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 ( fig1 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 ( fig2 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 ( fig2 c ). in this promoter region a consensus binding site for nfat was also detected by computational analysis ( fig2 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 ( fig2 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 ( fig2 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 . 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 fig1 a ) 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 fig1 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 fig1 b ). some of the consensus binding sites for transcription factors were seen to be conserved between all three species in the proximal promoter ( fig3 ). 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 fig2 a ) were transformed in competent dh5α cells . 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 fig2 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 fig2 c and 2d . the results show an increase in luciferase activity of tpa and ionomycin stimulated mnf5 ′ 0 . 3 - luc transfected cells ( fig2 c and d ) and an increase in luciferase activity in tpa and ionomycin and lps stimulated mnf50 . 6 ′- luc transfected cells . 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 fig3 . 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 fig4 . 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 ( fig3 ). 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 . 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 . gtatct atg aat ata gct ctg ttc tat atc ctt ttg aca tgt agc aat 48 acc tct cca tcc tca agg aac tca acc cag tct ggg tct ccc cag gct 96 cca gtg gta gac tct gac agg tgg gag gat aca gtg ctc tgg gct gtt 144 ttg tta caa aag tgt ctt ctg tcc ttt ccc tcc tcc caa ttc agc atg 192 acc cct gtg agc agg ctc tca caa tct cct ggg gca ggg ctg agg cag 240 ggg ctt tca gct ctt ctc cat aac tat ccc ttc ttc ctt ccc cca tgc 288 cat tta gca gtt atc acc cag cct tgc ctt ctc cct cca tcc ctt gcc 336 ctg aca tat act gtg cct tat tta tgc tgc aaa tat aac att aaa cta 384 tyr thr val pro tyr leu cys cys lys tyr asn ile lys leu ser arg
the present invention relates to isolating , sequencing and cloning of the 5 ′- flanking region of neuropeptide ff promoter . the characterized neuropeptide ff promoter is useful in gene therapy and in dna analyses , and in production of gene - modified animals .