DNA SEQUENCE ENCODING THE CPG-1 NEURAL POLYPEPTIDE AND THE POLYPEPTIDE ENCODED THEREBY

Abstract: A novel Candidate Plasticity-Related cDNA designated CPG-1 and a polypeptide encoded thereby which is believed to be a novel neuropeptide or a neurotrophic factor or a precursor of one of them.

Description: DNA SEQUENCE ENCODING THE CPG- 1 NEURAL POLYPEPTIDE AND THE POLYPEPTIDE ENCODED THEREBY FIELD OF THE INVENTION The present invention concerns a new neural-derived plasticity related gene inducible in the hippocampus dentate gyrus and the polypeptide encoded thereby. Genes encoding polypcptidcs of this kind arc herein designated as "Candidate Plasticity -related Genes" (CPGs) and the term "cpg- 1 " will be used hereto to denote the novel cpg derived cDNA. identified and isolated in accordance with the present invention The term "cpg- 1 Product" will be used herein to denote the product ol the cpg- 1 LIST OF REFERENCES The following is a list of references referred to in the following disclosure: ^ 1 . Bcn-Ari, Y. and Rcprcsa, A. ( 1990). Brief seizure episodes induce long-term potentiation and mossy fibre sprouting in the hippocampus. Trends Neurosci. , L3, 312-31 . 2. Bliss, T.V.P. and Lomo, T. (1973). Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the pcrforant path. J. Physioi. 232. 331- 356. 3. Chomczynski. P. and Sacchi, N. ( 1987). Single step method of RNA isolation by acid guanidinium thiocyanatc - phenol - chloroform extraction. Analyt. Biochem., 162, 156- 159. 4. Cole, A.J., Saffcn, D.W., Baraban, J.M.. and Worlcy, P.F. ( 1989). Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMD A receptor activation. Nature, 340, 474-476. 5. Gall, C. ( 1988). Seizures induce dramatic and distinctly different changes in enkephalin, dynorphin, and CCK immunorcactivitics in mouse hippocampal mossy fibers. ./. Neurosci. , 8, 1852- 1862. (.. Goclct, P., Castcllucci, V.F., Shachcr, S., and Kandcl, E.R. ( 1986). The long and the short of long-term memory - a molecular frame¬ work. Nature, 322, 419-422. 7. Gublcr, V., and Hoffman, B.J. (1983). A very simple and efficient method for generating cDNA libraries. Gene, 25, 263-269. 8. Madison, D.V., and Nicoll, R.A. (1988). Enkephalin hypcrpolarizcs intcrncuroncs in rat hippocampus. J. Physioi. , 398, 123- 130. 9. McNamara, J.O. (1988). Pursuit of the mechanisms of kindling. Trends Neurosci. , l , 33-36. 10. Morris, B.J., Fcascy, K.J., Bruggencate, G., Herz, A., and Hollt, V. ( 1988). Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells. Proc. Natl. Acad. Sci. USA, 85, 3226-3230. - J Pearson. W R . and Lipman. D J ( 1988) Improved tools for biological sequence comparison Proc Null Xtad Sc i USA. 85, 2444-2448 : ~ . Sambrook. J , Fπtsch, E F , am. Maniatis. 1 (1989 Molcc ulai Cloning 2nd Ed Cold Spring Harbor Laboratory Press π Seigelbaum, S A , Camardo, J S , and Kandcl. E R ( 1982) Serotonin and cyclic AMP close single K+ Channels in Aplv .ia sensory 10 neurons Nature, 299, 4132-4 47 14 Sheng, M and Greenberg, M E (1990) The regulation and function of c-fo ~ and other immediate early genes in the nervous system Neuron, 4, 477-485 1 5 15 Short, J M , Fernandez, J M , Sorge, J A and Husc, W D (1988) λZAP a bacteπophage λ expression vector with in vivo excision properties Nucl Acids Res , 16, 7583-7598 20 16 Sive, H.L and St John, T ( 1988) A simple subtractivc hybridiza¬ tion technique employing photoactivatablc biotin and phenol extraction Nucl Acids Res., 16, 10937 17 Sonncnbcrg, J L Rauschcr, F J , Morgan, J I , and Curran, T (1989) 5 Regulation of proenkephalin by Fos and Jun Science, 246, 1622- 1625 1 Stashcff, S F , Anderson, W W , Clarck, S . and Wilson W A ( 1989) NMDA antagonists differentiate cpilcptogcncsis from seizure ^0 expression in an in vitro model Science. 245 648-651 19 Wisden, W , Erπngton, M L., Williams, S , Dunnctt, S B , Waters. C , Hitchcock, D , Evan, G , Bliss, T V P , and Hunt, S P (1990) Differential expression of immediate early genes in the hippocampus " 55 and spinal cord New on, 4, 603-614 20 Wong, E H F , Kemp, J A , Priestley, T , Knight, A R . Woodruff, G N , and Ivcrscn, L L ( 1986) The anticonvulsant MK-801 is a potent N-mcthyl-D-aspartatc antagonist Pi oc Na M- ad Sc i 0 USA, 83, 7104-7108 BACKGROUND OF THE INVENTION Plasticity of the nervous system allows it to cope with changes in the outside world and to modify its response to an altered input. A high degree of plasticity is exhibited, for example, during CNS development and in learning and memory processes in the adult brain. One of the areas in the adult mammalian brain that exhibits high levels of plasticity is the hippo¬ campus, a region known to be involved in learning and memory, and plasticity has been extensively studied in this brain region. Long-term potentiation (LTP), which is believed by many to be involved in long-term memory mechanisms, was studied mainly in the hippocampus ( Bliss and Lomo, 1973; Madison et al., 1991). This brain region was also the focus of study of other plastic mechanisms which include kindling (McNamara, 1988) and other models for epileptic seizures (Bcn-Ari and Reprcsa, 1990; Stashcff et al., 1989). In LTP, kindling and epileptic seizure, ncuronal plasticity is manifested as a lasting potentiation along a pathway previously stimulated by a strong and/or recurring electrical stimulus. Short-term plastic changes, like those underlying short-term memory, involve post-translational modifications of proteins, such as for example, ion channels (Seigelbaum et al. , 1982). However, long-term persistence of plastic changes appears to require a de novo activation of gene expression. Treatments that induce LTP, seizure or kindling in the hippo¬ campus and those that produce other plasticity phenomena, activate transcription of a number of immediate early genes (IEGs) such as c-fos and zifl26 (Cole et al.. 1989; Wisdcn et al. , 1990; Sheng and Greenberg, 1990). Many of these IEGs encode transcription factors that control expression of "downstream " genes (Goclct et al., 1986; Sheng and Greenberg, 1990) whose products are believed to bring about long-term plastic changes. - -. SUMMARY OF THE INVENTION To identify genes that may participate in long-term plasticity, a highly sensitive differential cDNA cloning procedure was employed. Fii .y-two cDNAs of candidate plasticity-related genes (CPGs), genes that arc induced by the glutamate analog kainatc (kainic acid) in the hippocam¬ pus dentate gyrus (DG), were cloned. One of the cloned CPGs, cpg-1 , was found to be highly specific to the activated adult dentate gyrus and predicts a novel DG-spccific protein. The present invention provides by its first aspect a new cDNA sequence designated herein as "cpg- 1 ". By its second aspect, the present invention provides the polypeptide encoded thereby, designated herein as "cpg- 1 product", which is believed to be a novel neuropeptide or a ncurotrophic factor or precursor of one of them. In accordance with the present invention, there is thus provided a DNA molecule selected from the group consisting of: (i) a DNA molecule comprising the following coding nucleic acid sequence: 1 ATGGCCAAGAAGTCACATCCTTACCTACAC 31 TGGTATAAGAACGAAAACGGCCCATCAGTA 1 ATCAGGCCTGGAGGGAGGAG AGGTTCAGGG 91 AGCCCTAGCTCTACCGCTGCTCTCTTTGTC 121 TCTGTΛGATGCAAGCAGAGGGCGAACATTA 151 CCTTCAGTTATACACCCCTTGGTG ACCAAA 181 TCTAAAGGTCACACAGATGGTCCTGGTGAA 21 1 ACGAAATGCTCATTAAACCAAACCCCAAGC 241 CACGTTTGTGGAAAGGAACTGTTAGGGΛCA 271 AGGGGAGGGGGA; .. - (ii) a DNA molecule having the nucleic acid sequence of (i) in which one or more codons has been added, replaced or deleted in a manner that the polypeptide encoded by said sequence essentially retains the same biological properties as the polypeptide encoded by an unaltered DNA sequence; (iii) a DNA molecule encoding a polypeptide having an amino acid sequence of a polypeptide encoded by the DNA molecule of (i) or (ii) but which differs from the DNA sequences of (i) or (ii) in view of the degenerative nature of the genetic code; (iv) a DNA molecule having a coding nuclcotidc sequence, which is homologous to the DNA molecule of (i), (ii) or (iii), which is derived from a mammal other than rats and which encodes a polypeptide having a similar biological activity to that encoded by the sequences of (i), (ii) or (iii); (v) a fragment of the coding sequence of (i) - (iv) which encodes a peptide which essentially retains the biological properties of the peptide encoded by the unfragmented DNA molecule; (vi) a DNA molecule comprising the coding DNA sequence of fragments of (i) - (v) and additional DNA sequences in the 3' and 5' ends; and (vii) a recombinant DNA molecule comprising an encoding sequence according to any of (i) - (vi) above. The DNA molecule defined in (i) above is the cpg-1. In accordance with said second aspect of the present invention there is provided a polypeptide selected from the group consisting of: (i) a polypeptide having the following amino acid sequence: 1 M A K K S H P Y L H W Y K N E N G P S V 21 I R P G R R G S G S P S S T A A L F V 41 S V D A S R G R T L P S V I H P L V T K 61 S K G H T D G P G E T K C S L N O T P S (SI H V C G K E L L G T R G G G (ii) the polypeptide of (i) in which one or more amino acid residue has been added, deleted or replaced or chemically modified without substantially affecting the biological activity of the polypeptide; (iii) a biologically active fragment of the polypeptide of (i) or (ii); and (iv) a homologous polypeptide to that of (i), (ii) or (iii) derived from another mammal and which has a similar biological activity to that of (i), (ii) or (iii). The polypeptide as defined under (i) above, herein designated ""cpg- 1 product", is a highly basic small protein of 94 amino acids containing a repeated 6-amino-acid motif and two cysteine residues at positions 73 and 83 which are preceded by three amino acids (E, T and K at positions 70-72), that arc conserved in the ncurotrophin family. Said cpg-1 product may be prepared by cloning said DNA molecules in a suitable host cell within an appropriate expression vector. Methods of cloning and expression of DNA molecules in an appropriate host are generally known per se and their description goes beyond the scope of the present writing. For general reference the reader may be referred to J. Sambrook, E.F. Fritsch and T. Maniatis in Molecular Cloning: a Laborato- ry Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, 1989. A specific example of a host cell is E.coli. Cpg-1 is a low-abundance gene and is not continuously expressed but rather activated only after specific stimulus. Similarly to other CPGs, cpg-1 is induced in the hippocampus DG 6 hours after kainate injection. The isolation of cpg- 1 was facilitated by a novel highly sensitive differential cDNA cloning method suitable for the detection and optional isolation of a cDNA coding for a CPG product (cpg cDNA), which method comprises: 5 (a) obtaining an RNA sample from a cell suspected of expressing a CPG product (CPG + cell) and incubating with a reverse transcriptase enzyme, thereby obtaining single-stranded cDNA; (b) reacting the single-stranded cDNA obtained in step (a) with non-specific mRNA obtained from cells which do not express the CPG ( ) product; (c) incubating the remaining, non-hybridized single-stranded cDNA with a DNA polymcrase, thereby obtaining a double-stranded cDNA; (d) preparing a cDNA library by introducing said double-stranded cDNA as an insert into a viral vector or plasmid; 5 (c) plating the viral vectors or plasmids on cultured cells susceptible to infection by them, whereby placquc or colonies arc obtained as a result of a viral infection or transfection by the plasmids, respectively; (f) picking individual placquc or colonies and rescuing the viral vector or DNA plasmid therefrom; 0 (g) digesting said plasmid with a restriction enzyme which recognizes restriction sites flanking the cDNA inserts included in the vector or plasmid, whereby said cDNA inserts arc freed; (h) subjecting two digested cDNA samples in parallel to gel elcctrophorcsis and then blotting each sample of separated DNA onto a 5 filter; (i) hybridizing one blot with a first DNA probe being cDNA from a CPG * cell and hybridizing the other blot with a second DNA probe being cDNA from a cell not expressing a CPG product, a cDNA which has positively hybridized with the first DNA probe and less with the second DNA probe, being said cpg cDNA: and (j) optionally isolating said cpg CDNA. In the following, the known and accepted term "blotting " and "blots" will be used to denote the process of separating DNA by gel clectrophoresis, blotting it onto a filter and then hybridizing with a DNA or RNA probe, and the blots obtained, respectively. In accordance with prior art methods, blots were prepared directly from the plate and the DNA contents of the entire plaques was then hybridized with the probe. A positive hybridization then indicated that the plaques contained a DNA of interest. However, the problem of prior art methods were that at times even if a DNA sequence was present in the plasmid it did not hybridize owing to limited sensitivity resulting from a variety of factors including low amounts of DNA on the filter, low- abundance of RNA, where the probe is prepared from total tissue RNA, non specific background "noise" (non-specific hybridization) masking "true" signals. In accordance with the method used in the present invention these problems are overcome by initially isolating and purifying the DNA from the plaques or colonies, digesting the DNA so that the inserts arc freed, separating the inserts from the other components of the vector by clectro¬ phoresis whereby they will typically form a distinct band in the elcctropho- rctogram. Following blotting, differential hybridization of the cDNA probes to filters containing library clone is conducted. Thus, the differential cDNA cloning method used in the invention is unique in that it is significantly more sensitive than prior art differential cloning. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a scheme of the general strategy for subtractivc and differential cDNA cloning in the method of isolating CPGs; Fig. 2 shows a scheme of the screening procedure of CPGs; Fig. 3 shows Northern blotting of RNA from different organs showing tissue pattern of cpg-1 expression; Fig. 4 shows in situ hybridization of cpg-1 to a rat brain section; Fig. 5 shows the cpg-1 full-length cDNA sequence including sequences flanking 5' and 3' to the coding region (the underlined portion denotes the coding sequence). Fig. 6 shows a cpg-1 cDNA nuclcotidc sequence and the expected amino acid sequence of the encoded polypeptide; and Fig. 7 shows the partial predicted amino acid sequence of the cpg- 1 product compared to the partial amino acid sequence of three other neurotrophic peptides. GENERAL DESCRIPTION OF THE INVENTION In accordance with the present invention, a new DNA sequence which is derived from the candidate plasticity-related gene cpg-1 has been identified and isolated from kainate-stimulatcd rat hippocampus brain cells. As may no doubt be appreciated, on the basis of this newly discovered sequence, the artisan will have no difficulty in obtaining other DNA molecules coding for peptides having a similar biological activity. For example, the newly discovered sequence or portions thereof, may be used to "fish" homologous DNA sequences from brains of other mammals including humans. Furthermore, any such identified DNA sequence may be modified in a manner that the resulting DNA sequence will code for a polypeptide which has a similar biological activity to the unmodified DNA sequence: for example, adding one or more codons to cither or both the C or N ends of the DNA sequence; by deleting or replacing certain codons in the sequence; etc. Furthermore, on the basis of the degenerative nature of genetic codes, it is possible to prepare the very wide variety of DNA sequences coding for the same polypeptide. Finally, fragments of the coded polypeptidcs may be prepared, as well as DNA sequences encoding them, which likely essentially retain the biological activity of the entire polypeptide. The cpg- 1 product is believed to be a neural polypeptide or a neurotrophic factor or a precursor of one of them or of a smaller ncuro- peptide and may thus have therapeutic, diagnostic, as well as scientific significance. Cpg- 1 was isolated from the hippocampus dentate gyrus known to be related to various memory functions of the brain, and the cpg-1 product of the invention, may thus, for example, have potential uses in the treatment of various dementias, e.g. Alzheimer's disease, and for mediation or prevention of epileptic seizures and glutamate excitoxicity. The cpg-1 DNA sequence of the invention is useful for producing, by means of genetic engineering, the cpg-1 product. For this purpose, suitable expression vectors expressing the cpg-1 DNA and other sequences, e.g. for promoting expression in prokaryotic or eukaryotic cells, are prepared by methods known in the art. Host cells, including bacterial, insect and mammalian cells arc transfected with such recombinant vectors and used for production of the cpg-1 product. The cpg-1 DNA may also have uses per se, e.g. as a marker for identifying various normal or disease related phenomenas in cells or tissue culture, in histological samples, etc. The detection and isolation of DNA sequences encoding cpg- 1 was facilitated by the use of the method used herein which represents a considerable advance over conventional cDNA library screening procedures which are limited in their sensitivity. The invention will now be illustrated further by the following examples and annexed drawings. EXAMPLE I - Experimental procedures DG RNA Male rats (Wistar of The Wcizmann Institute), 8-10 wks old (230-300 gr body weight) were injected intrapcritoncally (IP) with kainate I D ( 8mg/kg body weight; stock solution 5mg/ml in phosphate saline buffer). Six hours later the animals were sacrificed, the dentate gyrus (DG) dissected out and stored in liquid N,. DG from about 100 animals were pooled and RNA prepared by a modification of the guanidinium thiocyanatc (GTC) method (Chomczynski and Sacchi, 1987): after lysis in GTC, 2 phenol and 15 one chloroform extractions were performed and the RNA was precipitated and resuspended in H 2 O. For subtraction, library construction, and cDNA probing, the RNA was treated with DNasc (Promcga, RNasc free) to eliminate contaminating genomic DNA. Poly (A)+ RNA was selected using oligo-(dT)-ccllulose columns (Clontcch). Poly (A)+ RNA from control 0 non-active DG or from other tissues was prepared by the same methods. Subtracted activated-DG cDNA library 1. First strand cDNA - First strand cDNA from activatcd-DG poly 5 (A)+ RNA was synthesized in two 50 μl reactions each containing 5 ug RNA, 30 μl RT buffer (Gublcr and Hoffman, 1983), 1 μl RNasc (4u/μl, Promega), 1 μg oligo-(dT)-Xbal primer adapter (Promega), 30 μCi , P- cxdCTP (3,000 Ci/mmolc, Amcrsham) and 400 u MLV cloned reverse transcriptase (BRL). After 60 min at 37°C, the RNA was hydrolyzcd for 20 mm at 68°C bv adding 10 ul NaOH ( I N), 2 ul rDT A (0 5M ) and H,O up lo 100 ul, removed to ice and then neutralized with 10 ul 1 M HC1 5 ug transfer RNA was added and the reaction was spun through a Scphadcx G- X) spin-column Tne recovery of cDNA was determined bv comparing radioactivity in the column cluatc to that in the applied sample Typicallv, l rom 10 ug RNA, 1 5 ug first strand cDNA was recovered at this stage The two cDNA samples were pooled, ethanol precipitated with N Acctatc and resuspended to 10 ng/μl in H ^ O 2. Subtraction - The cDNA was mixed with equal volume of total rat brain poly (A)+ RNA (l μg/μl) coupled to biotin (by 2 rounds of photo- biotinylation, Clontcch as described [Sivc and St John. 1988]) and ethanol precipitated with NH^cctate After rcsuspcnsion (to 100 ng/ul cDNA, 10 g/ul RNA) in formamidc buffer (40% formamidc, 50mM Hcpes pH 7 6, 0 5 M NaCl, 2mM EDTA) the solution was divided into glass capillaries (25 μl each) that were scaled and incubated 3 mm at 68°C and then 2 days at 52°C The capillaries were broken open and each added to 180 μl buffer (Hcpes pH 7 6 50 mM, NaCl 0 5 M, EDTA 2mM) Streptavidin (Vector) was added 1 μg/cach μg of biotinvlatcd RNA, mixed and incubated 10 mm at room temperature Two phenol/chloroform ( 1 1 , vol vol) and one chloroform extractions then followed The recovered aqueous phase typically contained 1 -20% of the cDNA taken for subtraction The cDNA was ethanol precipitated and resuspended in 16 ul H-,0 3. Second strand synthesis - 1 ul dATP ( 10 mM), 2 ul Mg 2+ buffer (for TdT, IBI), were added to the cDNA After 3 mm at 100°C and cooling on ice, terminal deoxynucleotidyl transferase ( 17 u, Kodak) was added for 2 hrs at 37°C Two μg olιgo-(dT)-Xbal primer adapter was added and the mixture incubated 5 mm at 60°C Second strand synthesis was in 50 ul volume containing 90 mM Hcpes buffer pH 6.6, MgCl, 10 mM, all 4 dcoxynucleotidc triphosphatcs 0.5 mM each, DTT 10 mM and 10 u Klcnow ( Boehringcr, sequencing grade). Af ter 6 hrs at room temperature another aliquot of enzyme was added for 3 additional hrs. The reaction was stopped 5 with phenol and chloroform extractions, 5 μg transfer RNA was added and ihc cDNA was ethanol precipitated with NH 4 Acctatc. 4. Cloning - The double strand cDNA was resuspended in 9.6 μl H 2 O and digested for 5 hrs at 37°C with 10 u of the restriction enzyme Xba 1 I D (Boehringer), after which it was loaded on a thin 1 % agarose gel, next to DNA size markers. A gel-slice containing cDNA molecules >550 bp in size was excised, the cDNA extracted using QIAEX (Qiagcn) and recovery (about 10% of subtracted cDNA) determined by radioactive counting. This cDNA was ligatcd into λZAP (Stratagenc) arms that were previously 1 digested with Xbal and treated with Calf Intestinal Phosphatasc (Boehringcr). Ligations were conducted with 1 μg phage arms and various concentrations of cDNA (3-20 ng). The ligations were packaged (Gigapack. Stratagene) and phage titer determined. 0 Screening the subtracted activatcd-DG library The library was plated at low density so that individual placquc could be picked separately and used for pBluescript plasmid 'rescue' (Short et al.. 1988). Two to 4 μg plasmid miniprep DNA was digested with Xbal and Southern blotted onto Hybond N+ (Amcrsham) filters (Sambrook et al. , 5 1989). The filters were hybridized to kainate-activatcd vs. control non- activated DG first strand cDNA probes. 100 ng first strand cDNA (as above) was labelled to very high specific radioactivity using 12 ul random primers (Boehringer, 90 A 2 «, u/ml), 2 mCi 2 P- dCTP (3,000 Ci/mmolc, Amcrsham), 0.6 mM dcoxynuclcotidcs (except dCTP) and 40 ul Klenow - --. - ( Boehringcr, 2u/μl) in 800 μl of the buffer used for second strand cDNA synthesis. Incubation was (in 2x400 μl aliquot) overnight at room temperature; such a reaction resulted in a probe of 1.2xl() cpm (Ccrcnkoff). After more than 6 hrs prehybridization, blot hybridization was performed for 48 hrs in a shaking 42°C water bath in 50% formamidc, 5X SSCPE (Sambrook et al., 1989), 1 X Denhardt's, 0.5% SDS, 0.5 mg/ml herring sperm carrier DNA and the cDNA probe at a concentration of l() h cpm/ml hybridization solution. The blots were washed with 0.1XSSC, 0.2% SDS, 3 times for 1 hr at 68°C. Northern blots were similarly hybridized and washed, except that individual cDNA probes were separated from plasmid DNA on agarosc gels and labelled with random primers (Sambrook et al. , 1989). Hybridization was for 16-24 hrs and probe concentration was l-3x l0 ϋ cpm/ml. MK-801 treatment Rats were injected I. P. with 5 mg/kg body weight of the NMDA receptor antagonist MK-801 (Wong et ai , 1986; RBI, Natick, MA; 5 mg ml in 25% ethanol), 30 min before the kainate injection. In some experiments, MK-801 injection was repeated 2 hrs after the kainate injection. Full-length cDNA cloning To obtain clones spanning the full-length of the cpg-1 transcript, 2 additional cDNA libraries were constructed from activated DG RNA in a fashion similar to that described above. One library was made, in addition to the o!igo-(dT)-Xbal. with 2 internal primers derived from sequences at the 5' side of the original clone 406 and it was selected to include cDNAs larger than 2 kb. The second library was made in the same way only that Oligo-(dT)-Notl primer adapters (Promega) were used instead of the Xbal primers. This measure was taken in order to span artificial termination of clones due to internal Xbal site(s). The strong specificity of cpg- 1 to activated DG was also reflected by an initial screen of a postnatal rat brain library ( P7) for cpg- 1 that resulted in only 1 positive clone/500,000 rccombinants. Screening the new activated and size-selected DG library with clone 406 resulted in 179 positive clones. These were further analyzed with more 5' probes until clones that span the entire sequence were isolated and subjected to DNA sequence analysis on both strands using scquenasc (USB). Restriction mapping of a large number of clones indicated that the only alternative splicing events of cpg-1 transcripts occur in the 5' untrans¬ lated region. EXAMPLE 2 - Isolation of cpg-1 CPG induction The dentate gyrus (DG) of the rat hippocampus was stimulated by injecting kainate into the peritoneum of rats, a treatment known to produce electrical stimulation that induces seizures and LTP-likc potentiations. This treatment results in higher excitability and toxicity than that observed with tetanic stimulations that induce LTP. It is therefore likely to induce, not only 'LTP-inducible' genes, but also genes that mediate more severe and perhaps longer-lasting potentiations such as seizures and kindling. To determine the kinetics and concentrations of kainate needed for optimal CPG induction, the behavior of Procnkcphalin (PE), a gene known to be induced in the DG by electrical activations producing LTP (Morris et al. , 1988) or seizures (Gall, 1988), was followed. PE is a suitable marker for CPGs, being a 'second wave' gene whose transcription is activated by previously induced IEGs (Sonncnberg et al., 1 89). Maximal PE induction in the DG occurred when animals were injected with 8 mg/kg (body weight) of kainate and sacrificed 6 hrs after injection (not shown) CPG cDNA cloning 5 CPG cDNAs were operationally defined as complementary to transcripts that arc induced in the DG 6 hrs after kainate treatment To clone these, a subtracted cDNA library was constructed from DG of kainatc-injcctcd rats (sec Fig 1 for outline of procedure). For subtraction, poly (A)+ RNA from total rat brain (rather than from non-activated DG) I D was used, to avoid subtraction of cDNAs that arc present in the DG but become further induced by kainate treatment The cDNA library was differentially screened for kainatc-induccd cDNAs by comparing hybridiza¬ tion signals obtained with a cDNA probe made from poly (A)+ RNA of kainatc-activatcd DG to those of a probe made from non-activated DG 15 (Fig. 1 ) Limited sensitivity of the conventional library screening procedure prevents detection of most of the cDNA clones of the library. To overcome this problem a novel screening procedure was employed, shown schematically in Fig. 2. The library was screened clone by clone: individual 20 plaques of a primary (unamplified) library were picked and "rescued" as (pBluescπpt) plasmid from the λZAP vector, using M 13 as helper phage (Fig. 2). The plasmids were digested with the restriction enzyme Xbal (the cloning site of the cDNA library) to release the cDNA insert Duplicate Southern blots, wherein each lane contains DNA of an individual clone, "> were prepared and differentially screened for kainate induction (Fig. 2). One blot was hybridized to the activated DG cDNA probe (left blot in the scheme) and its duplicate to the non-activated probe (right blot) Clones appearing stronger on the first as compared to the latter blot were considered potential CPGs. (The sensitivity of this procedure allowed detection of about 80% of the clones that were screened). All cDNAs showing activation by kainate in 2 independent screens (made with 2 independent sets of DG cDNA probes) were scquenccd from both ends (-200-300 bp) and subjected to a search for homologics in the DNA sequence data bases (Pearson and Lipman, 1 88). To verify that cDNAs showing kainate activation in the Southern screen represent kainatc-induciblc RNAs in the DG, the clones were hybridized to Northern blots containing poly (A)+ RNA from whole rat brain (T), non-activated DG (D), and kainate-activated DG (D+) (not shown). To date, 1000 clones from the subtracted, kainate-activated, DG library were screened in this manner. Fifty-two (-5%) clones were found to be of CPGs (according to the definition above). One third of them were of known genes and the rest appeared to be novel. EXAMPLE 3 - cpg-1 - a novel dentate gyrus-specific CPG Tissue distribution of cpg-1 A novel CPG that is transcribed from a single copy gene designated here cpg-1 , was further analyzed. This clone was intriguing because of its high specificity to the activated DG. Fig. 3 shows the Northern blotting results evidencing the specificity of cpg- 1 gene expression to the activated DG. A Northern blot containing 5 μg poly (A)+ RNA from total rat brain of untreated (B), or kainatc-trcatcd animals (B+), control untreated DGs (D), kainate-activated DGs (D+), kidney (K), heart (H), liver (Li), spleen (S), lung (Lu), and tcstis (Ts) was probed with cpg-1 cDNA and subsequently with a G3PD probe (lower panel). The glyceraldehydc-3- phosphatc dehydrogenase (G3P.D) gene was expressed in all tissues tested. The G3PD is used to normalize RNA levels. As can be seen from the results in Fig. 3, cpg-1 RNA was absent from all other tissues tested, including untreated or kainate-activated adult brain. It also exhibited strict dependence on NMDA receptor activation as described below by in situ hybridization. 5 In situ hybridization The glutamatc receptor of the NMDA type is, more than any other known protein, associated with induction of plasticity changes. To determine whether induction of cpg-1 is dependent on NMDA receptor I D activation, probes were prepared from rat brain sections for in situ hybridizations. Untreated rats (left sections in Fig. 4), rats treated with kainate (middle sections) or rats treated with kainate and the NMDA receptor blockcr MK-801 (right sections) were sacrificed 6 hrs after injection and the brains sectioned and processed for in situ hybridizations. 15 The brains were frozen and coronal cryostat sections ( 15 μm) were made through the hippocampus and processed. Sections were probed with 2 P- labelled DNA of cpg-1. according to known procedures. As seen in Fig. 4, induction of the cpg-1 gene was completely blocked by MK-801. 0 Sequence of cpg- 1 cDN A The cpg-1 transcript appeared to be about 4.5 kb RNA on Northern blots. Characterization of a large number of cpg-1 cDNA clones showed that 293 nucleotides of its most 5' end arc alternatively spliced in at least 4 ways into the rest of the mRNA (not shown). The DNA sequence 5 of cDNAs spanning the entire major (4,207 bp) (Fig. 5) and a few minor variants of cpg-1 transcripts were determined. The major cpg-1 cDNA contains a very small open reading frame flanked by very long untranslated sequences on both sides (839bp on the 5' end and 3086 on the 3' end; not shown). The sequence oi ~ the open reading frame and the small protein predicted to be encoded by it arc shown in Fig. 6. EXAMPLE 4 - Recombinant cpg- 1 protein expression and purifica- 5 tion to homogeneity The small open reading frame found in all cDNAs of cpg- 1 that were cloned is presented in the single-letter amino acid code (Fig. 6). The 2 pairs of basic amino acids found in the predicted protein arc I D underlined. The repeated motif (PSVI[R/H ]P) is underlined by asterisks. In addition, (as can be seen in Fig. 7), the predicted protein contains two cysteine residues at positions 73 and 83 (Fig. 6). The cysteine at position 73 is preceded by three amino acids (ETK) at positions 70-72. A comparison of the predicted amino acid sequence of the cpg- 1 product to 15 the amino acid sequence of three other known neurotrophins is shown in Fig. 7 (NGF -nerve growth factor; NT3 - ncurotrophin-3; BDNF - brain derived neurotrophic factor) reveals that the cysteine residues present in cpg-1 at positions 73 and 83 as well as the three amino acids (ETK) present at positions 70-72 arc also found in proteins of the ncurotrophin family. 0 Thus, the coding sequence predicts a small protein with features suggesting it may be a novel neuropeptide or neurotrophic factor or a precursor of one of them. There arc other small open reading frames that cither do not contain an initiator mcthioninc or arc downstream to the one presented here. Whether they arc used in the context of other transcripts is not yet clear. 5 Over 30 independent clones were mapped and used for sequencing; none of them exhibited alternative splicing events that affect the predicted open reading frame. The cpg- 1 product was obtained by expression in E.coli cells. PCR primers flanking the coding region of cpg- 1 were used to produce D copies of a 380 base pair fragment from the cpg- 1 cDNA clone. The 5' primer incorporated single BamHI and Ndcl restriction sites The 3 pπmcr incorporated a single EcoRl site T he PCR fragment was cloned into Bluescript SK plasmid using the BamHI and EcoRI sites, and subsequently subcloned into the bacterial expression vector pCTM 1 56 (Amgcn) using the Ndcl and EcoRI sites This clone (pCcpg- 1 ) was scqucnccd to confirm proper insertion and conservation of the reading frame To isolate cpg- 1 protein, 2 L of L c oli cells containing pCcpg-1 were grown 6- 10 hours at 30°C, then induced overnight at 42°C Abundant protein expression resulted m the formation of inclusion bodies, visible at 1000X under light microscope Cell lvsis and inclusion bodies isolation were performed using the protocol of Maniaiis (Maniatis, T , et al . Molecular Cloning) Isolated inclusion bodies were resuspended in about 5 mis of water. Using the Bradford assay the protein concentration was determined to be 16 mgs/ml. SDS polyacrylamide gels of this sample, when Coomassic stained, showed a strong band, comprising about 50% of the protein present, around 11 kDa (not shown) Subsequently purification of the protein was performed using size exclusion and ion exchange resins A 250 mi size exclusion column (Pharmacia 200HR rcsin) run under denaturing conditions ( 1 % SDS) was used to purify cpg- 1 product away from higher molecular weight bacterial contaminants The column was run at a flow rate of 0 67 ml/min Additional separation was made using cation exchange rcsin (Pharmacia SP Scpharose Fast Flow) The cpg- 1 protein fractions taken from the size exclusion column in 1 % SDS were dialyzcd to 8 M urea A 25 mL cation exchange column was used with a flow rate of 2 5 mls/min The sample was run in 8 M urea, 50 mM Tricinc, pH 8 1 solution A salt gradient ranging from 0 to 0.5 M NaCl was used to clutc proteins from the column cpg-1 product clutcd at around 0 3 M, well separated from other proteins Coomassic stained gels of cpg- 1 product containing fractions showed no contaminating bands. The collected cpg- 1 product fraction volume was 12 mis. with a protein concentration of 0.34 mg/ml yielding 4. 1 mgs of >99% pure cpg- 1 product.

Claims: CLAIMS: A DNA molecule selected from the group consisting of (i) a DNA molecule comprising the following coding nucleic acid sequence 1 ATGGCCAAGAAGTCACATCCTTACCTACAC 31 TGGTATAAGAACG AAAACGGCCCATCAGTA 61 ATCAGGCCTGGAGGGAGGAGAGGTTCAGGG 9i AGCCCΓAGCΓCΓACCGCTGCTCTCTTTGTC 121 TCTGTAGATGCAAGCAGAGGGCGAACATTA 151 CCTTCAGTTATACACCCCTTGGTGACCAAA 181 TCTAAAGGTCACACAG ATGGTCCTGGTG AA 211 ACGAAATGCTCATTAAACCAAACCCCAAGC 241 CACGTTTGTGGAAAGGAACTGTTAGGGACA 271 AGGGGAGGGGGA, (ii) a DNA molecule having the nucleic acid sequence of (i) in which one or more codons has been added, replaced or deleted in a manner that the polypeptide encoded by said sequence essentially retains the same biological properties as the polypeptide encoded by an unaltered DNA sequence, (iii) a DNA molecule encoding a polypeptide having an ammo acid sequence of a polypeptide encoded by the DNA molecule of (i) or (ii) but which differs from the DNA sequences of a polypeptide encoded by the DNA molecule of (i) or (ii) in view of the degenerative nature of the genetic code, (iv) a DNA molecule having a coding nucleotide sequence, which is homologous to the DNA molecule of (i), (ii) or (in), which is derived from a mammal other than rats and which encodes a polypeptide having a similar biological activity to that encoded by the sequences of (i), (ii) or (iii); (v) a fragment of the coding sequence of (i) - (iv) which encodes a pepti e which essentially retains the biological properties of the polypeptide encoded by unfragmented DNA molecule; (vi) a DNA molecule comprising the coding DNA sequence of fragments of (i) - (v) and additional DNA sequences in the 3' and 5' ends; and (vii) a recombinant DNA molecule comprising an encoding sequence according to any of (i) - (v) above. 2. A DNA molecule according to Claim l(i) herein designated cpg-1. 3. A DNA molecule according to Claim 1 or 2, encoding a neural polypeptide, a neurotrophic factor, a precursor of one of them or a precursor of a smaller ncuropeptidc. 4. A polypeptide selected from the group consisting of: (i) a polypeptide having the following amino acid sequence: 1 M A K K S H P Y L H W Y K N E N G P S V 21 I R P G R R G S G S P S S T A A L F V 41 S V D A S R G R T L P S V I H P L V T K 61 S K G H T D G P G E T K C S L N O T P S 81 H V C G K E L L G T R G G G (ii) the polypeptide of (i) in which one or more amino acid residue has been added, deleted, replaced or chemically modified without substantially affecting the biological activity of the polypeptide; (iii) a biologically active fragment of the polypeptide of (i) or (ii); and (iv) an homologous polypeptide to that of (i), (ii) or (iii) derived from another mammal and which has a similar biological 5 activity to that of (i), (ii) or (iii). 5. A polypeptide according to Claim 4(i) herein designated "cpg-1 product". 6. A polypeptide according to Claim 4 or 5 being a neural polypeptide, a neurotrophic factor, a precursor of one of them or a precursor I D of a smaller neuropeptide.

Inventors: CITRI YOAV, CITRI, YOAV
Applicants: CITRI YOAV, RYCUS AVIGAIL, YEDA RES & DEV, CITRI, YOAV, RYCUS, AVIGAIL, YEDA RESEARCH AND DEVELOPMENT CO., LTD.
Application year: 1993
Main classification: C12N15
IPC codes: C07K14, C12N1, C12N15, C12P21, C12R1