Gene Structure of the Rat Kainate Receptor Subunit KA2 and Characterization of an Intronic Negative Regulatory Region*

(Received for publication, August 19, 1996, and in revised form, November 1, 1996)

Fei Huang and Vittorio Gallo Dagger

From the Section on Molecular Neurobiology of Glia, Laboratory of Cellular and Molecular Neurophysiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have isolated and analyzed the structure of the gene grik5 (lutamate eceptor onotropic ainate ), encoding the rat kainate receptor subunit KA2. Six overlapping DNA fragments containing the entire grik5 gene were identified in a rat genomic library. grik5 is a unique gene composed of 20 exons that together span over 54 kilobases (kb). Reporter gene analysis demonstrated that 2 kb of grik5 5'-flanking sequence confers tissue-specific expression on a chloramphenicol acetyltransferase gene in vitro. We show that (i) the first intron of grik5 (3.4 kb) inhibited transcription of the chloramphenicol acetyltransferase gene driven by the 2-kb grik5 5'-flanking region; (ii) the negative regulatory element was located within 500 bp of the 3'-end of intron 1, and this 500-bp fragment selectively bound nuclear proteins isolated from neural and nonneural cells; (iii) the effect of the negative regulatory element on grik5 transcription was orientation- and distance-independent; and (iv) a 24-nucleotide sequence (CTTTCTGTGGCCTCTGACCTTTCC) was identified as the binding site for nuclear proteins within the 500-bp fragment, as determined by footprinting and gel shift assays. We conclude that an intronic element that displays features of a silencer modulates grik5 transcription.


INTRODUCTION

Glutamate is a ubiquitous excitatory neurotransmitter in the mammalian central nervous system (1). Glutamate receptors (GluRs)1 have been classified into N-methyl-D-aspartate, (R,S)-alpha -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate subtypes, according to agonist binding selectivity (2). Cloning of glutamate receptor subunit cDNAs has revealed that the molecular diversity of the gene families encoding the various receptor subunits is responsible for the pharmacological and functional heterogeneity of glutamate receptors in the brain (3, 4). Analysis of genomic DNA also identified RNA editing and alternative splicing as molecular mechanisms that increase functional diversity of GluR subunits (for review, see Refs. 3 and 4).

Two kainate-preferring GluR subunit gene families have been described, GluR 5-7 and KA1-2 (3, 4). The kainate-preferring subunit KA2 (5) is encoded by the gene grik5 (lutamate eceptor onotropic ainate ), which has been mapped to chromosomes 1, 7, and 19 in rat, mouse, and human, respectively (6). The subunit KA2 is closely related to KA1 (5, 7), displaying 68% identity in amino acid sequence. Like KA1, KA2 does not form functional homomeric ion channels but interacts with GluR5 and GluR6 to form heteromeric, agonist-sensitive channels (5). The grik5 gene is widely expressed in the adult brain and is especially abundant in the cerebral cortex, pyriform cortex, caudate-putamen, hippocampal complex, medial habenula, and granule cell layer of the cerebellum (5, 8). Little is known about the function and physiological role of the KA2 subunit, or how the grik5 gene is regulated during brain development.

Analysis of the 5'-untranslated region revealed that grik5 is a TATA-less gene, whose transcription can initiate at several sites within a 500-bp region flanking the receptor subunit coding sequence (9). The DNA elements required for tissue-specific expression of grik5 in vitro and in vivo reside within 2 kb of the 5'-flanking region (9). We have now isolated the entire grik5 gene, determined its exon-intron organization, and identified a negative regulatory region within the first intron, which inhibits reporter gene expression driven by the 5'-flanking region of grik5. We also established that, within this negative regulatory region, nuclear proteins bind to a 24-nucleotide sequence. The negative regulatory element within the first intron exerts its inhibitory effect in a distance- and orientation-independent manner and therefore displays features of a silencer.


EXPERIMENTAL PROCEDURES

Isolation and Characterization of grik5 Genomic Clones

A Harlan Sprague Dawley rat genomic library in lambda DASH (Stratagene, La Jolla, CA) was screened for kainate receptor subunit grik5 gene sequences, using restriction fragments of the rat KA2 cDNA as probes (5). A 0.6-kb SmaI/ClaI restriction fragment (cDNA positions +1 to +647) was used to identify the 5'-region. Two restriction fragments, 1.2-kb ClaI/SmaI and 0.9-kb SmaI (cDNA positions +648 to +1837 and +1838 to +2736, respectively), derived from middle portions of KA2 cDNA, were used to isolate recombinant phage carrying exons for the middle region and the transmembrane regions. Recombinant phage covering the 3'-end part of the grik5 gene were isolated using a 1.0-kb EcoRI fragment (cDNA position +2677 to +3662) derived from the 3'-end coding and noncoding sequences of KA2 cDNA. All probes were labeled with [alpha -32P]dCTP (DuPont NEN) using a random primer labeling kit (Stratagene). Overlap of clones was determined by Southern blot analysis of lambda  DNA. Single or combinatorial BamHI, EcoRI, and SmaI digests were blotted onto Nytran membrane (Schleicher & Schuell, Keene, NH) and hybridized with the probes used for the phage isolation. Restriction fragments of genomic clones were subcloned in pBluescript SK- (Stratagene) and sequenced using a Sequenase II kit (U. S. Biochemical Corp.). Some of the sequencing was performed by Lark Sequencing Technologies, Inc. (Houston, TX).

Cell Cultures and Transient Transfections

Cells of the rat glial cell line CG-4 were cultured as described previously (10, 11) and plated on poly-D-ornithine-coated plates (0.1 mg/ml). HeLa and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (both from Life Technologies, Inc.). PC12 cells were grown in Dulbecco's modified Eagle's medium with 10% horse serum and 5% fetal bovine serum. Differentiated PC12 cells were grown on collagen-coated plates and treated with nerve growth factor (50 ng/ml) every other day for 5 days.

All expression plasmid DNAs used for transfections were purified with the Qiagen DNA purification kit (Qiagen Inc., Chatsworth, CA). A LacZ gene driven by the RNA polymerase II gene promoter (pPolIIplacF.beta gal) was simultaneously transfected with all the grik5-CAT constructs, to correct for possible variations in transfection efficiency. CAT constructs (10 µg) and pPolIIplacF.beta gal (2 µg) were transiently transfected using LipofectAMINE reagent (Life Technologies) according to the manufacturer's directions. Cells were transfected for 8 h in serum-free Dulbecco's modified Eagle's medium and then returned to serum-containing medium except for PC12 cells, which were transfected for 20 h in Opti-MEM I medium (Life Technologies). All cells were harvested 38 h after transfection and assayed for CAT and beta -galactosidase activities as described (12). Levels of CAT activity were determined on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). All transfection experiments were repeated 3-6 times, and averages (± S.E.) of the results are presented.

Construction of Plasmids

All restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Beverly, MA).

CAT Constructs

2-CAT

The 2-kb 5'-flanking sequence of the grik5 gene was first cloned into pBluescriptSK- by inserting the 2-kb EcoRI-BamHI fragment from the 5'-end of lambda 5 into the EcoRI-BamHI site of pBluescriptSK-. The 2-kb fragment was then excised by HindIII and XbaI digestion and cloned into the HindIII/XbaI site of pCAT-basic (Promega, Madison, WI).

4.3-CAT

The 4.3-kb BamHI DNA fragment of grik5 5'-flanking sequence was isolated from the genomic lambda A11 clone (Fig. 1A) and subcloned into pBluescript SK-. A clone with a 3' right-arrow 5' orientation was selected, and the DNA was digested with HindIII/XbaI. The resulting 4.3-kb fragment (HindIII site at the 5'-end and XbaI site at 3'-end) was cloned into the pCAT-basic vector to form the 4.3-CAT construct.


Fig. 1. Exon-intron organization of the rat grik5 gene. A, schematic diagram of the six overlapping lambda  clones comprising the entire grik5 gene and restriction map of the grik5 gene. The resolution of the restriction map is 100-200 nucleotides. B, schematic diagram of grik5 exon-intron organization. Exons are boxed, and the untranslated regions of exons 1, 2, and 20 are hatched. The position of the translational start (ATG) and stop (TGA) codons are marked by arrowheads. Coding sequences for the putative transmembrane regions TMI-TMIV are shown by black boxes. The size of the exons and the introns (given in base pairs and in kilobases, respectively) are not drawn to scale. C, nucleotide sequences of all intron-exon boundaries and all exon sizes. All the splice donor (GT) and splice acceptor (AG) sites are underlined.
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5.4-CAT

This construct contains 2 kb of 5'-flanking sequence and the 3.4-kb first intron of grik5. It was prepared from two genomic fragments joined by multiple subcloning steps. First, a 2.3-kb XbaI/HindIII fragment from lambda A11 was subcloned into LITMUS-28 vector (New England Biolabs) to form LITMUS-2.3 kb. A 3.1-kb HindIII/MvnI lambda A11 fragment was subcloned into pBluescript SK- at HindIII/SmaI sites and excised by HindIII/SpeI digestion. This 3.1-kb HindIII/SpeI fragment with a SpeI site at its 3' end was then ligated with LITMUS-2.3 kb plasmid DNA at HindIII/SpeI sites to obtain a 5.4-kb fragment linked by HindIII resulting in the LITMUS-5.4 kb construct. Finally, the 5.4-kb grik5 fragment was isolated by SalI and SpeI digestion and cloned into SalI/XbaI sites of the pCAT-basic vector to generate the 5.4-CAT construct.

7.7-CAT

This construct containing 4.3 kb of grik5 5'-flanking sequences and the 3.4-kb first intron was prepared similarly to the 5.4-CAT construct. A 4.6-kb XbaI/HindIII genomic DNA fragment was subcloned into the LITMUS-28 vector and then digested by HindIII/SpeI and ligated with a 3.1-kb HindIII/SpeI fragment to form LITMUS-7.7 kb construct. This 7.7-kb fragment was isolated by SalI/SpeI digestion and cloned into pCAT-basic vector at SalI/XbaI sites to generate the 7.7-CAT construct.

Fusion of grik5 First Intron with the Chick beta -Actin Promoter

A 494-bp HindIII DNA fragment containing the chicken beta -actin promoter region was cloned into the HindIII site of the pCAT-basic vector to form the beta -actin-CAT construct. To clone intron 1 downstream of the beta -actin promoter in the CAT vector, the 3.4-kb SpeI DNA fragment of intron 1 was cloned into the XbaI site of beta -actin CAT to generate the beta -actin-I-CAT.

Fusion of grik5 First Intron with the SV40-CAT Transcriptional Unit

grik5 intron 1 was cloned downstream of the SV40-CAT transcriptional unit (p-CAT control vector; Promega), containing SV40 promoter and enhancer sequences. The 3.4-kb SpeI DNA fragment of grik5 intron 1 was cloned in the XbaI site of p-CAT control, 3' of the SV40 enhancer. A clone with a 5' right-arrow 3' orientation of the intron was selected to generate the pSV-CAT-intron construct.

Intron Deletion Constructs

The 5.4-CAT plasmid was used to make all intron deletion constructs (Fig. 4). Plasmids Delta SacI-CAT and 2-I-R-SacI-CAT were made by digesting 5.4-CAT with SacI, religating, and selecting clones with the 2.0-kb SacI fragment deleted (Delta SacI-CAT) or reversed (2-I-R-SacI-CAT). BglII/SacI deletions were obtained by complete digestion of 5.4-CAT DNA with BglII and partial digestion with SacI and then blunt-ended with Klenow and ligated. The Delta BglII/SacI-CAT has a 1.3-kb deletion, whereas Delta SacI/BglII-CAT has a 0.7-kb deletion. To construct plasmid Delta HindIII/SacI, the plasmid Delta SacI-CAT DNA was digested with HindIII and SacI. Two of the three resulting DNA fragments (SacI/HindIII, 4.3 kb; HindIII, 2.3 kb) were isolated, blunt-ended with Klenow, and ligated. The clone with the correct orientation was selected. This plasmid was designed to delete intron 1 except for the splicing sites. The HindIII/AccI deletion was made by digestion of Delta AccI-CAT with AccI and EcoR V. The AccI/EcoRV fragment was isolated, blunt-ended, and ligated with the blunt-ended HindIII fragment. The other deletion constructs were generated by digestion with selected restriction enzymes, blunt-ending with Klenow, and religating the ends. The resulting plasmids have corresponding restriction enzyme fragment deletions (Fig. 4). All deletion junctions were analyzed by sequencing to confirm that correct deletions were obtained.


Fig. 4. Deletion analysis identifies a negative regulatory region within the 3'-end of intron 1. A, schematic diagram of intron 1 deletion constructs made by internal deletion with different restriction enzymes (indicated on the left) followed by blunt-end ligation (see "Experimental Procedures"). The nucleotide numbers in parentheses are relative to the 5'-end of intron 1 (taken as +1). B, CAT reporter gene activities (averages ± S.E.) measured after transient transfection (3-8 independent experiments) of the constructs in CG-4 cells. Data are corrected for transfection efficiency and expressed as percentage of 2-CAT (set at 100%). In this set of experiments, CAT activity of the 2-CAT construct was approximately 20-fold higher than that of CAT-basic.
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LacZ Constructs

A promoterless LacZ vector, galscript-ES4, containing the nuclear localization signal, was used to construct LacZ expression plasmids (Fig. 3C). 2-LacZ and 4.3-LacZ constructs were prepared by separately cloning the 2-kb NotI/BamHI and 4.3-kb BamHI fragments, both excised from the 5'-flanking region of grik5 lambda A11, into a galscript-ES4 vector. 5.4-LacZ and 7.7-LacZ constructs were obtained as follows: LITMUS-5.4 kb and LITMUS-7.7 kb plasmids were digested with NotI and SpeI, and the 5.4- and 7.7-kb DNA fragments were isolated. The galscript-ES4 vector DNA was digested with NotI/XbaI and ligated with 5.4- or 7.7-kb NotI/SpeI DNA fragments to form 5.4-LacZ or 7.7-LacZ constructs, respectively.


Fig. 3. The inhibitory effect of intron 1 on CAT reporter gene expression is not due to aberrant RNA splicing. A, schematic diagram of the grik5-CAT constructs used for transient transfection in CG-4 cells and of the RT-PCR primers (arrowheads). The sense primer (S) is located 200 bp from the 3'-end of the 2-kb 5'-flanking region of grik5; the two antisense primers AS1 and AS2 are located within the SV40 small T antigen coding region. B, RT-PCR analysis of RNA isolated from CG-4 cells transfected with different DNA constructs. Lanes 1 and 14 are DNA size markers (indicated on the left); lanes 2-7 are amplified with S and AS1 primers; lanes 8-13 are amplified with S and AS2 primers; lanes 2 and 8 are PCR reactions from the 2-CAT plasmid DNA serving as a positive control for PCR. Lanes 3-7 and 9-13 are RT-PCR of RNAs isolated from transfected cells. Lane 3, pCAT-basic, negative control (the S primer could not anneal to this cDNA); lane 9, reverse transcriptase was omitted in the reverse transcription reaction to test for plasmid DNA contamination; lanes 4, 9, and 10, 2-CAT; lanes 5 and 11, 5.4-CAT; lanes 6 and 12, 4.3-CAT; lanes 7 and 13, 7.7-CAT. The RT-PCR products are indicated by arrowheads, and their size is expressed in kilobases. C, beta -galactosidase activities of grik5-LacZ reporter gene constructs. Histograms represent Lac-Z activities (averages ± S.E.) from three independent transfection experiments and are corrected for transfection efficiency by co-transfection with a CAT plasmid. The activities are calculated relative to the activity of galscript-ES4 (set arbitrarily as 100%).
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Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Plasmids pCAT-basic, 2-CAT, 4.3-CAT, 5.4-CAT, and 7.7-CAT were used to transfect CG-4 cells as described above. Total RNA was isolated from transfected cells by RNAzol (Tel-Test, Inc., Friendswood, TX) and treated with RNase-free DNase (Ambion, Inc., Austin, TX). For each RT reaction, 2 µg of total RNA isolated as outlined above, was combined with 100 pmol of random hexamers (Life Technologies) and denatured at 80 °C for 10 min. The reactions were then assembled by adding 1 × PCR buffer, 1.0 mM MgCl2, 333 µM each dNTP, 10 mM dithiothreitol, 100 mg/ml acetylated BSA, 1 unit/ml RNasin, and 200 units of Superscript II RT in a volume of 30 µl. Reactions were incubated at 25 °C for 10 min and then at 42 °C for 50 min. After heat inactivation, 30 units of RNase H was added, and incubation continued at 37 °C for 20 min.

For PCR reactions, 10 µl of each RT reaction was added to a tube containing 1 × PCR buffer, 50 pmol of sense primer (5'-TTGCTTTTCCACCTGTCC-3', located at the 3'-end of the 5' noncoding region of grik5), 50 pmol of either one of the two antisense primers (located in the SV40 small T antigen coding region; Fig. 3) AS1 (5'-CAGCTTTTTCCTTTGTGGTG-3') or AS2 (5'-ATGTTTCAGGTTCAGGGGG-3'), 200 µM each dNTP, and 2.5 units of AmpliTaq in a volume of 100 µl. The PCR cycling profile was as follows: 95 °C for 5 min and then 30 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min with a 5-s time extension added per cycle and a final extension at 72 °C for 7 min. Products were separated on 1% agarose gels and visualized by ethidium bromide staining.

DNA Mobility Shift Assays

The 1.3-kb BglII/SacI DNA fragment from grik5 intron 1 was subcloned into LITMUS-28 vector. The resulting plasmid was grown in a methylase-deficient E. coli strain GM2163 (dam-, dcm-; New England Biolabs). The 1.3-kb BglII/SacI DNA fragment was purified and digested with Sau3AI. The resulting restriction fragments of the Sau3AI digestion are shown in Fig. 6A. Fragments were purified and end-labeled with [alpha -32P]dCTP using Klenow fragment. Nuclear protein extracts from CG-4 and HeLa cells were prepared according to Dignam et al. (13). The labeled probes were used in gel shift assays with or without nuclear protein extracts. The reactions were carried out in a total volume of 20 µl of a binding buffer containing 25 mM HEPES, pH 7.5, 60 mM KCl, 10% glycerol, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 50 µg/ml poly(dI-dC) (Pharmacia Biotech Inc.). The binding reactions were incubated on ice for 60 min and directly loaded onto a 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (1 × TBE: 90 mM Tris borate, pH 8.3, 1 mM EDTA) and electrophoresed at 10 V/cm for 3-4 h at 4 °C. The gels were dried and autoradiographed on x-ray film (Eastman Kodak Co.).


Fig. 6. Nuclear factors bind to a 500-bp fragment within the 3'-end of intron 1. A, schematic diagram showing the restriction map of the 1.3-kb BglII/SacI DNA fragment derived from intron 1. Sau3AI digestion generated five DNA fragments of different sizes. The shifted fragment is indicated by a hatched box, and the numbers indicate the size of the DNA fragments in base pairs. B, gel shift assay with individual fragments derived from the Sau3AI-digested DNA and nuclear protein extracts from CG-4 cells. C, gel shift assay with fragment D (500 bp) and nuclear protein extracts from HeLa (lanes 1-7) and CG4 (lanes 8-14) cells. The assays contained 0.25 ng of end-labeled DNA fragment D and 0 µg (lanes 1 and 8), 2 µg (lanes 2 and 9), 4 µg (lanes 3 and 10), or 6 µg (lanes 4-7 and 11-14) of nuclear proteins. All reactions contained the nonspecific competitor poly(dI-dC). A 10-fold (lanes 5 and 12) or 50-fold (lanes 6 and 13) excess of unlabeled specific competitor fragment D, or a 10-fold excess nonspecific competitor poly(dI-dC) (lanes 7 and 14) was added to some reactions as a test of specificity.
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Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (Perkin-Elmer). Oligonucleotides ON1 (5'-CGGCGTCTTTACTTTTGCGG-3'), ON2 (5'-GTCAGATACTTTTTCTTTTCCTTCCGTT-3'), and ON3 (5'-CCTGTCCTTTCTGTGGCCTCTGACCTTTCCTTGTTCCAGA-3') are derived from different regions of intron 1, corresponding to nucleotide positions 2779-2798, 2805-2832, and 3202-3241, respectively (Fig. 8A). The complementary oligonucleotide pairs were annealed and labeled using [gamma -32P]ATP (DuPont NEN). The double-stranded oligonucleotides were then gel-purified and used for gel shift assays, as described above.


Fig. 8. Nuclear factors specifically bind to a DNase I-protected region of intron 1. Gel shift assays were performed with labeled oligonucleotides from the the DNase I-protected region of intron 1 and from other regions of the same intron. Nuclear extracts from both CG-4 and HeLa cells were used. A, map of the oligonucleotides ON1, ON2, and ON3 used for the assays. B, reactions contained nuclear extracts from CG-4 cells and labeled ON1, ON2, and ON3 without (lanes 1, 2, 5, 6, 9, and 10), or with 50-fold (lanes 3, 4, 7, 8, 11, and 12) molar excess of the competitors indicated. Oligonucleotide UR was used as an unrelated competitor. The specifically bound complex was only obtained with oligonucleotide ON3. Identical results were also obtained with HeLa cell extracts (not shown). Note that the labeled unbound probe ON1 was eluted from the gel (lanes 1-4) because of its smaller size, compared with ON2 (lanes 4-8) and ON3 (lanes 5-12). The gel was exposed to x-ray film for 1 day. A nonspecific complex similar to that observed in panel C was also seen in this experiment at longer exposure times. C, reactions contained nuclear extracts from CG-4 and HeLa cells and labeled oligonucleotide ON3, without (lanes 4, 5, 7, and 8) or with unlabeled ON3 (50-100-fold molar excess; lanes 2 and 3 and lanes 9 and 10) or the unrelated oliogonucleotide UR (100-fold molar excess; lanes 1 and 11). The gel was exposed to x-ray film for 3 days.
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DNase I Footprinting

Four pairs of primers were made to PCR-amplify four overlapping DNA fragments (153-220-bp size range) spanning the entire Sau3AI/SacI region of intron 1, corresponding to nucleotide positions 2660-2879, 2778-2963, 2938-3090, and 3040-3244, respectively (Fig. 7A). The PCR products were cloned into pCRTM 2.1 vector using the TA cloning kit (Invitrogen, San Diego, CA), excised by cutting with HindIII/XbaI (these sites flank the BamHI-EcoRV insertion sites), and treated with CIP. The fragments were then labeled at both ends using [gamma -32P]ATP (DuPont NEN) and T4 polynucleotide kinase (New England Biolabs). A singly end-labeled probe was then generated by digesting the label off one end with EcoRV (upper strand labeled), or with BamHI (lower strand labeled). The labeled probes were gel-purified. For DNase I footprinting, nuclear extracts (5-20 µg) were incubated on ice for 15 min in 50 µl, containing 25 mM HEPES, 60 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 40 µg/ml poly(dI-dC), pH 7.5. The singly 32P-end-labeled probes were then added, and the samples were incubated on ice for 30 min. Fifty microliters of Ca2+/Mg2+ solution (5 mM CaCl2, 10 mM MgCl2, at room temperature) were added to the reactions. Diluted RQ1 RNase-free DNase (Promega) was added to the samples at 0.25 units/reaction and incubated at room temperature for 1 min. The reactions were terminated by adding 90 µl of prewarmed (37 °C) stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast tRNA) and extracted with phenol/chloroform/isoamyl alcohol. The reaction products were precipitated and analyzed on 6% sequencing gels. Dideoxy sequencing reactions were performed with a specific upper strand primer (initiating at the HindIII site) and a specific lower strand primer (initiating at the XbaI site) to serve as size standards.


Fig. 7.

Localization of the nuclear factor binding site by DNase I footprinting. DNase I footprinting reactions were performed by using nuclear protein fractions prepared from CG-4 and HeLa cells. A, unidirectionally labeled probes were generated for the upper and lower strand by 5'-end labeling of four DNA overlapping fragments (size range: 153-220 nucleotides) obtained by PCR amplification of the Sau3AI/SacI region of intron 1. B, DNase I footprinting of the 205-bp PCR product. The footprinting reactions of upper and lower strands were performed with (lanes 2-4, 6-8, 11-13, and 15-17) or without (lanes 5 and14) nuclear proteins (see "Experimental Procedures"), and products were separated on sequencing gels. Dideoxy sequencing reactions were performed using specific upper strand (left panel) and lower strand (right panel) primers. Protected regions are marked, and the corresponding sequence positions are indicated. C, nucleotide sequence of the 24-nucleotide protected region (nucleotides 3207-3231), as determined by DNase I footprinting.


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RESULTS

Gene Structure of the Rat Kainate Receptor Subunit KA2

Four different rat KA2 cDNA restriction fragments were used as probes to screen a rat lambda DASH genomic library (see "Experimental Procedures"). Six overlapping recombinant lambda  clones were isolated, which contained the entire grik5 gene (Fig. 1A). The restriction map of each DNA insert was determined by electrophoretic analysis of restriction fragments (Fig. 1A). To determine the exon-intron organization, the size, and exact location of the grik5 gene introns, Southern blot analysis was performed on the clones to identify restriction fragments containing coding sequence. These were subcloned into pBluescript SK- and sequenced. Restriction mapping and sequence analysis indicated that grik5 is distributed over 54 kb of genomic sequence. As shown in Fig. 1B, the gene is composed of 19 translated exons and one untranslated exon, ranging in size from 54 bp (exon 7) to 944 bp (exon 20). The size of introns varies between 0.1 and 12 kb. All of the exon-intron boundaries were determined by DNA sequencing and conform to consensus splice donor/splice acceptor sequences (Fig. 1C; Ref. 14). Exon 20 (944 bp) of grik5 contains 423 bp of coding and 521 bp of 3'-untranslated region (a TGA translational stop codon is present 424 nucleotides downstream of the intron-exon boundary). The total length of the grik5 coding region (3.78 kb) deduced from the genomic sequence is consistent with the 4-kb mRNA identified by Northern blot analysis of tissue and cultured neural cell poly(A+) RNAs (Ref. 9; data not shown). Based on the previously cloned rat cDNA sequence (5), polyadenylation of grik5 mRNA occurs 23 nucleotides downstream of a canonical signal sequence (AATAAA) in exon 20 (nucleotides 917-922 from the 5'-end of exon 20).

Intragenic Sequences Inhibit grik5 Expression

grik5 is a TATA-less gene, whose transcription can initiate at several sites within 500 bp upstream of the exon1/intron 1 boundary (9). It was previously found that 2 kb of grik5 5'-flanking region confers tissue-specific expression on the CAT reporter gene both in vitro and in vivo (9). grik5-CAT reporter gene constructs were tested by transient transfection assays in neural (CG-4 and PC12) and nonneural (HeLa and NIH3T3) cells. Either 2 or 4.3 kb of grik5 5'-flanking region were able to selectively direct CAT gene expression in neural cells (Fig. 2, B-D). In nonneural cells, CAT activity of both grik5 gene constructs was below that of promoterless pCAT-basic. As shown in Fig. 1B, 3.4-kb intron 1 interrupts the grik5 5'-untranslated region. To examine if the presence of intron 1 altered the expression of the CAT gene driven by the 5'-flanking region of grik5, we tested reporter gene constructs that contained the two different segments of grik5 5'-flanking region, either with the intron (5.4-CAT and 7.7-CAT) or without it (2-CAT and 4.3-CAT) (Fig. 2A). The results showed that in CG-4 and PC12 cells the activities of the constructs containing intron 1 (5.4-CAT and 7.7-CAT) were reduced approximately 3-fold when compared with the activities of the constructs without the intron (2-CAT and 4.3-CAT) (Fig. 2, B and D). Thus, the presence of intron 1 inhibited expression of the CAT reporter gene driven by the grik5 promoter.


Fig. 2. Intron 1 inhibits reporter gene expression driven by the grik5 promoter in cultured neural cells. A, schematic diagram of the grik5-CAT reporter gene constructs containing 2 or 4.3 kb of 5'-flanking sequences with (5.4-CAT and 7.7-CAT) or without (2-CAT and 4.3-CAT) the 3.4-kb intron 1. The 5'-flanking regions of grik5 and the beta -actin promoter are boxed, intron 1 is indicated by hatched boxes, and the CAT gene is shown by a black box. Constructs were transiently transfected in neural (CG-4 and PC12), and nonneural (HeLa and NIH3T3) cells. B, representative CAT assay from transfected CG-4 cells. C, representative CAT assay from transfected HeLa cells. D, histograms derived from quantitation of CAT assays from transfected CG-4, PC12, HeLa, and NIH3T3 cells. Data are corrected for transfection efficiency and are averages (± S.E.) obtained from four (CG-4 and PC12 cells) or three (HeLa and 3T3 cells) independent transfection experiments. All data are expressed as the percentage increase over pCAT-basic (set to 100%).
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To exclude the possibility that grik5 promoter activity was diminished by intron 1 as a result of aberrant splicing (15), two sets of experiments were performed. First, RNA was isolated from CG-4 cells transfected with different grik5-CAT constructs. RT-PCR was carried out with two different sets of specific primers corresponding to sequences in the grik5 2-kb 5'-flanking region (sense, S primer) and in the SV40 small T antigen sequence (antisense, AS primers; Fig. 3A). If aberrant splicing occurred, a different size of RT-PCR products would be observed in cells transfected with grik5 constructs containing intron 1 than with constructs that did not contain it. On the other hand, if intron 1 were spliced out of transcripts properly, the length of RT-PCR products would be independent of its presence in the DNA constructs. Fig. 3B shows that single bands with the expected sizes of 1.4 and 1.7 kb were produced with primers S and AS1 and primers S and AS2, respectively. Identical results were obtained from RNAs of cells transfected by grik5 constructs with or without intron 1 (compare 5.4-CAT with 2-CAT and 7.7-CAT with 4.3-CAT), indicating that aberrant splicing likely did not occur. This conclusion is further supported by transient transfection experiments performed with the 5.4-CAT Delta HindIII/SacI construct (Fig. 4A), which only contained the 5' splice donor and 3' splice acceptor sequences of intron 1 and displayed reporter gene activity identical to 2-CAT (Fig. 4B).

In a second set of experiments, the same DNA fragments from grik5 were subcloned into a different promoterless vector, galscript-ES4, to form LacZ reporter gene constructs (see "Experimental Procedures"). The results in Fig. 3C indicate that intron 1 inhibited the beta -galactosidase expression by a factor of 3 when 2-LacZ and 4.3-LacZ were compared with 5.4-LacZ and 7.7-LacZ, respectively. Since intron 1 conferred inhibition independent of the sequence of the transcriptional unit (i.e. CAT versus LacZ), we conclude that aberrant splicing does not contribute to the inhibition of reporter gene expression observed in grik5 constructs containing intron 1.

The Negative Regulatory Element Is Located within the 3'-End of Intron 1

If the inhibitory effect of intron 1 on grik5 reporter expression was due to the presence of a negative cis-acting element, elimination of this element should abolish the inhibitory effect. To explore this possibility, a series of intron 1 deletion constructs were made in order to define the essential negative regulatory region. All constructs were generated by internal deletions of intron 1 in the 5.4-CAT construct (Fig. 4A). Both the 5' splice donor and 3' splice acceptor sequences were maintained in all constructs (Fig. 4A), which were tested by transient transfection assays in CG-4 cells.

The activity of 2-CAT was defined as 100%. Deletion of 2,936 nucleotides within intron 1 (construct Delta HindIII/SacI) completely eliminated the inhibition on reporter gene activity (Fig. 4B). Furthermore, when a 2-kb SacI fragment was deleted from intron 1 in the 5.4-CAT construct (Delta SacI), reporter gene activity was completely recovered (97% of 2-CAT level; Fig. 4B). This result indicated that the inhibitory region resides within the SacI fragment. The position of the inhibitory region was further defined by deletion mapping of the SacI fragment (Fig. 4A). The activities of constructs containing deletions in the 5' portion of intron 1 (Delta HindIII/BglII, Delta SacI/BglII, and Delta KpnI/BglII) were still significantly inhibited. On the other hand, a deletion in the 3'-end region of intron 1 (Delta BglII/SacI) relieved the inhibition (83% of 2-CAT). Finally, deletion of the smaller BglII/AccI or AccI fragments did not modify the inhibitory activity of intron 1. Therefore, we concluded that one or more negative regulatory elements are located at the 3'-end of intron 1, within the AccI/SacI fragment (nucleotides 2625-3245 of intron 1). To further support this conclusion, in a construct that only contained the AccI/SacI region of intron 1 (Delta HindIII/AccI; Fig. 4A), CAT activity was reduced to the same extent as if the the entire intron were present (Fig. 4B).

The Effect of the Negative Acting Element Is Orientation- and Distance-independent

Deletion analysis indicated that the negative regulatory region of intron 1 is located within the SacI fragment (Fig. 4). To test if the inhibitory effect of intron 1 was orientation-dependent, we made constructs with the SacI fragment in a reverse (3' right-arrow 5') orientation (Fig. 5A). Results obtained from transiently transfected CG-4 cells showed that there was no significant difference in CAT activities between the constructs containing the SacI fragment cloned in a reverse (2-I-R-SacI-CAT) or forward orientation (2-I-F-CAT; Fig. 5A). These results indicate that the inhibitory effect of intron 1 is independent of its orientation relative to the site of transcription initiation.


Fig. 5. The inhibitory effect of intron 1 is orientation- and distance-independent. A, grik5 intron 1 (hatched box) was placed either upstream (I-F-2-CAT) or downstream (2-I-F-CAT) of the grik5 2-kb 5'-flanking DNA fragment (empty box) in CAT (black box) constructs. The SacI fragment of intron 1 (lightly hatched box) was cloned in the CAT constructs either in the forward 5' right-arrow 3' (2-I-F-CAT, I-F-2-CAT) or the reversed 3' right-arrow 5' (2-I-R-SacI-CAT, I-R-SacI-2-CAT) orientation. All constructs were assayed by transient transfection assays in CG-4 cells (3-5 independent experiments). Histograms represent CAT activity (averages ± S.E.) corrected for transfection efficiency and are expressed as percentages of the 2-CAT construct. B, grik5 intron 1 (hatched box) was cloned downstream (actin-I-CAT) of the beta -actin promoter (empty box). Constructs were tested by transient transfection assays in CG-4, HeLa, and NIH3T3 cells (3-5 independent experiments). Histograms represent CAT activity (averages ± S.E.) corrected for transfection efficiency and are expressed as percentages of the actin-CAT (without intron 1) construct.
[View Larger Version of this Image (26K GIF file)]


To test if the inhibitory effect of intron 1 was position-dependent, we also made constructs in which intron 1 was cloned upstream of the grik5 promoter. Results indicated that when intron 1 was placed upstream of the grik5 promoter (I-F-2-CAT) its inhibitory effect was weaker but still significant (Fig. 5A). A similar construct containing the SacI fragment cloned in a reverse orientation (I-R-SacI-2-CAT) also reduced promoter activity (Fig. 5A). These results indicate that the negative regulatory element in intron 1 is orientation- and distance-independent and acts in a relatively position-independent manner.

Since intron 1 of the grik5 gene inhibited expression of its own promoter, we sought to examine whether the negative regulatory sequences could also regulate expression of heterologous promoters. As shown in Fig. 5B, intron 1 cloned downstream of the chicken beta -actin promoter in a 5' right-arrow 3' orientation (actin-I-CAT) strongly inhibited beta -actin expression in transfected CG-4, HeLa, and NIH3T3 cells. We also analyzed whether intron 1 was able to limit SV40 promoter activity in the presence of SV40 enhancer, which confers high reporter gene expression in many types of eukaryotic cells. CG-4 cells were transfected with a plasmid containing grik5 intron 1 placed 3' of the SV40 promoter-CAT-SV40 enhancer construct (pCAT-control). The resulting CAT activity of the SV40 construct containing grik5 intron 1 was 55 ± 10% of pCAT-control (three separate transfection experiments; data not shown). In conclusion, grik5 intron 1 significantly inhibited heterologous promoters.

Nuclear Proteins Bind to the Negative Regulatory Region of Intron 1

The transcriptional analysis of intron 1 in CG-4 cells (Fig. 4) indicated that deletion of the 500-bp AccI/SacI fragment in intron 1 resulted in loss of inhibitory activity, whereas its presence was sufficient to inhibit grik5 expression to a similar extent of the entire intron. To begin to identify the factors responsible for this inhibition, we first asked whether protein binding sites were present within this 500-bp DNA fragment.

Gel shift assays were then performed with individual Sau3AI-digested DNA fragments (Fig. 6A). As shown in Fig. 6B, the DNA fragments A and B were not shifted by CG-4 nuclear proteins, but both fragments C and D were complexed and up-shifted. Because fragment C comprised both B and D and fragment B was not shifted, it can be concluded that fragment D contained nuclear protein binding sites. This 500-bp fragment formed complexes and was shifted with nuclear proteins from both CG4 and HeLa cells (Fig. 6C), and the shift was competed by unlabeled specific DNA fragment D (Fig. 6C). In conclusion, the results from the band shift assays were consistent with the intron deletion analysis (Fig. 4) and indicated that nuclear factors specifically bound to the 500-bp region at the 3'-end of intron 1 might inhibit grik5 promoter activity.

Identification of the Negative Regulatory Region within Intron 1

The nuclear factor binding site in intron 1 was defined by DNase I footprinting experiments. The deletion analysis (Fig. 4) and the gel shift experiments (Fig. 6) indicated that the binding site was contained within the Sau3AI/SacI region of intron 1; therefore, four overlapping PCR-amplified DNA fragments spanning the whole Sau3AI/SacI region were obtained (Fig. 7A) and subcloned into pCRTM 2.1 vector. Unilaterally labeled fragments were generated by 5'-end labeling of the upper or lower strand and analyzed by DNase I footprinting (Fig. 7B). Footprinting reactions were performed with increasing amounts of CG-4 and HeLa cell nuclear extracts, and products were separated on sequencing gels. One protected region of 24 or 25 nucleotides could be recognized in both the coding and noncoding strands of the 3040-3244 fragment, with both CG-4 and HeLa nuclear extracts (Fig. 7B). This region corresponds to nucleotide positions 3206-3231, as shown in Fig. 7C. No protected sequences could be found in any of the other three fragments (2660-2879, 2778-2963, and 2938-3090; data not shown).

An oligonucleotide probe, ON3 (5'-CCTGTCCTTTCTGTGGCCTCTGACCTTTCCTTGTTCCAGA-3'; Fig. 8A), corresponding to the protected region shown in Fig. 7, was tested for its ability to bind CG-4 and HeLa cell nuclear factors by gel retardation assays (Fig. 8). Binding to ON3 resulted in the formation of an easily detectable retarded complex (Fig. 8B). Identical results were obtained by using an ON3-related 27-bp oligonucleotide (5'-TCCTTTCTGTGGCCTCTGACCTTTCCT-3'). Conversely, the oligonucleotides ON1 and ON2, corresponding to sequences contained within the 2778-2963 fragment, did not form any detectable complex with cell nuclear factors (Fig. 8B). This finding is consistent with DNase I footprinting experiments that showed no protected sequences within the 2778-2963 DNA fragment (data not shown). The specificity of the retarded complex formed by ON3 and nuclear proteins was also tested by competition experiments, using a molar excess of either unlabeled ON3 or an unrelated competitor (Fig. 8, B and C). The complex was specifically competed by the addition of a molar excess of ON3 but not by the unrelated competitor (Fig. 8, B and C). This complex is therefore formed by the specific interaction of the DNA sequence CTTTCTGTGGCCTCTGACCTTTCC with a nuclear factor(s). Similar results were also obtained with nuclear extracts isolated from HeLa cells (Fig. 8C).


DISCUSSION

grik5 is the first kainate receptor subunit gene for which the structure, with respect to exon-intron organization, has been determined. grik5 contains more exons than the rat gluR-B and mouse nr2C (17 and 15 exons, respectively; Refs. 17 and 18), but fewer than the rat nr1 gene (22 exons; Ref. 19). The exon-intron organization of grik5 is different from that of gluR-B, nr1, and nr2C. In particular, the grik5 intron positions are not conserved, compared with the other GluR subunit genes cloned so far, except for the three introns that separate the TMIII and TMIV containing exons (17-19). A unique feature of the rat grik5 gene is that the TMIII coding sequence is split by an intron between exons 15 and 16. The TMI and TMII regions are encoded in two separate exons in the kainate receptor subunit genes rat grik5 and mouse gluR-5 and -6 but are both encoded in a single exon in the AMPA receptor genes gluR-A to -D (20). In some genes, introns separate functional or structural domains of the proteins encoded by exons, suggesting that introns may accelerate evolution of proteins with different functional properties (21). Therefore, differences in gene intron/exon structure may reflect the evolutionary origin as well as the distinct functional properties of different GluR classes. The cloning and characterization of other kainate receptor subunit genes, in particular KA1, will confirm a close evolutionary origin of the corresponding subunits.

Previous studies have demonstrated that kainate and AMPA receptor subunit genes can exist in alternative spliced isoforms (for reviews, see Refs. 3 and 4). Additionally, differences were found between genomic and mRNA nucleotide sequences of GluR2-4, -5, and -6 in codons contained within the TMI and TMII regions as well as in an extracellular loop regulating receptor desensitization (22, 23). These differences in one nucleotide are due to RNA editing and significantly alter the functional properties of the corresponding GluR channels (22, 23). Our analysis of the nucleotide sequence of grik5 exons offers no evidence for alternative splicing or RNA editing, when compared with the rat and mouse KA2 cDNAs (5, 24). A rigorous proof will require more thorough analysis of RNA species expressed in different cell types at distinct developmental stages. Our findings with the grik5 gene, however, raise the possibility that in view of its physiological relevance (23) RNA editing is limited only to GluR genes that encode subunits capable of forming functional homomeric channels.

We have previously demonstrated that DNA elements residing within 2 kb upstream of the 5'-end of intron 1 are necessary for tissue-specific expression of grik5 in the brain (9). This was determined by reporter gene analysis in transgenic mice as well as in the glial cell line CG-4. We now have used CG-4 and PC12 cells (the latter as a neuronal model) and found that inclusion of an additional 2.3 kb of sequence further upstream of the previously characterized 5'-flanking region did not modify reporter gene expression in neural and nonneural cells (Fig. 2). These results indicate that, at least in vitro, this distal sequence is not involved in regulating grik5 transcription and tissue-specific expression. Transcriptional regulatory elements of eukaryotic genes are found not only in their 5' upstream region, but also within their introns (25-34). These sequences can act as transcriptional enhancers (27, 28, 30, 33) or can be required for appropriate tissue- or cell-specific expression (25, 26, 34). Intragenic negative regulatory elements were initially described in yeast as cis-acting elements that repress transcription, were able to function in either orientation, and were relatively independent of position (35). Similar DNA elements have also been characterized in other eukaryotic genes (25, 26, 31, 36-38). In the present study, we provide evidence that the first intron of the neural gene grik5 negatively regulates transcription without altering the tissue-specific expression pattern of the gene in vitro. In transiently transfected CG-4 and PC12 neural cells, reporter gene activity was significantly reduced in CAT constructs containing intron 1. Deletion analysis of intron 1 (Fig. 4), combined with gel retardation assays (Fig. 6), indicated that the DNA element responsible for inhibiting grik5 transcription resided within a 500-bp Sau3AI fragment.

The inhibitory effect of intron 1 on grik5 transcription was orientation-independent, as shown by experiments in which the SacI intronic fragment was cloned in a reverse orientation in the intron 1-containing grik5-CAT constructs (Fig. 5). Also, intron 1 exerted its inhibitory effect in a distance-independent fashion, as demonstrated by the significant inhibition of reporter gene expression found with the Delta HindIII/BglII and Delta SacI/BglII constructs (Fig. 4). Finally, the effect of intron 1 was relatively position-independent, because partial inhibition was observed when the intron was placed upstream of the grik5 promoter. In view of its orientation independence and distance independence from the transcription initiation site, the negative regulatory element of intron 1 displays the features of a transcriptional silencer (29). The presence of an intronic silencer is not unprecedented for neural genes (25, 26), but to our knowledge grik5 represents the first case of a neurotransmitter receptor gene.

In agreement with the deletion analysis experiments in transiently transfected cells, DNase I footprinting and gel shift assays demonstrated that nuclear factors bind to a 24-nucleotide sequence (5'-CTTTCTGTGGCCTCTGACCTTTCC-3') within the region of intron 1 (500-bp Sau3AI fragment) responsible for the inhibition of grik5 transcription. Comparison of this 24-nucleotide region with published sequences in data bases revealed no obvious homologies to known motifs. Mutation experiments are in progress to determine the sequence of the minimal intronic element involved in the binding to nuclear proteins and in the inhibitory effect on grik5 transcription.

The identity of the nuclear factors binding to the silencing element of intron 1 remains to be determined, but it appears that their expression occurs in cells of neural and nonneural origin. Similar findings were reported with nuclear proteins isolated from different cell types that bind to the first intron of the human alpha 1(I) collagen gene (31), or to the immunoglobulin heavy chain enhancer (39). Our experiments in HeLa and 3T3 cells transiently transfected with grik5 intron 1 linked to a heterologous beta -actin or SV40 promoter are consistent with the idea that the nuclear factors expressed in nonneural cells are capable of binding to one or more intragenic regulatory elements and may thereby inhibit gene transcription.

The effect of intron 1 on the production of reporter gene mRNA may occur through any of several mechanisms. The intron might contain sequences that block transcriptional elongation, target the primary transcript for rapid turnover, or prevent efficient processing. Alternatively, the silencer in this intron may interact with promoter and upstream elements through DNA-binding proteins. This rather distant interaction could occur through the formation of DNA loops (40). It can therefore be hypothesized that protein binding to the intronic silencing element may directly interfere with formation of the transcriptional complex and/or with the binding of trans-acting factors to the promoter region. Based on this hypothesis, it could be speculated that the inhibitory potential of the intronic region would depend on the levels of relevant DNA binding proteins in the cell. These could be different in distinct neural cell types and therefore determine the cell-specific expression pattern of the GluR gene grik5 that occurs in several brain regions (8). Alternatively, higher levels of intron-binding proteins might contribute to maintain the grik5 RNA levels at a plateau in the postnatal rat brain (41). To address these questions more directly, we are currently investigating the effects of intron 1 on grik5 expression in transgenic mice during development.

In conclusion, our results indicate that expression of the kainate receptor subunit gene grik5 is modulated by an intronic element that displays features typical of a silencer. These findings represent the first demonstration that, similar to the other neural genes encoding the glial fibrillary acidic protein and the cell adhesion molecule Ng-CAM (25, 26), promoter activity of a GluR gene can be modulated by intragenic DNA elements. This will provide further impetus to identify negative regulatory elements in introns of other neurotransmitter receptor genes.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81010[GenBank].


Dagger    To whom correspondence should be addressed: Laboratory of Cellular and Molecular Neurophysiology, NICHD, National Institutes of Health Bldg. 49, Room 5A-78, 49 Convent Dr., Bethesda, MD 20892. Tel.: 301-402-4776; Fax: 301-402-4777; E-mail: vgallo{at}helix.nih.gov.
1   The abbreviations used are: GluR, glutamate receptor; kb, kilobase(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase; RT-PCR, reverse transcription-polymerase chain reaction; AMPA, (R,S)-alpha -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid.

ACKNOWLEDGEMENTS

We thank Montse Molne for help during the first phase of this project and for generating the lambda 5 clone and the grik5-2.CAT construct. We are grateful to Xiaoqing Yuan and Jiamin Zhou for assistance. We thank Arther Sands for the gift of plasmid beta -actin-CAT, Kathleen Mahon and Maria Morasso for the galscript-ES4 plasmid, and Grant Mac Gregor for the pPolIIplacF.beta gal plasmid. We thank Michael Whalin and Gordon Guroff for PC12 cells. We are particularly indebted to Gretchen Gibney for discussion and for critically reading the manuscript. We thank Steve Scherer and Albert Dobi for technical advice and Sharmila Banjeree Basu, Michael Brenner, and Steve Scherer for critically reading the manuscript.


REFERENCES

  1. Mayer, M. L., and Westbrook, G. (1987) Progr. Neurobiol. 28, 197-276 [CrossRef][Medline] [Order article via Infotrieve]
  2. Pharmacol. Rev. 40, 143-210Collingridge, G. L., and Lester, R. A. J. Pharmacol. Rev. 40, 143-210
  3. Hollman, M., and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31-108 [CrossRef][Medline] [Order article via Infotrieve]
  4. Seeburg, P. H. (1993) Trends Neurosci. 16, 359-365 [CrossRef][Medline] [Order article via Infotrieve]
  5. Herb, A., Burnashev, N., Werner, P., Sakmann, B., Wisden, W., and Seeburg, P. H. (1992) Neuron 8, 775-785 [Medline] [Order article via Infotrieve]
  6. Szpirer, C., Molne, M., Antonacci, R., Jenkins, N. A., Finelli, P., Szpirer, J., Riviere, M., Rocchi, M., Gilbert, D. J., Copeland, N. G., and Gallo, V. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11849-11853 [Abstract/Free Full Text]
  7. Werner, P., Voigt, M., Keinanen, K., Wisden, W., and Seeburg, P. H. (1991) Nature 351, 741-744
  8. Wisden, W., and Seeburg, P. H. (1993) J. Neurosci. 13, 3582-3596 [Abstract]
  9. Molne, M., Huang, F., Scherer, S., and Gallo, V. (1995) Soc. Neurosci. Abstr. 21, 52
  10. Louis, J. C., Magal, E., Muir, D., Manthorpe, M., and Varon, S. (1992) J. Neurosci. Res. 31, 193-204 [Medline] [Order article via Infotrieve]
  11. Patneau, D. K., Wright, P. W., Winters, C., Mayer, M. L., and Gallo, V. (1994) Neuron 12, 357-371 [Medline] [Order article via Infotrieve]
  12. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  13. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  14. Mount, S. M. (1982) Nucleic Acid Res. 10, 459-472 [Abstract]
  15. Huang, M. T. F., and Gorman, C. M. (1990) Mol. Cell Biol. 10, 1805-1810 [Medline] [Order article via Infotrieve]
  16. Kozmík, Z., and Paces, V. (1990) Gene (Amst.) 90, 287-291 [Medline] [Order article via Infotrieve]
  17. Suchanek, B., Seeburg, P. H., and Sprengel, R. (1995) J. Biol. Chem. 270, 41-44 [Abstract/Free Full Text]
  18. Köhler, M., Kornau, H.-C., and Seeburg, P. H. (1994) J. Biol. Chem. 269, 17367-17370 [Abstract/Free Full Text]
  19. Hollmann, M., Boulter, J., Maron, C., Beasleg, L., Sullvan, J., Pecht, G., and Heinemann, S. (1993) Neuron 12, 943-954
  20. Sommer, B., Köhler, M., Sprengel, R., and Seeburg, P. H. (1991) Cell 67, 11-19 [Medline] [Order article via Infotrieve]
  21. Duester, G., Jornvall, H., and Hatfield, G. W. (1986) Nucleic Acids Res. 14, 1931-1941 [Abstract]
  22. Köhler, M., Burnashev, N., Sakmann, B., and Seeburg, P. H. (1993) Neuron 10, 491-500 [Medline] [Order article via Infotrieve]
  23. Seeburg, P. H. (1996) J. Neurochem. 66, 1-5 [Medline] [Order article via Infotrieve]
  24. Sakimura, K., Morita, T., Kushiya, E., and Mishina, M. (1992) Neuron 8, 267-274 [Medline] [Order article via Infotrieve]
  25. Sarkar, S., and Cowan, N. J. (1991) J. Neurochem. 57, 675-684 [Medline] [Order article via Infotrieve]
  26. Kallunki, P., Jenkinson, S., Edelman, G. M., and Jones, F. S. (1995) J. Biol. Chem. 270, 21291-21298 [Abstract/Free Full Text]
  27. Rossi, P., and Crombrugghe, B. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5590-5594 [Abstract]
  28. Horton, W., Miyashita, T., Kohno, K., Hassell, J. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8864-8868 [Abstract]
  29. Bornstein, P., McKay, J., Morishima, J. K., Devarayalu, S., and Gelinas, R. E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8869-8873 [Abstract]
  30. Buchman, A. R., and Berg, P. (1988) Mol. Cell. Biol. 8, 4395-4405 [Medline] [Order article via Infotrieve]
  31. Bornstein, P., and McKay, J. (1988) J. Biol. Chem. 263, 1603-1606 [Abstract/Free Full Text]
  32. Deng, T., Li, Y., and Johnson, L. F. (1989) Nucleic Acids Res. 17, 645-658 [Abstract]
  33. Jallat, S., Perraud, F., Dalemans, W., Balland, A., Dieterle, A., Faure, T., Meulien, P., and Pavirani, A. (1990) EMBO J. 9, 3295-3301 [Abstract]
  34. Belecky-Adams, T., Wight, D. C., Kopchick, J. J., and Parysek, L. M. (1993) J. Neurosci. 13, 5056-5065 [Abstract]
  35. Brand, A. H., Breeden, L., Abraham, J., Sternglanz, R., and Nasmyth, K. (1985) Cell 41, 41-48 [Medline] [Order article via Infotrieve]
  36. Imler, J-L., Lemaire, C., Wasylyk, C., and Wasylyk, B. (1987) Mol Cell. Biol. 7, 2558-2567 [Medline] [Order article via Infotrieve]
  37. Hewitt, S. M., Fraizer, G. C., and Saunders, G. F. (1995) J. Biol. Chem. 270, 17908-17912 [Abstract/Free Full Text]
  38. Haniel, A., Welge-Lüßen, U., Kühn, K., and Pöschl, E. (1995) J. Biol. Chem. 270, 11209-11215 [Abstract/Free Full Text]
  39. Schlokat, U., Bohmann, D., Scholer, H., and Gruss, P. (1986) EMBO J. 5, 3251-3258 [Abstract]
  40. Ptashne, M. (1986) Nature 322, 697-701 [Medline] [Order article via Infotrieve]
  41. Bahn, S., Volk, B., and Wisden, W. (1994) J. Neurosci. 14, 5525-5547 [Abstract]

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