Abstract:
The present invention relates to isolating, sequencing and cloning of the 5′-flanking region of neuropeptide FF (NPFF) promoter. The characterized neuropeptide FF (NPFF) promoter is useful in gene therapy and in DNA analyses, and in production of gene-modified animals.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of application Ser. No. 09/365,755, filed Aug. 3, 1999, now abandoned, which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to sequencing and cloning of the 5′-flanking region of neuropeptide FF (NPFF) promoter. A neuropeptide FF (NPFF) promoter region for mouse, rat and human has been cloned and sequenced. The characterized neuropeptide FF (NPFF) promoter is useful in screening and treating for genetic diseases associated with the promoter area of the NPFF gene by modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area. The promoter can also be used as a marker for its locus in the corresponding chromosome. Thus, the characterized promoter is of considerable diagnostic value and can be used in gene therapy and in DNA analyses. Further, the promoter can be used for developing genetically modified animals. 
     BACKGROUND OF THE INVENTION 
     Neuropeptide FF was originally identified as a mammalian counterpart of the molluscan cardioactive peptide FMRF-amide (Yang et al., 1985), found in the superficial dorsal horn of the spinal cord, hypothalamus, medulla and pituitary gland (Kivipelto et al. 1989). The findings that the peptide is present in the hypothalamo-pituitary system, decreases during salt-loading and is deficient in the pituitary gland of vasopressin-deficient Brattleboro rats, implicate NPFF involvement in hypothalamic regulation of pituitary functions (Majane and Yang, 1991; Majane and Yang, 1990; Majane et al., 1993). Peripherally administered NPFF raises blood pressure in rats, an effect mediated by both peripheral and central mechanisms (Allard et al., 1995; Laguzi et al., 1996). NPFF has also been implicated in sensory systems, most notably pain and morphine analgesia (Yang et al., 1985). Intracerebroventricular NPFF has been reported to induce a vigorous abstinence syndrome in morphine-tolerant rats. NPFF has attenuated the antinociceptive effects of morphine when administered in the third ventricle, whereas intrathecal NPFF produces long-lasting antinociception (Gouarderes et al., 1993). 
     The NPFF gene is located in the human chromosome locus 12q13 (Burke et al. 1998), which is known to be associated with a severe condition referred to as Allgrove syndrome (triple-A syndrome). The current NPFF promoter area is an evident region where mutations responsible for triple A syndrome are located. It serves as a useful marker for the appropriate area of chromosome 12, and has diagnostic and therapeutical value in treatment of a triple-A syndrome. 
     SUMMARY OF THE INVENTION 
     Accordingly, it is an object of this invention to clone and sequence the 5′-flanking region of the NPFF promoter in human, rat and mouse. 
     The promoter region cloned and characterized here plays an essential role in etiology and/or pathogenesis of CNS disorders involving NPFF, including those associated with deficient regulation of autonomic function, pain conditions, and hormonal dysfunction. Accordingly, another object of the invention is to provide potential methods of screening and treating for genetic diseases associated with the promoter area of the NPFF gene by modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area. The sequence can also be used as a marker for its locus in the corresponding chromosome. Thus, the characterized promoter is of considerable diagnostic value and can be used in gene therapy and in DNA analyses. 
     Another object of the invention is to provide genetically modified animals. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1A is a schematic representation of the gene structure and cloning strategy for the mouse NPFF gene. Cutting sites for restriction enzymes are indicated by the following abbreviations: B=BamHI, H=HindIII, RI=EcoRI, X=XbaI and Xh=XhoI. The cloning strategy is marked by numbers 1-4. Bar 1 indicates the 4.3 kb EcoRI DNA fragment, bar 2 indicates the 4.5 kb BamHI-EcoRI DNA fragment, bar 3 indicates the 5.5 kb BamHI DNA fragment and bar 4 indicates the 7 kb XbaI DNA fragment. 
     FIG. 1B shows the nucleotide sequence of the 5′-flanking region of the mouse NPFF gene (SEQ ID NO:6). Sequence is numbered relative to the translational start site (indicated by a bent arrow). The consensus TATA box is shaded. The putative regulatory elements are indicated by arrows underneath the sequences. The AC dinucleotide repeat is indicated by a thick bar under the sequence. 
     FIG  1 C shows: Mouse NPFF promoter sequences. Nucleotide sequence from −9840 bp to −1 bp relative to the translational start site. cDNA sequence is underlined (SEQ ID NO: 1). 
     FIG  1 D shows: Mouse genomic NPFF sequences from coding region and the 3′ end of the gene. Nucleotide sequence from +1 relative to the translational start site. cDNA sequence is underlined (SEQ ID: 2). 
     FIG. 2A shows a map of mouse NPFF promoter deletions fused to luciferase reporter gene. The numbers indicate the nucleotide positions of the 5′-untranslated region with the first nucleotide of the codon for initiation of translation as +1. The promoter fragments were subcloned in NheI-SmaI site of the promotorless luciferase vector pGL3 basic. The MNF5′1.8-LUC reporter construct contains a dinucleotide repeat. Consensus binding sites for transcription factors are indicated by gray-scale colours. 
     FIG. 2B shows: Basal activity of mouse NPFF promoter to drive luciferase expression. A549 cells were transiently transfected for 48 hours with 5′-promoter deletion constructs fused with a promoterless luciferase vector (pGL3 basic). Transfections were done in duplicate and repeated at least three times. The activity of the promoter was seen to vary with the highest activity in the shortest construct MNF5′0.3-LUC. The activity was seen to drop to almost unmeasurable levels in the MNF5′1.5-LUC construct. The activity was still minimal in the following MNF5′1.8-LUC construct. 
     FIGS. 2C and 2D show: Activity analysis of the two shortest mouse NPFF promoter constructs MNF5′0.3-LUC and MNF5′0.6-LUC. A549 cells were transiently transfected for 24 hours whereafter the transfectants were stimulated for 16 hours with 10 nM TPA, 10 nM TPA and 1μM ionomycin (IM), 15 μg/ml LPS or 10 μM forskolin. Unstimulated transfectants serve as control. Activity was seen to increase with ˜30% in TPA+IM stimulated MNF5′0.3-LUC transfected cells. A slight increase in activity in the same transfectants was also seen with LPS stimulation. In cells transfected with MNF5′0.6-LUC a ˜50% increase in activity was seen after TPA+IM stimulation. LPS stimulated transfectants also gave an at least ˜40% increase in activity. 
     FIG. 3 shows a comparison between the first 400 bp of the NPFF promoter from mouse (SEQ ID NO: 7), rat (SEQ ID NO: 8) and human (SEQ ID NO: 9) and a consensus sequence (SEQ ID NO: 10). Sequence similarity is about 90% between mouse and rat and about 70% between rat and human. Conserved consensus binding sites for transcription factors are marked under the sequence. The translational start site is marked by a bent arrow and the TATA-box is marked by a box. M=mouse, R=rat and H=human. 
     FIG. 4 shows: Nucleotide sequence of the human NPFF promoter. Nucleotide sequence from −2480 bp to −1 bp relative to the translational start site (SEQ ID NO: 3). 
     FIG. 5 shows: A new, previously unidentified open-reading frame was found in the mouse promoter residing from −4236 to −3841 relative to the translational start site. Amino acids are marked under the sequence (SEQ ID NO: 4). This protein (SEQ ID NO:5) may have multiple functions and it may give rise to a previously unidentified bioactive peptides. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     We report here the cloning of the 5′-flanking region of the NPFF gene from mouse, rat and human. Totally 9.8 kb, 1.5 kb and 1.3 kb of the NPFF promoter from mouse, rat and human was cloned and sequenced. Comparisons between the promoter region from all species showed a high sequence similarity. Such a high sequence homology and well-conserved structure could indicate an important physiological function and a need for a similar, tightly regulated transcription of the gene. We have previously shown that the NPFF gene is expressed in specific regions in the brain and in the spinal cord and is induced upon inflammatory stimulus (Vilim et al., 1999). In agreement with this several inflammation related transcription factor consensus sites were found (FIG.  1 B), amongst them e.g. NFκB, which is considered as an immediate early mediator of immune and inflammatory responses (Lenardo &amp; Baltimore, 1989). Also several consensus sites for the nuclear factor of activated T-cells (NFAT) were found (for review see Kel et al., 1999). Transcription factors belonging to this growing family play key roles in the regulation of cytokine and other genes during immune response. An interesting finding was also the consensus site in mouse and rat for heat shock factor 1 (HSF1). HSF1 is activated in cells exposed to elevated temperatures and other environmental stress conditions (Sarge et al., 1993). We also focused on the potential involvement of AP1, STAT1 and CREB. 
     By using transiently transfected cells with deletional series of mouse NPFF promoter fused with firefly luciferase cDNA we could show that the basal activity of the promoter changes with increasing promoter size (FIG.  2 B). This indicates that the promoter is tightly regulated is affected by several transcriptional factors and apparently has a complex transcriptional control. To study the possible involvement of some transcription factors our attempt was to a) activate transcription factors by protein kinase C (PKC) b) activate NFATp by PKC and Ca 2+ -levels with TPA and ionomycin (Verweij et al., 1990), c) activate transcription factors by the bacterial endotoxin LPS and d) activate CREB by increasing intracellular cAMP values with forskolin. TPA and ionomycin increased promoter activity in MNF5′0.3-LUC transiently transfected cells (FIG.  2 C). In this promoter region a consensus binding site for NFAT was also detected by computational analysis (FIG.  2 A). It seems likely that this consensus binding site is active and that the transcription factor NFAT contributes to the transcriptional regulation of the NPFF gene. When MNF5′0.6-LUC was transiently transfected in cells and transfectants were stimulated both TPA and ionomycin and LPS stimulated cells showed higher promoter activity compared to control (FIG.  2 D). This still confirms the involvement of NFATp and also possibly confirms the function of a NFκB or a STAT1 site residing in this construct (FIG.  2 A). An interesting and contradictory finding was that although LPS seems to increase promoter activity, TPA does not. It seems likely that the transcriptional regulation is dependent on a synergistic mechanism involving several transcriptional factors affecting the gene expression. 
     A possible silencer element was also found in the proximal mouse promoter residing between 1.5 kb to 1.8 kb. Promoter activity dropped dramatically in cells transiently transfected with MNF5′1.5-LUC if compared with cells transfected with the construct MNF5′1.3-LUC, which is only about 200 bp shorter than MNF5′1.5-LUC. A possible silencer could accordingly reside between 1.3 and 1.5 kb of the mouse NPFF promoter. This finding is also confirmed by that cells transiently transfected with the longest construct. MNF5′1.8-LUC, also exhibits a similarly low promoter activity. It would be interesting to see if a longer promoter construct than the ones used in this study could again raise the promoter activity. Since we have cloned and sequenced also the corresponding region in the rat NPFF promoter and the sequence similarity was about 90%, it is possible that this region could be a functional, cell-specific silencer. It still remains unknown if the human NPFF promoter contains similar sequence. The possibility also remains that the AC-dinucleotide repeat in the MNF5′1.8-LUC construct adds additional regulatory effects. Additional roles of the AC-repeat might also be possible since an almost complementary GT-repeat was found approx. 6.5 kb upstream from the TATA-box in the mouse promoter. These partially complementary elements might interact to form complex secondary structures that may in turn contribute to the regulation of this promoter. Similar AC-repeat structures have been characterized in some other brain specific promoters such as PAX-6 gene (Okladnova et al., 1998) and the GLYT-1 gene (Borowsky &amp; Hoffman, 1998). Proteins bound to repeat elements has been reported (Xu &amp; Goodridge, 1998). Thus it is possible to identify this/these proteins bound to the AC-repeat which may play a role in control of the function of the AC-repeat. These proteins might control the accessability of the AC-segment to the complementary GT-repeat. 
     To study the endogenous NPFF promoter activity in vitro, we tried to affect the transcription of the gene by stimulating a cell-line, A549, which has been found to endogenously express NPFF mRNA. The results are in accordance with the transfection results; the expression of NPFF increased with LPS stimulation, which also was seen in the transfection results. This additionally emphasises the role of NF κ B in the transcriptional regulation of the NPFF gene. The involvement of NF κ B was also confirmed by decreased expression in cells stimulated first with TPA and then with the known NF κ B inhibitor PGA 1 . 
     As a conclusion, current data shows that the transcriptional regulation of the NPFF gene is highly complex and probably involves several transcription factors in synergy in control of the gene expression. 
     It is obvious that key elements involved in regulation of the NPFF gene are included in the characterized promoter, which has not been reported earlier. NPFF is involved in autonomic regulation including blood pressure and heart functions, analgesia and morphine tolerance, learning, and its expression is increased in the spinal cord after peripheral inflammation. Modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area may be a part of pathophysiology in disease conditions related to these conditions. 
     The NPFF gene is located in the human chromosome locus 12q13 (Burke et al. 1998), which is known to be associated with a severe condition referred to as Allgrove syndrome (triple-A syndrome) characterized by a triad of adrenocorticotropic hormone (ACTH), resistant adrenal insufficiency, achalasia and alacrima, hypoglycaemia and sensory impairment and autonomic neuropathy. The current NPFF promoter area is an evident region where mutations responsible for triple A syndrome are located. Thus, the characterized promoter serves as a useful marker for the appropriate area of chromosome 12. Also in other applications it is of considerable diagnostic value and can be used in gene therapy and in DNA analyses. 
     The invention will be further described with reference to the following non-limiting examples. 
     EXAMPLE 1 
     Cloning of the Mouse NPFF Promoter 
     Stratagene mouse genomic library was screened with mouse NPFF cDNA (Vilim et al., 1999). DNA from positive clones were isolated, digested with a panel of restriction enzymes and analysed by Southern blotting. A 4.3 kb EcoRI DNA fragment (see FIG. 1A) that hybridized with the mouse NPFF cDNA probe was subcloned in pBluescript KS+/− vector (Stratagene). The cloned 4.3 kb EcoRI fragment was sequenced by automated sequencer (ABI Prism Automated Fluorescence Sequencer) by using universal primers (T3, T7, Promega) and gene specific primers. Restriction enzyme map of the resulting sequence was computed by using the University of Wisconsin GCG Sequence Analysis Software Package. A 0.8 kb BamHI-EcoRI fragment at the 3′-end of the 4.3 kb EcoRI fragment was subsequently used to probe the Southern blot of NPFF lambda-DNA. This probe hybridized to a 4.4 kb fragment of BamHI-EcoRI digested NPFF lambda-DNA. The 4.4 kb BamHI-EcoRI fragment was thereafter subcloned in pBluescript KS+/− and sequenced as described previously. A 3.5 kb EcoRI-BamHI fragment located at the 5′ end of the 4.3 kb EcoRI fragment was then used to reprobe the NPFF Southern. This probe hybrized to a 5.5 kb BamHI fragment, which in turn, was subcloned in pBluescript KS+/− and sequenced. A 2.5 kb BamHI-XbaI fragment at the 5′-end of the 5.5 kb BamHI fragment was finally used to reprobe the NPFF Souther filter and it hybridized to a 7 kb XbaI fragment. The 7 kb XbaI fragment was subsequently subcloned in pBluescript KS+/− and sequenced. The resulting nucleotide sequences were assembled using the GCG Sequence Analysis Software Package. Total of 10.3 kb of the NPFF promoter was cloned and sequenced. The search for the consensus transcription factor recognition sites was performed by MatInspector Professional from Transfac Database at NCBI/NIH. 
     By using the strategies presented in examples 1 and 4 a 9.8 kb, 1.5 kb and 1.3 kb piece of the NPFF promoter region was cloned and sequenced from mouse, rat and human, respectively. An illustration of the cloning strategy of the mouse NPFF promoter is shown in FIG.  1 A. The homology between the proximal promoter based on sequence similarity was seen to be high between all species when doing comparisons based on computorial software (GCG, Wisconsin/CSC, see FIG.  1 B). Some of the consensus binding sites for transcription factors were seen to be conserved between all three species in the proximal promoter (FIG.  3 ). 
     EXAMPLE 2 
     Luciferase Reporter Constructs 
     To prepare 5′-deletion constructs of the proximal mouse promoter a 325 bp, 579 bp, 805 bp, 1085 bp, 1289 bp, 1527 bp and a 1861 bp fragment were amplified by PCR with the antisense primer 5′-TGG AGT CCA TGC TGC CAT-3′ (SEQ ID NO: 11) and the sense primers (cutting site for NheI is underlined) 5′-GT G CTA GC A ATC TGT TGA AGG ATT GG-3′ (SEQ ID NO: 12), 5′-GT G CTA GC A GTC TCC TAT CTC TCA CT-3′ (SEQ ID NO: 13), 5′-GT G CTA GC A GAC GGA ACT GGA AAA AT-3′ (SEQ ID NO: 14), 5′-GT G CTA GC T CTC CTA GCA AGT AAT TC-3′ (SEQ ID NO: 15), 5′-GT G CTA GC T ACA TAT GAC TGA GAG AT-3′ (SEQ ID NO: 16), 5′-GT G CTA GC A GCC TGG ATG CAT TGT AT-3′ (SEQ ID NO: 17) and 5′-GT G CTA GC A CAG AGT CTC AGG CTT AG-3′ (SEQ ID NO: 18), respectively. PCR reactions were performed with Pfu DNA polymerase (Promega) using an Eppendorf Mastercycler gradient machine with the following program: 1. 95° C. for 2 min, 2. 95° C. for 45 sek, 3. 53.6° C. for 30 sek, 4. 72° C. for 3 min with a 1 sek addition after each cycle, 5. Steps 2-4 for 35 cycles followed by a 72° C. 5 min final extension. The PCR products were cleaned from primers and nucleotides with QIAquick PCR purification kit (Qiagen) and subsequently cut with NheI. The cut products were run on a 1.2% agarose gel, excised from the gel and extracted by QIAquick agarose gel extraction kit. The PCR products were ligated in NheI-SmaI cut promoterless pGL3-basic vector (Promega) upstream of the firefly luciferase cDNA in sense orientation. The yielded constructs MNF5′0.3-LUC, MNF5′0.6-LUC, MNF5′0.8-LUC, MNF5′1.0-LUC, MNF5′1.3-LUC, MNF5′1.5-LUC and MNF5′1.8LUC (see FIG. 2A) were transformed in competent DH5α cells. 
     EXAMPLE 3 
     Transfection and Luciferase Assay 
     The functioning of the promoter in a cell was shown. 
     The human lung carcinoma A549 cell-line was kept in a humidified cell-incubator at 37° C. and with 5% CO 2 . The cell-line was grown in Dulbecco&#39;s modified eagle medium (Gibco) containing 10% fetal calf serum (Gibco), 1×Glutamax (Gibco), 50 μg/ml penicillin and 50 IU/ml streptomycin (Gibco) and was regularly passed. 16-24 hours before transfection cells were plated at a density of 1×10 5  cells in 1 ml medium in 12-well dishes (Nunc). Reporter constructs (1 μg) or promoterless pGL3-basic vector (1 μg, as negative control) were co-transfected with pSV-β-galactosidase vector (0.5 μg, Promega) by first mixing 3 μl of Fugene 6 (Roche) transfection reagent with 57 μl of serum-free medium, which was incubated at RT for 5 minutes. The mix was added to the DNA and incubated at RT for 15 minutes and then added to the cells. The transfections were done in duplicates and repeated at least three times. Cells were transiently transfected for a total time of 48 h and before collecting the transfectants some cells were left unstimulated or stimulated for 16 hours with TPA (10 nM), TPA (10 nM) and ionomycin (1 μM), lipopolysaccharide (LPS, 15 μg/ml) or forskolin (10 μM). Cells were collected and lysed with 140 μl of reporter lysis buffer (Promega) according to the manufacturer&#39;s manual. The luciferase assay was performed on 20 μl of cleared cell extract and 100 μl of luciferase assay reagent (Promega) using a Luminoscan luminometer (Labsystems). Transfection efficiencies were determined by using β-Galactosidase enzyme assay system (Promega) with 50 μl of cell extract. The luciferase acitivity of each transfection was expressed as luciferase activity/β-galactosidase activity. 
     The ability of a deletional series of the NPFF promoter to drive the transcription of firefly luciferase cDNA is presented in FIG.  2 B. The highest expression of luciferase was seen in the shortest construct (MNF5′0.3-LUC) with decreasing activity in the two following constructs (MNF5′0.6-LUC and MNF5′0.8-LUC, respectively). The luciferase expression started increasing again with the two following constructs (MNF5′1.0-LUC and MNF5′1.3-LUC, respectively) whereafter the expression decreased to barely measurable level with the last two constructs (MNF5′1.5-LUC and MNF5′1.8-LUC). The last construct also contained a 29×(AC) dinucleotide repeat. 
     To study the involvement of some known pro-inflammatory transcriptional factors the two shortest constructs (MNF5′0.3-LUC and MNF5′0.6-LUC) were transfected in cells and stimulated with a phorbol ester, a phorbol ester together with a Ca 2+ -ionophore, a bacteria endotoxin and a cAMP activator. These compounds should all affect several transcription factors, amongst them many known pro-inflammatory transcription factors eg. NF κ B, NFAT and CREB. The results are presented in FIGS. 2C and 2D. The results show an increase in luciferase activity of TPA and ionomycin stimulated MNF5′0.3-LUC transfected cells (FIGS. 2C and D) and an increase in luciferase activity in TPA and ionomycin and LPS stimulated MNF50.6′-LUC transfected cells. 
     EXAMPLE 4 
     Cloning of the 5′-flanking Region of the Human and Rat NPFF Gene Promoter 
     To clone the promoter region of the human and rat NPFF gene, a Genome Walker kit (Clontech, Palo Alto, Calif.) was used according to manufacturer&#39;s instructions. The NPFF specific human and rat antisense primers used for the primary PCR reaction was 5′-GCT GCC ACC ACC TAC CCT CCT AC-3′ (SEQ ID NO: 19) and 5′-CAC CCC AGC TCC CTG CCT CTT-3′ (SEQ ID NO: 20), respectively. The antisense primer for the nested human and rat PCR was 5′-GTG GAT CCA TCT AGA GCA GGC AAA TG-3′ (SEQ ID NO: 21) and 5′-CGT GGC CCC AGT TCC TCA GCA-3′ (SEQ ID NO: 22), respectively. The PCR reactions were performed using a MJ Research MiniCycler. The primary PCR reactions was performed with the following conditions: 94° C. for 25 sec and 72° C. for 4 min×7 cycles; 94° C. for 25 sec and 67° C. for 4 min×32 cycles, followed by a 67° C. 4 min final extension. The nested PCR reactions was conducted using a 50 times diluted primary PCR product as a template, the same reaction composition and cycle parameters, except that nested primers were used and that 20 thermocycles were performed instead of 32. Using this technique, we were able to identify a single major PCR product in one of the human libraries (PvuII) and two major PCR products in the rat libraries (DraI and PvuII) provided in the Genome Walker kit. The products were purified with the QIAquick PCR purification kit (Qiagen) and cloned into pGEM T-vector (Promega) and sequenced automatically in both directions (ABI Prism Automated Fluorescence Sequencer). Using this approach 1.3 kb of the human NPFF promoter and 1.5 kb of the rat NPFF promoter was cloned and sequenced. We still continued our cloning efforts conserning the human NPFF promoter and by using a second round of the same approach as above we were able to clone an additional 4 kb fragment of the human promoter. Sequencing is now in progress. A comparison between the NPFF promoters from all species is presented in FIG.  3 . The search for the consensus transcription factor recognition sites was performed by MatInspector and MatInspector Professional from Transfac Database at NCBI/NIH. Comparisonal studies to study homology between the NPFF promoters were performed using Seqweb software package available at CSC web site (www.csc.fi). 
     The human sequences have been presented in FIG.  4 . 
     EXAMPLE 5 
     Comparison of Proximal Promoter of NPFF Gene Between Mouse, Rat and Human 
     A comparison between the first 400 bp of the NPFF promoter from mouse, rat and human. Sequence similarity is about 90% between mouse and rat and about 70% between rat and human. Conserved consensus binding sites for transcription factors are marked under the sequence. The translational start site is marked by a bent arrow and the TATA-box is marked by a box (FIG.  3 ). M=mouse, R=rat and H=human. 
     The invention has been illustrated by examples and embodiments, but it may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the enclosed claims. 
     References 
     Allard, M., Labrouche, S., Nosjean, A. &amp; Laguzzi, R. (1995). Mechanisms underlying the cardiovascular responses to peripheral administration of NPFF in the rat.  J. Pharmacol. Exp. Ther.  274: 577-583. 
     Burke, J. F., Clarke, A. J. H., James, M. R. &amp; Perry, S. J. (1998). Physical mapping of the human NPFF gene and investigation of its candidacy as a disease gene locus.  Soc. Neurosci. Abstr.  24(2): 2046. 
     Gouarderes, C., Sutak, M., Zajac, J. M. &amp; Jhamandas, K. (1993). Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat.  Eur. J. Pharmacol.  237:73-81. 
     Kel, A., Kel-Margoulis, O., Babenko, V. &amp; Wingender, E. (1999). Recognition of NFATp/AP-1 composite elements within genes induced upon the activation of immune cells.  J. Mol. Biol.  288: 353-376. 
     Kivipelto, L., Majane, E. A., Yang, H. Y. &amp; Panula, P. (1989). Immunohistochemical distribution and partial characterization of FLFQPQRFamidelike peptides in the rat central nervous system of rats.  J. Comp. Neurol.  286: 269-287. 
     Laguzzi, R., Nosjean, A., Mazarguil, H. &amp; Allard, M. (1996). Cardiovascular effects induced by the stimulation of neuropeptide FF receptors in the drosal vagal complex: an autoradiographic and pharmacological study in the rat.  Brain Res.  711: 193-202. 
     Lenardo, M. J. &amp; Baltimore, D. (1989). NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control.  Cell  58(2): 227-229. 
     Majane E. A. &amp; Yang, H. Y. (1991). Mammalian FMRF-NH 2 -like peptide in rat pituitary: decrease by osmotic stimulus.  Peptides  12: 1303-1308. 
     Majane E. A., Zhu, J., Aarnisalo, A. A., Panula, P. &amp; Yang, H. Y. (1993). Origin of neurohypophyseal neuropeptide-FF (FLFQPQRF-NH 2 ).  Endocrinology  133: 1578-1584. 
     Okladnova, O., Syagailo, Y. V., Tranitz, M., Stöber, G., Rieder, P., Mössner, R. &amp; Lesch, K.-P. (1998). A promoter-associated polymorphic repeat modulates PAX-6 expression in human brain.  Biochem. Biophys. Res. Commun.  248: 402-405. 
     Sarge K. D., Murphy, S. P. &amp; Morimoto, R. I. (1993). Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress.  Mol. Cell Biol.  13(3): 1329-13407. 
     Verweij, C. L., Guidos, C. &amp; Crabtree, G. R. (1990). Cell type specificity and activation requirements for NF-AT-1 transcriptional activity determined by a new method using transgenic mice to assay transcriptional activity if an individual nuclear factor.  J. Biol. Chem.  265: 15788-15795. 
     Vilim, F. S., Aarnisalo, A. A., Nieminen, M.-L., Lintunen, M., Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P. &amp; Ziff, E. (1999). Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.  Mol. Pharmacol.  55: 804-811. 
     Yang, H. Y. T., Fratta, W., Majane, E. A. &amp; Costa, E. (1985). Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine.  Proc. Natl. Acad. Sci. USA  82: 7757-7761. 
     
       
         
           
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             1 
             9840 
             DNA 
             Mouse 
           
            1
tctagacaga taccaggtac caatgttaaa tcatgatctt acgttttgtg tcagaactta     60
ttttgcatgc cattcatgac tctggagagt tttctccaaa aggaaagatg agattaattt    120
tcttttgcat tctttgtaat tatgacaaag aagagtcctt tctgttcctc tgtcctattt    180
tctaaccact tgctctctac tgtttgccct ccttcctggc agaaagtccc aaggaaagct    240
cagagccaac gggttctcca gccccagtga ttcagcacag ttccgcatca gcccctagca    300
atggccttag tgttcgatct gcagctgaag ctgtggccac ctcggtcctc actcagatgg    360
ccagccaaag gacagaactg agcatgccca tacagtcaca tgtgatcatg accccacagt    420
cgcagtctgc gggcagatga tgcctcccat gatggagagg tccccagcca gagctcgccc    480
gcctgagcca agagagagat caacttaacc cctcctgtct gtcttggaat tgggcagaat    540
ggagagggag aaattgtgtg ttgtgagtat ggacagatgt ggggcttttc tctttagcca    600
catgatcatg tacgattaac acctgtacta ggcgcttctg gccccaaagc cacatagatc    660
atcccccaag ggtagggttt cttatgtacc tacagccaaa ggcctttcca tatggtctga    720
gcagtcatcc ctggccctga tgcatgtgtg cctgtctctt aacactaacc cttgcacttg    780
taggtttggg gtttctcagt gtcccctcct cccattgacc tgtggctgtt accttcttat    840
ttcttattag catgccactt cctgccagat ggagggaggt gagatttgat ggcgtgttac    900
ttgcttgtcc cacccctagc caccacatgc actcaggact ttgtctccca atagctgctt    960
atttgtctct ttcctccttc ctaaactgca gtgaaaacat tcacttgttg agaatgtaat   1020
acatttaatg tatgaggtag cagtgtttcc cacctcttcc atgtgcttta ttcctatctg   1080
ctaccaaaaa caaacacaaa caaaaaaagc aaaactctga gagtttgaat catttttcat   1140
ttccaaatct attggtacct catttcgtct cttgactttc cttagtctag tggtggggtc   1200
tttcctactt ccccactagg ccttgggaac ctcatcttgt ggccttattg ttagtggcaa   1260
tgaaaaagga gagagctgga gacctaactg ggcttccctg tctccttccc taccctccag   1320
ttctaatcat cagggcagac aggaacagtg taatttaaaa cttgttccat caggttactg   1380
gattaactga ttcttttatg ttttacaaga gtttactggc caaagtctac atattgtcat   1440
ctttggtcat ctgtgctcct gcctctccac tgtgcttccc tgtccacatc actgcctgtt   1500
ccactataac tactttagct cagacctcct agagttgtca gacagtagct gcttccattc   1560
tctgtccctc ctacagctct cagcctccct tcttttctaa ggtatgtttt gtacccacca   1620
gtgtccagca ctctctcaac aaccttttca tcttctcacc cagttcttag cctacccact   1680
ggttgtctct agtcctgaaa tttcgttgaa cttgtccatg gtacaggctc cagggctctg   1740
aagcatcatg gttgaggtca tggatgcttt tgaatataag actgagtgga gagaggaggt   1800
atacattttc ctatgtataa tctcagcaga gcactcagga tatggcctgt ctgtgagatt   1860
gctgtctgtg gctgttggct gtccttattt tatgggttac agagagaaaa tacacccctc   1920
ctcttcttca tcttcccttg gctctgagct atgcaggctc ttccagagcc agaggcctgt   1980
ggatagatag gtcagcttta atagctcttg agttgagaat ccttcccatt gtcctagaac   2040
caccttctgc catctgctca agccaagtcc tcttttctag ctccatctta agtatgcaga   2100
ataattgctt gcgtctttgt cttccaaaat tcactgtcgt ggaggaatgg aaatgtcgtg   2160
tagcttatat gattatttcc tcagagttta gactagtgag cccatcctgt gacatgtttg   2220
tatgttttat gtaaatttcc ctcctgctct ttagagtcaa tgctgaacag gccacaccca   2280
gtgaaaacta ggaactggtt ttatagtctt ctcccacagg gtcttaacaa aaacatcccc   2340
tgaggtgaca aggatagcaa atgccacagc agatggttga gggcaagcca ccatctccag   2400
gggtttcact tggccttaga aactcacagc catagtttga gctcaggact tctttagatg   2460
gctgcttcct aggatttttt tttcctgctt atgaattttg tttctttttt tttaattgtc   2520
ttgatttccc agtagcagcc ttacactaaa atatgactga gcttatagct tccaagggcc   2580
ccccttggct attttcttcc tccatcagtc aagtgtttaa ttcagtgtaa cctaccagtc   2640
tgtcctggtt gcatgtctag tatacgtgga ggttcttttt cactttcttg acccttcatg   2700
tctgcttctc ttgagtcttt gtttttatag caggaagtta gtattggggg cttgaatgat   2760
gcagggcacc aacagaacca ttgcaggact gaaatcccca gactaccgat accttggtgg   2820
tcggttctca gcttcactaa gaaagcagaa cggctgctta tgctgaagcc tctgtgacag   2880
tcaagggggt catcacctac attattgctg ccaggggtca cagccctgac ctttgccttc   2940
cagacttaac tgaaccagaa ccagatacca tagaggatag caataaaccc ttcttgacat   3000
ctgactatga tgtgtcatag cggggtctct ggtcatgtct atttggggtt caatatgcct   3060
cttgtatttg aatgtcaact cctgtcccta ggtataggaa attttgtgat ataatttcat   3120
taaatagttt gactaattcc tttttaattt atctcaggtc ctccttctcc ccagtggctt   3180
ctgaggtttg tttggtcttt taagtatatt ccaagcttct tgaaaatact ggtaatatct   3240
atctctctct ttctctctct ctctctctgt ctctgtgtgt gtgtgtgtct gtctgtctgt   3300
ctgtctgtct gtctgtgtgt gtctgagtat atgattgttc caactttgtg ctccactgca   3360
gaactgcatg atcttaatct cttggggaca ctttctagtg agggttttca ctttcatgtt   3420
tctttcatgt taatttctat cttgttcctt ttccatgttt gaatgccctc atcaaaatct   3480
ccctccatat atctgacttt ctcctccaac ttacccactg ttttgttcat tttggaggtt   3540
ttgtatacag ttcctagttc atgcatttgt atctgagact tctacatcaa ctctgaactc   3600
tttctcagac ttctcagtac ttttggtttt cattaaggag aggtttgtag tatgccctgg   3660
tctgctgttg tctcatgttc cttgtgtttc tctgtggaat tttgtacatc agtcaggatg   3720
gctatgtcct ccagttttaa ctcatctttt cccaactctg ttttgtttca tgtaactaag   3780
cccttcttca gaggtctgtc agaaaggcgc tgagagtgtg atgtctcagg cccaggtgtg   3840
gtcagaggcc tggtctgtgc tcttgccacc cactccatat aacacagagt gttcagagtc   3900
tcaaaggtga gaagccgccc aacacgcagg gagagctggg aaggacacgg gccccttcat   3960
tcactttcac tcccttcctg ttggctgaga tgccagatct gcctgcttcc ttcctccact   4020
ccttcccatg gctgtaagca ccagaccaca tacttaaggg agctgtgctg gtgaaccttg   4080
ccttgctgcc tgccttccct ctcccattct gtagctcttg actccttcct tgattcacct   4140
cttccgtctc ctcagggacc cgccctccag caccatcatc ctgagagata taactgtact   4200
ttgtacagcc tgaaccgcca aaaagacaca catgcaattc ttcctctggc ttctgagagg   4260
ctccttaaag gtgctaactg ctcagctcac tctccccggc gtgtccccat cctcagaaca   4320
catttccatt atctattgta cccaccaaaa agaaatatgt acttcttatg aaaagaaaac   4380
cctagtctgt tcagatgtgt ctcacagctg tgtgacacgt gccttcgttg ctatgctttc   4440
tcctttcttt agccatgttt gaccagggtg ggagggtgga tcctaaagcc tatcaaaaga   4500
ccctacccca ctccagtcca gctagacatt cttcttacaa attctgtttc tgtctgtata   4560
tgtgcatatg catagacaaa tccccctatt ccaccagcct ggtgatccat aggaatgagc   4620
agtgcctgct ggccacattc ccaccgtttg cactgttact ttgaggtaaa atcctaccct   4680
agaatgaaca aaggctggtg aaagtagggc agattaaggc agcttatgtt cttgtaaatg   4740
caagtttcta tttcagctag taggtgtttt ctttttctgt tttgtttttt aatgtagagc   4800
tggtaggcta taagccagca attgtgagta aggctttagc tattagtggc tgtgagcact   4860
agtttcattg actttacctt agggcagtgg ttctcagaac atggtccaag aacaacatca   4920
gcagcaccat cacctgaaga gcttgctaga aatgtacact ctgggccatc ccaacctcct   4980
gagttggcca ctctgaaggt gagctttaac taacagtctc tgctgctagt tcacactaat   5040
gcatgacagt ccctagcgga caggctggga gcatcttagc tctgggatga caacgattac   5100
tttaaatgtc ttctctgcct tagaattgat atttttattt cccccagtcc ttccttcctc   5160
ttccattaaa acagccacca ccacattact catctcaaat tctaggttgg tcttccttct   5220
agtcttagct ctaaaactct tgcctgcatt ggtctggact catatttcct tgcaggctac   5280
tagttctgca ttcttggtga ctttagccag caggaaggcc aggagtggtg gtggcatatg   5340
cctttgatcc cagcactttt gagggaagca gaggcaggca gatctctttg tgttcaagac   5400
cagtctggtc tacataggga gttcaaagcc aatcatagct atgcagtgag accctgtctc   5460
aaaacaaaca aacaaaatca gcaggagcct tagttgtcca tttcttccct gtgcacacac   5520
cacatctctt acaggaagat tagcctccac ccccacagtg gagcctccta catcctgata   5580
gagtatatgt tgagaagcca tgtgtatcta tgaatatagc tctgttctat atccttttga   5640
catgtagcaa tacctctcca tcctcaagga actcaaccca gtctgggtct ccccaggctc   5700
cagtggtaga ctctgacagg tgggaggata cagtgctctg ggctgttttg ttacaaaagt   5760
gtcttctgtc ctttccctcc tcccaattca gcatgacccc tgtgagcagg ctctcacaat   5820
ctcctggggc agggctgagg caggggcttt cagctcttct ccataactat cccttcttcc   5880
ttcccccatg ccatttagca gttatcaccc agccttgcct tctccctcca tcccttgccc   5940
tgacatatac tgtgccttat ttatgctgca aatataacat taaactatca agagaatgac   6000
tggtatgttt ggtgcttccc tacgcagact catggggccc attggtcact cctagagact   6060
cagtaggcat ttgtgtctga ccatcctcct ccttccactt cttagggcag aactagcagg   6120
ctctctctgc tttcagtaag taacatggtg ttggaaaagg cacagagttc agatctttaa   6180
actgcctcag agccaaggca tcacaaaaag actgaccaat gggaatactg aacaccctgg   6240
ctctttcagt gttttatgct cacccacttc caacaattga aaggaagaaa aagtcctact   6300
cccaagaaag gggcttggga gtgtacaaag aggtagacaa agtcaagctt tctccagaga   6360
ctagaaggaa tagctgaaga gatggctcag tagttaagag gaaaaactgc tcttgtctca   6420
aaagatccgt ttggtcccca cacccatgtt ggctagttta ccaccaccct taactccagc   6480
tgcaagggat ctggtgccct cttttggccc ccacaggcac tgcactcact tgcataaccc   6540
ttcccccagc acacatatac acaattaaaa agttaaaaaa aaaaaaatta aaaagagaag   6600
aagattggaa actcgaggca aactttgtaa aagcagatta aagctcacag gagaacaggt   6660
aatgatcagg gtgaggaagc ggacaggtga gccactgatc ctttctgtgt ctgtgtcttc   6720
cactaaaagt ggaaaccacc aaggagacag actgaagaac ctgacaaaag acagaacagg   6780
tacctctaag gttccttggt agaacagatc tactgggttg gtgtctggtg aaggactcag   6840
agcctcctta ggaaatggaa acacttactg gccatcactg tgtgggcccc agcaattaag   6900
gtacttactg ccaagcctga agacctgagc ttgatccctg gatacatgtg gtgggaagag   6960
aactggcaag gtgttctctg agctccacat atgctctgtg gggcatgtgt ctccttctcc   7020
ccaggtaaat aaatgaatga gaaagtgggt ggggagcaca cagtatgtcc aagaaagaga   7080
gaacattacc aaaagctaag acagagtctg gaggaagact ggagaggtgg ctcagtggtt   7140
aagagcactt gtgttctaga ggacttgagt tccgttccat ttaggtggct cacatctgga   7200
attctggact ttcggaagaa cagtcaggtg ctcttaccca ctgagccatc tcaccagccc   7260
caatattttt tttttttttt tttgagacag ggtttctctg tgtagctctc actgtcctgg   7320
aactcattct gtagaccagg ctggccttga actcagagat ttgccctacc tcccaagtgc   7380
tgggactaaa ggcatatgtc accacaggcc agctgagatc ctgtatttaa ataaataagt   7440
ctggaaggtg ataaataaaa ctaagtctag aagatgagaa tcctagcaca caggttagga   7500
tgattaattt ttgttgaggt tagaagtgaa ccagcttctt tgtgaactta gtagcttcag   7560
cccagactcc ggtactgaag cagcagtgca gtgaacacag ggtggctgct gtgagactgc   7620
tgtgtacgca acccatctgc tgttcaggac agcttcctgt tcacagggtt aggttttttt   7680
attgttctct gggtgctgga gattgtgctc aggggcctcc agcaagttgc ctctttgttg   7740
tttttttgtt tgtttgtttt gttttgaggc agggtttctc tgtgtagcct tggctgtttt   7800
ggaactagct ctgtagaaga gcctggcctc gactcaaaaa gatcagcctg cctctgaagt   7860
cacacagtga atttcgagag caaagataaa atacgaaatt tctgccaggt gtggtggtgc   7920
acatctgtaa tcccagcact tgagaaacta aaaacagagg ccagcatatt gggctacata   7980
cagagtctca ggcttagaaa accagacaag ctgggcactg tgacacacac tttaatccca   8040
gcacttggga ggcagaggca tcctaatcta catagtaaat tctagaccag ccaaggctaa   8100
atggtgacac ctgtttacac acacacacac acacacacac acacacacac acacacacac   8160
acacacacac acacagtgga gacaggaaaa gagagaggtg gggagagaaa ccgagaagac   8220
ccacaacaaa agcagcagtg aaatatttca actataatgt atgacaagct ctacaggaaa   8280
tcttgagaca aaccttacag aaaggttcac ccaagcctgg atgcattgta ttctagaaca   8340
tcagaaacct gatctagaag gtctccctct gcagtagagc acctgcctac catacacaag   8400
gctcttcatt tggacttgat tcccaagaaa gaaattttaa aatgccactt atccgcaaac   8460
ctaaaatgta agttggttcc aataaagtac ttccctgttg agcaaagaag atgtggatac   8520
atggtatttc tccaaatatg tgaggggctt ttacatatga ctgagagata acctgagact   8580
ccattgaaca agctaggacc tctggtagag gccaccaaga agctaaagcc cactcagcac   8640
tctctggaga tggtaagttc cccaggactg gagtgggagg acagcaaagg gaatcacagt   8700
tgacattttg aaaacaccgg gtctgtgctt tcctacaaaa tgcatcccaa atgtttctcc   8760
tagcaagtaa ttcattttac tgttcccata tgtaagtgag gaaaaaaaga gtgtgagcag   8820
cttgctttgc tcatggggta gaaccccgac agtccttctt ctgtttaggc tagagacatg   8880
gtactctgac acctggattt gcaagtgagg ttaggactag ctcctttaaa ggacccttcc   8940
ctgaactgga gtgattgtct gtccctaaag cagaacccta gtcgccagct ccagtagtat   9000
attagaacca gaaccaggca gagcccatgc tgaccagacg gaactggaaa aatgtcacaa   9060
ttctgggccc caaagaacta ggtcctcaag tcctagacaa aatgtatgga aagggaaatg   9120
gctggacgtg gcagtgaaga gtagtggcca caaggtggca gcagagtttc agctgtggag   9180
gcccaatccc cagttctctt gcaaagatgg gcctgtccac aaaatttaca ggccacctct   9240
actcagtaag gctccaaaaa gagtctccta tctctcactt aactattcac aggtaaatct   9300
taaagggtag tgaacccaca tttaacctga ctagaagcag tggggattga aatggggctg   9360
tggtcctgat cacccattcc aggcaggagt agggaccaag ctggttcacc ctagcctgca   9420
cttaacacta gttccttccc atccaggaca taatgcccaa ttctgacagg agtttctcca   9480
gtcaggaaca agaggtgatc aattgaagct tctccaatct gttgaaggat tggaggttct   9540
tctaaggttc ccccagggtc taactctgac aaactgtctg caattaatga tgcttcctga   9600
gctccggaga caagatttat gcatctaata aagtctatat aactccaggc ttaggctggg   9660
ggggaggaag ctaagagcag agagtcccca ggggagtacg ggaggggggg tcccaggtgg   9720
ctcttaatag agccatgcat ttccattgct tgtctagatt tccccccagg ctgccggtga   9780
ggtgggggta gggacatcag gtataagaag accgtgggca ctcaggaggc agatggcagc   9840 
           
             2 
             5124 
             DNA 
             Mouse 
           
            2
atggactcca agtgggctgc tctgctgctg ctgctactgc tgctgctgaa ttggggccac     60
actgaagagg cagggagctg gggtgaagac caagtctttg cagtgagtga acatcctgct    120
gccccatcca cccagccgcc atctctcctt cagctgaaac ttatgataca gggtttggtg    180
ttccccaggc tgctaaggtc ttcaaaaagt ctggcttcta agggagggaa atggcttctc    240
agcccacgca agtttcccct agaagccatt ctttccctcc atgtcttcag tgtacaagac    300
actgccaggt tccttccctg ccactctctc atcacaggga gaagataagg gaccccaccc    360
accacagtat gcccacattc cagacaggat ccagactcct gggtccctct ttcgtgttct    420
gctccaggcc atggacacac ctagaaggag cccagccttc ctgtttcagc cccagaggtg    480
agttccaaag ggaagaggct gagaagggtg gagggcagag gaagatgtga gagaaggtga    540
gatggaggga gtgccaacta aggatgaatc tctcctaagg tttggcagaa gtgcatgggg    600
gtcctggagc aaggaacagc taaatccgca ggccagacag ttctggagcc tggccgctcc    660
tcagcgcttt gggaagaagt agcatcgtca gctgtgatgc ctgcatgcaa aaccacttcc    720
ccatgttccc tgtgtgcccc caaataaaaa tggtccggct ggcttcagaa tccctgtgtt    780
tggacaagac tgtcagggag caggtgggag cccaagggca atagctgtag ccccccttca    840
cctccactca gtctctagcc attctgttgt taaggatccc caaggctact actgcacctt    900
gcctcctctc ggtaacaaaa aagaacaagg ggttcaaaag gagaacaagc tcaccatgtt    960
tattccttat accctcatga cccaaggcca gagagagcag ggttttggaa gccaaagagc   1020
agcatttatt caggactcca atagattcat ccatcaccca cggaatgagg acaaatcctg   1080
tgctggctgg ggccctgtgg ttcatggctc cttgcttgcc tgtgccttcc tcagtctcaa   1140
ggcagacagg ctgtgtcaga ggtagagatg gcacttctgg agggtaccag agctaggtgg   1200
atacatggac ccaggggcag aaggagcaag aagtaaaaga tgcatatcca tcactgcagt   1260
gggatgctac ttgctacccg ccatgatcct gaggtactgt agggcgcggt gagcagcatc   1320
acctcgggct gcctccctgg tggttgcaga accataacac acagtggctg gctgggtgga   1380
cagttccact aggcactggc agagcccact caggctcagt tcctctggaa accaagaaag   1440
agaagggagg cactggtggg gaaggggcac caaacagtgt acacatcacc catctcccaa   1500
ctccttcata cttgctttga tccagtcccc tccatcccca gtctctagtt ggccatacca   1560
atatccagat agctgacatg gaaagcctgc tcctcagaga gctcactgag gacactgcag   1620
caggcagagc ccagagcccc tagagagccc acggagcaac tgcgaaggga taggatcttt   1680
tctcccacag aattccgcaa ggaatcccag gtgcagcctg gcccacgatt cctcagtcca   1740
tccaggcggg agctcacgcc ctaatgagat aagagtgaga aagcagctaa ggactgtggg   1800
acccaagaac tgggtattat gctctgactc ctggcagcct tcctcatccc aggaaaagtt   1860
gcctataggg gctttgccct aggccagctt tcactagtga ccctacatat aggctgaggg   1920
gacttctggg cgcacaggtt caaggagccc tgggaaggaa agcaggccca tactcacttc   1980
caaaccacag acccaaccca attccgtacc atgtcctgca gtgtgaacca catgtcgagc   2040
ccacacaagc cacttacaat ggaaaaatga tcgtcatcag gctctgcctc attgccatcc   2100
cgggcatcca gaggtacaag tgtgcactcg aaggagcatc ttagctgctg cgttacgctt   2160
tgccagcttt ttggaagtgc cactgcctga gggtggggga aggaattgtt caacttgggg   2220
attcctaaca gaccaatgct ttcctctagg gtaacttaga cagtcaccca atgagatacc   2280
actgcaaaga ggagctcaag gcctgatctt tttaactgag gagttggtga gccagataag   2340
agggggaaag atgggcaggg acaagggaga gacaggaagg caggagctga gcaaggacct   2400
gcaaagcaaa ggaagctggg acagagcaca gccaggcaaa gaaccacctt gtgtttgggg   2460
ttacagtgtg gcaaagagag aaggtgagtg aacccccttt tccccagtta ccaatctcaa   2520
tgaaacgctc cacccggcaa gtcatggtga actctttgcg gtgagcaggc ccagactctt   2580
gggtcaccat gtactctggc aaacgccagc ctttttgcac caccagctcc ttgaaagaaa   2640
accataagag agtccaaggc ttagtgagga aagggtcctt agccaatggc cttctaccaa   2700
gagcctccac ctttgagtga gaaggaggac caccctaccc acaagtgcac acgcccctgc   2760
ctcagcccta cttcagagaa gatagcacac atgacaatga agacgaggca cacctgcaga   2820
gcaccgacgg ggttgcactc agactgctga ggagagacag ggggctgcat ctccatggga   2880
gggctcctaa agaaaaaagg gcccaggcca gtgctgagga aaatgcagag gttcctcctg   2940
tcctggtcat ggatcaagtg ccagccacta ccacctccca attcctcacc aggcaggcag   3000
ctatctgtcc aaacatctag cctttctcct ccctccccac accccaccaa gtgaggctct   3060
gctacttccc aggagcactt agatgtggaa atccttcagc aatgtccctg tgatggaggt   3120
aaaggagctg tacacacagt cctggaagta ccttttgggt ctcaaacatg gagcctctaa   3180
gatgtttaaa caatctaaca ttcattgctt ggctatttaa tcatcataaa ttacctccta   3240
gctcaggctt accaaccagc tggtgcaaat ttttcaatta caccagtctg tctttactct   3300
caggcttcct catcctgacc tgcctgagtc caagagctat gagactagaa gccaagaccc   3360
cctccttcct ccaagacgtc aggggactca agatacctgg ttagtacagc agatggaaca   3420
ggggcagcag cttctgcagc aacgacagga gtgtcctcag gcggtgaaga gtctaggaga   3480
gaaaaagaac ttcccgaggg gaagaaggtc ttggattcac acccatgtag aagggaatga   3540
gggtgtgagg tccttctgac ctttccatcc cctgagaact ccttcccccc gatgcagact   3600
cggagagcct cagcaacagc tcttcctcct gggatcctct gctgacccca cactggctca   3660
gaagcactga ggttagggtg ggggctcaga aaagagtctc tttctaccca acaagctgca   3720
acctgaatgt ggaagaggga aagccatctt gctagaaaga gggggcatgc caggaccaac   3780
tttatcagtc tttgcctcca ccccctatgc ccttttctca tgccccagag cagccaccac   3840
ccatcaaagg gggctggcta ttactctgag gcactctaga aacaccctgt tcagcttcag   3900
aggaaattct caggaagggg ccacgagagg gtggccatga gtcaccagac caggcttagg   3960
gagagaagta gatagtgcag aggcctgggt tctcagcggg cttctagtgt gccttggctg   4020
ttcctccctc acctgctgtc ctccagggct ggttccagca tgctcccccc tttgaggtgt   4080
ttgagggcca cctcagctgc cttgtgcttg gctgccttct tgctggggcc ctgacctaag   4140
aaaaggggcg tgggcaatac tggaggtcac cggcaggaca gcagagaaac cagcccccac   4200
agcctatctc ctcatagcca gagggagtga gaagagccct ctgaccaacc tcccctgtca   4260
gaactggaag ggttctacta attgttggac tagccttttt cctagttagc attaacagtc   4320
aacttggcac aacctaggat ctaaaaggat ctgaaaggag agccttaact gaggaattgc   4380
ctacatcagc ctggcctgag ggcatgtctg ggggtgggtg ggggggatgg actgatagtt   4440
aattgatgct gatatgcaca gcccactatt gtggtaccac ccctaggcag gtggtcctga   4500
actgtgtaag aaatctagta gagtataaat aagccagcca gccagcatgg agtatctgcc   4560
attgttcctg ctgtgcttct gtggctgtga agtgagttcc ttagtcagaa gtagcaagaa   4620
tcagattatt ttctttggtc ttgactgata atgtgagttt cttccttagc ttcccttagt   4680
catgaatgta acctgaactg taaacctatc aacccttttc ctcctctaaa ctgcttttag   4740
tttcggagcc ttactacagc aacagaaatg ggccttgaac acctcctctt tcagtcagct   4800
cccgtaatga aactgtgcca ggcaggattt ttgagctcca acactggtgg gttgcggagg   4860
gatgttttgc tctgtctttc tttccccttt tctcttccct cggtaaatag gaaatagaga   4920
caactgaggg tctgaagtca aagctctgct ctgccactta ttacatgtga actggagcct   4980
accatttaat ttctcatact aagttcttcg tgttcaatgt gaggcatgaa ggctagcaga   5040
gtatgtgctc cccctaatac ccacgaggag ttcaatgtct catttactct tcctctgggg   5100
tgataaagta gcagatccga attc                                          5124 
           
             3 
             2480 
             DNA 
             Homo sapiens 
           
            3
tggcttagtc cgcattaaca gaaatatggt atctggagca tccagatcca aacctgctct     60
aatctacgtg tctaaaggat aagttggttc ctagatgcct tgccttaaag agtggggtgc    120
ttccccttga gcaaaaaaga ctatggagtg ggccaggtgc ggtggctcac gcctgtaatc    180
ccaacacttt gggatgccaa ggtgggtgga ttgcttgagg tcaggagttc aagaccagcc    240
tggccaacat ggtgaaaccc catctctact ccccctacaa aaattagccg ggcatagtgg    300
tgtgtgcctg taatcccagc tacttgggag gctgaggcag gagaatcgcc tgaacccggg    360
aggcagaggg gttgcggtga gccgagatcg caccactgca ctccagcctg ggtgacagag    420
ccagactcca tcgtaaaaaa aaaaaaaaaa aagctggagt gtaagaatga actctctctt    480
caaaaatatg aagagctttt acacatgaaa gagatgagat gttctgttga gacttaatgg    540
agcaaactaa gaccccgagt agaagtcaca ggaagcccaa agccccctcg gcacaaggat    600
aaatattgtc cgaagactgt ctgaggagat ggtaagttcc ccgactgggg cacatgaggc    660
aatcacagct aacgttttga ggatacctgt tctatgcaag ctgttttatt gaatggatta    720
caaaaagtat gttaagtaat gcacacccta ggaaggcgtt ccttgtaaaa tgggcccatg    780
tttcaggtga ggaaactgaa gctcagagcg ggagacagct tgctcaagca gaggccaagg    840
cctttacgtc atggctcttc ctcagcacaa gctagagacc cacactggtg gggatgctcc    900
aaaagggact cgaggattag aaggaacctg caccatatgg ctttgaaggc ctctccctga    960
ggattagtga ttctgatttt tcttaagaca ggattctagg ctactctata tgagagccag   1020
ggccaggcag aggctgtgga gaccagagtc ggggtcagaa tgatagtcca gccccacagt   1080
agcctgctcc tgtccttctg ggactccctg gagactggac cctagcacct ccactcagcc   1140
tcaccctcct cacttcctct gcagaggtct attctaggaa aaggaaatgg ccggagctgg   1200
caatgaggag ctgtgaccac agggtggcag cagtgcttca gctgtggaga gtgactatag   1260
gaagggcgat gaggcaggtc ggaagcagtg ctctctagtg aggatggggg tctgtctgca   1320
aagattggaa gccacactca ttcagtgggc tccaaaatcc tgtagcctcc ctctatatct   1380
taataatttt ttttttttga gacagagttt ctctttttgc caaggctgga gtgtagtggt   1440
gccatctcag ctcactgcaa cctctgcctc cccgattcaa gcgattctcc tgcctcagcc   1500
tcctgagtag ctgggattac aggtgcctac caccacgccc agctaatttt tgtattttta   1560
gtagacaggg gtttcaccat gttggccagg ctggtctcga actcctgccc tcaggtgatc   1620
cacccgcctt ggtctcccaa agtgctgggg ttacaggtgt gaggcactgc acccggcaaa   1680
aaaaaaatgg tttttaatta aaaaaaaaaa gatacaggct gggcatggtg gttgacgcct   1740
gtagtcccag ctacttggga ggctgaggca ggagaatcac ttgaacccag gagccagagg   1800
ttgcagtgag ccgagatcgc gccactgcac tccagcctgg gcaaaaagag cgaaactcca   1860
tctcaaaaag aaaaaaagtt aaattctctc catcatcatg aagttgaata tattttttct   1920
atccacaggc aaatctgagt agcctccaag aggcacacaa gcagaggatg ggctgtgttg   1980
ccctgactgc cagccccagg cacagaggac caggcctggt catcctcaca gactctgacc   2040
ctggctcttc ccactcctct tccactccag gacatcctac ttaacccctc ctgacatgag   2100
tttcttgtgc tttagtctac aggttaggaa agaggggaag tgataaacaa gctctccaac   2160
ctgttgaggg attaggggtt cgtctaaggc tccccagggc ctggctctga caaagcgtct   2220
gcaactaatg atgcttcttg agctctggag acaggattta tgcatctaat aaagtctgta   2280
actccaggct taggggccgg gggcaggagg ctgagagcat gaagtcctgg gggcgccatg   2340
ggaggagatc ccaggtggct cctaatgagc cctgcatttc atttgcctgc tctagattcc   2400
cctaaggcta ctgtgaggct gggggtgggg gaacagcagg tataagaggt tggggtggct   2460
gtaggagggt aggtggcagc                                               2480 
           
             4 
             396 
             DNA 
             Mouse 
             
               CDS 
               (7)..(393) 
             
           
            4
gtatct atg aat ata gct ctg ttc tat atc ctt ttg aca tgt agc aat        48
       Met Asn Ile Ala Leu Phe Tyr Ile Leu Leu Thr Cys Ser Asn
         1               5                  10
acc tct cca tcc tca agg aac tca acc cag tct ggg tct ccc cag gct       96
Thr Ser Pro Ser Ser Arg Asn Ser Thr Gln Ser Gly Ser Pro Gln Ala
 15                  20                  25                  30
cca gtg gta gac tct gac agg tgg gag gat aca gtg ctc tgg gct gtt      144
Pro Val Val Asp Ser Asp Arg Trp Glu Asp Thr Val Leu Trp Ala Val
                 35                  40                  45
ttg tta caa aag tgt ctt ctg tcc ttt ccc tcc tcc caa ttc agc atg      192
Leu Leu Gln Lys Cys Leu Leu Ser Phe Pro Ser Ser Gln Phe Ser Met
             50                  55                  60
acc cct gtg agc agg ctc tca caa tct cct ggg gca ggg ctg agg cag      240
Thr Pro Val Ser Arg Leu Ser Gln Ser Pro Gly Ala Gly Leu Arg Gln
         65                  70                  75
ggg ctt tca gct ctt ctc cat aac tat ccc ttc ttc ctt ccc cca tgc      288
Gly Leu Ser Ala Leu Leu His Asn Tyr Pro Phe Phe Leu Pro Pro Cys
     80                  85                  90
cat tta gca gtt atc acc cag cct tgc ctt ctc cct cca tcc ctt gcc      336
His Leu Ala Val Ile Thr Gln Pro Cys Leu Leu Pro Pro Ser Leu Ala
 95                 100                 105                 110
ctg aca tat act gtg cct tat tta tgc tgc aaa tat aac att aaa cta      384
Leu Thr Tyr Thr Val Pro Tyr Leu Cys Cys Lys Tyr Asn Ile Lys Leu
                115                 120                 125
tca aga gaa tga                                                      396
Ser Arg Glu 
           
             5 
             129 
             PRT 
             Mouse 
           
            5
Met Asn Ile Ala Leu Phe Tyr Ile Leu Leu Thr Cys Ser Asn Thr Ser
  1               5                  10                  15
Pro Ser Ser Arg Asn Ser Thr Gln Ser Gly Ser Pro Gln Ala Pro Val
             20                  25                  30
Val Asp Ser Asp Arg Trp Glu Asp Thr Val Leu Trp Ala Val Leu Leu
         35                  40                  45
Gln Lys Cys Leu Leu Ser Phe Pro Ser Ser Gln Phe Ser Met Thr Pro
     50                  55                  60
Val Ser Arg Leu Ser Gln Ser Pro Gly Ala Gly Leu Arg Gln Gly Leu
 65                  70                  75                  80
Ser Ala Leu Leu His Asn Tyr Pro Phe Phe Leu Pro Pro Cys His Leu
                 85                  90                  95
Ala Val Ile Thr Gln Pro Cys Leu Leu Pro Pro Ser Leu Ala Leu Thr
            100                 105                 110
Tyr Thr Val Pro Tyr Leu Cys Cys Lys Tyr Asn Ile Lys Leu Ser Arg
        115                 120                 125
Glu 
           
             6 
             1920 
             DNA 
             Mouse 
           
            6
tacagagtct caggcttaga aaaccagaca agctgggcac tgtgacacac actttaatcc     60
cagcacttgg gaggcagagg catcctaatc tacatagtaa attctagacc agccaaggct    120
aaatggtgac acctgtttac acacacacac acacacacac acacacacac acacacacac    180
acacacacac acacacagtg gagacaggaa aagagagagg tggggagaga aaccgagaag    240
acccacaaca aaagcagcag tgaaatattt caactataat gtatgacaag ctctacagga    300
aatcttgaga caaaccttac agaaaggttc acccaagcct ggatgcattg tattctagaa    360
catcagaaac ctgatctaga aggtctccct ctgcagtaga gcacctgcct accatacaca    420
aggctcttca tttggacttg attcccaaga aagaaatttt aaaatgccac ttatccgcaa    480
acctaaaatg taagttggtt ccaataaagt acttccctgt tgagcaaaga agatgtggat    540
acatggtatt tctccaaata tgtgaggggc ttttacatat gactgagaga taacctgaga    600
ctccattgaa caagctagga cctctggtag aggccaccaa gaagctaaag cccactcagc    660
actctctgga gatggtaagt tccccaggac tggagtggga ggacagcaaa gggaatcaca    720
gttgacattt tgaaaacacc gggtctgtgc tttcctacaa aatgcatccc aaatgtttct    780
cctagcaagt aattcatttt actgttccca tatgtaagtg aggaaaaaaa gagtgtgagc    840
agcttgcttt gctcatgggg tagaaccccg acagtccttc ttctgtttag gctagagaca    900
tggtactctg acacctggat ttgcaagtga ggttaggact agctccttta aaggaccctt    960
ccctgaactg gagtgattgt ctgtccctaa agcagaaccc tagtcgccag ctccagtagt   1020
atattagaac cagaaccagg cagagcccat gctgaccaga cggaactgga aaaatgtcac   1080
aattctgggc cccaaagaac taggtcctca agtcctagac aaaatgtatg gaaagggaaa   1140
tggctggacg tggcagtgaa gagtagtggc cacaaggtgg cagcagagtt tcagctgtgg   1200
aggcccaatc cccagttctc ttgcaaagat gggcctgtcc acaaaattta caggccacct   1260
ctactcagta aggctccaaa aagagtctcc tatctctcac ttaactattc acaggtaaat   1320
cttaaagggt agtgaaccca catttaacct gactagaagc agtggggatt gaaatggggc   1380
tgtggtcctg atcacccatt ccaggcagga gtagggacca agctggttca ccctagcctg   1440
cacttaacac tagttccttc ccatccagga cataatgccc aattctgaca ggagtttctc   1500
cagtcaggaa caagaggtga tcaattgaag cttctccaat ctgttgaagg attggaggtt   1560
cttctaaggt tcccccaggg tctaactctg acaaactgtc tgcaattaat gatgcttcct   1620
gagctccgga gacaagattt atgcatctaa taaagtctat ataactccag gcttaggctg   1680
ggggggagga agctaagagc agagagtccc caggggagta cgggaggggg ggtcccaggt   1740
ggctcttaat agagccatgc atttccattg cttgtctaga tttcccccca ggctgccggt   1800
gaggtggggg tagggacatc aggtataaga agaccgtggg cactcaggag gcagatggca   1860
gcatggactc caagtgggct gctctgctgc tgctgctact gctgctgctg aattggggcc   1920 
           
             7 
             411 
             DNA 
             Mouse 
           
            7
tccaggacat aatgcccaat tctgacagga gtttctccag tcaggaacaa gaggtgatca     60
attgaagctt ctccaatctg ttgaaggatt ggaggttctt ctaaggttcc cccagggtct    120
aactctgaca aactgtctgc aattaatgat gcttcctgag ctccggagac aagatttatg    180
catctaataa agtctatata actccaggct taggctgggg gggaggaagc taagagcaga    240
gagtccccag gggagtacgg gagggggggt cccaggtggc tcttaataga gccatgcatt    300
tccattgctt gtctagattt ccccccaggc tgccggtgag gtgggggtag ggacatcagg    360
tataagaaga ccgtgggcac tcaggaggca gatggcagca tggactccaa g             411 
           
             8 
             410 
             DNA 
             Rat 
           
            8
tccaggacta taatgctcaa tcctgacagg agtttcttca gtcaggaaca agaggtgatc     60
aattgaagct tctccaacct gttgaaggat tggaggttct tgtaagactc ctccagggcc    120
tagctctgac aaactgtctg caattaataa tgcttcctga gctctggaga caagatttat    180
gcatctaata aagtctataa ctccaggctc atgctggggg tagagaactg agagcagaaa    240
gtctcccagg gcggtatggg aggggggtcc caggtggctc ttaatagagc catgcatttc    300
cattgcctgt ctagatttcc cccaggctgc tgatgaggtg ggggtagggg acatcaggta    360
taagaagccc gtgtgccacg gaggaggcag atggcagcat ggattccaag               410 
           
             9 
             411 
             DNA 
             Homo sapiens 
           
            9
taacccctcc tgacatgagt ttcttgtgct ttagtctaca ggttaggaaa gaggggaagt     60
gataaacaag ctctccaacc tgttgaggga ttaggggttc gtctaaggct ccccagggcc    120
tggctctgac aaagcgtctg caactaatga tgcttcttga gctctggaga caggatttat    180
gcatctaata aagtctgtaa ctccaggctt aggggccggg ggcaggaggc tgagagcatg    240
aagtcctggg ggcgccatgg gaggagatcc caggtggctc ctaatgagcc ctgcatttca    300
tttgcctgct ctagattccc ctaaggctac tgtgaggctg ggggtggggg aacagcaggt    360
ataagaggtt ggggtggctg taggagggta ggtggcagca tggattctag g             411 
           
             10 
             417 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Consensus
      sequence for NpFF promotor region 
             
           
            10
tccaggacna taatgcncaa ttctgacagg agtttctnca ngtcaggaac aagaggtgat     60
caattgaagc ttctccaacc tgttgaagga ttggaggttc ttctaaggct ccnccagggc    120
ctagctctga caaactgtct gcaattaatg atgcttcctg agctctggag acaagattta    180
tgcatctaat aaagtcnnta taactccagg cttanggctn gggggnagga agctgagagc    240
agaaagtcnc cnggggnggn cngggggggg ggtcccaggt ggctcttaat agagccatgc    300
atttccattg cctgtctaga tttcccccca ggctgctgnt gaggtggggg taggggnaca    360
tcaggtataa gaagnccgtg tggcactnag gaggcagatg gcagcatgga ttccaag       417 
           
             11 
             18 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence antisense
      primer for mouse 
             
           
            11
tggagtccat gctgccat                                                   18 
           
             12 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 325 bp mouse promoter 
             
           
            12
gtgctagcaa tctgttgaag gattgg                                          26 
           
             13 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 579bp  mouse promoter 
             
           
            13
gtgctagcag tctcctatct ctcact                                          26 
           
             14 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 805bp mouse promoter 
             
           
            14
gtgctagcag acggaactgg aaaaat                                          26 
           
             15 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 1085bp  mouse promoter 
             
           
            15
gtgctagctc tcctagcaag taattc                                          26 
           
             16 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 1289bp mouse promoter 
             
           
            16
gtgctagcta catatgactg agagat                                          26 
           
             17 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for1527bp  mouse promoter 
             
           
            17
gtgctagcag cctggatgca ttgtat                                          26 
           
             18 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesense primer
      for 1861bp mouse promoter 
             
           
            18
gtgctagcac agagtctcag gcttag                                          26 
           
             19 
             23 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence antisense
      primer for human 
             
           
            19
gctgccacca cctaccctcc tac                                             23 
           
             20 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence antisense
      primer for rat 
             
           
            20
caccccagct ccctgcctct t                                               21 
           
             21 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence antisense
      primer for nested human 
             
           
            21
gtggatccat ctagagcagg caaatg                                          26 
           
             22 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence antisense
      primer for nested rat 
             
           
            22
cgtggcccca gttcctcagc a                                               21