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Two olfactory marker proteins inXenopus laevis

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THE JOURNAL OF COMPARATIVE NEUROLOGY 395:273–280 (1998)
Two Olfactory Marker Proteins
in Xenopus laevis
PATRICIA RÖSSLER, MARIO MEZLER, AND HEINZ BREER*
University Stuttgart-Hohenheim, Institute of Physiology, D-70593 Stuttgart, Germany
ABSTRACT
Mature olfactory receptor neurons of mammals are characterized by the expression of the
highly conserved olfactory marker protein (OMP) encoded by single copy genes. In Xenopus
laevis, two homologous genes encoding olfactory marker proteins have been identified that
share a sequence identity with mammalian OMPs of about 50%. Sequence comparison
revealed significant variability in the N-terminus and C-terminus regions; in contrast, two
internal domains were highly conserved between amphibian and mammalian OMPs, suggesting some functional relevance. The two OMP subtypes were regionally expressed in the
olfactory nasal epithelium of Xenopus. XOMP1 transcripts were more abundant in the lateral
diverticulum and XOMP2 in the medial diverticulum. The lateral location of XOMP1 and
medial location of XOMP2 correspond to the suggested locations of olfactory receptor neurons
responsive to water-borne and air-borne odorants, respectively. J. Comp. Neurol. 395:273–
280, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: mature olfactory receptor neurons; amphibia; subtypes; expression pattern
The olfactory marker protein (OMP) is an abundant
19-kDa cytoplasmic protein that is almost exclusively
expressed in vertebrate mature olfactory sensory neurons
(Margolis, 1985, 1988). Although the tissue-specific expression and some recent observations on OMP-deficient mice
(Buiakova et al., 1996) imply an involvement in chemosensory signal transduction, the biological function of OMP
remains enigmatic. Deciphering the amino acid sequence
of rat OMP revealed a unique structure; there is no
evidence for any related protein (Margolis, 1985). A comparison of the sequences of OMP from rat, mouse, and
human revealed high conservation among the mammalian
OMPs (Buiakova et al., 1994) but provided no hints to
some conserved and potentially important functional domains. Determining the primary structure of OMP in
nonmammalian vertebrate species may be more promising
for discovering selective conservation of distinct domains
during vertebrate evolution. Because some previous studies have demonstrated that antibodies against rat OMP
showed weak but significant immunoreactivity in the
nasal epithelium of nonmammalian vertebrates (Keller
and Margolis, 1975; Margolis, 1980; Rama Krishna et al.,
1992; Riddle and Oakley, 1992), we have set out to identify
the primary structure of OMP in Xenopus laevis.
MATERIALS AND METHODS
The experiments comply with the Principles of Animal
Care, Publication 85–23 (revised 1985) of the National
r 1998 WILEY-LISS, INC.
Institute of Health and also with the current laws of
Germany.
Screening of an olfactory cDNA library
of Xenopus laevis
Olfactory tissue was isolated from nasal cavities of 50
adult Xenopus laevis (Kähler, Hamburg). The tissue comprised sensory epithelium of the medial diverticulum
(MD), the lateral diverticulum (LD), and the vomeronasal
organ (VNO). The tissue was used for isolation of poly(A)1
RNA and subsequent construction of a directional oligo
(dT)-primed Xenopus olfactory library in phage vector l
Zap Express (Stratagene, La Jolla, CA). DNA of 3.6 3 105
independent recombinant phage clones was transferred to
nylon membranes (Pall, East Hills, NY) and were subsequently prehybridized at 37°C for 3 hours in a solution
containing 30% formamide, 53 standard saline citrate
(SSC; 13 SSC 5 150 mM sodium chloride, 15 mM sodium
citrate, pH 7.5), 0.02% sodium dodecylsulfate (SDS), 2%
blocking reagent (Boehringer, Mannheim, Germany), 0.1%
laurylsarcosin, and 100 µg/ml herring sperm DNA. Hy-
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number:
Br 712/16–1; Grant sponsor: Human Science Frontier Program; Grant
sponsor: Fond der Chemischen Industrie.
*Correspondence to: Prof. Dr. Heinz Breer, University StuttgartHohenheim, Institute of Physiology, Garbenstrasse 30, D-70593 Stuttgart,
Germany. E-mail: physiol1@uni-hohenheim.de
Received 6 October 1997; Revised 7 January 1998; Accepted 23 January
1998
274
P. RÖSSLER ET AL.
Fig. 1. A: Deduced amino acid sequences of Xenopus olfactory
marker protein (OMP) genes XOMP1 and XOMP2. Differences in the
amino acid sequences are shaded. Most of the 14 amino acid substitutions are conservative exchanges and are scattered over the sequence.
B: Alignment of Xenopus OMPs XOMP1 and XOMP2 with OMP
proteins from human, mouse, and rat. Amino acid residues conserved
in all sequences are indicated by an asterisk. Amino acids sharing
.50% identity in all sequences are shaded. Dashes represent gaps in
the amino acid sequence.
bridization was performed overnight at 37°C in the same
buffer with 2.5 ng/ml of digoxigenin (Dig)-labeled DNA
encoding the complete coding region of the rat OMP
(Rogers et al., 1987). The DNA was labeled by using the
method of Feinberg and Vogelstein (1983) with the random
priming Dig-DNA labeling kit (Boehringer). After hybridization, filters were rinsed for 2 3 2 minutes in 53 SSC,
0.1% SDS at room temperature, followed by a wash in 23
SSC, 0.1% SDS at 37°C for 4 3 20 minutes. Chemiluminescent detection was performed by following the Dig system
user’s guide for filter hybridization of Boehringer. Filters
were exposed to Fuji RX films for 16 hours.
corresponded to Xenopus XOMP1. Four clones were identical and corresponded to Xenopus XOMP2. Sequencing
primers were T3 primer—58 ATT AAC CCT CAC TAA AGG
GA 38, T7 primer—58 TAA TAC GAC TCA CTA TAG GG 38,
primer position 551—58 AAT (G/A)TT CCA TTG GCT TCA
38, primer position 843 for XOMP1, 815 for XOMP2—58
GT(A/G) TGT TAC (C/T)AA GTG TAG 38, primer position
1358 specific for XOMP1—58 GAC TAC TGG TGG GTT
CAG 38, and primer position 1339 specific for XOMP2—58
GGT ACC ATC TCC CAC TG 38.
Analysis of sequence data was performed by using the
HUSAR 3.0 software package based on the sequence
analysis software package 7.2 from the Genetic Group
(Madison, WI).
Isolation of full-length cDNA clones for DNA
sequencing and analysis of sequence data
Eleven positive phage plaques were isolated; of these,
five l clones showed positive hybridization signals in the
rescreen. These were recovered as phagemid clones by
using the ExAssist helper phage, and DNA was prepared
by alkaline lysis by using the Qiagen Midi kit (Chatsworth, CA). Both strands of each recombinant phagemid
were sequenced by using the RR Dye Deoxy Terminator
cycle sequencing kit (Perkin Elmer, Weiterstadt, Germany). Automatic sequencing was performed on an ABI
310 genetic analyzer. One of the five analyzed clones
Southern blot analysis
High-molecular-weight DNA was prepared from muscle
tissue of an individual adult Xenopus according to the
method of Ausubel et al. (1987). Ten micrograms of genomic DNA were digested with restriction endonucleases
EcoRI and KpnI, size fractionated on a 1% agarose gel, and
transferred to Hybond N1 nylon membrane (Amersham,
Braunschweig, Germany) by using standard protocols
(Sambrook et al., 1989). A 382-bp fragment of XOMP1
cDNA encoding amino acids 19–146 was labeled by using
XENOPUS OLFACTORY MARKER PROTEINS
275
the Dig DNA labeling kit (Boehringer). The blot was
hybridized under high stringency conditions to the
Dig-labeled DNA probe, as described by Engler-Blum et al.
(1993), after the optimized hybridization and detection
protocol (blocking reagent and anti-Dig AP-Fab fragments
were obtained from Boehringer). Subsequently, the membrane was exposed to Fuji RX film for 12 hours.
Northern blot analysis
For the analysis of tissue-specific expression of XOMP1
and XOMP2, total RNA from Xenopus olfactory epithelium
(OE), olfactory bulb (OB), brain (B), and liver (L) was
prepared by tissue homogenization in TRIzol reagent
(Gibco BRL, Eggenstein, Germany) according to the manufacturer’s instructions. Five micrograms of total RNA were
separated on a 1.2% agarose gel containing formaldehyde
and were blotted (Sambrook et al., 1989). Prehybridization
and hybridization was performed at 68°C by using Dig
easy hyb buffer (Boehringer). The hybridization buffer
contained 100 ng/ml Dig-labeled RNA of either XOMP1 or
XOMP2 full-length clones. The antisense RNA probes were
generated by using the T3/T7 RNA transcription system
according to the manufacturer’s specifications (Boehringer), with recombinant pBK-CMV phagemid vectors as a
template. In brief, 2 micrograms of linearized vector were
transcribed in the presence of 70 nmol digoxigenin-11uridine-58-trisphosphate, followed by partial alkaline hydrolysis of the RNA according to Angerer and Angerer
(1992), thereby producing fragments of about 200 bases in
length. After hybridization, the membrane was washed in
0.13 SSC, 0.1% SDS at 68°C. After chemiluminescent
detection, films were exposed to Fuji RX film for 11 hours.
Reverse transcriptase–polymerase chain
reaction (RT-PCR)
To analyze the expression of both XOMP subtypes in the
different subcompartments of the nasal cavity, total RNA
was extracted from the olfactory bulb and sensory epithelia of the VNO, LD, and MD of single animals, as described
above. Total RNA was dissolved in 20 µl of RNase-free
water followed by a DNase digestion (DNase I, Gibco
BRL). After the spectrometric determination of the amount
of total RNA, equal amounts of mRNA were isolated
(Dynabeads, Dynal, Oslo, Norway). Messenger RNA redissolved in 10 µl RNase-free water was reverse transcribed
by using a first-strand cDNA synthesis kit according to the
manufacturer’s instructions (Pharmacia, Freiburg, Germany). To compare the amount of template used in each
reaction, empirical tests were performed basically as described previously (Rappolee et al., 1988; Deutsch Murphy
et al., 1990; Wilson and Melton, 1994). To make sure that
equal amounts of cDNA were used as a template throughout all tissue samples in subsequent PCR reactions, additional PCR experiments were performed by using the
housekeeping gene L8 as endogenous control. To amplify
either XOMP1 or XOMP2, specific oligonucleotide antisense primers corresponding to the 38 untranslated region
of each subtype were combined with a general XOMP
sense primer. The primers were sense for XOMP1 and
XOMP2 (position 469)—58 CTT TCT TAG ATG GCG CTG
ACC 38, antisense for XOMP1 (position 872)—58 GTG GTT
ATT TCT CTA CAC TTG G 38, antisense for XOMP2
(1017)—58 ACA CAC TTT TTT GTC TTG GG 38, primers
for L8 sense—58 TTG CAT TCC GTG ATC CTT ACA GG 38,
Fig. 2. Southern blot analysis of Xenopus XOMP genes. Xenopus
genomic DNA isolated from muscle tissue was digested with restriction endonuclease EcoRI (lane 1) and KpnI (lane 2), electrophoresed
on a 1% agarose gel, and blotted on a nylon filter. The blot was
hydridized with a Dig-labeled probe that corresponded to a 382-bp
fragment from XOMP1 cDNA encoding amino acids 19–146, which
shows no internal recogntion sight for EcoRI or KpnI. Hybridizing
fragments share a sequence homology of .90%. The position of
HindIII and HindIII/EcoRI digested lambda-DNA is shown on the left
in kilobase pairs.
antisense—58 ATC TCT CCT GAT GGT TGA GGG 38. To
have a linear correlation between template concentration
and PCR products, the optimal PCR cycle number was
determined. Amplification was carried out in 50 µl of 10
mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, with
200 µM of each dNTP, 100 pmol of each primer, and 2 U of
Taq DNA polymerase (Gibco BRL). PCR was performed by
using the following conditions: 94°C for 2 minutes, 57–
60°C for 2 minutes (depending on the primers), and 72°C
for 4 minutes (1 cycle); 94°C for 30 seconds, 65°C for 1
minutes, and 72°C for 50 seconds (20–35 cycles); and 72°C
for 8 minutes. The optimal number of cycles were 20 cycles
for L8 at an annealing temperature of 60°C and 35 cycles
for both XOMP subtypes at 57°C. To determine the specificity of the PCR primers and for quantification purposes, the
PCR products were separated on 1% agarose gels and
blotted, as described above. Dig-labeled probes corresponding to the 38 untranslated region of either XOMP1 or
276
P. RÖSSLER ET AL.
Fig. 3. A: Northern blot analysis of XOMP1 and XOMP2 expression in the nasal organ, the olfactory bulb, and nonolfactory tissues of
Xenopus. Hybridization was performed under high stringency conditions with 5 µg of total RNA from Xenopus nasal cavity, olfactory bulb,
brain, and liver by using antisense riboprobes of both full-length
clones. The blots were exposed for 11 hours. The position of the 18-S
and 28-S ribosomal RNA bands are indicated. XOMP mRNA of both
subtypes is expressed in the olfactory epithelium (OE), whereas no
expression of either XOMP1 or XOMP2 is detectable in the olfactory
bulb (OB), brain (B), and liver (L). In the left blot, the native 1.8-kb
XOMP1 mRNA is visible; in the right blot, expression of XOMP2
mRNA of a slightly smaller size (1.6 kb) is detectable. Note the higher
level of XOMP2 mRNA. B: Compartment-specific expression of XOMP
genes revealed by semiquantitative reverse transcriptase–polymerase
chain reaction (RT-PCR). RNA from all three compartments including
vomeronasal organ (VNO), lateral diverticulum (LD), medial diverticulum (MD), and olfactory bulb (OB) of one Xenopus nasal cavity was
subjected to RT-PCR analysis by using primer pairs specific for the
38-untranslated region of either XOMP1 or XOMP2. To compare equal
amounts of cDNA, additional PCR experiments were performed by
using the housekeeping gene L8 as an endogenous control. The PCR
samples of L8 were separated by electophoresis to compare band
intensities (lower panel). The linearly amplified XOMP PCR products
were analyzed on Southern blots by using labeled XOMP-subtype
specific probes corresponding to the 38-untranslated regions. The
amount of probe was similar for both XOMP subtype probes. In the
LD, a preferential expression of XOMP1 was recognized. In the MD,
XOMP2 was preferentially expressed. In the VNO, small amounts of
XOMP1 and XOMP2 transcripts were observed. In the OB, no XOMP
expression was detected.
XOMP2 were hydridized as described by Engler-Blum et
al. (1993). The PCR products of the housekeeping gene L8
were measured with ethidium bromide staining.
and rapidly frozen in an N2 cooled isopentane bath.
Coronal sections 10 µm thick were cut on a Reichert &
Jung cryostat (model 2800 E) at 230°C, adhered to
Superfrost plus (Fisher, Orangeburg, NY) microslides, and
air dried for 2 hours. For in situ hybridization, tissue
sections were covered with 10 µl of hybridization solution
containing 50% deionized formamide and 3–5 ng Diglabeled antisense RNA, and coverslipped. Hybridization
was carried out at 55°C for 16 hours in closed humid boxes.
After incubation, sections were washed twice for 30 min-
In situ hybridization
Adult Xenopus laevis were decapitated, and the upper
jaws were fixed in 4% paraformaldehyde, 1% picric acid in
phosphate buffered saline, pH 7.3, for 2 hours. After
cryoprotection in 25% sucrose buffer overnight at 4°C, the
jaws were embedded in Tissue Tek (Miles, Elkhart, IN)
XENOPUS OLFACTORY MARKER PROTEINS
277
utes in 0.13 SSC at 60°C. Hybridization was visualized by
using an anti-Dig AP antibody (1:750, Boehringer) for 30
minutes at 37°C, followed by two washes in Tris-buffered
saline (100 mM Tris-HCl, 150 mM NaCl, pH 7.0) for 15
minutes. Bound antibodies were visualized by using nitroblue tetrazolium and bromo-chloro-indolyl phosphate (Biomol, Hamburg, Germany) as substrates. Subsequently,
sections were mounted in Euparal (Roth, Karlsruhe, Germany) and examined under a Zeiss Axiphot microscope by
using Nomarski phase-contrast optics. In control experiments, sense RNAs were transcribed and hybridized to
tissue sections, as described for antisense probes. No
signals were observed in any of these controls.
RESULTS
An olfactory cDNA library from Xenopus laevis was
screened by means of a rat OMP DNA probe. Two different
full-length cDNA clones, XOMP1 and XOMP2, were isolated under low stringency conditions. The complete nucleotide sequences of these cDNA clones are deposited in the
database. The open reading frames of both clones comprise
474 bp and are flanked by 50 bp of untranslated region on
the 58 side. The 38 flanking regions differ in length;
whereas XOMP1 comprises a 38 tail of 1,275 nucleotides,
XOMP2 contains a 38 untranslated region of 1,048 bp
(including the stop codon). The open reading frames of
XOMP1 and XOMP2 encode polypeptide chains of 158
amino acids with calculated molecular masses of 18,724
Da (XOMP1) and 18,445 Da (XOMP2). A comparison of the
amino acid sequences of XOMP1 and XOMP2 (Fig. 1A)
demonstrated an identity of 91%, which is similar to the
sequence identity among OMPs from mammalian species.
However, the sequence identities between Xenopus and
mammalian OMPs ranged only between 51% and 55%.
Aligning the amino acid sequences of Xenopus OMPs and
OMPs from mammals (rat, mouse, human) demonstrated
a striking conservation of two domains (Fig. 1B). These
regions, ranging from positions 75 to 100 and from 118 to
141, were highly conserved and demonstrated a significantly higher sequence identity of 92% and 83%; in contrast, both the N-terminus and the C-terminus differed
significantly between OMPs from mammals and amphibia. Furthermore, both Xenopus OMPs exhibited three
deletions in the N-terminus and also missed the final
leucine at the C-terminus.
In rat, OMP is encoded by a single copy gene (Danciger
et al., 1989). To investigate how many OMP genes may
exist in Xenopus, Southern blot analyses were performed
by probing genomic DNA with a 382-bp fragment from
XOMP1 cDNA encoding amino acids 19–146, which does
not contain sites for EcoRI or KpnI. Thus, because the
OMP genes are not supposed to contain introns within the
coding regions, the number of hybridizing bands should
indicate the number of OMP genes. Under high stringency
conditions in each restriction endonuclease lane, four
bands of different intensity were visualized, suggesting
the existence of at least four different OMP genes in the
Xenopus genome (Fig. 2).
To examine, whether both XOMP genes are actually
transcribed and whether the two XOMP subtypes display
any tissue- or region-specific expression, riboprobes corresponding to the full-length clones of both XOMP1 and
XOMP2 were hybridized to Northern blots from Xenopus
Fig. 4. Cellular localization of XOMP gene expression in adult
Xenopus olfactory epithelium by in situ hybridization. A: Section
through the medial diverticulum (MD) incubated with a digoxigeninlabeled antisense riboprobe of XOMP2. Labeled cells are confined to
the sensory part of the mucosa; no signals were detected in the
nonsensory part. Arrowheads indicate the transition between the
olfactory epithelium (OE) and adjacent respiratory epithelium (RE).
B: A section through the lateral diverticulum (LD) probed with
XOMP1. Within the epithelium, specific hybridization is restricted to
olfactory receptor neurons (ORN) but not to sustentacular cells (SC) or
basal cells (BC). Signals are absent from the lamina propria (LP).
Scale bars 5 100 µm in A, 40 µm in B.
olfactory epithelium, olfactory bulb, brain, and liver under
high stringency conditions. The results (Fig. 3A) indicate
that both probes only hybridized to RNA from the olfactory
epithelium. A strong hybridization signal was obtained for
the XOMP2 probe, whereas the XOMP1 probe only produced a weak response. Because both probes produced
equal signal intensities on standardized templates, this
result suggests that XOMP2 is more strongly expressed in
the olfactory tissue. No hybridization was observed with
RNA from the olfactory bulb, brain, and liver. The size of
XOMP2 mRNA is slightly smaller than that of XOMP1
mRNA. The length of the hybridizing RNA is about 1.8 kb
for XOMP1 and 1.6 kb for XOMP2; this is in good agreement with the size of the cloned cDNA, thus supporting the
notion that XOMP1 and XOMP2 represent full-length
cDNAs.
278
Fig. 5. In situ hybridization of adult Xenopus coronal sections
through the sensory epithelia of all subcompartments probed with
either XOMP1-specific or XOMP2-specific antisense RNA. A,B: Adjacent sections through the lateral diverticulum (LD) hybridized with
XOMP1 and XOMP2. XOMP1 is strongly expressed in the sensory
cells of the LD (A). The expression of XOMP2 is significantly weaker
(B). C,D: Consecutive sections through the medial diverticulum (MD)
probed with XOMP1 and XOMP2. C: XOMP1 strongly hybridized to
only single neurons randomly distributed within the MD. The major-
P. RÖSSLER ET AL.
ity of neurons are weakly stained. D: Hybridization with the XOMP2specific probe intensively labeled many cells in the olfactory neuron
layer. E: Coronal section of the vomeronasal organ (VNO) annealed
with XOMP1. The probe labeled single, randomly distributed chemosensory cells in the VNO. These XOMP1-expressing neurons are
surrounded by cells with a diffuse and very weak staining. Arrowhead
indicates a single mature VNO neuron expressing XOMP1. Scale
bars 5 50 µm.
XENOPUS OLFACTORY MARKER PROTEINS
The olfactory system of Xenopus consists of three separate and functionally different compartments: the main
olfactory cavity, which is subdivided into the lateral diverticulum (LD) and the medial diverticulum (MD), and the
vomeronasal organ (VNO; Föske, 1934; Altner, 1962). To
approach the question if the two OMP subtypes were
expressed differently in the nasal subcompartments, the
appropriate olfactory tissue from an individual animal was
dissected. Because of the very small quanitites of tissue,
samples were analyzed by following a semiquantitative
RT-PCR protocol (Zamorano et al., 1996) with specific
primers corresponding to the 38 untranslated region of
XOMP1 and XOMP2. The results shown in Figure 3B
indicate that the endogenous internal standard, the housekeeping gene L8 (Shi and Liang, 1994), was amplified at
constant levels throughout all tissue samples, demonstrating the suitability of the procedure. XOMP1 was expressed
in all three cavities, but the highest level of expression was
found in the lateral diverticulum; XOMP2 transcripts
were also detected in the three cavities, but XOMP2 was
preferentially expressed in the medial diverticulum. In
contrast to mammals (Wensley et al., 1995), in Xenopus
there was no indication of XOMP mRNA in the olfactory
bulb (Fig. 3B).
To analyze the topographic localization of XOMPexpressing cells in the nasal epithelium of adult Xenopus,
in situ hybridization experiments were performed by using
digoxigenin-labeled antisense probes from the full-length
clones under high stringency conditions. Labeled cells
were only found in the chemosensory epithelium of the
nasal cavity. No hybridization was detected in the adjacent
respiratory epithelium (Fig. 4A). Within the olfactory
epithelium, reactive cells were situated in the layer of the
neuroepithelium, which consists of mature receptor neurons (Fig. 4B). The stretch of mucosa above the basal
membrane, where immature neurons are located, was
devoid of labeled cells. Sustentacular cells in the apical
layer of the neuroepithelium showed no staining. Hybridization signals were also absent from the lamina propria
beneath the epithelium (Fig. 4B). In detail analysis of
adjacent sections through the lateral diverticulum demonstrated that XOMP1 is strongly expressed in many cells of
the LD epithelium (Fig. 5A), whereas a significantly
weaker staining was obtained with the XOMP2 probe (Fig.
5B). Employing a XOMP1-specific probe to coronal sections through the medial diverticulum demonstrated only
few intensely hybridizing neurons randomly distributed
within the epithelium; the majority of cells produced very
weak signals (Fig. 5C). Probing adjacent sections through
the epithelium of the medial diverticulum with a XOMP2specific riboprobe resulted in hybridization signals of high
intensity in the layer where mature neurons are situated
(Fig. 5D). These observations are in line with the RT-PCR
results. In the vomeronasal organ, expression of both
XOMP subtypes appeared to be restricted to very few cells
on consecutive sections. The punctate hybridization pattern of XOMP1 in the VNO is shown in Figure 5E. The
specific probe for XOMP2 produced similar results (data
not shown). The significant differences concerning the
number and intensity of hybridization signals in all three
subcompartments are consistent with the different amounts
of transcripts determined by semiquantitative RT-PCR
experiments.
279
DISCUSSION
In the present study, two subtypes of olfactory marker
protein were identified in Xenopus laevis. Sequence comparison demonstrated a moderate overall homology with
the highly conserved OMPs from three mammalian species; however, two domains emerged that are identical in
all OMP subtypes. Conservation of the primary structure
during vertebrate evolution suggests that these domains
may be of considerable relevance for structure and/or
function of the OMPs. Furthermore, elucidation of these
highly conserved domains may open the way for novel
experimental approaches, such as the yeast two-hybrid
system for identification of target proteins, and ultimately
disclose the functional role of these proteins in olfactory
neurons.
Whereas rat OMP seems to be encoded by a single copy
gene (Danciger et al., 1989), Southern blot analysis of
Xenopus genomic DNA demonstrated four bands. This
result suggests that four different OMP genes exist in
Xenopus, possibly as a consequence of genome duplication
as a result of tetraploidization. Because intensive screening of the Xenopus olfactory cDNA library led to the
isolation of only two different OMP cDNA clones and both
XOMP subtypes generated a single band in Northern
blots, it seems likely that only two XOMP genes are
expressed within the nose of the frog. However, the
observation that in the VNO only very few cells express
XOMP1 or XOMP2 may indicate that additional XOMP
subtypes are expressed. The preferential expression of
XOMP1 in mature olfactory neurons of the lateral diverticulum and XOMP2 in the sensory cells of the medial diverticulum may contribute to the functional compartmentalization of the olfactory system. In Xenopus, the lateral
diverticulum is supposed to be specialized for watersoluble odors (Altner, 1962) and selectively express ‘‘fishlike’’ receptors, whereas the medial diverticulum seems to
be responsible for the reception of volatile odorants and is
equipped with ‘‘mammalian like’’ receptors (Freitag et al.,
1995). Therefore, each XOMP subtype may be expressed in
a distinct subset of functionally specialized olfactory neurons. Although the functional role of OMP in mature
olfactory neurons still awaits elucidation, the identification of OMP from nonmammalian vertebrates may shed
some new light on the evolution of OMP genes.
LITERATURE CITED
Altner, H. (1962) Untersuchungen über die Leistungen und Bau der Nase
des südafrikanischen Krallenfrosches Xenopus laevis (Daudin, 1803). Z.
Vergl. Physiol. 45:272–306.
Angerer, L.M. and R.C. Angerer (1992) In situ hybridization to cellular RNA
with radiolabelled RNA probes. In D.G. Wilkinson (ed): In Situ Hybridization. Oxford, England: IRL, pp. 15–32.
Ausubel, F.M., R. Brent, R.F. Kingston, D.D. Moore, J.G. Seidmann, J.A.
Smith, and K. Struhl (1987) Current Protocols in Molecular Biology.
New York: Greene and Wiley.
Buiakova, O.I., N.S. Rama Krishna, T.V. Getchell, and F.L. Margolis (1994)
Human and rodent OMP genes: Conservation of structural and regulatory motifs and cellular localization. Genomics 20:452–462.
Buiakova, O.I., H. Baker, J.W. Scott, A. Farbman, R. Kream, M. Grillo, L.
Franzen, M. Richman, L.M. Davis, S. Abbondanzo, C.L. Stewart, and
F.L. Margolis (1996) Olfactory marker protein (OMP) gene deletion
causes altered physiological activity of olfactory sensory neurons. Proc.
Natl. Acad. Sci. U.S.A. 93:9858–9863.
Danciger, E., C. Mettling, M. Vidal, R. Morris, and F.L. Margolis (1989)
Olfactory marker protein gene: Its structure and olfactory neuronspecific expression in transgenic mice. Proc. Natl. Acad. Sci. U.S.A.
86:8565–8569.
280
Deutsch Murphy, L., C.E. Herzog, J.B. Rudick, A.T. Foje, and S.E. Bates
(1990) Use of the polymerase chain reaction in the quantification of
mdr-1 gene expression. Biochemistry 29:10351–10356.
Engler-Blum, G., M. Meier, J. Frank, and G.A. Müller (1993) Reduction of
background problems in nonradioaktive Northern and Southern blot
analyses enables higher sensitivity than 32P-based hybridizations.
Anal. Biochem. 210:235–244.
Feinberg, A.P. and B. Vogelstein (1983) A technique for radiolabelling DNA
restriction fragments to high specific activity. Anal. Biochem. 132:6–13.
Föske, H. (1934) Das Geruchsorgan von Xenopus laevis. Z. Anat. Entweil.
103:519–550.
Freitag, J., J. Krieger, J. Strotmann, and H. Breer (1995) Two classes of
olfactory receptors in Xenopus laevis. Neuron 15:1383–1392.
Keller, A. and F.L. Margolis (1975) Immunological studies of the rat
olfactory marker protein. J. Neurochem. 24:1101–1106.
Margolis, F.L. (1980) A marker protein for the olfactory chemoreceptor
neuron. In R.A. Bradshaw and D.M. Schneider (eds): Proteins of the
Nervous System, 2nd ed. New York: Raven Press, pp. 59–84.
Margolis, F.L. (1985) Olfactory marker protein: From PAGE band to cDNA
clone. Trends Neurosci. 8:542–546.
Margolis, F.L. (1988) Molecular cloning of olfactory specific gene products.
In F.L. Margolis and T.V. Getchell (eds): Molecular Neurobiology of the
Olfactory System. New York: Plenum, pp. 237–265.
Rama Krishna, N.S., T.V. Getchell, F.L. Margolis, and M.L. Getchell (1992)
P. RÖSSLER ET AL.
Amphibian olfactory receptor neurons express olfactory marker protein. Brain Res. 593:295–398.
Rappolee, D.A., C.A. Brenner, R. Schultz, D. Mark, and Z. Werb (1988)
Develompental expression of PDGF, TGF-a, and TGF-b genes in
preimplantation mouse embryos. Science 241:1823–1825.
Riddle, D.R. and B. Oakley (1992) Immunocytochemical identification of
primary olfactory afferents in rainbow trout. J. Comp. Neurol. 324:575–
585.
Rogers, K.E., P. Dasgupta, U. Gubler, M. Grillo, Y.S. Khew-Goodall, and F.L.
Margolis (1987) Molecular cloning and sequencing of a cDNA for
olfactory marker protein. Proc. Natl. Acad. Sci. U.S.A. 82:5218–5222.
Sambrook, J., E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.
Shi, Y.-B. and V.C.-T. Liang (1994) Cloning and characterization of the
ribosomal protein L8 gene from Xenopus laevis. Biochim. Biophys. Acta
1217:227–228.
Wensley, C.H., D.M. Stone, H. Baker, J.S. Kauer, F.L. Margolis, and D.M.
Chikaraishi (1995) Olfactory marker protein mRNA is found in axons of
the olfactory receptor neurons. J. Neurosci. 15:4827–4837.
Wilson, P.A. and D.A. Melton (1994) Mesodermal patterning by an inducer
gradient depends on secondary cell–cell communication. Curr. Biol.
4:676–686.
Zamorano, P.L., V.B Mahesh, and D.W. Brann (1996) Quantitative RT-PCR
for neuroendocrine studies. Neuroendocrinology 63:397–407.
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