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• W1999700412 abstract "Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced bydepolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation.KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex. Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced bydepolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation.KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex. Neurons respond to stimuli through a variety of overlapping signal transduction pathways. Activation of these pathways leads to changes in gene expression that ultimately result in changes in neuronal function. One stimulus, membrane depolarization leading to a rise in [Ca2+]i, activates Ca2+/calmodulin kinase- and adenylyl cyclase-dependent (1Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9491) Google Scholar) signal transduction pathways that are involved in the synaptic plasticity necessary for brain development (2Murphy D.D. Segal M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1482-1487Crossref PubMed Scopus (259) Google Scholar) and for learning and memory (3Wu Z.-L. Thomas S.A. Villacres E.C. Xia Z. Simmons M.L. Chavkin C. Palmiter R.D. Storm D.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 220-224Crossref PubMed Scopus (302) Google Scholar, 4Tsien J.Z. Huerta P.T. Tonegawa S. Cell. 1996; 87: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar, 5Mayford M. Bach M.E. Huang Y.-Y. Wang L. Hawkins R.D. Kandel E.R. Science. 1996; 274: 1678-1683Crossref PubMed Scopus (1096) Google Scholar, 6Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1240) Google Scholar). We and others are searching for genes induced in neurons by depolarization to identify candidate genes whose expression may mediate neuronal plasticity. Studies using classical differential and subtractive screening procedures have identified many previously known immediate-early genes (IEGs) 1The abbreviations used are: IEG, immediate-early gene; NGF, nerve growth factor; RDA, representational difference analysis; EGF, epidermal growth factor; GST, glutathioneS-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; bp, base pair; KA, kainic acid; ECS, electroconvulsive shock. 1The abbreviations used are: IEG, immediate-early gene; NGF, nerve growth factor; RDA, representational difference analysis; EGF, epidermal growth factor; GST, glutathioneS-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; bp, base pair; KA, kainic acid; ECS, electroconvulsive shock.(7Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Kuhl D. Nature. 1993; 361: 453-457Crossref PubMed Scopus (639) Google Scholar, 8Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-721Crossref PubMed Scopus (434) Google Scholar, 9Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Neuron. 1993; 11: 371-386Abstract Full Text PDF PubMed Scopus (1090) Google Scholar), one novel IEG, rheb, enriched in depolarized brain (10Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar), and two novel neuron-specific, depolarization-induced genes, cpg1 (8Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-721Crossref PubMed Scopus (434) Google Scholar) and synaptotagmin IV (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar). Most recently, differential display was used to identify synaptotagmin X, a gene induced in degenerating brain following kainic acid seizures (12Babity J.M. Armstrong J.N. Plumier J.-C.L. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar). Many of these genes are induced not only by depolarization but also by other stimuli such as nerve growth factor (NGF) (10Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). The products of genes induced by both types of stimuli presumably function not only during periods of neuronal plasticity or seizure activity but also during neuronal growth and development. Our goal is to identify genes that are induced in neurons by depolarization and not by neurotrophins and/or other growth factors. We suggest that the products of such genes may have roles specific to neuronal plasticity and/or seizure activity but not to growth and development. To perform this task, we have utilized representational difference analysis (RDA) (13Lisitsyn N. Lisitsyn N. Wigler M. Science. 1993; 259: 946-951Crossref PubMed Scopus (1164) Google Scholar, 14Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar, 15Braun B.S. Frieden R. Lessnick S.L. May W.A. Denny C.T. Mol. Cell. Biol. 1995; 15: 4623-4630Crossref PubMed Scopus (154) Google Scholar) to identify cDNAs for messages induced in PC12 cells by depolarization but not by NGF or epidermal growth factor (EGF). PC12 cells are widely used as a system for studying both presynaptic events involving synaptic vesicles and neuronal differentiation in response to NGF. PC12 cells respond to membrane depolarization by releasing neurotransmitters from synaptic-like vesicles (16Melega W.P. Howard B.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6536-6538Crossref Scopus (54) Google Scholar, 17Bauerfeind R. Regneir-Vigouroux A. Flatmark T. Huttner W.B. Neuron. 1993; 11: 105-121Abstract Full Text PDF PubMed Scopus (118) Google Scholar). In contrast, they respond to NGF by differentiating into a more neuronal cell type (18Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4839) Google Scholar) and to EGF by proliferation (19Huff K. End D. Guroff G. J. Cell Biol. 1981; 88: 189-198Crossref PubMed Scopus (206) Google Scholar). We (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar, 20Altin J.G. Kujubu D.A. Raffioni S. Eveleth D.D. Herschman H.R. Bradshaw R.A. J. Biol. Chem. 1991; 266: 5401-5406Abstract Full Text PDF PubMed Google Scholar, 21Kujubu D.A. Lim R.W. Varnum B.C. Herschman H.R. Oncogene. 1987; 1: 257-262PubMed Google Scholar) and others (22Bartel D.P. Sheng M. Lau L.F. Greenberg M.E. Genes Dev. 1989; 3: 304-313Crossref PubMed Scopus (395) Google Scholar, 23Milbrandt J. Neuron. 1988; 1: 183-188Abstract Full Text PDF PubMed Scopus (530) Google Scholar, 24Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Crossref PubMed Scopus (177) Google Scholar) have previously used PC12 cells to study the induction of IEGs by various stimuli that include both depolarization and NGF. For these reasons, we chose PC12 cells to identify genes induced specifically by depolarization. RDA is a rapid, sensitive method for identifying differences between two populations of DNA. RDA was originally developed to identify DNA sequences unique to one of two genomic populations (13Lisitsyn N. Lisitsyn N. Wigler M. Science. 1993; 259: 946-951Crossref PubMed Scopus (1164) Google Scholar). The RDA procedure was subsequently modified for comparing cDNA populations (14Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar) and detecting differentially expressed sequences (14Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar, 15Braun B.S. Frieden R. Lessnick S.L. May W.A. Denny C.T. Mol. Cell. Biol. 1995; 15: 4623-4630Crossref PubMed Scopus (154) Google Scholar, 25Chu C.C. Paul W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2507-2512Crossref PubMed Scopus (72) Google Scholar, 26Edman C.F. Prigent S.A. Schipper A. Feramisco J.R. Biochem. J. 1997; 323: 113-118Crossref PubMed Scopus (21) Google Scholar). We have recently described the use of RDA to isolate genes preferentially induced in PC12 cells by the neurotrophin NGFversus the mitogen EGF (27Vician L. Basconcillo R. Herschman H.R. J. Neurosci. Res. 1997; 50: 32-43Crossref PubMed Scopus (34) Google Scholar). In this report we again applied RDA to cDNAs from PC12 cells and isolated a number of cDNA fragments that identify messages induced by depolarization but not by NGF or EGF. We describe in detail the identification and characterization of KID-1, a proteinkinase induced by depolarization, in PC12 cells and in brain. KID-1 expression is low in unstimulated PC12 cells and is rapidly induced by membrane depolarization. The cDNA sequence of KID-1 predicts a serine/threonine protein kinase with strong homology to the proto-oncogene PIM-1. When expressed as a glutathioneS-transferase (GST) fusion protein, KID-1 is able to catalyze both histone phosphorylation and autophosphorylation.KID-1 is an IEG; its expression is induced PC12 cells in the presence of cycloheximide, a protein synthesis inhibitor.KID-1 is not induced by NGF or EGF. In brain,KID-1 expression is induced after generalized seizures and is limited to specific areas of the hippocampus and temporal lobe. PC12 cells, obtained from B. Howard (UCLA), were cultured in RPMI with 10% heat-inactivated horse serum and 5% fetal calf serum in T75 flasks. The cells were passaged, at a 1:4 to 1:5 ratio, by trituration when they reached 75–80% confluence. Experiments were performed in cells at 50–60% confluence. KCl (55 mm), forskolin (50 mm), NGF (50 ng/ml), or EGF (10 ng/ml) was added to the cultures for the times indicated in the text or the figures. Growth factors were purchased from Collaborative Biomedical Products (Bedford, MA). Forskolin was purchased from Sigma. For each experiment, paired flasks were treated with KCl, forskolin, NGF, EGF, or no ligand (control) for 0.75, 1.5, 2, or 4 h. For the synthesis of cDNA to be analyzed by RDA, RNAs from all eight flasks treated with each ligand and from three separate experiments (a total of 24 flasks) were pooled. For subsequent experiments, RNAs from the paired flasks treated with each ligand at each time point were pooled. RNAs from control flasks at all time points were pooled. To determine whether a message is the product of a primary response gene, cycloheximide (10 μg/ml) (Sigma) was added, either alone or along with the specific ligand, for 1.5 h. After ligand treatment, medium was removed, and the cells were washed twice with ice-cold phosphate-buffered saline. The cells were lysed by adding 1.2 ml of cold lysis buffer (5 m guanidinium thiocyanate/10 mm EDTA/50 mm Tris, pH 7.5/8% 2-mercaptoethanol) directly to the flask, and total RNA was isolated by precipitation with lithium chloride (28Cathala G. Savouret J.F. Mendez B. West B.L. Karin M. Martial J.A. Baxter J.D. DNA. 1983; 2: 329-335Crossref PubMed Scopus (1228) Google Scholar). Poly(A)+ RNA was isolated using an Oligotex mRNA kit (Qiagen, Chatsworth, CA). The final concentration of RNA was determined using a GeneQuant RNA/DNA calculator with a 10-μl cuvette (Amersham Pharmacia Biotech). 2 μg each of the KCl and forskolin poly(A)+ RNA pools were pooled to make “tester” cDNA. Similarly, 2 μg each of the NGF and EGF poly(A)+ RNA pools were pooled to make “driver” cDNA. Double-stranded cDNA was synthesized from the pooled poly(A)+ RNAs, using a SuperScript Choice cDNA Synthesis kit (Life Technologies, Inc.), with a combination of 1.0 μg of oligo (dT)12–18 and 100 ng of random hexamers. To evaluate the induction of known genes, Northern blots prepared with 50 ng of tester, driver, and control poly(A)+ RNA were probed with: arc (provided by P. Worley, Johns Hopkins University, Baltimore, MD), NGFI-B (provided by J. Milbrandt, Washington University, St. Louis, MO), secretogranin I (provided by P. Danielson, Scripps Research Institute, La Jolla, CA), synaptotagmin IV, and TIS21. Blots were also probed with rat GAPDH (provided by R. Bravo, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) to normalize RNA loading. Hybridization signals were quantitated with an Ambis Radio-Imager (Scanalytics, Billerica, MA). Based on the relative intensities of the signals from the specific probes to that from GAPDH, and our decision to dope the driver with a 10-fold excess, 1.2, 12.7, and 5.5 ng of NGFI-B, secretogranin I, and synaptotagmin IV cDNAs, respectively, were added to 1.5 μg of driver cDNA prior to the preparation of driver amplicons. RDA was performed as described previously (27Vician L. Basconcillo R. Herschman H.R. J. Neurosci. Res. 1997; 50: 32-43Crossref PubMed Scopus (34) Google Scholar), with the following modifications: Tfl polymerase (Promega, Madison, WI), 5 units, was used in all PCR reactions. PCR products were purified using a PCR Purification kit (Qiagen). Three rounds of RDA reactions were performed. Tester:driver hybridization ratios were 1:100 for the first round, 1:1000 for the second round, and 1:50,000 for the third round. Hybridizations were maintained for approximately 60 h. The difference product of the third round of RDA demonstrated a small number of distinct bands on agarose gel. Gel plugs were taken from each of these bands, using the small end of a Pasteur pipette, and the DNA was eluted in 25–50 μl of TE (10 mm Tris, pH 8.0, 1 mmEDTA, pH 8.0) at 4 °C overnight. The DNAs were amplified using 5 units of Taq polymerase in a 100-μl PCR reaction, cloned into pCRII using a TA Cloning Kit (Invitrogen, Carlsbad, CA), and used to transform DH11-S Escherichia coli. Plasmid DNA was isolated from individual colonies from among the transformation reactions using a QIAprep Spin Plasmid Miniprep kit (Qiagen). The cloned fragments were used to prepare a dot blot, which was probed with the third round RDA difference product. Fragments that yielded strong hybridization signals were used as probe on Northern blots to confirm their preferential induction. Northern analysis was carried out as described previously (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar). The progress of RDA was analyzed by separating 100 ng of the difference products on 0.5% agarose/1.5% NuSieve (FMC Bioproducts, Rockland, ME) gels and staining with ethidium bromide. Southern analysis was performed by standard methods (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequencing was performed by the core facility of the Department of Pediatrics, University of California, Los Angeles, on an Applied Biosystems instrument. The DNA sequences obtained were compared with the current version of the nonredundant, updated GenBank™ and EMBL data base using BlastN (30Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar). Analysis was performed using MacVector and AssemblyLign (Eastman Kodak, New Haven, CT) and MegAlign (DNAStar, Madison, WI) software. The full-length cDNA clone was obtained by screening a PC12 cDNA library constructed in λZAP II (Stratagene, La Jolla, CA) in our laboratory. Plaque lifts were probed with the cloned fragment by standard methods (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Positive clones were converted into plasmid using Exassist helper phage (Stratagene). By performing PCR with the full-length KID-1 cDNA as template, and the oligonucleotides 5′-GGATCCATGCTGCTGTCCAAGTTCGGCTCCCTGG-3′ and 5′-CTCGAGTCACAAGCTCTCACTGCTGGAAGTGGTACTGG-3′ as primers, the KID-1 open reading frame was modified to create a 5′ BamHI site and a 3′ XhoI site. The PCR product was cloned into pCRII using a TA Cloning Kit (Invitrogen) and sequenced to confirm that no mutations were introduced during PCR. The KID-1/pCRII plasmid was digested with BamHI (New England Biolabs, Beverly, MA) andXhoI (New England Biolabs), and the 1000-bp fragment containing the modified KID-1 open reading frame was isolated from an agarose gel. This fragment was ligated into the BamHI andXhoI sites of the bacterial expression vector pGEX-4T-3 (Amersham Pharmacia Biotech). This insertion created an “in-frame” fusion of the KID-1 open reading frame cDNA with the isopropyl-1-thio-β-d-galactoside-inducible GST gene. The JM109 strain of E. coli was transformed with the GST-KID-1 plasmid using standard methods (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). As a control, anE. coli strain containing a protein-arginineN-methyltransferase (GST-PRMT1) plasmid (31Lin W.-J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar) was processed in parallel with the GST-KID-1 strain. Overnight cultures of the transformed bacteria were diluted 1:5 in 2× YT (16 g of bacto-tryptone, 10 g of bacto-yeast extract, 5 g of NaCl/liter of H2O) containing 100 μg/ml ampicillin and grown for 3 h with shaking at 37 °C. Protein expression was induced by adding 500 μm isopropyl-1-thio-β-d-galactoside, and growth was continued for an additional 4 h at 37 °C. The bacteria were pelleted by centrifugation, resuspended in phosphate-buffered saline containing 50 μm3,4-dichloroisocoumarin (Sigma), 1 mm phenylmethylsulfonyl fluoride (Sigma), 10 μml-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane (Sigma), and 0.1 μg/ml pepstatin A (Sigma), and lysed by sonication. The proteins were purified by binding to glutathione-Sepharose beads as described by Amersham Pharmacia Biotech, except that all steps were performed at 4 °C. The proteins were eluted from the glutathione-Sepharose beads with 30 mm glutathione and frozen at −20 °C. Aliquots of the purified fusion proteins were subjected to SDS-polyacrylamide gel electrophoresis on 8% acrylamide and stained with Coomassie Brilliant Blue. For Western analysis, the proteins were transferred from acrylamide to nitrocellulose using a Trans-Blot SD semi-dry transfer cell (Bio-Rad) at 16 V for 40 min. The filter was treated by standard methods (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The primary antibody, anti-GST (Amersham Pharmacia Biotech) was used at 1:1000 dilution, and the secondary antibody, anti-goat horseradish peroxidase (Biosource, Camarillo, CA) was used at 1:4000 dilution. Horseradish peroxidase was detected by ECL (Amersham Pharmacia Biotech). The in vitro kinase assays were performed as described previously (32Hoover D. Friedmann M. Reeves R. Magnuson N.S. J. Biol. Chem. 1991; 266: 14018-14023Abstract Full Text PDF PubMed Google Scholar). Reactions containing 10 μl of protein, 19 μl of kinase buffer (50 mm Tris, pH 8.0/150 mmNaCl/10 mm MgCl2/2 mmMnCl2) and 10 μg of histone Hl (Sigma) were set up on ice. The reactions were initiated by the addition of 10 μCi of [γ-32P]ATP (Amersham Pharmacia Biotech) and incubated at room temperature for 30 min. The reactions were terminated by the addition of 6 μl of 6× SDS protein loading buffer. The samples were heated to 98 °C for 5 min and subjected to electrophoresis on 8% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. The gels were dried under vacuum and exposed to film at room temperature. RNAs from tissues were prepared by a modification of the AGPC protocol (33Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). Immediately upon removal from the animal, tissues were weighed, placed in 10 volumes of cold denaturing solution (4 m guanidinium thiocyanate/25 mm sodium citrate, pH 7/0.5% sarcosyl/0.1 m2-mercaptoethanol), and homogenized using an electric tissue homogenizer. Next, 0.1 volume of 2 m sodium acetate, pH 4, 1 volume of acid phenol, and 0.2 volume of chloroform-iso-amyl alcohol mixture (24:1) were added to the homogenate. After mixing and centrifugation, the aqueous phase was transferred to a new tube, and the extraction was repeated. The aqueous phase was mixed with 1 volume of isopropanol and placed at −20 °C overnight to precipitate RNA. The RNA was pelleted, washed with 80% ethanol, and resuspended in 400 μl of RNA solubilization buffer (0.1% SDS/1 mm EDTA/10 mm Tris, pH 7.5). The solution was frozen, thawed, and extracted twice with equilibrated phenol and twice with chloroform. The aqueous phase was mixed with 40 μl of 3 m sodium acetate, pH 5.2, and extracted once with chloroform-iso-amyl alcohol mixture. This aqueous phase was mixed with 1 ml of ethanol and placed at −80 °C to precipitate RNA. Finally, the RNA was pelleted, washed with 80% ethanol, and resuspended in TE, pH 8.0, at concentrations appropriate for Northern analysis. Wistar rats (270 to 300 g) were housed with free access to rat chow and water for at least 24 h before the experiment. KA treatment was carried out as described previously (34Marcheselli V.L. Bazan N.G. J. Biol. Chem. 1996; 271: 24794-24799Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Briefly, rats were injected with KA (10 mg/kg) intraperitoneally and kept in isolated cages under surveillance to classify the seizure class. Animals not developing seizures above class III (35Racine R.J. Electroencephalograph. Clin. Neurophysiol. 1972; 32: 281-294Abstract Full Text PDF PubMed Scopus (5705) Google Scholar) by 30 min after injection were discarded. Animals were sacrificed at 0 (control), 0.5, 1, 2, and 4 h after injection. ECS treatment was delivered through two platinum electrodes implanted in the scalp 1 cm apart. A single stimulus was delivered with a S48 Stimulator (Grass Instruments, Quincy, MA) set at 110 V, 150 pulses/s, 0.5 ms in duration, at a train rate of 0.75 trains/s, and a train duration of 250 ms. Each animal developed a tonic-clonic seizure that lasted 12–15 s and recovered in 40–50 s. Control animals had electrodes implanted but did not receive ECS. ECS-treated animals were sacrificed at 0.5, 1, 2, and 4 h after seizure. KA- and ECS-treated rats were killed by decapitation. The hippocampus and cortex were dissected, and RNA was isolated as described previously (36Marcheselli V.L. Bazan N.G. J. Neurosci. Res. 1994; 37: 54-61Crossref PubMed Scopus (59) Google Scholar). Two separate experiments were performed with three animals in each condition analyzed. Kainic acid treatment, preparation of brain sections, and in situ hybridization were carried out as described previously (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar, 37Tocco G. Bi X. Vician L. Lim I.K. Herschman H. Baudry M. Mol. Brain Res. 1996; 40: 229-239Crossref PubMed Scopus (51) Google Scholar) with the following modifications. Male Sprague-Dawley rats were injected with kainic acid (12 mg/kg) subcutaneously and sacrificed at 1, 4, and 8 h after the initiation of seizure activity. Control animals did not receive any handling. For statistical analysis, four animals were prepared for each treatment group and for control. Following hybridization, the sections were washed for 20 min in 4× SSC, followed by 2× SSC for a few hours. The high stringency wash was performed at 55 °C for 30 min in 0.2 × SSC. The antisense (5′-GGAGCAGCGTTCAAAAAGGCACTCAAAGCAAAGGAAACAG-3′) and the sense (5′-GCCTGGCGGCGTGGACCACCTCCCAGTGAAGATCCTACAG-3′) oligonucleotides were selected from nucleotides 227–266 of the KID-1cDNA sequence. To prepare the starting mRNA populations to be used to identify cDNAs for messages induced by depolarization, but not by growth factors, we employed four populations of PC12 cells: cells treated individually with KCl (55 mm), forskolin (50 mm), NGF (50 ng/ml), or EGF (10 ng/ml). We used forskolin-treated cells as well as KCl-treated cells to prepare our tester mRNA populations (13Lisitsyn N. Lisitsyn N. Wigler M. Science. 1993; 259: 946-951Crossref PubMed Scopus (1164) Google Scholar), from which we planned to clone cDNAs for genes preferentially induced by depolarization, because many genes strongly induced by depolarization in brain are also strongly induced by forskolin in PC12 cells (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar, 38Thompson M.E. Zimmer W.E. Wear L.B. MacMillan L.A. Thompson W.J. Huttner W.B. Hidaka H. Scammel J.G. Mol. Brain Res. 1992; 12: 195-202Crossref PubMed Scopus (32) Google Scholar, 39Laslop A. Tschernitz C. Eiter C. Neuroscience. 1994; 59: 477-485Crossref PubMed Scopus (15) Google Scholar). Because we had no prior knowledge of the kinetics of induction of differentially expressed messages, different time points were pooled for each inducer to prepare RNAs for the tester and driver populations to be used for RDA (see “Experimental Procedures” for details). One of the advantages of the RDA procedure is that the driver population, which in this case was derived from mRNA prepared from NGF- and EGF-treated cells, can be artificially enriched or “doped” with cDNAs for genes known to be more strongly expressed in the tester population. Fragments of genes known to be preferentially expressed in the tester population can be eliminated in the RDA procedure as a consequence of this doping. As a result, restriction fragments from other preferentially induced messages in the tester population can be selected during the amplification steps. Preliminary experiments, using Northern blots to evaluate the induction of several genes in PC12 cells by the four inducers (KCl, forskolin, NGF, and EGF), confirmed that secretogranin I (38Thompson M.E. Zimmer W.E. Wear L.B. MacMillan L.A. Thompson W.J. Huttner W.B. Hidaka H. Scammel J.G. Mol. Brain Res. 1992; 12: 195-202Crossref PubMed Scopus (32) Google Scholar, 39Laslop A. Tschernitz C. Eiter C. Neuroscience. 1994; 59: 477-485Crossref PubMed Scopus (15) Google Scholar), synaptotagmin IV (11Vician L. Lim I.K. Ferguson G. Tocco G. Baudry M. Herschman H.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2164-2168Crossref PubMed Scopus (137) Google Scholar), and NGFI-B/nur77/TIS1 (22Bartel D.P. Sheng M. Lau L.F. Greenberg M.E. Genes Dev. 1989; 3: 304-313Crossref PubMed Scopus (395) Google Scholar, 23Milbrandt J. Neuron. 1988; 1: 183-188Abstract Full Text PDF PubMed Scopus (530) Google Scholar) were indeed preferentially induced by KCl and forskolin. Moreover, the approximate levels of these messages in the pooled tester mRNA populations could be estimated from these Northern blots. The driver cDNA, prepared from NGF- and EGF-treated cells, was doped with an approximately 10-fold excess of the cDNAs for each of these rat genes prior to preparation of the driver amplicons. Tester amplicons, derived from the cDNA prepared from the pooled KCl- and forskolin-treated cells, and driver amplicons, derived from the doped cDNA prepared from the pooled NGF- and EGF-treated cells, were used to carry out RDA reactions using procedures we described previously (27Vician L. Basconcillo R. Herschman H.R. J. Neurosci. Res. 1997; 50: 32-43Crossref PubMed Scopus (34) Google Scholar) with modifications (see “Experimental Procedures” for details). After three rounds of RDA reactions, the initial tester cDNA population becomes greatly simplified (Fig. 1 A). Ethidium bromide staining of the initial tester and driver cDNAs demonstrates a homogenous distribution of DNA throughout the range of 0.4–0.8 kilobases, without distinct bands. In contrast, staining of the third round RDA difference product demonstrates a small number of distinct bands with a faint background of diffuse signal. Thus, by the third round of RDA reactions, a small number of DNA fragments have become a large proportion of the total DNA population. One clone from the 718-bp band (Fig. 1 A) demonstrated a pattern of preferential induction in a Northern analysis of tester versus driver poly(A)+ RNA (Fig. 1 B). The 5′ sequence of this fragment shares strong sequence similarity with a portion of theXenopus laevis PIM-1 cDNA (40Palaty C.K. Kalmar G. Tai G. Oh S. Amankawa L. Affolter M. Aebersold" @default.
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