Chem. Senses 26: 1157-1166,
2001
© Oxford University Press 2001
New GPCRs from a Human Lingual cDNA Library
Laboratoire d'Etude des Interactions des Molécules Alimentaires, Institut National de la Recherche Agronomique, BP 71627, 44316 Nantes, Cedex 3, France
Correspondence to be sent to: T. Haertlé, Laboratoire d'Etude des Interactions des Molécules Alimentaires, Institut National de la Recherche Agronomique, BP 71627, 44316 Nantes, Cedex 3, France. e-mail: haertle{at}nantes.inra.fr
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Abstract |
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Sweet and bitter taste perception involve G protein coupled receptors (GPCRs) present at the taste receptor cell surface. It is likely that various mechanisms are active and various families of GPCRs are involved in the perception of these tastes. The expression of GPCRs in human tongue was studied using degenerated primers corresponding to transmembrane domains 2 or 3 (for 5' primer), 6 or 7 (for 3' primer) of olfactory-like receptors in reverse transcription-polymerase chain reaction experiments. It was demonstrated that four previously identified, eight new olfactory-like receptor genes, three previously known and eight new olfactory-like receptor pseudogenes, mostly located on chromosome 11, are expressed in adult tongue and/or in fetal tongue. Previously identified genes include HGMP071, HTPCR06, TPCR120 and TPCR85 whose cDNAs were originally isolated from male germinal cells. New genes were named JCG1, JCG2, JCG3, JCG4, JCG5, JCG6, JCG9 and JCG10. HGMP071, HTPCR06, TPCR120, JCG3 and JCG5 are also expressed in the epithelium of adult tongue, whereas all these genes are expressed in fetal tongue. Although functional studies are needed before definitive conclusions are made, the obtained results imply that lingual olfactory-like receptors could be involved in taste perception.
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Introduction |
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TopAbstractIntroductionMaterial and methodsRT-PCRResultsDiscussionReferences |
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Mammals are able to discriminate between five basic tastes: sweet, salty, sour, bitter and umami. Taste receptor cells (TRCs) are localized in small specialized organs called taste buds distributed on the surface of the tongue and palate, mainly in circumvallate, fungiform and foliate papillae. It is well known that salty and sour perception involve membrane ion channels of TRCs. In rats, umami taste, the taste elicited by sodium glutamate, is mediated by a truncated form of the metabotropic glutamate receptor 4 (mGluR4) (Chaudhari et al., 2000
) and probably also by ionotropic glutamate receptors
(Chaudhari and Roper, 1998;
Lin and Kinnamon, 1999).
mGluR4 is a G protein coupled receptor (GPCR). Although still not very
precisely defined, it is generally accepted that at least a part of sweet and
bitter taste signals is also transduced by GPCRs. Recent and independent
cloning, in two laboratories, of a family of candidate receptors for bitter
and sweet compounds confirms this hypothesis
(Adler et al., 2000;
Matsunami et al.,
2000). Among this family of receptors forming a new class of
GPCRs, three members named mT2R-5 and mT2R-8 in mice and hT2R-4 in humans
function as bitter taste receptors in transfected cells
(Chandrashekar et al.,
2000). Good candidate receptors for sweet taste named Tas1r3
(Max et al., 2001) or
T1R3 (Kitagawa et al.,
2001; Montmayeur et
al., 2001; Sainz et
al., 2001) have been cloned in four independent laboratories
recently. This receptor is also a GPCR belonging to another class of GPCRs
containing T1R1 and T1R2 taste receptors
(Hoon et al.,
1999).
Before the cloning of members of these classes of GPCR, it had been
proposed that receptors for sweet and bitter molecules may display a high
degree of homology with olfactory receptors. Abe et al. showed using
reverse transcription-polymerase chain reaction (RT-PCR) strategy that a
family of olfactory-like receptor (OLR) mRNAs is expressed in rat tongue
(Abe et al., 1993a).
One of these mRNAs, coding a protein named GUST27, is present in epithelial
cells of rat tongue, including taste buds
(Abe et al., 1993b).
Additionally, GUST27 was found to be expressed in a taste bud area where
-gustducin, a taste-specific G protein, is also present. These data
suggested that GUST27 could be involved in taste transduction
(Kusakabe et al.,
1996). A RT-PCR approach also allowed Matsuoka et al.
(Matsuoka et al.,
1993) to obtain cDNA clones highly similar to olfactory receptors
from bovine taste tissue.
The present study reports the cloning of several cDNAs corresponding to OLR mRNAs expressed in human tongue and the identification of the corresponding genes.
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Material and methods |
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Total RNA extraction
Total RNA was prepared from a sample of adult human tongue epithelium. The
sample originated from a 52-year-old Caucasian male subject to tongue
ablation. It was quickly washed in PBS and frozen in liquid nitrogen.
Epithelial and sub-epithelial layers were separated from the muscular layer by
dissection using a scalpel. Tissue sample still frozen (
100 mg) was
broken in liquid nitrogen with a freezer/mill Spex 6700 apparatus. The powder
obtained was suspended in 350 µl of lytic solution (Qiagen SA, Courtaboeuf,
France) and homogenized for 30 s using a polytron apparatus (Kinematica,
Luzern, Switzerland). Total RNA was extracted using Qiagen RNeasy extraction
kit and DNase I treatment (Qiagen SA). The integrity of RNA was
checked by 1.2% agarose gel electrophoresis.
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RT-PCR |
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Reverse transcription (RT) was performed on 1-2 µg of total RNA incubated 1 h at 37°C in 40 µl of a reaction mixture containing 1x buffer, 0.625 µM each dNTP, 3.5 µM oligo-dT primer, 1 µM random nonamer primer, 0.5 units of RNase inhibitor (Eurogentec, Seraing, Belgium) and 4 units of Omniscript Reverse TranscriptaseTM (Qiagen).
PCR primers were synthesized by Cybergene (Evry, France). Degenerated
primers RS1
5'-CA(AGCT)AC(AGCT)CC(AGCT)ATGTA(CT)(ACT)(AGCT)TT(CT)(CT)T-3',
RS2
5'-ATGGC(AGCT)TA(CT)GA(CT)(AC)G(AGCT)TA(CT)GT(AGCT)GC-3',
RAS3
5'-(GC)(CT)(AG)CA(AGCT)GT(AGCT)(GC)(AT)(AG)AA(AGCT)GC(CT)TT-3',
RAS4
5'-TA(AGT)AT(AGCT)A(AG)(AGCT)GG(AG)TT(AGCT)A(AG)CAT(AGCT)GG-3'
were chosen according to the amino-acid sequences (H/Q)TPMY(F/L/I)FL,
MAYDRYVA(I/V), KAFSTC(G/T/A), PMLNP(F/L)IY(S/T), which are highly conserved
among the olfactory receptors family and situated close to transmembrane
domains 2, 3, 6 and 7, respectively (Horn
et al., 1998). RS1 and RS2 were used as
5' primers, RAS3 and RAS4 as 3' primers.
Specific primers were: JCG1—5 5'-ATGGGGACTGGAAATGA-3' and JCG1—3 5'-TCAAGAAAATATTTTTATTCTAAG-3' for full length JCG1 cDNA amplification; JCG2—5 5'-ATGGCTACTTCAAACCATTCTTC-3' and JCG2—3 5'-TCAGGATGACTGCCTTCCC-3' for full length JCG2 cDNA amplification; JCG3—5 5'-ATGAATTCCCTGAAGGACG-3' and JCG3—3 5'-CTATGTAATATCATTATTTGAAGTTC-3' for full length JCG3 cDNA amplification; JCG5—5 5'-ATGATGTGGGAAAACTGG-3' and JCG5—3 5'-TCATAGTTTCTGAGAGCC-3' for full length JCG5 cDNA amplification; JCG6—5 5'-ATGGCTATAGGAAACTGG-3' and JCG6—3 5'-CTATGGGATACAGTTTCTG-3' for full length JCG6 cDNA amplification; JCG9—5 5'-ATGACCATGGAAAATTATTCTA-3' and JCG9—3 5'-TCATTTTCCTACTAAGACCT-3' for full length JCG9 cDNA amplification; OR1E1—5 5'-ATGATGGGACAAAATCAAAC-3' and OR1E1—3 5'-TCAGAGAGAGAAGAAAGTT-3' for full length HGMP07I/OR1E1 cDNA amplification; JCG8—5 5'-ATGGCTGCTGAGAATTC-3' and JCG8—3 5'-TCAGGAGAATGCATTTTTG-3' for full length TPCR85 cDNA amplification; HTPCR-5 5'-ATGCAAGGAGAAAACTTCAC-3' and HTPCR-3 5'-TCAGAGATGTTCGTGTGTTT-3' for full length HTPCR06 cDNA amplification, GAPDH-5 5'-GAAATCCCATCACCATCT-3' and GAPDH-3 5'-TCCACAGTCTTCTGGGTG-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA amplification.
PCRs were performed on either 2 µl of RT reaction mixture, 1 µl of human fetal tongue Gene PoolTM (Invitrogen, Groningen, The Netherlands), 0.05-0.1 µg of total RNA from human adult tongue epithelium or 100 ng of human placenta genomic DNA. The PCR mixture (final volume 50 µl) contained 1x PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.3 µM of each specific primer (1 µM in the case of degenerated primers RS1, RS2, RAS3 and RAS4) and 2.5 U HotStartTaqTM DNA polymerase (Qiagen SA). An initial step 15 min at 95°C was followed by 35 or 40 cycles: 30 s at 94°C for denaturation, 1 min 30 s at 55°C (45°C in the case of degenerated primers RS1, RS2, RAS3 and RAS4) for annealing and 2 min at 72°C for elongation. After this, a final step of 7 min at 72°C was carried out. Thirty-five cycles were applied in PCR using degenerated primers and 40 cycles were applied in PCR using specific primers. In few cases, 2 µl of the first PCR reaction product were subject to 30 additional cycles. According to the sample, 5-20 µl of PCR products were analysed on 1.5% agarose gel.
Cloning and sequencing
According to the instructions of the supplier (Promega, Madison, WI, USA), PCR fragments were inserted into pGEM®-T Easy vector and characterized by restriction analysis. Restriction enzymes were purchased from Eurogentec (Seraing, Belgium). Plasmid DNA of selected clones was purified using Plasmid MidiTM columns (Qiagen SA) and sequenced from T7 and SP6 primers by E.S.G.S. (Groupe Cybergene, Evry, France).
Sequence homology searches were done with BLAST 2.1 (NCBI, National Library
of Medicine, Bethesda, MD, USA) in either nr database (`all
non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PR') or
htgs [`Unfinished High Throughput Genomic Sequences: phases 0, 1 and
2 (finished, phase 3 HTG sequences are in nr database)'], or with
FASTA (Pearson and Lipman,
1988) in the Human Olfactory Receptor Data Exploratorium (HORDE)
database (Weizmann Institute of Science, Rehovot, Israel). Sequence alignments
were done with ClustalW (EMBLEBI, Cambridge, UK). Data from the ClustalW
alignment were treated with the NEIGHBOR program from the PHYLIP package
(http://evolution.genetics.washington.edu/phylip.html) to construct a
phylogenetic tree.
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Results |
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Cloning of partial olfactory-like receptors cDNA from human tongue
In order to investigate the expression of GPCRs in human tongue,
degenerated primers RS1 or RS2 as 5' primer and
RAS3 or RAS4 as 3' primer were used in attempt to
amplify OLR cDNAs in RT-PCR experiments. Taking advantage of the fact that all
known OLR genes are intronless, different couples of primers (RS1/RAS3,
RS1/RAS4, RS2/RAS3 and RS2/RAS4) were tested first on human
genomic DNA. The best results were obtained with RS2/RAS4, which
allowed the amplification of a single-band amplicon product of 520 bp
(data not shown), a size corresponding to that which could be expected.
Therefore, primers RS2 and RAS4 annealing with transmembrane
domain 3 (TM3)/intracellular loop II junction and with TM7 coding sequence,
respectively, were used for further experiments. PCR experiments were carried
out on both human fetal tongue Gene PoolTM (ready-to-use RT reaction
realized on polyA+ RNAs) provided by Invitrogen and on RT reaction
realized on total RNA from human adult lingual epithelial and sub-epithelial
layers.
Because all the OLR genes known are intronless, it was important to verify
the absence of contaminating genomic DNA. PCR using RS2/RAS4 realized
directly on adult tongue RNA without reverse transcription step did not show
any product (Figure 1D, line
1), even after two successive rounds of PCR
(Figure 1D, line 3). This
result showed that the RNA extracted from adult tongue was DNA-free. In the
case of fetal tongue, a Gene PoolTM provided by Invitrogen was used. This
product is the result of a reverse transcription reaction made on
polyA+ RNAs by the supplier, therefore it was impossible to perform
a PCR without reverse transcription as a control. The absence of contaminating
genomic DNA in the Invitrogen Gene PoolTM was double-checked by
amplification of ß-actin, clathrin and GAPDH cDNAs. DNA-containing
introns was absent for these three genes. Additionally, we used amplification
of the ubiquitously expressed housekeeping gene GAPDH as another test for
genomic DNA presence/absence on both cDNA sources. GAPDH cDNA was amplified
using primers GAPDH-5 and GAPDH-3. These primers were chosen
because they allow the amplification of a band of 355 bp in the case of cDNA
and a band of 855 bp in the case of contamination with genomic DNA. These two
bands were observed when a PCR was performed on human genomic DNA (data not
shown). A single band of 350 bp was amplified from both fetal and adult
tongue after 35 PCR cycles (Figure
1A,C). These results confirmed the absence of contaminating
genomic DNA in both cases.
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PCR using RS2/RAS4 realized on human fetal tongue Gene PoolTM
allowed the amplification of a single band of 520 bp after 35 cycles
(Figure 1B) whereas PCR
realized on total RNA from human adult tongue epithelium led to the
amplification of two major bands at 520 and 900 bp and two very thin
bands at 380 and 700 bp (Figure
1D, line 2). In the second case, because the intensity of the
bands was low, the PCR product was subject to 30 additional PCR cycles using
the same primers. The products corresponding to all four bands were slightly
re-amplified (Figure 1D, line
4) and were cloned separately into pGEM®-T Easy vector as well as the
product obtained from the fetal tongue.
Sequencing of 380, 700 and 900 bp inserts and the screening of BLAST nr database showed that they constitute artefactual amplification due to the use of degenerated primers.
Inserts of size neighbouring 520 bp were analysed by restriction digestions. Their analysis demonstrated the presence of several different fragments. A total of 42 independent clones contained 35 different inserts (23 partial cDNA sequences from fetal tongue and 12 from adult tongue) were sequenced. Four inserts were present in two clones and one insert was present in four clones The sequences were translated according to six coding frames.
OLR prototype sequence was defined using an alignment of 88 members of the
olfactory receptor family found in the GPCR Database (GPCRDB)
(Horn et al., 1998).
The frequency of amino acid residues at a given position was analysed. This
prototype sequence is shown in Figure
2A,2B
where conserved residues are reported and non-conserved residues are indicated
by a star. Homology search of nucleotidic and deduced peptide sequences with
BLAST in nr database and comparison with the prototype OLR sequence
revealed that among cloned sequences, 12 (35%) were sequences unrelated to OLR
(data not shown), 7 (20%) contained already published OLR sequences (cDNAs,
genes or pseudogenes) and 16 (45%) were new cDNA sequences (Figure
2A,2B).
Presence of artefactual fragments (e.g. KIAA-0782 protein, DNA polymerase
alpha subunit, plakoglobin partial cDNAs) was mainly due to the use of
degenerated primers leading to mispriming and amplification of non-OLR-related
sequences. It was found that the sequence of clone RC86 was identical to
partial HGMP07I cDNA sequence (Parmentier
et al., 1992) and to a part of the complete OR1E1 gene
sequence (Glusman et al.,
2000); clone JCG8 sequence was identical to partial TPCR85 cDNA
sequence (Vanderhaeghen et al.,
1997); clone RC254 sequence was identical to partial HTPCR06 cDNA
sequence (Parmentier et al.,
1992); clone RC212 sequence was identical to partial TPCR120 cDNA
sequence (Vanderhaeghen et al.,
1997); clone RC70 was identical to partial TPCR24 pseudogene cDNA
sequence (Vanderhaeghen et al.,
1997); clone RC95 sequence was identical to partial OR7E13P
pseudogene sequence (Buettner et
al., 1998) and clone RC183 sequence was identical to partial
OR7-86 pseudogene sequence (Rouquier
et al., 1998). All five sequences of TPCR24, TPCR85,
TPCR120, HGMP07I and HTPCR06 were originally cloned by RT-PCR from male
germinal cell mRNA whereas OR7E13P and OR7-86 sequences were originally cloned
or identified from chromosome 11 genomic DNA. All the other clones named JCG1,
JCG2, JCG3, JCG4, JCG5, JCG6, JCG9, JCG10, PJCG1, PJCG3, PJCG4, PJCG5, PJCG6,
PJCG7, PJCG8 and PJCG9 contained previously unreported new partial cDNA
sequences. [GenBank accession number of the sequences: HGMP07I/OR10E1
(AF087916, X64994); HTPCR06 (X64977); TPCR120 (X89669); TPCR24 (AF309699,
AF309702); OR7-86 (U86282); OR7E13P (AF065855, AF238487); JCG1 (AF158377);
JCG2 (AF162668, AF162669); JCG3 (AF173006); JCG4 (AF220494); JCG5 (AF209506);
JCG6 (AF065874); TPCR85 (AF238488); JCG9 (AF238488); JCG10 (AF308814); PJCG1
(AF220493); PJCG3 (AF309700, AF309701); PJCG4 (AF359415); PJCG5 (AF209507);
PJCG6 (AF356416); PJCG7 (AF359417); PJCG8 (AF359418) and PJCG9
(AF359419).]
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Partial OLR peptide sequences obtained by translation of RS2/RAS4
cDNA fragments were compared to the OLR prototype sequence. They could be
classified in two categories (Figure
2A): (i) sequences containing uninterrupted open reading frames
(ORF) homologous to OLR prototype: TPCR24, HGMP07I/OR1E1, OR7E13P, TPCR85,
OR7-86, TPCR120, HTPCR06, JCG1, JCG2, JCG3, JCG4, JCG5, JCG6, JCG9, JCG10 and
PJCG1; and (ii) sequences containing interrupted ORF or ORF with changed
reading frame in respect to the defined OLR prototype: PJCG6, PJCG4, PJCG5,
PJCG6, PJCG7, PJCG8 and PJCG9. The former probably constitute parts of
expressed pseudogenes. It is important to highlight that, in spite of their
classification in the first category, sequences OR7E13P and OR7-86 were
already reported as pseudogenes (Buettner
et al., 1998;
Rouquier et al.,
1998). Indeed, stop codons were found outside of the cloned
portion of their cDNA.
Homology searches in htgs and HORDE databases were used in order
to identify full-length sequences of the genes or pseudogenes corresponding to
partial cDNA fragments. The genes corresponding to JCG4, JCG10, PJCG8 and
PJCG9 (all of them new) could not be found. PJCG8 and PJCG9 are pseudogenes
very similar to previously described OR2-53, OR2-75, OR2-4 and OR2-52
(Rouquier et al.,
1998). All these pseudogenes are very similar to each other
(98-99% identity). Partial JCG4 is very similar to partial JCG3, showing 96%
identity at the protein level. However, full-length coding sequences of the
genes or pseudogenes corresponding to all the other cloned fragments were
identified.
Full-length coding sequences of HGMP071/OR1E1, TPCR85, HTPCR06; JCG1, JCG2, JCG3, JCG5, JCG6 and JCG9 cDNAs were cloned from the fetal tongue Gene PoolTM. They ranged from 927 bp to 969 bp, so the deduced protein sequences ranged from 308 amino acids (aa) to 322 aa (Figure 2A). All found sequences agreed well with the theoretical sequences extracted from human genome sequencing data. Nevertheless, in the case of HGMP07I/OR1E1 a point mutations was observed between the sequences described in this paper and the sequences available in databanks.
The full-length coding sequence of HGMP07I/OR1E1 (HORDE name OR1E1) is 945 bp long. The deduced protein sequence is 314 aa long. The cDNA sequence cloned from fetal tongue contains one point mutation at position 771 in respect to genomic one (OR1E1 gene and clone RP11-587F22). Because this was found in partial cDNA clones from embryonic tongue, in full-length cDNA clones and by direct sequencing of PCR products from adult tongue it can be considered to be a result of polymorphism. Additionally, this silent mutation does not modify the sequence of the corresponding protein.
The full-length coding sequence of TPCR85 (HORDE name OR8B8) is 936 bp
long. It is identical to the genomic sequence contained in clones RP11-728D14
and RP11-164A10. Its deduced protein sequence is 311 aa long. Nevertheless,
its partial cDNA clone obtained by Vanderhaeghen and co-workers contains two
point mutations when compared to the genomic sequence leading to
Gly204 
Ser conversion.
The full-length coding sequence of HTPCR06 (HORDE name OR2K2) is 951 bp
long. Its deduced protein sequence is 316 aa long. Its cDNA sequence cloned
from fetal tongue is identical to the genomic sequence contained in clones
RP11-17E20 and RP11-386D8, whereas the partial cDNA sequence cloned by
Parmentier et al. contains one point mutation at position 632
(Leu211 Pro).
The full-length coding sequence of JCG1 (HORDE name OR5P3) is 936 bp long. Its deduced protein sequence is 311 aa long. Its full length cDNA sequence is identical to those found in genomic clones RP11-799H15, RP11-494M8 and RP11-399N15.
The full-length coding sequence of JCG2 (HORDE name OR8D2) is 936 bp long. Its deduced protein sequence is 311 aa long. cDNA sequence cloned from fetal tongue is identical to genomic sequence found in clones pDJ9j14 and RP11-164A10.
The full-length coding sequence of JCG3 (HORDE name OR5P2) is 969 bp long. Its deduced protein sequence is 322 aa long. The full-length cDNA sequence is identical to the sequence found in clones RP11-799H15, RP11-494M8 and RP11-399N15. There are 12 differences at the nucleotide level and six differences at the protein level between JCG4 partial cDNA sequence and JCG3 sequence in an area that does not include the sequence of the degenerated primers. Even if point mutations could be introduced by the use of a non-proofreading Taq-polymerase, the introduction of 12 mutations in a 472 bp sequence is very surprising. Because PCR fragments containing full-length JCG4 were not found, it can be suspected that differences between JCG4 and JCG3 are probably due to PCR artefacts. Nevertheless, it can not be completely excluded that JCG4 could be a different gene whose genomic sequence is still unknown.
The full-length coding sequence of JCG5 (HORDE name OR10A4) is 948 bp long.
Its deduced protein sequence is 315 aa long. The full-length cDNA clone
sequence contains one point mutation at position 880 in respect to the
sequence of genomic clone RP11-560B16. By direct sequencing of the PCR
fragment, two bases, A and C, were detected at this position in similar
amounts. Consequently, the difference observed at position 880 is probably due
to polymorphism, two different alleles being expressed. Nevertheless, this
difference represents silent polymorphism because the protein sequence is
unchanged. At position 617 the base found in genomic clone pDJ610i20 is
different than in all other sequences leading to Leu206 Pro
conversion. If the sequence of clone PDJ610i20 is correct, a point mutation at
position 617 can be assigned to polymorphism at this position as well. As well
as this difference between the two genomic clones, clone RP11-560B16 contains
deletion at position 351 when compared to PDJ610i20. This deletion is probably
due to a sequencing mistake.
The full-length coding sequence of JCG6 (HORDE name OR10A5) is 954 bp long. The deduced protein sequence is 317 aa long. This sequence is identical to genomic sequence found in genomic clones RP11-560B16 and PDJ610i20.
The full-length coding sequence of JCG9 (HORDE name OR8D1) is 927 bp long. Its deduced protein sequence is 308 aa long. The full-length cDNA sequence is identical to genomic sequence contained in clone RP11-164A10.
All these sequences, when compared to the OLR prototype, could be classified in three groups (Figure 2A):
- Previously identified genes encoding OLR proteins: TPCR85, HGMP07I/OR1E1,
HTPCR06 and TPCR120 and new OLR genes: JCG1, JCG2, JCG3, JCG5, JCG6 and
JCG9.
- OLR pseudogenes sequences presenting the same reading frame as OLR genes
but containing one or several stop codons located upstream from the stop codon
normally used: TPCR24, PJCG5 and OR7-86.
- OLR pseudogenes containing insertions or deletions leading to changes of
reading frame: OR7E13P, PJCG1, PJCG3, PJCG4, PJCG6, PJCG7, PJCG8 and
PJCG9.
Remarkably, 15 out of 19 genes or pseudogenes corresponding to cloned cDNAs are located on chromosome 11, some very close to each other. For instance, JCG2, TPCR85, JCG9 and TPCR120 are located on 11q25 between 137.68 megabases (Mb) and 137.96 Mb. JCG2, TPCR85 and JCG9 are localized in the same genomic clone RP11-164A10. The interval between JCG2 and JCG9 amounts to only 8640 bp. A phylogenetic tree based on nucleotide sequence conservation showed that they are closely related and derive from the same common ancestor gene (Figure 2B). JCG1, JCG3, JCG5, JCG6, TPCR24, PJCG1, PJCG4, PJCG5 and PJCG7 are located between 2.78 Mb and 6.93 Mb on chromosome 11. JCG1, JCG3 and TPCR24 are found in the same genomic clones (RP11-799H15, RP11-494M8 and RP11-399N15). This could indicate that one or several clusters of OLR genes located on chromosome 11 are expressed in tongue epithelium. OR13E7P and PJCG3 are also located on this chromosome.
Expression of OLR genes in adult and fetal human tongue
Expression of TPCR85, HGMP07I/OR1E1, HTPCR06, JCG1, JCG2, JCG3, JCG5, JCG6 and JCG9 was studied by RT-PCR on both human fetal and human adult tongue (Figure 3). For each gene, specific primers allowing the amplification of the complete theoretical coding sequence were used (the 5' primer included the start codon and the 3' primer included the stop codon). Because JCG3 was found to be very similar to JCG4 and because the JCG4 full-length coding sequence was not identified, primers chosen for JCG3 amplification would also have allowed the amplification of the full-length coding sequence. The sizes of the expected bands range from 927 to 969 bp. GAPDH was used as a positive control of the PCR.
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During the experiment, PCRs allowed the amplification of fragments representing the expected size. However, in the case of JCG5 with fetal mRNA, TPCR85/JCG8, JCG1, JCG3 with adult mRNA and JCG6 with both mRNA, additional bands of lower molecular weight were observed. Cloning, restriction analysis and sequencing of these products showed that they are artefactual PCR fragments due to mispriming (e.g. myosin heavy chain in the case of JCG6).
It was found that all the tested genes are expressed in fetal tongue whereas only HTPCR06, OR1E1/HGMP07I, JCG3/JCG4 and JCG5 are expressed in epithelium of adult human tongue. The bands amplified from adult tissues were very thin, indicating that these mRNAs are probably very poorly expressed. The band for HTPCR06 had the lowest intensity on the gel corresponding to fetal tissues and the highest intensity on the gel corresponding to the adult tissues, suggesting that HTPCR06 is preferentially expressed in adults.
In order to confirm that the bands observed between 900 and 1000 bp markers in the previous experiment correspond well to cDNA of the genes of interest, PCR fragments obtained from fetal tongue were cloned into pGEM®-T Easy vector and sequenced. The cDNA sequences obtained were compared to both genomic and partial cDNA corresponding sequences. The results confirmed that the obtained PCR fragments correspond to the genes of interest. In the case of the amplification of JCG3/JCG4, only full-length coding sequence of JCG3 was found among three independent clones and by direct sequencing of the PCR product.
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Discussion |
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TopAbstractIntroductionMaterial and methodsRT-PCRResultsDiscussionReferences |
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The present paper describes the cloning of a subset of previously described and new OLR cDNAs expressed in tongue. It was shown that several OLR genes and pseudogenes are expressed in human fetal and adult tongue. Nevertheless, it was found that in adult human tongue, the number of these genes or pseudogenes expressed is much lower than in fetal lingual tissue. One possible reason for the observed differences could be differentiation of gene expression during maturation of this tissue. It was proposed that olfactory receptors might have other functions in addition to simple odorant detection [for a review see Dryer and Berghard (Dryer and Berghard, 1999
)]. One of these functions could be axon guidance. Such a
phenomenon could occur in tongue tissues during the establishment of neuronal
connections between fetal taste receptors. However, this does not exclude the
possibility that OLR proteins could also play a role in the recognition of
sapid compounds. It is also possible that the observed differences are related to the origins of the samples used for RNA extraction. Available fetal tongue Gene PoolTM was prepared from total tongue, hence it was not the optimal source to search for genes expressed specifically in epithelium or in a precise area of the tongue. So, it is also possible that some of the cloned genes are expressed, for instance, in the muscular layers underneath the epithelial layer of the tongue, in its laropharyngeal part or in distinct areas of the lingual epithelium. Available sample from adult tongue was histologically much more precise since it contained only epithelial and sub-epithelial tissues sampled at the extremity of the tongue.
Surprisingly, some of the OLR mRNAs described in this paper found in tongue
(HGMP07I, HTPCR06, TPCR24, TPCR85, TPCR120) were isolated for the first time
from mammalian male germinal cells
(Parmentier et al.,
1992; Vanderhaeghen et
al., 1997). Expression of the OLR gene in both taste and
reproductive tissues was also reported in rats
(Thomas et al.,
1996). This is also the case of adenylate cyclase type 3 (AC3) and
the olfactory G protein subunit Golf
(Defer et al., 1998).
Both are involved in olfactory signalling pathway. Even if several elements of
the olfactory signalling pathway are present in male germ cells of several
mammalian species, the function of OLR proteins in these cells remains
unclear.
Point mutations were observed between new, partial or full-length cloned
sequences and previously cloned sequences or genomic sequences—this is
the case for HGMP-07I/OR10E1, TPCR85, HTPCR06 and JCG5. In the case of JCG5,
the difference observed is most probably due to genetic polymorphism whose
intensity was already reported within a cluster of 15 olfactory receptor genes
located on chromosome 17 (Gilad et
al., 2000; Sharon et
al., 2000). Another illustration of such polymorphisms is
that, in some cases, the same gene contained in two genomic clones exhibited
one or several point mutations. In the case of HGMP07I/OR10E1, TPCR85 and
HTPCR06, their sequences found are identical to the genomic one, whereas they
vary slightly from previously cloned partial cDNA sequences. These point
mutations can also be due to PCR artefacts.
The results presented show that some OLR pseudogenes are expressed in human
tongue. Expression of pseudogenes is a rather uncommon phenomenon, which,
however, has already been reported in the case of OLR. An OLR pseudogene
located on chromosome 17 was found to be expressed in human olfactory tissue
(Crowe et al., 1996).
TPCR24 from male germ cells mRNA first cloned in 1997 by Vanderhaeghen et
al. (Vanderhaeghen et al.,
1997) and described as an OLR gene is in fact a pseudogene. One of
the models of OLR regulation proposes that entire clusters of OLR genes could
be regulated by common cis-acting elements. Because OLR pseudogenes
are included mostly in such clusters, it is conceivable that the expression of
OLR pseudogenes can be maintained by such a mechanism of regulation.
Among the genes and pseudogenes cloned in this study, 15 (eight genes and seven pseudogenes) are located on chromosome 11. They are probably parts of one or of several clusters. The fact that they are expressed together in fetal tongue could support this hypothesis. Nevertheless, out of all of them, only expression of JCG3 and JCG5 was detected in adult lingual tissues. This suggests that other regulation mechanisms could occur and/or that the expression levels of the other genes were too low to be detected in applied experimental conditions.
Recently, a new family of GPCRs expressed specifically in lingual tissue
and containing receptors for bitter taste in humans was described
(Hoon et al., 1999;
Adler et al., 2000;
Chandrashekar et al.,
2000; Matsunami et
al., 2000). Lingual OLRs identified in this study do not
display significant homologies with any member of this class of bitter taste
receptors. Nevertheless, the possibility of involvement of OLR proteins in
taste perception can not be excluded. More intensive work would be needed
confirm such a possibility. A histological localization study of the OLR
transcripts, especially in taste bud cells, would be of great help in
assessing their true functional relevance.
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TopAbstractIntroductionMaterial and methodsRT-PCRResultsDiscussionReferences |
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Accepted July 14, 2001
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