©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Nicotinic Acetylcholine Receptor 3 Gene
ITS REGULATION WITHIN THE AVIAN NERVOUS SYSTEM IS EFFECTED BY A PROMOTER 143 BASE PAIRS IN LENGTH (*)

(Received for publication, August 19, 1994; and in revised form, November 3, 1994)

Maria-Clemencia Hernandez (§) Linda Erkman (¶) Lidia Matter-Sadzinski Tomas Roztocil Marc Ballivet (**) Jean-Marc Matter (**)

From the Department of Biochemistry, Sciences II, University of Geneva, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genomic and cDNA clones encoding the chicken neuronal nicotinic acetylcholine receptor beta3 subunit were isolated and sequenced. The beta3 gene consists of six protein-encoding exons and the deduced protein has the structural features found in all other members of the neuronal nicotinic acetylcholine receptor subunit family. Although they are undetectable in most brain compartments, beta3 mRNAs are relatively abundant in the developing retina and in the trigeminal ganglion. In situ hybridization and immunohistochemical analysis demonstrated that in retina, beta3 transcripts and protein are confined to subpopulations of cells in the inner nuclear and ganglion cell layers. beta3 is expressed in the proximal and distal regions of the developing trigeminal ganglion, i.e. in both placode- and neural crest-derived neurons. Transient transfection assays in cells freshly dissociated from selected regions of the central nervous system at different developmental stages allowed the identification of genetic elements involved in the neuronal-selective expression of the beta3 gene. A promoter fragment 143 base pairs in length and containing TATA, CAAT, and other consensus sequences is sufficient to restrict reporter gene expression to a subpopulation of retinal neurons. This promoter is totally inactive upon transfection into neuronal and non-neuronal cells from other regions of the central nervous system.


INTRODUCTION

Nicotinic acetylcholine receptor (nAChR) (^1)subunits constitute a family of closely related proteins whose members are assembled in various combinations at the neuromuscular junction and in different regions of the central and peripheral nervous systems. Muscle nAChR is a pentamer composed of four distinct subunits, alpha, beta, , and ; the subunit in mammals being replaced by an subunit during development (reviewed in Schuetze and Role(1987) and Galzi et al. (1991)).

cDNAs encoding seven distinct ligand-binding subunits (alpha2-alpha8) and two structural subunits (beta2, beta4) have been isolated from chicken neural tissues, and most of the corresponding cDNAs have also been cloned in the rat. In Xenopus oocytes, functional neuronal receptors are produced upon coinjection of cRNAs or cDNAs encoding a beta subunit (beta2 or beta4) and any one of the alpha2, alpha3, or alpha4 subunits (reviewed in Deneris et al.(1991), Role(1992), and Sargent(1993). The alpha7 and alpha8 subunits assemble into fully functional homomeric nAChRs (Couturier et al. 1990b; Séguéla et al., 1993; Gerzanich et al., l994) whose properties are being investigated by site-directed mutagenesis (Revah et al., 1991; Bertrand et al., 1992). Recently, it was demonstrated that neuronal nAChR have a pentameric stoichiometry, like their muscle siblings (Cooper et al., 1991; Anand et al., 1991).

The different neuronal nAChR subunit genes are expressed in distinct areas of the developing and adult nervous system, as demonstrated by in situ hybridization and Northern blot analysis. In the CNS, alpha4 and beta2 mRNAs are widely distributed, whereas the other subunit mRNAs and proteins are more restricted, yet there are many instances of overlapping expression (Wada et al., 1989; Morris et al., 1990; Hill et al., 1993; Keyser et al., 1993). Recent studies have shown that subunit mRNAs which are rare in the CNS are relatively abundant in several peripheral ganglia (Boyd et al., 1988, 1991; Couturier et al., 1990a; Listerud et al., 1991; Corriveau and Berg, 1993). Some neuronal nAChR mRNAs are developmentally regulated; both the alpha7 and beta2 transcripts transiently accumulate in the avian optic tectum between embryonic day 7 (E7) and E16 (Matter et al., 1990; Couturier et al., 1990b).

Very little is known about the molecular mechanisms controlling gene expression in neurons. Two different cis-acting regulatory elements that mediate specific neuronal expression have been identified. One element activates promoters in neuronal cells (Yoon and Chikaraishi, 1992), whereas the other represses promoter activity in non-neuronal cells (Mori et al., 1992; Kraner et al., 1992). Although a number of transcription factors have been found in the nervous system, the identity of their target genes remains unknown (reviewed in Mandel and McKinnon(1993)), except for a transcriptional activator that binds to the regulatory sequence of several genes specifically expressed in olfactory neurons (Wang and Reed, 1993).

Because its individual members have very different expression patterns in the chick nervous system, the nAChR gene family is an excellent model system to study the regulatory mechanisms that restrict gene expression to particular classes of central or peripheral neurons. We have previously identified the upstream sequence conferring transcriptional specificity on the alpha7 gene (Matter-Sadzinski et al., 1992). We describe here the molecular cloning of the beta3 gene, a member of the neuronal nAChR gene family in the chick. We show that this gene has a highly restricted pattern of expression within the developing central and peripheral nervous system, and we define the 5`-flanking region containing the regulatory sequences required for specific neuronal expression.


MATERIALS AND METHODS

Standard Molecular Biological Procedures

Construction of a chicken genomic library, radioactive probe synthesis, screening procedures, bacteriophage purification, subcloning, and sequencing protocols were as described in Nef et al.(1988) and Couturier et al. (1990a).

Extraction of RNA

Chick embryos were staged according to Hamburger and Hamilton(1951). For simplicity, the developmental stages are reported in E. E5, 6, 8, 10, 12, 13, 14, 16, and 18 correspond to stages 27, 29, 34, 36, 38, 39, 40, 42, and 44, respectively. Trigeminal ganglia, retinae, optic tecta and other neural or non-neural control tissues were dissected at appropriate developmental stages, immediately frozen in liquid nitrogen and stored at -70 °C. The RNA extraction procedure was adapted from Maniatis et al.(1982). In order to obtain undegraded RNA, it was essential to disrupt the ganglia quickly and thoroughly. This was achieved by vigorous vortexing of 10-50 ganglia in 1.5-ml microcentrifuge tubes containing 0.5 ml of baked glass beads (mean diameter 0.45 mm) and 250 µl of lysis buffer (10 mM Tris-Cl, pH 7.4, 1 mM MgCl(2), 10 mM NaCl, 1% SDS, 1 mg/ml proteinase K).

Isolation of Genomic and cDNA beta3 Clones

The initial beta3 genomic clones were isolated from a chicken genomic library (Ballivet et al., 1983) screened at low stringency as described in Couturier et al. (1990a). The probe was a fragment of the chick neuronal alpha5 gene (HincII-XbaI fragment encoding residues Asp-Ser) (Couturier et al., 1990a).

Total RNA from E10 trigeminal ganglia was extracted as described above. Double-stranded cDNA was synthetized from 5 µg of poly(A) RNA using a Pharmacia cDNA synthesis kit and inserted in gt10 (Promega). Packaging (Gigapack, Stratagene) of a fraction of the ligation reaction yielded 10^7 independent virus particles. Screening performed as in Couturier et al. (1990a) yielded two full-length cDNAs from 10^6 independent plaques. The cDNAs were subcloned in pBluescript SK (Stratagene). Partial DNA sequencing and restriction mapping established that they were accurate transcripts of the beta3 gene.

Probes

For Northern blot analysis, we used a beta3 genomic fragment containing 250 bp of exon 5 (encoding amino acids Met-Gln) and 240 bp of intron 5. To increase the intensity of the signal in some experiments with the trigeminal ganglion, we used a 1700-bp cDNA fragment encoding the distal part of the subunit (amino acids Met-Leu) plus 3` noncoding sequences. These two probes, and a 396-bp fragment containing exon 1 and 209 bp of intron 1 gave the same hybridization patterns in Northern blots. Double-stranded genomic or cDNA fragments were labeled with [alpha-P]dATP (Amersham Corp.) using a random-primed DNA labeling kit (Boehringer-Mannheim).

For in situ hybridization studies, we used 45-mer antisense and sense oligonucleotides corresponding to amino acids Val-Tyr of the beta3 subunit. Oligonucleotides least likely to cross-hybridize to other chick neuronal nAChR mRNAs were selected by aligning with all known chick nAChR transcripts. On control Northern blots of retina poly(A) RNA, the P-labeled antisense oligonucleotide hybridized (Beyer, 1991) to the same major band as the cDNA probes. The minor mRNA band was not detected due to the very low specific activity of radiolabeled oligonucleotides as compared to homogeneously labeled cDNA fragments. Neither liver nor muscle poly(A) RNA gave detectable signals under the same conditions.

The antisense and sense oligonucleotides were labeled to a final specific activity of 5-10 times 10^6 cpm/pmol by 3` tailing, using alpha-S-dATP (DuPont-NEN; 1200-1400 Ci/mmol). Briefly, 4 pmol of oligonucleotide were incubated 1 h at 37 °C with 45-50 pmol of alpha-S-dATP in labeling buffer containing 100 mM sodium cacodylate, pH 7.2, 8 mM MgCl(2), 1 mM beta-mercaptoethanol, 200 µg/ml bovine serum albumin, and 13 units of Terminal Transferase (Amersham Corp.) in a final volume of 10 µl. The reaction was stopped by adding 80 µl of STE (0.1 mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA) and 100 µg of yeast tRNA. The probe was purified on a 2-ml Sephadex G-50 (Pharmacia Biotech Inc.) column equilibrated with STE. S-Labeled oligonucleotides were stored at -70 °C and used within 2 weeks.

Northern Blots

Total RNA (0.5 µg) from different developmental stages or 0.5 µg of poly(A) RNA from E12 retina and E10 trigeminal ganglion were denatured by heating 20 min at 65 °C in MOPS buffer (40 mM MOPS, 10 mM sodium acetate, 2 mM EDTA, pH 7.0), in the presence of 10% formaldehyde. Denatured samples were fractioned by electrophoresis in 1.5% agarose minigels in MOPS buffer containing 6.7% formaldehyde. Gel-fractionated RNA was blotted and hybridized as described by Khandjian(1986) and by Matter et al.(1990).

Preparation of Tissue Sections

Whole eyes and trigeminal ganglia at different developmental stages were dissected, embedded in OCT compound (Miles) and molded in Lab-Tek tissue culture chambers (Miles) or microcentrifuge tubes, frozen in isopentane cooled to -25 °C on dry ice, and stored at -70 °C. 10-µm cryostat sections were mounted on poly-L-lysine (200 µg/ml)-coated slides, thawed for a few seconds on a heating block at 40 °C, immediately transferred to -20 °C, and stored for a maximum of 2 weeks at -20 °C. All steps in the procedure were done under sterile conditions.

In Situ Hybridization

We followed the procedure described by Aubry et al.(1993). The hybridization medium was composed of 50% deionized formamide, 4 times SSC, 1 times Denhardt's solution, (10%) dextran sulfate (Pharmacia), 5 mM dithiothreitol, 250 µg/ml yeast tRNA, 5 µg/ml poly(A) (Boehringer Mannheim), 500 µg/ml sonicated salmon sperm DNA, and 10^7 cpm/ml radiolabeled oligonucleotide. The sections were covered with 60 µl of hybridization medium under a 4 cm^2 parafilm ``coverslip'' and incubated overnight at 42 °C in a humidified chamber. They were washed twice for 30 min each in 1 times SSC at 42 °C, 30 min in 0.1 times SSC at 42 °C, and 30 min in 0.1 times SSC at room temperature. The washed sections were air-dried (7 min) after dehydration in 70 and 100% ethanol, and processed for autoradiography as described in Matter et al.(1990).

Expression of the beta3 Cytoplasmic Loop

The system for high level production in Escherichia coli and rapid purification of recombinant proteins developed by Stüber et al. (1990) was utilized to express the subunit-specific cytoplasmic loop of the chicken beta3 subunit. A restriction fragment of the beta3 gene encoding Met-Ala and bounded by an NcoI site in 5` and a PvuII site in 3` was blunt-ended with Klenow DNA polymerase and ligated to the blunted BamHI site of expression vector pDS56/RBSII,6xHis. Plasmids bearing the insert in the proper orientation were selected in the E. coli host strain M15 harboring plasmid pREP4, a lac repressor overproducer. Cultures were grown at 37 °C to an OD of 0.6-0.7 in NZY medium containing 100 µg/µl ampicillin and 25 µg/µl kanamycin. Expression of recombinant protein was induced by the addition of isopropyl-beta-D-thiogalactoside to a final concentration of 2 mM. The recombinant protein containing the (His)(6) affinity tail (termed FPbeta3in) was purified by Ni-chelate affinity chromatography, using Ni-nitrilotriacetic acid-agarose (Qiagen).

Generation of Antibodies

Purified FPbeta3in cytoplasmic loop protein (50-100 µg in 150 µl of phosphate-buffered saline, PBS) was emulsified with Freund's complete adjuvant (1:1) and injected subcutaneously to female Lewis rats. Two subsequent booster immunizations (50 µg in Freund's incomplete adjuvant) were made at 15-day intervals. Two days before sacrifice, the rats were boosted by intraperitoneal injection (100 µg in 100 µl of PBS). The titers of the antisera were determined by enzyme-linked immunosorbent assay and by Western blot analysis.

Immunohistological Detection of the beta3 Subunit

Sections from retina and trigeminal ganglion were prepared as described for in situ hybridization. They were incubated 15 min with normal goat serum diluted 1/20 in PBS at room temperature and washed twice with PBS. beta3 antiserum diluted 1/5 in PBS was added, and the sections were incubated for 2 h. After washing, the slides were incubated for 1 h with the second antibody, a 1/400 dilution of biotinylated goat anti-rat IgG (Amersham) in PBS. We used either an enzymatic or a fluorescent system to detect the bound antibodies. In the enzymatic detection system, a preformed complex of streptavidin was bound to biotinylated horseradish peroxidase (Amersham). In the fluorescent system, we used a preformed complex of streptavidin bound to biotinylated phycoerythrin (Amersham). After washing, a stabilizer solution (Amersham) was added for 20 min. The slides were then dried at 37 °C for 20 min and mounted for fluorescent microscopy.

Primer Extension

A 19-mer antisense oligonucleotide (complementary to nucleotides +4 to -15, relative to the initiator ATG) was phosphorylated with [-P]ATP and hybridized to 2 µg of poly(A) RNA from chick neuroretina (E12) or from chick fibroblast-like cells (CEFs). Annealings were carried out for 4 h at 42 °C in 40 mM PIPES (pH 6.4), 1 mM EDTA, 0.4 M NaCl. Reverse transcription by cloned Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) was done in 100 mM Tris-Cl (pH 8.5), 20 mM MgCl(2), 80 mM KCl, 2 mM dithiothreitol, 550 µM dNTPs, 50 ng/µl bovine serum albumin, and RNAsin (10 units/50 µl). The reaction was allowed to proceed for 1 h at 37 °C, then the extension products were fractionated on a sequencing gel and analyzed by autoradiography.

Plasmid Constructions

Plasmid pCAT00 (Crossley and Brownlee, 1990) was selected as a vector because of its very low background chloramphenicol acetyltransferase (CAT) activity in our cell systems, and plasmid pSVCAT construction was described in Matter-Sadzinski et al.(1992). Restriction fragments beginning 128 bases (-128, SphI) 5` of the beta3 initiator ATG and extending upstream to -199 (PvuII, fragment PvS), -271 (EcoRI, fragment RS), -343 (NsiI, fragment NS), -850 (PstI, fragment PS) and to approximately -2 kbp (HindIII, fragment HS) were inserted into the SmaI site of pCAT00, immediately upstream of the CAT gene. A fragment bounded by a PstI site at -850 and an EcoRI site at -271 (fragment PR) was also inserted into the SmaI site of pCAT00.

Plasmid plac00 was constructed by first inserting a NotI linker into the SalI site of pCMV-nlacZ (a gift from M. J. Weber, Université de Toulouse) and into the SmaI site of pCAT00, then the NotI-PstI fragment of pCAT00 was replaced by the NotI-PstI fragment from pCMV-nlacZ. Restriction fragments PS and RS were then inserted in both orientations, and fragment NS in the correct orientation into the blunted NotI site of plac00 immediately upstream of the nlacZ gene (Kalderon et al., 1984). pSVlac was constructed by inserting the blunted HindIII-PvuII restriction fragment containing the SV40 promoter at the blunted NotI site of plac00. Constructions were checked by restriction mapping and DNA sequencing. To test whether promoter activity is orientation-dependent, fragment PS was inserted in inverted orientation in the lac and CAT vectors, and transfected into cells from E5 retina. No beta-gal-positive cells were detected, and CAT activity was at the background level.

Cell Culture, Transfections, CAT, and beta-Galactosidase Assays

Tissue dissociation, cell culture, cell transfection, and CAT assay procedures were as described in Matter-Sadzinski et al.(1992).

For X-gal staining, the transfected cells were grown in a four-well cluster dish (Nunc) or in a four-chamber plastic slide (Lab-Tek) for 48 h. The culture medium was then removed and the cells were rinsed with PBS (pH 7.2) and fixed in 2% formaldehyde, 0.4% glutaraldehyde (prepared in PBS) for 5 min at room temperature. They were rinsed twice with PBS and stained for 3 h at 37 °C in PBS containing 1 mg/ml X-gal, 4 mM K(3)Fe(CN)(6), 4 mM K(4)Fe(CN)(6), and 2 mM MgCl(2). The cells were rinsed once with PBS and either mounted in fluoromount (Fisherbiotech) or stored in the fix solution (2% formaldehyde, 0.4% glutaraldehyde) at 4 °C.


RESULTS

Isolation of the beta3 Gene and cDNA

To isolate additional members of the neuronal nAChR gene family, we screened a chicken genomic library constructed in the vector L47 (Ballivet et al., 1983) with an alpha5 probe, under low stringency hybridization conditions. Duplicate filters spotted with DNA from positive recombinant phage were hybridized in highly stringent hybridization conditions with the alpha5 probe or with probes from other nAChR genes. Two clones (phage 1 and 11) out of 30 failed to hybridize under these conditions and their restriction maps were different from that of previously identified neuronal nAChR genes (Fig. 1). Convenient consecutive restriction fragments were subcloned and sequenced, and the exons were determined by homology. The deduced amino acid sequence was related to, but different from that of all other cloned nAChR subunits. The beta3 gene (Fig. 1) has exactly the same structure as most previously characterized neuronal nAChR genes (alpha7 and alpha8 are exceptions), consisting of six protein-encoding exons of which exon 5 encodes approximately two-thirds of the mature protein product (Nef et al., 1988; Couturier et al., 1990a). As genomic phage 1 and 11 did not encompass exon 1 and upstream sequences, a fragment containing exon 2 was used as a probe in high stringency conditions to isolate overlapping phage extending further in 5`. In this way, we isolated phage 17 which, in addition to exons 1 and 2, contains several kilobase pairs of 5`-flanking sequences (Fig. 1).


Figure 1: Partial restriction map of the neuronal nAChR beta3 gene. The overlapping recombinant phage 1, 11 and 17 (arrows) define a unique region of the chicken genome extending across 13 kbp and containing the beta3 gene. Numbered boxes locate the exons encoding the subunit. In addition to coding sequences (filled boxes), exons 1 and 6, respectively, contain 5`- and 3`-untranslated sequences (hashed boxes). H, HindIII; P, PstI; R, EcoRI; Bg, BglII; Ba, BamHI sites.



Two full-length beta3 subunit cDNA clones were isolated from an embryonic trigeminal ganglion library (see ``Materials and Methods''), using a genomic probe encoding the subunit-specific cytoplasmic region of the protein. Both cDNA clones were checked by restriction site analysis and partial DNA sequencing to ensure that they were accurate transcripts of the beta3 gene.

The beta3 Subunit Protein

The beta3 protein (Fig. 2) exhibits the standard features found in other members of the neuronal nAChR subunit family. It includes a signal peptide that is probably cleaved at the indicated site to generate a mature protein of 435 residues with a calculated, unglycosylated M(r) of 50036. There are four putative transmembrane domains (TM1-TM4) spaced exactly as in all other known nAChR subunits, and located such that residues 1-210 encompass the extracellular domain and residues 300-405 the cytoplasmic loop of the protein.


Figure 2: Nucleotide and deduced protein sequences of the chicken beta3 gene. Nucleotides are numbered from the translation start site on the proximal 5`-flanking region of the beta3 gene. The double underline identifies the oligonucleotide used for primer extension analysis and the transcription initiation site is marked with an arrow. The TATA and CAAT boxes are underlined; CACCC boxes are overlined; E-boxes and the AP-1 motif are labeled (bullet) and (ˆ), respectively. Lower case nucleotide symbols indicate donor and acceptor sites of intervening sequences; dashed underline, postulated transmembrane regions TM1-TM4. The coding sequence contains 52.5% AT. The mature beta3 subunit (residues 1-435) has a calculated molecular weight of 50036 daltons and has potential N-linked glycosylation sites at positions 28, 143, and 432 (*). Cysteines 130 and 144 and intervening sequence are highly conserved in all known subunits of ligand-gated ion channels belonging to the nAChR superfamily.



The beta3 protein has three potential N-linked glycosylation sites (N28, N143, and N432), the first and second of which are conserved in several neuronal nAChR subunits. Two cysteine residues that correspond to cysteine 128 and 142 (alpha1 subunit numbering) (Nef et al., 1988) are also present in the protein but the subunit lacks the pair of vicinal cysteines at position 192-193 that is the hallmark of all alpha subunits and forms part of the ACh-binding site (reviewed in Galzi et al.(1991)). Although beta3 is thus operationally defined as a beta subunit, alignment of its amino acid sequence with those of all other known avian neuronal nAChR subunits (Table 1) reveals that beta3 aligns significantly better with most of the alpha than with the beta subunits (e.g. 68.2% amino acid identity and 11.3% amino acid similarity with the alpha5 subunit). When the alignment was made between beta3 and related members of the superfamily in other species, the highest percentage of identity (83.6) was obtained with the rat beta3 subunit (Deneris et al., 1989) and with the goldfish nalpha2 (74.9) and nalpha3 (77.0) subunits (Cauley et al., 1989, 1990).



In the CNS, Expression of the beta3 Gene Is Restricted to Small Subpopulations of Neurons

Northern blot analysis was performed on total and polyadenylated RNA isolated from different regions of the central nervous system at different developmental stages. At E12, we observed relatively high levels of beta3 mRNA in the retina, but no detectable transcripts in the optic tectum (Fig. 3A), telencephalon, cerebellum, and spinal cord (not shown). During embryonic development of the retina, two mRNAs of about 5 and 3 kb were detected in total and poly(A) RNA, the shortest of these was the most abundant and both species hybridized with probes from the 5` and the 3` ends of the cDNA (Fig. 3B and Fig. 6B). The levels of beta3 mRNAs are very low at E6, they increase until E12 and remain relatively high from E12 until adulthood. In control experiments, no signal was observed when the probes were hybridized to liver poly(A) RNA (Fig. 3A). In situ hybridization histochemistry on retinae from newly hatched chick (P1) detected labeling in the large majority of cells in the ganglion cell layer (GCL) and in a subset of cells in the inner half of the inner nuclear cell layer (INL), but not in any of the other plexiform and cellular layers (Fig. 4, A and B). The same distributions of hybridization positive cells was detected on E13 (not shown).


Figure 3: Northern blot analysis of beta3 transcripts in chick retina and trigeminal ganglion. A, poly(A) RNA (500 ng/lane) isolated from E12 liver, optic tectum, retina, and E10 trigeminal ganglion was fractionated by gel electrophoresis, blotted to nitrocellulose, and hybridized with a P-labeled beta3 probe (genomic fragment NcoI-EcoRI) encoding part of the cytoplasmic region of the protein. Exposure times for the left and right panels were 24 h and 10 days, respectively. Arrowheads locate the 28 and 18 S rRNA bands. B, total RNA (500 ng/lane) isolated from the retina at different stages of development (the embryonic day is indicated by numbers and the newly hatched and adult stages are indicated by H and A, respectively) were hybridized to the beta3 probe as in A. Exposure time was 10 days. Lower panel, methylene blue staining of the blot.




Figure 6: Localization of the beta3 transcription start site. A, molecular map of the 5`-flanking region of the beta3 gene. The extent of the three restriction fragments used for Northern blot analysis is indicated by the horizontal lines below the map. The box indicates the position of coding (filled box) and non-coding sequences (hashed box) in exon 1. H, HindIII; P, PstI; Ns, NsiI; R, EcoRI; Pv, PvuII; S, SphI. B, Northern blot analysis was performed to map the 5`-end of the beta3 gene. P-Labeled fragments 1, 2, and 3 were hybridized to 1 µg of poly(A) RNA from E12 retina. C, mapping of the transcription initiation site by primer extension. Poly(A) RNA (2 µg) from E12 retina (R) and total RNA from primary cultures of chick embryo fibroblast-like cells (F) were hybridized to a P-end-labeled synthetic oligonucleotide complementary to nucleotides -15 to +4 from the 5`-flanking region of the beta3 gene (double underline in Fig. 2). The extended fragment synthesized by reverse transcriptase is marked (*) and maps a single transcription initiation site at position -111 of the translation start site. Adjacent is a dideoxy sequencing ladder (GATC) of a beta3 genomic clone primed with the same oligonucleotide.




Figure 4: Localization of beta3 transcripts and protein in the retina. Darkfield photomicrographs of neonatal (P1) retina sections hybridized with beta3 oligonucleotide antisense (A) and sense (B) probes. The sections were exposed for 3 weeks. In the retina, beta3 transcripts are detected in the ganglion cell layer (GCL) and in the inner half of the inner nuclear cell layer (INL). The beta3 protein was detected (C) by antiserum to the beta3 cytoplasmic domain (FPbeta3in). Bound antibodies are visualized using biotinylated goat anti-rat IgG and avidin-biotinylated HRP complex. Anti-beta3 immunolabeling is present in the GCL and the INL. Note the presence of heavily labeled cells within the GCL (arrows) and along the inner edge of the INL (arrowheads). Bar, 50 µm.



To determine which retinal cells accumulate detectable levels of the beta3 protein, antiserum to FPbeta3in was used on sections of neonatal retinae (P1). The antibodies specifically labeled subpopulations of cells from the GCL and INL (Fig. 4C). The anti-beta3 antibody failed to label the inner plexiform layer (IPL) although that layer contains processes from ganglion and amacrine cells.

These restricted distributions of hybridization and immunoreactivity suggest that beta3 transcripts and the beta3 protein accumulated in the large majority of ganglion cells and in a subset of amacrine neurons.

Expression of the beta3 Gene in the Developing Peripheral Nervous System

In the peripheral nervous system, relatively high levels of beta3 mRNA are in evidence in the developing trigeminal (Fig. 3A) and dorsal root ganglia (not shown), being about 2-fold higher in the trigeminal ganglion. Only the short mRNA species (3 kb) is detectable in both ganglia. Very low levels of beta3 mRNA are observed in the superior cervical and sympathetic ganglia, and it is undetectable in the petrosal, nodose, and ciliary ganglia. (^2)In situ hybridization on E8 (Fig. 5A) and E10 trigeminal ganglion (data not shown) reproducibly showed that the ganglion cells expressing beta3 transcripts are mainly localized in the distal and proximal regions of the ophthalmic and maxillo-mandibular lobes. More intense labeling occurs preferentially in large neuronal cell bodies of the distal regions (Fig. 5A). No labeling was observed on trigeminal ganglion sections probed with sense oligonucleotide (Fig. 5B).


Figure 5: Localization of beta3 transcripts and protein in the trigeminal ganglion. In situ hybridization of E8 trigeminal ganglion sections with beta3 oligonucleotide antisense (A) and sense probes (B). Sections were exposed for four weeks. Note the stronger labeling in the distal part of the ganglion. Immunohistological localization of the beta3 protein in E10 trigeminal ganglion (C) by an antiserum to the beta3 cytoplasmic domain (FPbeta3in). Bound antibodies are visualized using biotinylated goat anti-rat IgG and streptavidin-biotinylated phycoerythrin complex. Anti-beta3 immunolabeling is present in some but not all neurons. In D, a similar section was processed as in C, except ,that normal rat serum replaced the antibody to FPbeta3in. Bars, 70 µm (A and B), 60 µm (C and D).



Immunohistochemistry on E8 and E10 trigeminal ganglion reveals beta3 immunoreactive sites throughout the ganglion. Not all cell bodies are labeled (Fig. 5C), and immunoreactive cells from the distal and proximal regions of the ganglion show stronger labeling at E10 than at E8.

The 5`-Flanking Region of the beta3 Gene

Because the tissue-specific expression of many genes is due to cis-acting elements within 5`-flanking regions, we subcloned and sequenced a 2-kb HindIII fragment from phage 17 (Fig. 1) encompassing exon 1 and upstream sequences. The nucleotide sequence extending 850 bp upstream of the beta3 initiator ATG is shown in Fig. 2.

We next determined the approximate 5`-end of the beta3 transcript. Restriction fragments 1, 2, and 3 (Fig. 6A) were used as radioactive probes to detect beta3 transcripts in neuroretina RNA blots (Fig. 6B). The two mRNA species were only detected upon hybridization with fragment 3, suggesting that the transcription initiation site of both mRNAs is localized within the 128-bp immediately upstream of the initiator ATG. The precise transcription initiation site of beta3 mRNA was mapped by primer extension. A P-labeled 19-mer synthetic oligonucleotide complementary to nucleotides +4 to -15 (relative to the translation initiation site, Fig. 2) was used in the reaction as described under ``Materials and Methods.'' A single extended product was detected with poly(A) RNA from retina, demonstrating the presence of a single initiation site 111 bp upstream of the ATG (Fig. 6C).

beta3 Promoter Elements

The 5` sequence of the beta3 gene contains putative TATA and CAAT boxes located 49 and 76 bp upstream of the mRNA cap site (-160 and -187 bp upstream of the ATG initiator codon, Fig. 2). At positions -265, -276, and -585 relative to the translation initiation site, we find CACCC boxes, first identified in the beta-globin gene promoters (Myers et al., 1986) and also found in several neural specific genes (reviewed in Mandel and McKinnon (1993)). The sequence starting at position -626 fits the consensus binding sequence of the transcription factor AP-1 (TGACTCA). In addition, there are five CANNTG motifs (E boxes), which are potential target sites for the helix-loop-helix (HLH) family of transcription factors.

The 5`-Flanking Sequence Directs Cell-specific Reporter Activity in Transient Expression Assays

To determine whether the 5`-flanking sequence of the beta3 gene contains sufficient information for tissue- and stage-specific expression, we generated a series of constructs (Fig. 7A) fusing varying lengths of DNA to promoterless reporter genes encoding either bacterial CAT or beta-gal. The fusions were made at position -128 relative to the translation initiation site of the beta3 gene and the fragments extend upstream stepwise to approximately -2 kbp. To determine the promoter activity of the different fragments in cells freshly dissociated from selected regions of the chick CNS at different developmental stages (optic tectum at E5 and E10; retina at E5, E8, and E13), we used a liposome-mediated transient transfection assay developed in the laboratory (Matter-Sadzinski et al., 1992). Secondary cultures of chick astroglia were prepared from retina and optic tectum to serve as a source of differentiated, non-neuronal cells from within the nervous system. We used proliferating secondary cultures of CEFs as a source of undifferentiated mesodermic stem cells, and the transformed cell line CHO as an additional, non-neural control.


Figure 7: The 5`-flanking region of the beta3 gene contains regulatory elements which confer neuronal specificity. A, schematic structure of the chimeric plasmids tested for activity in transient transfection assays. Restriction fragments beginning 128 bp upstream of the initiator ATG and extending 5` to a distance of -199 (PvS); -271 (RS); -343 (NS); -850 (PS) and approximately -2 kbp (HS) were fused to the CAT gene in pCAT00, as was a fragment extending from -271 to -850 (PR). Fragments RS, NS, and PS were also fused to the bacterial beta-galactosidase gene (lacZ) in plac00. B, comparison of the restriction fragment promoter activities in neural cells, CEFs, and CHO cells. Constructs were transfected into neural cells dissociated from E5, E8, and E13 retinae, E5 and E10 optic tecta, and into glial cells, CEFs and CHO cells. 48 h after transfection, cells were processed for CAT assay. The CAT activity obtained with transfection of pSVCAT is arbitrarily set at 100 for each cell type. All other CAT activities are given relative to this value. Activity of the SV40 early promoter in arbitrary CAT units per µg protein was similar in all cell types at all stages of development (for details see Matter-Sadzinski et al., 1992).



In these different cell systems, promoter activity was determined by CAT assay or X-gal staining 48 h after transfection. In all cell types at all the studied stages, the SV40 promoter drove beta-gal expression in 10-15% of the cells. As shown in Table 2, the constructs driven by fragments PS, NS, and RS all yielded about the same fraction of beta-gal positive cells in E5 and E13 retinal cells. In contrast, no labeled nuclei were ever detected in E5 and E10 optic tectal cells, astroglia, and CHO cells (frequency <10 of beta-gal positive cells obtained upon transfection by the SV40 promoter).



Although the fragments PS, NS, and RS were active in similar fractions of transfected retinal cells, Fig. 7B demonstrates that these fragments have very different activity levels in CAT assays. The constructs containing the CAT gene fused to fragments HS, PS, NS, and RS were expressed at significant levels in cells from E5 retina, being about 3-fold higher in the RS construction. The promoter activity of fragments PR and PvS was at the background level. In E8 and E13 retina, the constructs were much less active than at E5, yet they maintained the same rank-order in potency. Putting together the beta-gal and CAT assays, we conclude that the much reduced CAT activity measured in E8 and E13 retina results from a reduced reporter gene expression in a stable number of cells. This decrease coincides with the period of development when the majority of retinal cells withdraw from the mitotic cycle. In agreement with the beta-gal results, none of the constructs expressed detectable CAT activity in E5 and E10 tectal cells, astroglia, CEFs or CHO cells. Thus, the RS fragment (143 bp) contains regulatory elements sufficient to restrict promoter activity to the retina, i.e. to the only region of the CNS where expression of the beta3 gene was detected.


DISCUSSION

The beta3 Gene and Protein

Most neuronal nAChR subunit genes identified so far in vertebrates are composed, like the beta3 gene, of six protein-encoding exons with exactly conserved donor and acceptor splice sites (Nef et al., 1988; Couturier et al., 1990a). In addition, the first four and last exons are conserved between neuronal and muscle nAChR genes. Their common structure suggests that the alpha2-alpha5 and beta2-beta4 genes derive from a single member of an ancestral set of related precursor genes. The recently isolated alpha7 and alpha8 genes are members of another class of neuronal nAChR genes whose structure profoundly differs from that of the beta3 gene (Couturier et al., 1990b). (^3)

The deduced sequence of the beta3 protein has features found in all neuronal nAChR subunits. Lack of the two adjacent cysteines corresponding to Cys-192 and -193 of the muscle alpha1 subunit classify this protein as a beta subunit. We did not succeed in reconstituting a functional receptor upon injection into Xenopus oocytes of beta3 cDNA either alone or in pairwise combinations with alpha2, alpha3, alpha4 or alpha6. (^4)alpha5 also fails to yield functional receptors when injected alone or in pairwise combination with any of the beta subunits (Couturier et al., 1990a). Likewise, the cognate rat beta3 and alpha5 cDNAs fail to reconstitute functional receptors in oocytes (Boulter et al., 1990). However, recent immunoprecipitation experiments using subunit specific monoclonal antibodies indicate that some native neuronal nAChRs may assemble from more than two subunits and that the alpha5 subunit appears capable of participating in several distinct neuronal nAChR subtypes (Conroy et al., 1992; Vernallis et al., 1993). We speculate that the beta3 subunit perhaps participates in similar co-assemblies to form functional receptors in the nervous system. Alternatively, beta3 may require an unidentified alpha-subunit for assembly, or it may require a processing step that does not take place in oocytes.

As illustrated in Table 3, the avian beta3 and alpha5 subunits share amino acid substitutions in functionally important regions which are otherwise highly conserved in all other members of the neuronal nAChR family. The same amino acid substitutions are also found in the rat beta3 and alpha5 subunits as well as in the goldfish na2 and na3 subunits. The changes at positions 93 and 94 may have important effects on ligand binding cooperativity, as suggested by mutagenesis analysis at the corresponding sites of the alpha1 and alpha7 subunits (reviewed in Changeux et al., 1992). Changes in the region TM1-TM3 may affect channel properties as TM2 lines the channel (Giraudat et al., 1987; Leonard et al., 1988) and as rings of charged amino acids on either side of TM2 play an important role in ion selectivity (Imoto et al., 1988). Moreover, mutations of L247 in TM2 of the homomeric alpha7 channel (L251 in alpha1 numbering) profoundly modify the properties of the channel (Revah et al., 1991; Bertrand et al., 1992).



Expression of the beta3 Gene

beta3 transcripts are undetectable in the chicken brain, but they are relatively abundant in the developing and adult retina and in several developing sensory ganglia. Two beta3 mRNA species were detected by non-overlapping probes in the retina, and only one species in trigeminal or dorsal root ganglia. We suggest that the two mRNAs may result from different choices of polyadenylation sites. Alternatively, the 5-kb species may result from the accumulation of an incompletely spliced beta3 mRNA.

In neonatal retina the beta3 transcripts are localized in the inner half of the INL and in the GCL. Immunohistochemical analysis demonstrates that the beta3 transcripts are indeed translated and that the protein product mainly accumulates in the INL and GCL. The localization of the transcripts and protein suggests that in the retina the beta3 gene is mainly expressed in ganglion and amacrine cells. In the trigeminal ganglion at E8, beta3 transcripts are present mainly in the distal and proximal regions of the ganglion, being clearly more abundant in certain large cells of the distal part. Cells that form the avian trigeminal ganglion arise from the neural crest and placodal epithelium. Neural crest cells are concentrated in the proximal and central parts of the bilobed ganglion while placode-derived cells are restricted to the distal aspects of each lobule (Hamburger, 1961). Based on these observations, we suggest that beta3 transcripts are present in cell subpopulations derived from both embryonic origins.

Regulation of the beta3 Gene

In this report, we begin to characterize the regulatory elements in the 5`-flanking region of the beta3 gene. In contrast to the alpha7 gene (Matter-Sadzinski et al., 1992), the beta3 gene promoter contains TATA and CAAT consensus sequences, has a moderate GC content (49%), and initiates transcription at a single site. We analyzed by transient transfection the promoter activities of progressively larger restriction fragments extending upstream from a fixed point in the 5`-flanking region. We observed that within 143 bp (fragment RS) of 5`-flanking sequence, there is sufficient information to confer neuron-specific expression to the beta3 gene. Moreover, we found that the RS fragment has stronger promoter activity than larger fragments having the same 3` end, arguing for the presence of one or more transcriptional attenuators in 5` of the RS sequence. Consistent with the expression pattern of the beta3 gene within the retina, the RS fragment has a strong promoter activity in identified ganglion cells. (^5)

In addition to the appropriately spaced TATA and CAAT boxes, fragment RS contains one copy each of the CACCC and CANNTG (E-box) motifs. These motifs are known to regulate transcription in other genes (Myers et al., 1986; Murre et al., 1989). In particular, muscle nAChR transcription is regulated, at least in part, by the interaction between E-boxes and myogenic factors that are members of the HLH family of transcription factors. Finer mapping and mutagenesis studies are in progress to determine the number and function of the discrete cis-acting elements present in this regulatory region.

The alpha7 and beta3 promoters exhibit contrasting behaviors during development of the central nervous system. At early stages of embryogenesis, the alpha7 promoter is widely active in undifferentiated tissues of the developing CNS, and as development proceeds its activity becomes restricted to particular neuronal phenotypes (e.g. retinal ganglion cells) (Matter-Sadzinski et al., 1992). In contrast, activity of the beta3 promoter is restricted early on to a subset of neuroepithelial cells of the retina.^5 As judged by expression of two reporter genes, both promoters are very active in neuroepithelial cells of the retina on embryonic day 6, although the mRNAs encoded by these two genes are either very sparse (beta3) or undetectable (alpha7) at this early stage of development. This apparent discrepancy between transcriptional activity and mRNA levels may reflect a process of post-transcriptional regulation resulting in delayed accumulation of the transcripts at early stages of development. In transfection experiments, activity of both the beta3 and alpha7 promoters declines sharply in the retina between E5 and E13. In differentiated retina (E13), the beta3 and alpha7 promoters remain active, respectively, in about 10% and 1% of the cells, and that is consistent with RNA blot analysis showing that beta3 transcripts are much more abundant than alpha7 transcripts at this stage of development.

Although the alpha7 and beta3 genes use different strategies to achieve their stringent neuron specificity within the nervous system, both genes begin to be expressed in the retina several days before establishment of functional connections. The significance of this early expression remains unknown, but it suggests that the two corresponding receptor subunits may mediate regulation of cell differentiation during retina neurogenesis.


FOOTNOTES

*
This research was funded by the State of Geneva and by grants from the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) X83739 [GenBank]and X83740[GenBank].

§
Present address: Brain Tumor Research Center, The Preuss Laboratory, University of California, San Francisco, CA 94143-0520.

Present address: Dept. of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093-0648.

**
To whom correspondence should be addressed: Dept. of Biochemistry, Sciences II, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland. Tel.: 41-22-7026494; Fax: 41-22-7026483.

(^1)
The abbreviations used are: nAChR(s), nicotinic acetylcholine receptor(s); GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; HLH, helix-loop-helix; CAT, chloramphenicol acetyltransferase; beta-gal, beta-galactosidase; CEF(s), chick embryonic fibroblast-like cell(s); CHO, Chinese hamster ovary; MOPS, 3-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; X-gal, 5-bromo-4-chloro-3-indoyl beta-D-galactoside; E, embryonic day; bp, base pair; kbp, kilobase pair; CNS, central nervous system.

(^2)
L. Erkman, M. Gomez, J.-M. Matter, and M. Ballivet, submitted for publication.

(^3)
M.-C. Hernandez, L. Erkman, L. Matter-Sadzinski, T. Roztocil, M. Ballivet, and J.-M. Matter, unpublished results.

(^4)
S. Bertrand, N. Hussy, and D. Bertrand, unpublished results.

(^5)
J.-M. Matter, L. Matter-Sadzinski, and M. Ballivet, submitted for publication.


ACKNOWLEDGEMENTS

We are very grateful to Sonia Bertrand, Nicolas Hussy, and Daniel Bertrand (University of Geneva, Medical School) for functional assays in the Xenopus oocyte system. We thank Marie Gomez for help in the in situ hybridization experiments.


REFERENCES

  1. Anand, R., Conroy, W. G., Schoepfer, R., Whiting, P., and Lindstrom, J. (1991) J. Biol. Chem. 266, 11192-11198 [Abstract/Free Full Text]
  2. Aubry, J.-M., Schulz, M.-F., Pagliusi, S., Schulz, P. and Kiss, J. Z. (1993) Neuroscience 53, 417-424 [CrossRef][Medline] [Order article via Infotrieve]
  3. Ballivet, M., Nef, P., Stalder, R., and Fulpius, B. (1983) Cold Spring Harbor Symp. Quant. Biol. 48, 83-87 [Medline] [Order article via Infotrieve]
  4. Bertrand, D., Devillers-Thiery, A., Revah, F., Galzi, J-L., Hussy, N., Mulle, C., Bertrand, S., Ballivet, M., and Changeux, J.-P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1261-1265 [Abstract]
  5. Beyer, H. S. (1991) BioFeedback 11, 746-747
  6. Boulter, J., O'Shea-Greenfield, A., Duvoisin, R. M., Connolly, J. G., Wada, E., Jensen, A., Gardner, P. D., Ballivet, M., Deneris, E. S., McKinnon, D., Heinemann, S., and Patrick, J. (1990) J. Biol. Chem. 265, 4472-4482 [Abstract/Free Full Text]
  7. Boyd, R. T., Jacob, M. H., Couturier, S., Ballivet, M., and Berg, D. K. (1988) Neuron 1, 495-502 [Medline] [Order article via Infotrieve]
  8. Boyd, R. T., Jacob, M. H., McEachern, A. E., Caron, S., and Berg, D. K. (1991) J. Neurobiol. 22, 1-14 [Medline] [Order article via Infotrieve]
  9. Cauley, K., Agranoff, B. W., and Goldman, D. (1989) J. Cell Biol. 108, 637-645 [Abstract]
  10. Cauley, K., Agranoff, B. W., and Goldman, D. (1990) J. Neurosci. 10, 670-683 [Abstract]
  11. Changeux, J. P., Devillers-Thiery, A., Galzi, J. L., and Bertrand, D. (1992) Trends Pharmacol. Sci. 13, 299-301 [Medline] [Order article via Infotrieve]
  12. Conroy, W. G., Vernallis, A. B., and Berg, D. K. (1992) Neuron 9, 679-691 [Medline] [Order article via Infotrieve]
  13. Cooper, E., Couturier, S., and Ballivet, M. (1991) Nature 350, 235-238 [CrossRef][Medline] [Order article via Infotrieve]
  14. Corriveau, R. A., and Berg, D. K. J. Neurosci. 13, 2662-2671
  15. Couturier, S., Erkman, L., Valera, S., Rungger, D., Bertrand, S., Boulter, J., Ballivet, M., and Bertrand, D. (1990a) J. Biol. Chem. 265, 17560-17567 [Abstract/Free Full Text]
  16. Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M.-C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990b) Neuron 5, 847-856 [Medline] [Order article via Infotrieve]
  17. Crossley, M., and Brownlee, G. G. (1990) Nature 345, 444-446 [CrossRef][Medline] [Order article via Infotrieve]
  18. Deneris, E. S., Boulter, J., Swanson, L. W., Patrick, J., and Heinemann, S. (1989) J. Biol. Chem. 264, 6268-6272 [Abstract/Free Full Text]
  19. Deneris, E. S., Connolly, J., Rogers, S. W., and Duvoisin, R. (1991) Trends Pharmacol. Sci. 12, 34-40 [Medline] [Order article via Infotrieve]
  20. Galzi, J.-L., Revah, F., Bessis, A, and Changeux, J.-P. (1991) Annu. Rev. Pharmacol. 31, 37-72 [CrossRef][Medline] [Order article via Infotrieve]
  21. Gerzanich, V., Anand, R., and Lindstrom, J. (1994) Mol. Pharmacol. 45, 212-220 [Abstract]
  22. Giraudat, T., Dennis, M., Heidmann, T., Haumont, P. Y., Lederer, F., and Changeux, J.-P. (1987) Biochemistry 26, 2410-2418 [Medline] [Order article via Infotrieve]
  23. Hamburger, V. (1961) J. Exp. Zool. 148, 91-124
  24. Hamburger, V., and Hamilton H. L. (1951) J. Morphol. 88, 49-92
  25. Hill, J. A. Jr., Zoli, M., Bourgeois, J.-P., and Changeux, J-P. (1993) J. Neurosci. 13, 1551-1568 [Abstract]
  26. Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K., and Numa, S. (1988) Nature 335, 645-648 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) Cell 39, 499-509 [Medline] [Order article via Infotrieve]
  28. Keyser, K. T., Britto, L. R. G., Schoepfer, R., Whiting, P., Cooper, J., Conroy, W., Brozozowska-Prechtl, A., Karten, H. J., and Lindstrom, J. (1993) J. Neurosci. 13, 442-454 [Abstract]
  29. Khandjian, E. W. (1986) Mol. Biol. Rep. 11, 107-115 [Medline] [Order article via Infotrieve]
  30. Kraner, S. D., Chong, J. A., Tsay, H.-J., and Mandel, G. (1992) Neuron 9, 37-44 [Medline] [Order article via Infotrieve]
  31. Leonard, R. J., Labarca, C. G., Charnet, P., Davidson, N., and Lester, H. A. (1988) Science 242, 1578-1581 [Medline] [Order article via Infotrieve]
  32. Listerud, M., Brussaard, A. B., Devay, P., Colman, D. R., and Role, L. W. (1991) Science 254, 1518-1521 [Medline] [Order article via Infotrieve]
  33. Mandel, G., and McKinnon, D. (1993) Annu. Rev. Neurosci. 16, 323-345 [CrossRef][Medline] [Order article via Infotrieve]
  34. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Matter, J.-M., Matter-Sadzinski, L., and Ballivet, M. (1990) EMBO J. 9, 1021-1026 [Abstract]
  36. Matter-Sadzinski, L., Hernandez, M.-C., Roztocil, T., Ballivet, M., and Matter J.-M. (1992) EMBO J. 11, 4529-4538 [Abstract]
  37. Mori, N., Schoenherr, C., Vandenbergh, D. J., and Anderson D. J. (1992) Neuron 9, 45-54 [Medline] [Order article via Infotrieve]
  38. Morris, B. J., Hicks, A. A., Wisden, W., Darlison, M. G., Hunt, S. P., and Barnard, E. A. (1990) Mol. Brain Res. 7, 305-315 [Medline] [Order article via Infotrieve]
  39. Murre, C., McCaw, P. S., and Baltimore, D. (1989) Cell 56, 777-783 [Medline] [Order article via Infotrieve]
  40. Myers, R. M., Tilly, K., and Maniatis, T. (1986) Science 232, 613-618 [Medline] [Order article via Infotrieve]
  41. Nef, P., Oneyser, C., Alliod, C., Couturier, S., and Ballivet, M. (1988) EMBO J. 7, 595-601 [Abstract]
  42. Revah, F., Bertrand, D., Galzi, J.-L., Devillers-Thiery, A., Mulle, C., Hussy, N., Bertrand, S., Ballivet, M., and Changeux, J.-P. (1991) Nature 353, 399-402
  43. Role, L. W. (1992) Curr. Opin. Neurobiol. 2, 254-262 [CrossRef][Medline] [Order article via Infotrieve]
  44. Sargent, P. B. (1993) Annu. Rev. Neurosci. 16, 403-443. [CrossRef][Medline] [Order article via Infotrieve]
  45. Schuetze, S. M., and Role, L. W. (1987) Annu. Rev. Neurosci. 10, 403-457 [CrossRef][Medline] [Order article via Infotrieve]
  46. Séguéla, P., Wadiche, J., Dineley-Miller, K., Dani, J. A., and Patrick, J. (1993) J. Neurosci. 13, 596-604 [Abstract]
  47. Stüber, D., Matile, H., and Garotta, G. (1990) Immunol. Methods 4, 121-152
  48. Vernallis, A. B., Conroy, W. G., and Berg, D. K. (1993) Neuron 10, 451-464 [Medline] [Order article via Infotrieve]
  49. Wada, E., Wada, K., Boulter, J., Deneris, E., Heinemann, S., Patrick., J., and Swanson, L. W. (1989) J. Comp. Neurol. 284, 314-335 [Medline] [Order article via Infotrieve]
  50. Wang, M. M., and Reed, R. R. (1993) Nature 364, 121-126 [CrossRef][Medline] [Order article via Infotrieve]
  51. Yoon, S. O., and Chikaraishi, D. M. (1992) Neuron 9, 55-67 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.