From the Departments of Pharmacology and Molecular
Sciences, § Neuroscience, and
Psychiatry and
Behavioral Sciences, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In an effort to understand the molecular basis of olfaction, Buck
and Axel (1) cloned a family of putative odorant receptors (OdRs)1 predicted to contain
up to 1000 distinct gene products (2). The number and diversity of
putative receptors is consistent with the ability of mammals to
differentiate thousands of odors (2). Some of these receptors have been
discretely localized to olfactory neurons by in situ
hybridization (3-6). Despite the compelling circumstantial evidence
that this family of receptors functions in mammalian odor
discrimination, definitive data documenting ligand-receptor binding and
functional receptor expression are lacking. The possibility that these
orphan receptors may serve a more general function in chemodetection
comes from the discovery that OdR-like proteins are expressed in the
testis (7). Subsequently, we (8) and others (9) demonstrated the
localization of putative OdRs in spermatids of the testis and on the
midpiece of mature spermatozoa. Moreover, mammalian sperm tails possess
proteins involved in olfactory signal transduction including G-protein receptor kinase 3 (8), -arrestin2 (8), inositol 1,4,5-trisphosphate receptors (10), adenylate cyclase (11), and cyclic nucleotide-gated channels (12).2 In this
report, RNase protection assays likewise detect OdR transcripts in the
spleen. These findings suggest that sperm tails, olfactory cilia, and
immune cells may share common mechanisms to detect exogenous chemical
signals.
The hypothesis that OdRs function in mammalian sperm to regulate motility in response to exogenous signals derives from the existence of sperm-egg chemotaxis in invertebrates. The small peptides, speract and resact, are secreted by sea urchin eggs and attract spermatozoa in a species-specific manner by stimulating sperm motility and respiration (13-15). Recent evidence indicates that sperm-egg chemotaxis may likewise occur during mammalian fertilization (16, 17).
We were interested in clarifying the role of OdR expression in the testis. During the meiotic phase of spermatogenesis, gene promoters may become exposed as a result of altered chromatin structure, potentially leading to spurious transcription of a host of gene products lacking physiologic function. To avoid this confounding issue in studying whole testis, we restricted our investigation of OdR expression to round spermatids, the post-meiotic, differentiating cell population in the testis. We report the identification of two novel families of OdR-like proteins, termed spermatid chemoreceptors (SCRs), in spermatids of rat testis. In situ hybridization studies illustrate the selective localization of SCRs to spermatids in the testis and demonstrate that SCR expression is developmentally regulated, with transcripts first detectable during sexual maturation. RACE-PCR and sequencing of genomic clones led to the discovery that SCRs undergo 5'-splicing, which may regulate receptor expression during spermatogenesis. The 5'-splicing of OdR transcripts may influence the stability of OdR mRNAs and the expression of functional OdR proteins in olfactory, reproductive, immunologic, and experimental systems.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR of Putative SCRs-- Total RNA was prepared from testis, whole brain, and olfactory tissue using TRIzol reagant (Life Technologies, Inc.) and Oligotex poly(A)+ selection (Qiagen) according to manufacturer protocols. Round spermatids were purified from the testis by elutriation (18); the cell pellet was homogenized in guanidinium isothiocyanate and RNA pelleted through a cesium chloride gradient according to established procedures (19). RNA was treated with 10 units of RNase-free DNase (Life Technologies, Inc.) per µg of RNA. Five µg of RNA from each source was reverse-transcribed using oligo(dT) or random primers. PCR was performed on a 1/20 aliquot of the RT mixture for 35 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min using TAQ DNA polymerase (Life Technologies, Inc.).
Degenerate primers to the conserved LHTPMYLF (OdR 1) and STCASH (OdR 2) sequences of originally cloned OdRs (1) were employed. Buck and Axel clone F2 was used as a positive control for RT-PCR with OdR 1 + 2; negative control PCR reactions were run with mock reverse-transcribed RNA that was not treated with reverse transcriptase. The spermatid PCR product was TA-subcloned into pCRII (Invitrogen) and 14 clones (A-N) were sequenced. The testis PCR product was likewise subcloned and 7 clones were sequenced.Genomic Cloning of SCRs-- Two sets of PCR primers were designed for pSCRs D and G to generate probes corresponding to the two halves of each PCR product. PCR primer sequences were as follows: D.1 (EIGYTC, 5'-GAGATTGGCTATACTTGC); D.2 (GMGCTV, 3'-TACTGTGCATCCCATTCC); D.3 (GLGQTN, 5'-GGTCTGGGACAGACCAAT); D.4 (GRHKAL, 3'-AAGAGCTTTGTGGCGCCC); G.1 (MFSSVE, 5'-ATGTTCTCCTCTGTGGAG); G.2 (LGFLFM, 3'-CATGAAGAGGAACCCCAA); G.3 (ASPVVM, 5'-GCCAGTCCAGTTGTGATG); and G.4 (SARERQ, 3'-CTGTCGCTCCCTGGCTGA). Each half of the corresponding pSCR was amplified by PCR, gel-purified using QIAEX reagants (Life Technologies, Inc.), and labeled using a nick translation kit (Boehringer Mannheim). The probes were used to screen 2.5 × 106 phage plaques of an EMBL3 rat genomic library (CLONTECH) according to established procedures (20). To identify SCR D clones, duplicate lifts were taken, and one lift was screened with the D.1-D.2 probe and the other with the D.3-D.4 probe. Fifty-two co-hybridizing plaques were identified, and 10 plaques were purified by two additional rounds of screening. The same technique was used to identify SCR G clones. For the SCR G screen, 221 co-hybridizing plaques were identified, and 10 plaques were purified by two additional rounds of screening. Purified phage were used to infect K803 cells (CLONTECH), and phage DNA were isolated using the lambda prep kit (Promega). Phage DNA was double strand sequenced directly using the fluorescent terminator method of cycle sequencing on an Applied Biosystems 373a automated DNA sequencer at the DNA Analysis Facility of The Johns Hopkins University (21, 22). Oligonucleotides used for sequencing were synthesized on an ABI 394 synthesizer following ABI protocols. DNA sequence data was analyzed using Sequencher software (Gene Codes). SCR amino acid alignments were conducted using the MacVector program (Oxford Molecular Group). SCR hydropathy data were obtained by Kyte-Doolittle analysis (23) using MacVector. A putative phylogenetic tree relating SCRs to previously cloned full-length OdR sequences was constructed using the unweighted pair group method with arithmetic mean analysis (24, 25) of the Geneworks program (Oxford Molecular Group).
RNase Protection Assays--
Riboprobes for RNase protection
assays corresponding to D.1-D.4 and G.1-G.4 were in vitro
transcribed off of pCRII-pSCR D and pCRII-pSCR G using T7 RNA
polymerase (MAXIscript kit, Ambion) and [-32P]UTP (NEN
Life Science Products). Riboprobes were run on 6% polyacrylamide, 8 M urea precast TBE gels (Novex) and were gel-purified.
RNase protection assays were performed using the RPA II kit (Ambion) according to the manufacturer's protocol. To determine riboprobe sizes
on non-denaturing polyacrylamide gels for comparison with protected
fragments, the sense transcripts for pSCR D and pSCR G were in
vitro transcribed using SP6 RNA polymerase. Antisense riboprobes
were incubated for 24 h at 45 °C with sense RNA, 10 µg of
cesium chloride purified total RNA from pachytene spermatocytes, round
spermatids, and olfactory epithelium, and 0.5 µg of
poly(A)+ RNA (CLONTECH) from adult
testis, lung, brain, liver, and spleen. Ten µg of yeast RNA was used
as a control. The integrity of total and poly(A)+ RNA
employed was evaluated by running RPAs in parallel using a rat
-actin antisense riboprobe generated from the pTRI-
-actin-125-rat template (Ambion). RNase-treated samples were precipitated and run on
non-denaturing 6% polyacrylamide TBE gels (Novex). The gels were
transferred to Whatman filter paper and exposed to Kodak X-OMat film or
evaluated by phosphorimaging.
In Situ Hybridization-- In situ hybridization using digoxigenin-labeled probes was conducted using full-length SCRs D-2, D-8, and G-14 coding sequence and the novel 5'-SCR D RACE and SCR G RACE sequence. Fresh-frozen 20-mm cryostat sections of rat testis (2-, 6-, and 10-week-old rats as indicated) were cut onto Superfrost Plus slides (Fisher), allowed to air dry 1-3 h, and postfixed for 5 min in 4% paraformaldehyde/phosphate-buffered saline. When expression patterns in the testis were compared between probes, serial sections were utilized. The slides were washed 3 × for 3 min in TBS, treated 10 min in 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8.0, washed 3 × for 3 min in TBS, and prehybridized for 2 h at room temperature in hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's solution, 500 mg/ml sonicated herring sperm DNA, 250 mg/ml MRE tRNA). The tissue was incubated with 0.1 ml of buffer containing 40 ng of cRNA probe under a siliconized coverslip at 65 °C overnight. After removal of the coverslips in 5× SSC at 65 °C, sections were washed as follows: 2 × for 1 h in 0.2× SSC, 1 × for 5 min in 0.2× SSC, and 1 × for 5 min in TBS. Following a 1-h block at room temperature in 4% normal goat serum/TBS, the slides were incubated overnight at 4 °C in 1:5000 dilution of anti-DIG Fab fragment (Boehringer Mannheim) in 4% normal goat serum/TBS. Sections were then washed 3 × for 5 min in TBS and 1 × for 5 min in AP buffer (0.1 M Tris-Cl, pH 9.5, 0.1 M NaCl, 50 mM MgCl2). The color signal was developed in AP buffer containing 3.375 mg/ml NBT (Boehringer Mannheim), 3.5 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim), and 0.24 mg/ml levamisole. The color reaction was carried out in the dark for 24-72 h at room temperature and then terminated with a TE wash. The developed slides were coverslipped with Aquapolymount (Polysciences). Control sections were hybridized with identical quantities of sense cRNA, and no signal was observed. The in situ hybridization protocol has been utilized to distinguish the localizations of transcripts sharing as much as 85-90% nucleic acid identity (6, 26).
Rapid Amplification of cDNA Ends (RACE)-PCR-- 5'-RACE-PCR was conducted using the Life Technologies, Inc. 5'-RACE kit. One µg of adult testis poly(A)+ RNA (CLONTECH) was reverse-transcribed (Superscript II RT, Life Technologies, Inc.) using 25 ng of the SCR D specific 3'-primer D.2. First strand cDNA was purified and oligo(dC)-tailed according to the manufacturer's protocol. dC-tailed cDNA was amplified using the anchor primer and primer D.6 (PGECFL, 3'-GAGGAAGCATTCTCCAGG), a nested SCR D-specific 3'-primer. Products resulting from PCR reactions that contained dC-tailed cDNA, but not present in reactions using mock dC-tailed control cDNA, were gel-purified (QIAEX, Qiagen) and reamplified by PCR with the adapter primer and primer D.6. To confirm the presence of SCR D sequence in the RACE products, gel-purified bands were checked by PCR with D.5 (M(T/S)VNCS, 5'-ATGA(C/G)TGTCAACTGTTCT), a 5'-primer to the presumptive amino terminus of SCR D and D.6. If the appropriate 93-base pair D.5-D.6 product could be generated, the RACE product was subcloned into pCRII vector (Invitrogen) and sequenced with sp6 and T7 primers. The identical procedure was followed using the SCR G-specific 3'-primer G.2 to generate first strand cDNA, a nested SCR G-specific primer G.6 (YLASLMG, 3'-GCCCATGAGGGAGGCCAAGTA) for RACE-PCR with the anchor primer, and a 5'-primer to the presumptive amino terminus of SCR G, G.5 (MEWLM, 5'-ATGGAATGGCT(G/A)ATG), used with G.6 for PCR analysis of RACE products.
To investigate the origin of the novel sequence identified upstream of the starting methionines of SCRs, genomic clones containing SCRs G-14, G-15, and D-9 were sequenced in the 5' direction. PCR using primers designed against SCR D RACE sequence (5'-CGTGATGGGATCAGAAAT) and upstream SCR D-9 genomic sequence (3'-AGGATGTACCAAGATCAATGT) was also performed on SCR D-9 phage DNA to identify intervening sequence. To determine if SCR transcripts in spleen and olfactory tissue likewise undergo splicing, 1 µg of spleen poly(A)+ RNA and olfactory epithelium total RNA were reverse-transcribed as described above using primer G.2 to generate first strand cDNA. PCR was then conducted using a 5'-primer to the SCR G RACE sequence (5'-ACCACTCAAGATTTGCAC) and the 3'-G.6 primer; the products generated were TA-cloned and sequenced. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two Novel Spermatid Chemoreceptor (SCR) Families Are Identified in Purified Round Spermatids-- In a search for OdR-like proteins that may serve functional roles in reproduction, we reasoned that the post-meiotic and actively differentiating cells of the testis would be most likely to express chemoreceptors physiologically relevant to mature spermatozoa. Accordingly, we purified round spermatids from rat testis (Fig. 1a) to isolate total RNA from this select population of cells, and we conducted RT-PCR experiments using degenerate primers (OdR 1 and 2) designed against highly conserved regions of the OdR family.
|
|
|
Tissue Localization of SCR D and G Transcripts--
RNase
protection assays were used to confirm the presence of SCR D and G
transcripts in spermatids of the testis. Full-length protection of SCR
D and G riboprobes was observed with total RNA from round spermatids
but not with RNA purified from spermatocytes in the pachytene stage of
meiosis (Fig. 4). Full-length RNase protection was likewise achieved using total RNA from olfactory tissue,
with multiple partially protected products generated by the SCR G
riboprobe. Hybridization of antisense probes with the corresponding
sense cRNAs served as markers for full-length protection. The integrity
of template RNA was confirmed using a 125-bp -actin riboprobe. In
experiments utilizing poly(A+) RNA, we observed full-length
protection of SCR riboprobes in testis, with a less robust but
detectable product in spleen. No RNase protection occurred with the
yeast control RNA or with poly(A+) RNA from brain, lung, or
liver.
|
|
5'-Splicing of SCRs D and G-- Several observations prompted us to pursue a comparative analysis of SCR cDNA and 5'-genomic sequence. 1) The splice acceptor sequence (28), TTTCTTTCAGG, occurred immediately upstream of the presumptive starting methionine in the genomic sequence of SCRs D-2, -8, and -9. 2) In situ hybridization studies comparing the localization of SCR D and G transcripts in serial sections of sexually mature testis revealed overlapping but also non-overlapping mRNA patterns among seminiferous tubule cross-sections (data not shown). This finding, coupled with the observed developmental expression of SCRs, suggested that distinct transcriptional and/or translational controls may govern the production of SCRs. 3) The introduction of constructs containing OdR coding sequences into transgenic mice has failed to produce detectable expression of OdR proteins.3 We reasoned that the functional expression of OdR proteins may require 5'-translated or -untranslated spliced sequence for message stability, membrane targeting, and/or receptor function.
RACE-PCR experiments were conducted by employing SCR D- and SCR G-specific 3'-primers to generate first strand cDNAs from testis poly(A)+ RNA. RACE-PCR using the anchor primer and nested SCR 3'-primers yielded products for both SCRs D and G. SCR D RACE products contained sequence upstream of the SCR D-8 presumptive starting methionine that was distinct from the genomic clone, providing evidence of 5'-splicing (Fig. 6a). Interestingly, the novel sequence encodes an upstream open reading frame of 20 amino acids starting with a methionine at nt
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we have identified two families of putative OdRs in spermatids of rat testis, termed spermatid chemoreceptors (SCRs) D and G. The two classes of chemoreceptors are only 35% identical to each other, suggesting that they recognize disparate ligands and thereby subserve distinct functions. Within each family, the individual chemoreceptors are highly homologous, displaying at least 86% amino acid identity. The existence of nearly identical receptors may have resulted from gene duplication and may provide a biological back-up mechanism that ensures detection of signals important to the reproductive process. In contrast to the exclusive expression of one or at most a small number of OdRs by individual olfactory neurons (3, 6, 29), SCR D and G transcripts are abundant and widely detected among spermatids of the rat testis.
Whereas the identical pair of degenerate primers repeatedly identifies only two classes of chemoreceptors in rat spermatid cDNA, a much wider variety of partial OdR-like products are generated from whole testis cDNA. The physiologic relevance of OdRs in the testis has been questioned, because changes in chromatin structure during meiosis may generate spurious genetic transcripts. The identification of many partial receptor transcripts in whole testis compared with spermatids may lend support to this hypothesis or represent the requirement for multiple receptors to serve eclectic functions in spermatogenic cells. Nevertheless, the identification of SCRs D and G in the post-meiotic differentiating cells of the testis by RT-PCR, RNase protection assays, and in situ hybridization implies a physiologically relevant role for SCRs in mature spermatozoa. We previously used antibodies to conserved OdR peptide sequence to identify 64-kDa proteins in the membrane fractions of rat, hamster, and human spermatozoa (8). The molecular weights observed are roughly double the ~35-kDa mobility predicted from SCR amino acid sequence. Whereas glycosylation can account for some of the molecular weight disparities (30), others have detected multimeric forms of OdRs by immunoblot analysis (31). The latter observation may account for the approximate molecular weight doubling we observe in testis tissue and is believed to arise from the inability to completely disrupt inter-receptor hydrophobic interactions by denaturing gel electrophoresis. The localization of OdR protein to spermatids of the testis and to the midpiece of mature spermatozoa provides further support for the physiological relevance of such chemoreceptors in male reproductive function (8, 9). The localization of desensitization proteins (8) and inositol 1,4,5-trisphosphate receptor (10) to the tail midpiece, the respiratory center of mature sperm, provides additional intriguing support for the existence of a signaling mechanism to regulate mammalian sperm motility. Sengupta et al. (32) recently established an explicit functional role for Odr-10, a Caenorhabditis elegans olfactory receptor, in recognizing the molecule diacetyl, which results in worm chemotaxis toward the odorant. By analogy, liberation of a signaling molecule by the egg or female reproductive tract may activate sperm OdRs, modulating motility and directing locomotion toward the fertilization site.
In addition to olfactory tissue, mammalian OdR-like transcripts have now been identified in testis (7, 8), taste tissue (33), and developing heart (34). In this report, we also find OdR-like transcripts by RNase protection assays in the adult spleen. Preliminary in situ hybridization experiments show a selective localization of SCR transcripts to the periarterial lymphatic sheath region of the spleen, where B- and T-lymphocytes reside (data not shown). Thus, in addition to potential roles in odorant detection, olfactory neuron axonal guidance (35), sperm-egg signaling, taste sensation, and cardiac morphogenesis, OdR-like proteins may also function as immune cell chemoreceptors. By using RT-PCR, RNase protection assays, and/or in situ hybridization, we and others (33, 34, 36) have found identical or at least nearly identical receptor transcripts in multiple select tissues. In these cases, it is unclear whether the OdR receptors serve common chemosensory roles, are generated by leaky receptor transcription, or undergo post-translational modification to endow tissue-specific functions.
Our phylogenetic analysis demonstrates that SCRs D and G are no more homologous to testis OdRs of different species than they are to olfactory tissue-derived OdRs from the same or different species. Vanderhaeghen et al. (36) have recently completed a thorough analysis of partial olfactory receptor transcripts identified by PCR from human, rat, dog, and mouse whole testis cDNA. Across the different species evaluated, the testicular clones show no specific sequence characteristics compared with OdRs identified in olfactory tissue; testicular receptors within a given species likewise do not cluster. However, the investigators do find highly homologous and possibly orthologous receptors in two species, with three of the receptor couples both detected in testis by RPA. Theoretically, the diversity of testicular receptors between species may enable species-specific positive and negative regulation of sperm-egg signaling, whereas orthologous receptors may serve roles common to spermatogenesis and sperm function across species.
The location of splice acceptor sites upstream of the presumptive starting methionine in genomic SCR clones led to the discovery that SCRs undergo 5'-splicing. Novel 5'-SCR D and G sequences were identified by RACE-PCR and localized in corresponding SCR genomic clones by upstream sequencing. Excluding a 2-kilobase pair gap in SCR G-14, the intron sequences of SCRs G-14 and G-15 share regions of sequence homology and are likely to contain conserved elements involved in transcriptional control of SCR G receptors. Such regulation, for example, may account for the observed expression of SCRs in the sexually maturing and mature testis but not in the prepubertal testis. Furthermore, differences in transcriptional control may explain the instances where SCR D and G spermatid mRNA patterns are non-overlapping (data not shown).
Alternate use of upstream promoter sequences may confer tissue-specific control over receptor expression (37, 38). In a recent report, Asai et al. (39) compared the mouse OdR MOR 23 genomic sequence with corresponding RACE-PCR products derived from testis and olfactory cDNA. Based on the identification of a spliced RACE product from olfactory but not testis cDNA, they proposed that the two tissues differentially utilize transcription initiation sites. Use of alternate promoters has likewise been demonstrated within germ cells, and produces multiple transcripts with differential translation efficiencies (40). In the case of SCR G-14, the identical splice product was identified in testis, spleen, and olfactory epithelium, suggesting that this transcript is generated similarly in these tissues. Ultimately, DNase footprinting will determine which sequences are relevant in promoting and enhancing the transcription of OdRs in particular tissues.
The abundance of spermatid mRNAs detected by in situ hybridization that contain the novel 5'-SCR D RACE sequence as compared with the specific SCR D-8 receptor suggests that the spliced sequence plays a role in multiple SCR D (and perhaps other unrelated) transcripts. Indeed, 5'-sequencing of genomic clones confirmed the presence of homologous RACE sequences in distinct members of a given family. Whereas the novel SCR G RACE sequence likely represents spliced 5'-untranslated DNA, SCR D RACE could theoretically encode an alternate translation initiation site. Use of this site could produce a hydrophilic 20 amino acid amino-terminal extension, potentially affecting SCR function. In general, splicing of OdR transcripts may play important and perhaps tissue-specific roles in regulating mRNA stability, translation, membrane targeting, and functional receptor expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. William Wright for expert assistance in spermatid purification; Roxann Ashworth for DNA sequencing and analysis; Dr. Clark Riley for sequence alignments; and Drs. P. Vanderhaeghen and M. Parmentier for sharing unpublished data.
![]() |
Note Added in Proof |
---|
Feinstein and co-workers recently showed functional expression of mammalian odorant receptors (Zhao, H., Otaki, J. M., Hadimoto, M., Mikoshiba, K., and Feinstein, S. (1998) Science 279, 237-242).
![]() |
FOOTNOTES |
---|
* This work was supported by U. S. Public Health Service Grants MH-18501 and Research Scientist Award DA-00074, a grant from the W. M. Keck Foundation (to S. H. S.), National Institutes of Health Training Grant GM-07309 (to L. D. W.), and CNRS Grant UPR9040 (to M. R.).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.
We dedicate this work to Dr. Thomas S. K. Chang, in memory of his boundless encouragement and enthusiasm and his always helpful advice and conversation.
¶ Current address: Equipe ATIPE-96 CNRS, Laboratoire de Neurobiologie, Cellulaire et Moleculaire, 1 Avenue de la terrasse, 91198 Gif sur Yvette, Cedex, France.
** These authors contributed equally to this work.
To whom correspondence should be sent: Dept. of Neuroscience,
The Johns Hopkins University School of Medicine, 813 WBSB, 725 N. Wolfe
St., Baltimore, MD 21205. Tel.: 410-955-3024; Fax: 410-955-3623.
1 The abbreviations used are: OdR, odorant receptor; RACE, rapid amplification of cDNA ends; RPA, RNase protection assay; SCR, spermatid chemoreceptor; pSCR, partial spermatid chemoreceptors; TMD, transmembrane domain; TBS, Tris-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s).
2 I. Weyand and U. B. Kaupp, personal communication.
3 R. Reed, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|