in press, Peng et al. (Peng, Y., Mandai, K., Sakisaka, T., Okabe, N., Yamamoto, Y., Yokoyama, S., Mizoguchi, A., Shiozaki, H., Monden, M., and Takai, Y. (2000) Genes Cells, in press) independently characterized ankycorbin, a novel actin cytoskeleton-associated protein whose sequence is identical to that reported in this paper for mouse Norpeg protein.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF155135 and AF274866.
** Supported by Grant NIH-EY 13089 from the National Institutes of Health and by Research to Prevent Blindness.
§ To whom correspondence and reprint requests should be addressed: LRCMB, Rm. 338, National Eye Institute, NIH, 6 Center Dr., Bethesda, MD 20892-2740. Tel.: 301-496-5809; Fax: 301-402-1883; E-mail: krishnan@helix.nih.gov.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007421200
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ABBREVIATIONS |
---|
The abbreviations used are: RPE, retinal pigment epithelium; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; NORPEG, novel retinal pigment epithelial cell gene; bp, base pair(s); BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase(s).
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1. | Bok, D. (1993) J. Cell Sci. 17, 189-195 |
2. | Marmor, M. F., and Wolfensberger, T. J. (1998) The Retinal Pigment Epithelium: Function and Disease , Oxford University Press, New York |
3. | Crouch, R. K., Chader, G. J., Wiggert, B., and Pepperberg, D. R. (1996) Photochem. Photobiol. 64, 613-621[Medline] [Order article via Infotrieve] |
4. | Rao, N. A., and Wu, G. S. (2000) Prog. Retin. Eye Res. 19, 41-68[CrossRef][Medline] [Order article via Infotrieve] |
5. | Young, R. W. (1987) Surv. Ophthalmol. 31, 291-306[Medline] [Order article via Infotrieve] |
6. |
Fine, S. L.,
Berger, J. W.,
Maguire, M. G.,
and Ho, A. C.
(2000)
N. Engl. J. Med.
342,
483-492 |
7. | Davis, A. A., Bernstein, P. S., Bok, D., Turner, J., Nachtigal, M., and Hunt, R. C. (1995) Invest. Ophthalmol. Visual Sci. 36, 955-964[Abstract] |
8. | Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., and Hjelmeland, L. M. (1996) Exp. Eye Res. 62, 155-169[CrossRef][Medline] [Order article via Infotrieve] |
9. | Jiang, X. R., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A. G., Wahl, G. M., Tlsty, T. D., and Chiu, C. P. (1999) Nat. Genet. 21, 111-114[CrossRef][Medline] [Order article via Infotrieve] |
10. | Dunn, K. C., Marmorstein, A. D., Bonilha, V. L., Rodriguez-Boulan, E., Giordano, F., and Hjelmeland, L. M. (1998) Invest. Ophthalmol. Visual Sci. 39, 2744-2749[Abstract] |
11. |
Finnemann, S. C.,
Bonilha, V. L.,
Marmorstein, A. D.,
and Rodriguez-Boulan, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12932-12937 |
12. |
Chancy, C. D.,
Kekuda, R.,
Huang, W.,
Prasad, P. D.,
Kuhnel, J. M.,
Sirotnak, F. M.,
Roon, P.,
Ganapathy, V.,
and Smith, S. B.
(2000)
J. Biol. Chem.
275,
20676-20684 |
13. | Holtkamp, G. M., Van Rossem, M., de Vos, A. F., Willekens, B., Peek, R., and Kijlstra, A. (1998) Clin. Exp. Immunol. 112, 34-43[CrossRef][Medline] [Order article via Infotrieve] |
14. | Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002[Abstract] |
15. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
16. | Berger, B., Wilson, D. B., Wolf, E., Tonchev, T., Milla, M., and Kim, P. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8259-8263[Abstract] |
17. | Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13[Medline] [Order article via Infotrieve] |
18. | Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P., and Buckingham, M. (1990) J. Cell Biol. 111, 2427-2436[Abstract] |
19. |
Hu, M. C. T.,
Wang, Y. P.,
Mikhail, A.,
Qiu, W. R.,
and Tan, T. H.
(1999)
J. Biol. Chem.
274,
7095-7102 |
20. |
Fritzler, M. J.,
Lung, C. C.,
Hamel, J. C.,
Griffith, K. J.,
and Chan, E. K. L.
(1995)
J. Biol. Chem.
270,
31262-31268 |
21. | Bilbe, G., Delabie, J., Bruggen, J., Richener, H., Asselbergs, F. A. M., Cerletti, N., Sorg, C., Odink, K., Tarcsay, L., Wiesendanger, W., Dewolf-Peeters, C., and Shipman, R. (1992) EMBO J. 11, 2103-2113[Abstract] |
22. | Yamauchi-Takihara, K., Sole, M. J., Liew, J., Ing, D., and Liew, C. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3504-3508[Abstract] |
23. | Michaely, P., and Bennett, V. (1992) Trends Cell Biol. 2, 127-129[CrossRef] |
24. | Lux, S. E., John, K. M., and Bennett, V. (1990) Nature 344, 36-42[CrossRef][Medline] [Order article via Infotrieve] |
25. | Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289[Medline] [Order article via Infotrieve] |
26. | Thompson, C. C., Brown, T. A., and Mcknight, S. L. (1991) Science 253, 762-768[Medline] [Order article via Infotrieve] |
27. | Franke, W. W., Schiller, D. L., Moll, R., Winter, S., Schmid, E., Engelbrecht, I., Denk, H., Krepler, R., and Platzer, B. (1981) J. Mol. Biol. 153, 933-959[Medline] [Order article via Infotrieve] |
28. | Fawcett, D. W. (1975) in Handbook of Physiology (Hamilton D. W., and Greep, R. O., eds) Section 7, Vol. 5, pp. 21-55, American Physiological Society, Washington, D. C. |
29. | Sibley, C. P., and Boyd, R. D. H. (1998) in Fetal and Neonatal Physiology (Polin, R. A. , and Fox, W. W., eds) , pp. 77-89, Saunders, Philadelphia, PA |
30. | Faber, J. J., and Thornburg, K. L. (1983) Placental Physiology , Raven Press, Ltd., New York |
31. | Schmitt, M. C., and Ong, D. E. (1993) Biol. Reprod. 49, 972-979[Abstract] |
32. | Sapin, V., Ward, S. J., Bronner, S., Chambon, P., and Dolle, P. (1997) Dev. Dyn. 208, 199-210[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Edwalds-Gilbert, G.,
Veraldi, K. L.,
and Milcarek, C.
(1997)
Nucleic Acids Res.
25,
2547-2561 |
34. | Ross, J. (1996) Trends Genet. 12, 171-175[CrossRef][Medline] [Order article via Infotrieve] |
35. | Xu, N., Chen, C. Y., and Shyu, A. B. (1997) Mol. Cell. Biol. 17, 4611-4621[Abstract] |
36. | Krebber, H., and Ponstingl, H. (1996) Gene (Amst.) 180, 7-11[CrossRef][Medline] [Order article via Infotrieve] |
37. | Lu, Y., and Riegel, A. T. (1994) Gene (Amst.) 145, 261-265[Medline] [Order article via Infotrieve] |
38. |
Mishima, K.,
Price, S. R.,
Nightingale, M. S.,
Kousvelari, E.,
Moss, J.,
and Vaughan, M.
(1992)
J. Biol. Chem.
267,
24109-24116 |
39. | Grinberg, D., Thurlow, J., Watson, R., Smith, R., Peters, G., and Dickson, C. (1991) Cell Growth Differ. 2, 137-143[Abstract] |
40. | Sporn, M. B., Roberts, A. B., and Goodman, D. S. (1994) The Retinoids: Biology, Chemistry and Medicine , Raven Press, Ltd., New York |
41. | Giguere, V., Ong, E. S., Segui, P., and Evans, R. M. (1987) Nature 330, 624-629[CrossRef][Medline] [Order article via Infotrieve] |
42. | Petkovitch, M., Brand, N. J., Krust, A., and Chambon, P. (1987) Nature 330, 444-450[CrossRef][Medline] [Order article via Infotrieve] |
43. | Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229[CrossRef][Medline] [Order article via Infotrieve] |
44. | Janssen, J. J., Kuhlmann, E. D., van Vugt, A. H., Winkens, H. J., Janssen, B. P., Deutman, A. F., and Driessen, C. A. (2000) Neuroreport 11, 1571-1579[Medline] [Order article via Infotrieve] |
45. | Conlon, R. A. (1995) Trends Genet. 11, 314-319[CrossRef][Medline] [Order article via Infotrieve] |
46. | Niederreither, K., Subbarayan, V., Dolle, P., and Chambon, P. (1999) Nat. Genet. 21, 444-448[CrossRef][Medline] [Order article via Infotrieve] |
47. | McCaffery, P., and Drager, U. C. (2000) Cytokine Growth Factor Rev. 11, 233-249[CrossRef][Medline] [Order article via Infotrieve] |
48. | van Pelt, A. M. M., and de Rooij, D. G. (1991) Endocrinology 128, 697-704[Abstract] |
49. |
Komada, M.,
McLean, D. J.,
Griswold, M. D.,
Russell, L. D.,
and Soriano, P.
(2000)
Genes Dev.
14,
1332-1342 |
50. |
McCaffery, P.,
Mey, J.,
and Drager, U. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12570-12574 |
51. | McCaffery, P., Posch, K. C., Napoli, J. L., Gudas, L., and Drager, U. C. (1993) Dev. Biol. 158, 390-399[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Chen, F.,
Figueroa, D. J.,
Marmorstein, A. D.,
Zhang, Q.,
Petrukhin, K.,
Caskey, C. T.,
and Austin, C. P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
15149-15154 |