Proc. Natl. Acad. Sci. USAVol. 84, pp. 2068-2072, April 1987Neurobiology

Structure and expression of the rat neuropeptide Y gene(gene famfly/polymorphism/mRNA)

DAN LARHAMMAR, ANDERS ERICSSON, AND HAKAN PERSSONDepartment of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, Sweden

Communicated by Bengt Samuelsson, November 24, 1986 (receivedfor review October 13, 1986)

ABSTRACT Neuropeptide Y is a 36-amino acid peptidethat is abundant throughout the mammalian nervous system.It belongs to the same family of carboxyl-terminally amidatedpeptides as pancreatic polypeptide and peptide YY. We de-scribe here the gene encoding the rat neuropeptide Y precur-sor. The gene spans 7.2 kilobase pairs and contains four exons.The exon organization is identical to the pancreatic polypeptidegene, although the amino acid sequences of the neuropeptide Yand pancreatic polypeptide precursors differ extensively. Thepredicted amino acid sequence of mature rat neuropeptide Y isidentical to the human sequence. Also the sequence of the30-amino acid carboxyl-terminal peptide of preproneuropep-tide Y is highly conserved, which suggests that it is functionallyimportant. Two neuropeptide Y alleles were found to differ atnine positions in 2.5 kilobase pairs at the 5' portion of the gene.No exon difference was found. One nucleotide substitution closeto the gene promoter may influence the regulation of expres-sion. Neuropeptide Y mRNA was found in all rat brainsubregions tested, which shows that neuropeptide Y is synthe-sized throughout the brain. Developmentally, mRNA wasdetected in the rat brain as early as embryonic day 16 andincreased rapidly to adult levels. The level of neuropeptide YmRNA was also studied in several rat peripheral organs.Unexpectedly high levels were observed in heart and spleen.This mRNA may be synthesized in intrinsic ganglia andnon-neuronal cells, respectively.

Neuropeptide Y is one of the most abundant and widespreadpeptides in the mammalian nervous system (1, 2). It is a36-amino acid peptide that shares considerable sequencehomology with the endocrine gut peptides pancreatic poly-peptide and peptide YY. All three members of this peptidefamily have a carboxyl-terminal amide group. This propertyinitially led to the discovery of neuropeptide Y and peptideYY (3, 4). The widespread distribution of neuropeptide Y inthe brain and the findings that it coexists with catecholaminetransmitters in many neurones (5) and in adrenal medulla (6)suggest that this peptide serves as a neurotransmitter orneuromodulator. Indeed, neuropeptide Y potentiates thevasoconstrictor effect of noradrenaline on blood vessels (7).Also, neuropeptide Y coexists with epinephrine in neurons oflocus coeruleus (5), which have been implicated in the centralregulation of blood pressure (8). Thus, there is extensiveevidence that neuropeptide Y participates in vasoregulation.Neuropeptide Y has also been found to increase food intake(9) and to influence sexual behavior (10) and circadianrhythms (11). The high densities of neuropeptide Y and itsreceptor in the cerebral cortex and the hippocampus suggestit has a role in higher cognitive functions (12-14).The amino acid structure of the human neuropeptide Y

precursor was deduced from a cDNA clone (15). Humanpreproneuropeptide Y has 97 amino acid residues and iscomposed of a signal peptide of 28 amino acids, the mature

neuropeptide Y peptide, a Gly-Lys-Arg processing site, anda 30-amino acid carboxyl-terminal peptide. The C-terminalpeptide is also present in abundance throughout the brain(16), but its role is as yet unknown. As neuropeptide Y seemsto act in concert with catecholamines, the coordination ofthesynthesis of neuropeptide Y and these classical transmittersis of prime interest. As a first step toward understanding theregulatory mechanisms governing neuropeptide Y expres-sion, we describe here the rat neuropeptide Y gene. Further-more, the neuropeptide Y gene structure provides informa-tion on the evolution of this peptide family, as well as theroles of the various domains ofthe neuropeptide Y precursor.Using a probe from this gene, expression of neuropeptide YmRNA was investigated in various adult and embryonic rattissues, including the brain.

EXPERIMENTAL PROCEDURESScreening of Genomic Libraries. Two rat genomic libraries

in bacteriophage X Charon 4A were kindly provided by JamesBonner (California Institute of Technology, Pasadena, CA).Each library was prepared from liver genomic DNA of oneSprague-Dawley rat. One library was constructed afterpartial digestion with Hae III and addition of EcoRI linkers(J. Bonner, personal communication). The other library wasconstructed after partial digestion with EcoRI (17). Thelibraries were screened with a radiolabeled (18) 294-base-pairAva I-EcoRI fragment covering most of the coding region ofa human neuropeptide Y cDNA clone kindly provided byCarolyn D. Minth and Jack E. Dixon (15).

Hybridizations were performed for 10-16 hr at 42°C in 25%(vol/vol) formamide, 1 M NaCl, 10% (wt/vol) dextransulfate, 1% NaDodSO4, and fish testis DNA at 0.1 mg/ml.Filters were washed twice for 5 min at room temperature in2x SSC/0.2% NaDodSO4 (lx SSC = 0.15 M NaCl/15 mMsodium citrate, pH 7.0) and once for 1 hr at 60°C in 2xSSC/0.5% NaDodSO4. Phage DNA was isolated with amini-prep procedure (19) and analyzed with restriction en-zymes. Subclones were prepared in pUC19.

Nucleotide Sequence Determination. Sequences were deter-mined using the chemical degradation method (20). Allrestriction sites were covered by overlapping sequenceexcept the Xho I site at position 526 in Fig. 2A and the ScaI site at position 794 in Fig. 2B.DNA Blot Analyses. Genomic DNA was prepared from rat

liver, digested with restriction enzyme, electrophoresed onagarose gel, and transferred to nitrocellulose. Hybridizationwas carried out for 12 hr at 42°C in 50% (vol/vol) formamide,1 M NaCl, 10% (wt/vol) dextran sulfate, 1% NaDodSO4, andfish testis DNA at 0.1 mg/ml. Filters were washed twice for5 min at room temperature in 2x SSC/0.2% NaDodSO4 andtwice for 1 hr at 60°C in 0.2x SSC/0.2% NaDodSO4.RNA Blot Analyses. The isolation ofmRNA from rat organs

and brain subregions, as well as hybridizations, were asdescribed (21). Polyadenylylated RNA (10 ,ug) isolated byoligo(dT)-cellulose chromatography (22) was run in each

Abbreviation: nt, nucleotide.

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lane. Unless otherwise indicated, we used as probe thenick-translated (18) 287-base-pair Xba I-Ava I fragmentcontaining exon 2 of the neuropeptide Y gene, i.e., the regionencoding the signal peptide and most of the mature neuro-peptide Y peptide (see Fig. 1).

RESULTS

Isolation of the Neuropeptide Y Gene. Two rat genomiclibraries were screened with a neuropeptide Y probe from ahuman cDNA clone (15). Eighteen phage clones were iso-lated from the Hae III library. They were found to be siblingsof two distinct clones (15 copies of clone Hae-17 and 3 copiesof clone Hae-24). Thirty-eight phage clones were found in theEcoRI library, also representing two different clones (18copies of clone Eco-5 and 20 copies of clone Eco-7). Restric-tion site mapping of the clones revealed one site differencebetween the two libraries: a Bgl II site present ==430 nucle-otides (nt) on the 3' side of exon 2 in the EcoRI phages isabsent in the Hae III phages (Fig. 1). As all clones isolatedfrom each library were equivalent, we presumed that thisdifference represents a polymorphism at the neuropeptide Ylocus. To rule out the possibility that the rat haploid genomecontains two closely related neuropeptide Y genes, genomicDNA was isolated from five Sprague-Dawley rats, digestedwith Bgl II, electrophoresed in parallel lanes on an agarosegel, and transferred to nitrocellulose. The filter was hybrid-ized with a radiolabeled fragment containing exon 2 of theneuropeptide Y gene (Fig. 1). None of the five rats had bothBgl II fragments (data not shown). We conclude thatSprague-Dawley rats have a single neuropeptide Y gene andthat at least two alleles occur. Polymorphisms at other locihave been noted between these two Sprague-Dawley librar-ies (J. Bonner, personal communication). The two rats thatserved as donors of DNA for the phage libraries wereprobably both homozygous for their respective allele, and thefive rats tested above were all homozygous for absence of thepolymorphic Bgl II site.

Nucleotide Sequence of the Rat Neuropeptide Y Gene. Theexons of the neuropeptide Y gene were localized by using thehuman neuropeptide Y cDNA clone as probe under low-stringency conditions. Nucleotide sequencing confirmed thefour-exon structure of the neuropeptide Y gene (Figs. 1 and2). The gene spans -7.2 kilobase pairs (kb). All exon-intronjunctions conform to the "GT-AG" rule (22). Exon 1 encodesthe 5'-untranslated region and the initiating methioninecodon. Exon 2 encodes the remainder of the signal peptideand most of mature neuropeptide Y. Exon 3 encodes theGly-Lys-Arg processing site and another 23 amino acids.

Exon 4 encodes the last 7 amino acids of the neuropeptide Yprecursor and the 3'-untranslated region.The presumed transcriptional start site agrees well with the

consensus sequence (23) as well as with the start of the humanneuropeptide Y cDNA clone. It is preceded 30 nt upstreamby an AT-rich "TATA-like" element (23), ATAAAA, whichprobably serves as promoter. The 5'-untranslated regioncontains 79 nt and displays 80% homology to the humansequence, not counting six insertions/deletions. The 3'-untranslated region of the gene spans 176 nt and is 81%homologous to the human sequence, excluding three minorinsertions/deletions. This overall homology is higher than formany other rat-human gene pairs (24), which may indicate adistinct function for this region, e.g., in the regulation ofgeneexpression.The three introns of the neuropeptide Y gene are 0.79, 4.1,

and 1.85 kb long. Introns 2 and 3 are much larger than thecorresponding introns of the related human pancreatic poly-peptide gene (25). The pancreatic polypeptide introns 2 and3 are only 0.24 and 0.19 kb long, respectively. No repeatedelements belonging to the B1, B2, or Li families (see ref. 26)were identified in the neuropeptide Y introns that couldaccount for their larger sizes (1.85 kb of intron 2 has not beensequenced). However, two long stretches of alternating nt arepresent in intron 2 (open boxes in Fig. 1).Comparison of Two Neuropeptide Y Alleles. As the phage

clones of the Hae III and EcoRI libraries were found to differwith regard to a Bgl II site, we decided to sequence the entire5' region of both alleles. Table 1 lists the differences found in2.5 kb, including 670 nt of 5'-flanking sequence, exon 1,intron 1, exon 2, and 780 nt of intron 2. In total, we noted sixsingle-base substitutions and three insertions/deletions (en-compassing 4, 5, and 10 nt). Two of the insertions/deletionsare differences in the number of copies of short repeats,which have been observed between other polymorphic alleles(27, 28). None of the differences between the neuropeptide Yalleles occur in exons. However, one substitution is locatedonly 80 nt on the 5' side of the "TATA-like" promoterelement (position 562 in Fig. 2) and may influence the levelof expression. Regulatory factors have been found to bind tothe corresponding region of other genes (see ref. 29), andsingle-base substitutions up to 200 nt on the 5' side of thetranscriptional start site of globin genes alter the level ofexpression (see ref. 30).

Predicted Amino Acid Sequence of Rat Neuropeptide Y.Twojuxtaposed methionine codons are present in frame withthe coding sequence, one at the end of exon 1 and the otherat the beginning of exon 2 (Fig. 2). We favor the first of thesefor initiation of translation because it agrees better with theconsensus sequence (31). The human sequence, by compar-

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Sequenced regions: iFig. 2A Fig. 2B-

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FIG. 1. Restriction map of therat neuropeptide Y gene. Theexons are shown as filled boxes.

kb Open boxes show two repeatscomposed of alternating nucleo-tides. Horizontal bars below thescale show the four bacteriophageX clones. Star indicates the BgI II

site present only in the Ecophages. The following enzymeswere mapped: BamHI, Bgl II, ClaI, EcoRI, HindIII, Kpn I, and XhoI.

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AAAGCTTACAGATAGGGGCTCGAATCGCTGCCACTTTCCGCTGTAGATACAGAAGCCTCCTGGACCGCCAGCTCCCCGTGGCAGCCTTGGGGAGTTTCTGGCTGAGGCCGAGCCTGGCTAG 120CAGCGTTGGGGAGTGTGCTTGGGGAAGGGTCCACCTTTGGTGGGGGAGACCAGTAGGTCCAGTAGGTCCAGTAGGTCTAAGAAAGCCGCTGGGGACCTTGCGGTCCGGGACACCTGCTCC 240GGGAGCGGGAAAAACCTTGCTCGACTGCTTCCCTCCCAGCGCTCGCAGTTGTCCCAGAGATGCTCCCCAAGTACAGTGTCTGGTCCCTACAGACCCGCGCGCAGACAGCAGGCAATTCCC 360GCGACAGGCAATCTAAGCGGTCCCTGCTTTATCTTTCTCTCTGGCAGCGGGACTCGACGGGGAGAAGTAAAGAGGGATCTGGGGGATGCTCACTCTTGGATGTTCC CTTCTCCTCTCAGA 480GCGGGCTGCCTGGAATTGGGGTGTGGGTGGCTCCAGACGCCGCCACTCGAGCGGCTGTGGCTCCAGCCTCCTCCCCCGCTGATGGGGGCGGGAAGTGGCTGTGGGAGTCACCCGGGCGTG 600

ACTGCCCCCGAGGCCCCTCCTGCCGCGACAAGGGCGCTCCATAAAAGCCCGTTGGCGACCCGCTCTACGCATCCCACCGGTGGAGCTCATTCCTCGCAGAGGCGCCCAGAGCAGAGCACC 720

MetI -29CGCTGCGCAGAGACCACAGCCCGCCCGCCATG GTGAGTGCCAGGACCAACTGGGACAGCGGTGCGGGCCCCTAGACTCCCTTGAACTTGCCCTGCAGCCGGCCCCCTGAGCTTGTTCT 838

GCCAACTTGACACCCAGCTCTTTGGGGCAGCTAAATTTCACTTGCTGGACTCTGGTTCGACCCTCCACCTGTC CTTCTCCGGAGCCCTCCTATCTAGGCGCTCCACCTCCACGCCGGTTA 958TCTTTTGAAGGAAGGGAGAAGAACGTGGGGACACCTATCCCTXGGCAGCGGTGGGGTTTTGTTTGTTTGTTTGTTTAGTTGGTTACAACCAGGAATCCAGATTGCATGCGTGGTCCTAGA 1078GGGAGATTCCCCCAAAGTATTTTCCCTGGCATGGGGAGCCCCGTGCCCAGTCACCTCCAAGATCTGAACTGAGCCTCTGGGTATCTGACTGTCACAGGATCTGTAGCCACACAGATCATA 1198AGCGACTCAGAAGAAAAGTTTCTCCTCCGAGGGTGATTTAGGAACAAGAGGAGAAAAGTGGTCTTTCTTTAAAATAGAGGGCAGAACTTACAATTCTATTCTAAAAGGGGGCCCTCTGCT 1318TCTCACTAGGAGCGTTGGAGGGAAGTTTCATGAACAATGGGGCGGAGCAGAGGGGCCAGGTCCGGGCAAGTGGTTCTCTAGGGCTCTGGAAGTGGAGCCTGCCCAATCTGGGCTTTTTTT 1438

.. . . . s~~~~~~~~~~~~~~~~~~~~~~~~~~~MetLeu Gly -26CCTAGGGTCTGGGATGGGAATAGATGAGGGTCTAGAGTGGTAGCTGGAATCGGGACAAAGGCGAGCATTCTCTGCATCTCCAAGTCTGAGCCTTCTGTATCCACAG ATG CTA GGT 1553

Asn Lys Arg Met Gly Leu Cys Gly Leu Thr Leu Ala LQU Ser Leu Leu Val Cys Leu Gly Ile Leu Ala Glu Gly|Tyr Pro Ser Lys Pro 5AAC AAA CGA ATG GGG CTG TGT GGA CTG ACC CTC GCT CTA TCC CTG CTC GTG TGT TTG GGC ATT CTG GCT GAG GGG|TAC CCC TCC AAG CCG 1643

Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arl 35GAC AAT CCG GGC GAG GAC GCG CCA GCA GAG GAC ATG GCC AGA TAC TAC TCC GCT CTG CGA CAC TAC ATC AAT CTC ATC ACC AGA CAG AG GT 1734

GGGTGTATCCGCGGCTGGTATCTCGAGCCCCAAAAAACTGCGGTTCTGGGAATCTTGGACGCCAGAGACCATTTCTTTCTCCTTGTTCTGTCCCAGAATAGGACAGGATCCGGCATATAT 1854TCAGCTCTAGATAAATATGTGAGATGGGTACACACGCCACAAACTGGATACATTGTCTTTGTCACTAGACTTTCTCTCCAACCCCCGAAACTGAGATTCAGTACCTCTGGGCTGTGTCTG 1974TCTCCCACCCACCTTTCCTGCCTCTTTTGCTTACATCAGTTGGATGTTGTACTTACCCAGCACCCTCCTTGGGAAAAGGCAGCATCAGGAGCAGGGGTGCAACAGTGTAAAGGTGTAACC 2094TAATCTAGAGTAAACAACCACCCCCAAAGAAAACCAAGGAGACAGTCCAGCTGTCAGAGAGTGGATAGACCTTCCAGCCTGCCCAGGGCAAAGGGAGTGGCAGCATTTAGGGAAATAAAT 2214AAATAAATAAATAAATAAAT----------GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAACG----AACCATGGGTTTCTTCTGAACTATCTC 2334AGCCCTACACTTGAAAATTGCTAGATGGTCTTAAAAAGCTGACTTCTCTCATTTTATAGCAATTGCGTTCTGAAAAGAAAGCGTCCCATGAAGAAGAAACAAAACAAAGCAAAGCCTTTC 2454CGCTAAGGACTCTGGTGGATACAGACAGCTCCACTGGCTGGGGCCTGAGAGACAGACGGGGATCTGTATTTTTAATGTCCAGAAAGTGGAAGAGAGGTGCCTTGCCTTTCCTCTCAGCAT 2574TAAAATCAAACAGAAAAAAATCAAATCCAAGCTATTTTACAAACAATATGGGACTTAGTTTGCAAAGTCAGTGCATTTTAGTTCTAAATCATTTAACAACACATTTCAATTACCGAGAAC 2694

BGAATTCAGGTTGTTTGCCCGAAGTGTGAAGATTAACAAAATCAACTGAAGGACAGGTTAACCAAGAGCAGGCCACTAGCTCTAGTCTAATCTTGCAGAAAATCTTATATGAGAGTTGGGT 120GGTGAGGACGAGAATTGTTTACTTTTGCTCCCCTTGAGTCCAGCACAGGAGCAATGAGAAATCTCACCAATGCATCTGGATTTAGTTCTGCTGGGTTGGCACTCAAAACATCTGTCAGCT 240TTGTCCAGCTGTTCTACCTACTTGTTAATCCATCCAGACXAGGCCAAGTTTTCACTTGGAAATGAAGTGGCTGCCGGTGCCCCATGCCTTTCCTGAGAATGGGGAAAATCATATCATTTG 360CTATCAACTTGTCAACCCCCTTAGTGTTCTGAAGCAGTTAGTCTATTTTATCTTTACAGAGGAATGCAACCTCAGGTCCATATATTGTTCTTACCATATCCTACTATTACTGTTGGTCAG 480TAGGACTTGGTCATGCCTCAGAGAAAGCATTAGTGCATCCAACAGAGAGGAGAGAGTGCAAGATCCAGGGTATGTAGGGATCTGTGAGGAGGGCAGTGATGACAGGGATGTTAGTGTGGG 600TGCATTCAGGCAGCAGCTTCCTTTGGGCATCACTGATGACCTGAGTTCAATCCAGGAACCCAAAGAAGAAACAAAGGACCAACTTTGACTTCCAACAGCTGAGGAAGGGGTATGTGGGAG 720GGTTGGTAGGGAGAAATAGAAAGGGGTTAAATGAAGTGATTATAATATAATCTCAAAAATAAAATATTTATAAAGTACTTCAGAAAAATTTAAATTGTATCTGTGTGTGTATGTGTGTGT 840GTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATGTATGGATACCCACACTTGCATATG 960GAAGTCAGAGGATGGTTTTTCCAGAGTTGATCTCTCTCCTCTTTGGGTTCTGCAGATTGAACTCAGGTCATTGGGCTTGCATGGCCAGCACTCTCACCCTCTGGGCCGCCTCACCTGGTT 1080GTATGTATATATGTATGCGTATGTGTATATGCATATGTGGTCATGATAAAGTTGCTATGACATATGTGTCACACTCTGAGAATACTTATTAGCTCATGAACAGCTGGGAATAACATTTGA 1200

CPON. . . . ~~~~~~~~~~~~~~~gTyr Gly Lys Arg Sor Ser Pro Glu Thr 44

GTTTTGTGGGATACGCTCCCTTTTTCTCCTATGGTTACTCCAAGCTTACTTTATAAAACCTTGATTTTACTTTCTTGTTTCAGIA TATIGGC AAG AGA TCC AGC CCT GAG ACA 1311

Lou Ile Ser Asp Lou Lou Met Arg Glu Ser Thr Glu Asn Ala Pro Arg Thr Ars 61CTG ATT TCA GAT CTC TTA ATG AGA GAA AGC ACA GAA AAT GCC CCC AGA ACA AG GTATGGCCAAGCTAGG'GATGGAGATGTTGCTACAGAGCTTAAGGTGCCA 1413

GGCAAGGAGATCTAGGGGAGTCTGTGTGAACATTAGGATAGCAGCATCCGGGAAAGAGGGGTTAGGGTAGACACAGGGGATGGAGAAGTATACATGAAAGACCTTGCATTTCATGCATTC 1533ACAGCAAATCTCAACTTTCAAGGCATATTGTTTTCTAATACATATTATTTGGTGTCACTTTTTTAAAATGGCTGACTTCTAGCTCAGAAATTATATTCTATAGAATGTCATGCCTTATTA 1653GAAGTAAGGAAGTATAGAGCAAGCCCCCAACTCTATGTTGAAATGACCAATCATCCATTTGGGCTGGGCAATTTAGTTTTTGTACACTAGGTGGCATTCTCAAATGTCCTAGAGCCCGTA 1773GACCTCATGCGTTTGACTCTGCCTTTCCTGGCTTTATCCAAAACTAAAGATAAGTCAGGCGTTCCTGGCTACCTTTTGGTGTATTTTAGGTCAATATACTGATCTATTTTATTCAATGAA 1893TCATTGTTGGGTAATGTATTACCCTTAGGTGTTTATCTATAGTCTGCAAACTTTCTTGGAGTATAGATTGGTCTTGAGACAGATATCTGCCCAGATCTAAGGGCTCATCATTGGGCTAAG 2013CAGGTGCCTTCTGGTCAGGGAAGGAATAATGCTGAAAGAGGCTATCTACCTCACTTTCTTAACCCCTAACCTCATTTGCTGACCTTGAGTCCAGCTCACATGCCGTATCACACCAAGATG 2133TCAGCACACCAGTCCTATTGCTGTCCTATCCCTAGTAGTTAGCTGGCTAGCTTTCTGAGATCCAGAACTCTACACCATCAAAAACCATGTTCCCCAGAAGAGGTCACTAGCACACTTATT 2253CAGTGAGTATGACTGGAGAGTGACAGGAGGTTCATGGAGGTTCCCCCTGGTGACCTCATGGGTGACAACAGGACCAGCCCCTCCAGATAGTTTCTCTGAGGGAAGCGAATAAAGTACAAT 2373GCTTCTGTCCCTGGAGGCATCAACTTAAAGTTAACCAGTGAGTGCTAGGGAATGGTGGGAAATTATAAGCATTGTTTTTGATGCTAAACAATAAGCATCCTTCTGTGTCTCATTGATATG 2493CATGGGATATTTGCCACAGCAAAAGTTTAGGCTAGGCTATGTATACTTAGGATTTGGAAGTGATGATCCAGACCTACTTAGAACTCTGCTGCCTGCTTTGTTTTGTAGCCTGAAATAACA 2613CAGTTAGTGCAGGAAATAAAGTTAATTGCTCCCTGACTGACAGACTCAATCATGCGCAATTAGAAGGAGACAAAAGTTGATAGCCTTGGAGTTGCATGTGAGTGCTGCTTTATTAAAAGG 2733GGCTCTTTATAGTCTAATAAATAGCATCTCACTGCATAAAAGGCTTGCTGGGAAACAACAGAGCATATTCAGAGTTGAATAACACTAACTTCTTGTTATAGTTTCATGTGATTCCTGTGG 2853TTTTGATCTTCAGAAGTACTTTGGAGTCCCTTAGCTCCTTTGATATGTACAGAGGCTTCTAAGTACTCCCTTAGAGACTGCAGTTCCCATGGGCAAAAGCTGATGAACTGGGTCTTCATT 2973GTGCTGCTGTTCTCCGTAGCCTCTCATTCAGAGTGCCAACAGGAGGCTTCACAATAGCTTCTGATTATCTTGTAGCTCAGGGTGGAGAGTCTCACTCTTGTAAGACCCCGGGAGCTCCCA 3093

. ~ ~~~~sg Lou Glu Asp Pro Ser Met Trp *** ...69TGTTTTGCCTCTTGTGTTTTACAG G CTT GAA GAC CCT TCC ATG TGG TGATGGGAAATGAAACTTGCTCTCCTGACTTTTCCTAGTTTCCCCCCACATCTCATCTCATCCTG 3204

TGAAACCAGTCTGCCTGTCCCACCAATGCATGCCACCACCAGGCTGGATTCCGACCCATTTCCCTTGTTGTCGTTGTATATATGTGTGTTTA TAAPrTATCATGCATTCAAAATTGTG 3324TTCTCTGTGAATAATCTGCTATCACAATAGAAAGGATTAGGTTAGCCCTTAAATTATAATCAGCCTCAAGTAACAAACTCAGTTTGTTTCACCAACATTAAGCAATGGTCATGAAGAGTA 3444AATAAATAATTATGCTGCCCTTGGAAGAATTTATCTCTTAGAACTTTGTGCAAAATGTCTCATGTCCATCTATCTACTATAGCAGGCATGAGTGGATGACTGAACACCTGGTGTGACTAC 3564

FIG. 2. Partial nucleotide sequence and deduced amino acid sequence of the rat neuropeptide Y gene from the Hae library. A and B are asidentified in Fig. 1. Arrows mark splice junctions. Dashes show gaps as compared to the allelic neuropeptide Y gene from the Eco library (seeTable 1). The vertical bar indicates the boundary between the signal peptide and the mature neuropeptide Y peptide. The Gly-Lys-Arg process'nsite is within a box. CPON indicates the C-terminal peptide of the neuropeptide Y precursor. The presumed TATA-like promoter element isunderlined, as are the 5'- and 3'-untranslated regions of the neuropeptide Y mRNA. Star denotes the putative start site for transcription. Thepolyadenylylation signal is within a box. The unsequenced segment between A and B in the figure is -1.85 kb.

ison, only has the second methionine codon. The predicted tions to mRNA from various rat organs (Fig. 3A). High levelsrat neuropeptide Y precursor has 98 amino acid residues. A were observed in brain, heart, spleen, and adrenal gland.signal peptide of 29 amino acids precedes the mature Considerably lower levels were found in ovary, thymus,neuropeptide Y peptide, which is followed by a Gly-Lys-Arg kidney, and liver whereas testis contained undetectableprocessing site. This leaves a carboxyl-terminal peptide of 30 amounts. Identical results were obtained using probes cov-amino acids in agreement with human preproneuropeptide Y ering exons 1 and 3 (data not shown), confirming the(15). specificity of the exon 2 probe. Further analyses of the brain

Levels of Neuropeptide Y mRNA in Peripheral Organs and revealed presence of neuropeptide Y mRNA in all subregionsin the Central Nervous System. A probe containing exon 2 of tested (Fig. 3B). The highest levels were found in cortex andthe neuropeptide Y gene (see Fig. 1) was used in hybridiza- olfactory bulb.

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Table 1. Nucleotide sequence differences between twoneuropeptide Y alleles

Base(s)Nucleotide Gene from Gene from

position in Fig. 2A Hae library Eco library67 G A562 A C1362 G A2020 ATGTT2024 T G2132 G C2164 C T2235 - AAATAAATGA2304 AACG

The organs and brain subregions that contain the highestlevels of neuropeptide Y mRNA display, in addition to themature 800-nt mRNA, also two weekly hybridizing largermRNA species (:3.5 and 7 kb, respectively; see Fig. 3). Theidentity of these transcripts is unknown. They do not appearto represent incompletely processed nuclear neuropeptide YRNA transcripts, as they are not detected with an intronprobe (data not shown).

Interestingly, the mature mRNA species seems to differslightly in size between organs. Gels with higher resolutionthan the one shown in Fig. 3A revealed that the mRNA ofheart, ovary, and spleen is -760 nt, whereas that of thymus,kidney, and adrenal gland is =820 nt. Brain contains bothmRNAs, and analysis ofthe various brain subregions showedthat pons/medulla has the short mRNA, whereas all othersubregions tested have the long mRNA. Similarly, mousespleen neuropeptide Y mRNA is shorter than the predomi-nant mouse brain mRNA (data not shown).

Developmentally, low neuropeptide Y mRNA levels weredetected in 16-day embryo brains (the first developmentalstage studied) (Fig. 3C). Levels increased drastically fromday 18 to day 20 and remained high in adult rats (10-12 weeksold).

DISCUSSIONThe rat neuropeptide Y gene described here emphasizesfurther the evolutionary relationship between neuropeptideY and pancreatic polypeptide, as the genes encoding theprecursors of these two peptides have introns in identicalpositions (25). The mature neuropeptide Y and pancreaticpolypeptide peptides are about 50%o homologous. However,the signal peptides and C-terminal peptides have divergedextensively. The amino acid sequence of neuropeptide Y isnow known in three species. They show a remarkably highdegree of conservation, since the rat and human sequencesare identical, and the porcine sequence has merely a con-servative methionine to leucine replacement. In comparison,the pancreatic polypeptide sequence of rat (32) has eight ornine differences from the pancreatic polypeptide sequencesof human, dog, pig, cow, and sheep. These species have oneto three differences among themselves.

Equally striking is the high conservation of the C-terminalpeptide of the neuropeptide Y precursor (amino acids 40-69).Only two replacements are found between rat and human,and both ofthese are ofconservative nature. The correspond-ing part of the pancreatic polypeptide precursor has divergedmuch more between rat and human, and the 20-amino acidsegment from position 40 to 59 of pancreatic polypeptide has6-10 differences between human, sheep, and dog. Thesequence conservation of this peptide suggests that it fulfillsan important function of its own, possibly as a neurotrans-mitter or neuromodulator.Immunohistochemical studies with antisera against neuro-

peptides (13) have primarily focused on the sites of storage,

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FIG. 3. Levels of neuropeptide Y mRNA in various rat organs(A), rat brain subregions (B), and embryonic rat brain (C) at differentstages ofdevelopment (E16, E18, and E20 are embryonic day 16, 18,and 20, respectively). Each lane contains 10 ,g of polyadenylylatedRNA. Abbreviations are as follows: total, total brain; p/m, pons andmedulla; hc, hippocampus; ctx, cerebral cortex; str, striatum; coll,colliculus; olf, olfactory bulb; thal, thalamus; and sept, septum.

i.e., nerve terminals. Hybridizations with radiolabeled DNAprobes, in contrast, reveal the sites of mRNA translation,i.e., the cell bodies. Using a probe from the rat neuropeptideY gene, we detected abundant neuropeptide Y mRNA in ratbrain, adrenal, heart, and spleen. The findings in brain extendthe immunochemical analyses by showing that neuropeptideY is not only stored in nerve terminals but is indeedsynthesized throughout the brain. These observations pro-vide further evidence for a role for neuropeptide Y in manyand/or general functions in the nervous system.

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Unexpectedly high levels ofmRNA were observed in heartand spleen. High levels of neuropeptide Y mRNA in heartcorroborates results showing that some neuropeptide Ypeptide remains in the heart after disruption of sympatheticnerve input by treatment with 6-hydroxydopamine (33, 34).The neuropeptide Y mRNA level in heart remains unalteredafter 6-hydroxydopamine treatment (data not shown). Theheart neuropeptide Y mRNA may be produced in intrinsicganglia (35-37). However, in the spleen there are no knownintrinsic neurones that can account for the high mRNA levelsobserved, although the spleen has neuropeptide Y after6-hydroxydopamine treatment (33, 34). The possibility re-mains that splenic neuropeptide Y is produced by non-neuronal cells, perhaps immune cells. Indeed, other neuro-peptides have been found in cells of the immune system (38).It should also be noted that unexpectedly high mRNA levels,as compared to protein levels, have been found for pre-proenkephalin in heart and testis (39, 40) and for prepro-opiomelanocortin in testis (41). A slightly shorter neuropep-tide Y mRNA is found in heart, ovary, spleen, and pons/medulla than in other organs and brain subregions. Thiscannot be explained by alternative use of polyadenylylationsignals, as the two AATAAA sequences that occur in the 3'part of the gene are located 140 nt apart, whereas the sizedifference between the two mRNA species is only about 60nt. Another possibility would be omission of exon 3, becausethe translational reading frame would still be maintained ifthis exon were left out. This is ruled out because an exon 3probe still hybridizes to the shorter mRNA species (data notshown). The size difference also cannot be accounted for bydifferential use of promoters and transcriptional start sites,because a probe containing the 5'-untranslated region, indi-cated in Fig. 2, hybridizes to both mRNAs (data not shown).Another possibility is that the size difference is merely due toa difference in the length of the poly(A) tail added to themRNA, as has been described for both vasopressin andoxytocin in hypothalamus as compared to peripheral organs(42, 43).Genes that are expressed in the same types of cells have

been found to possess highly conserved nucleotide segmentsin their promoter regions, e.g., globin genes (30) and histo-compatibility antigen genes (44). The nucleotide sequence ofthe neuropeptide Y gene reported here will be of importancefor comparisons with other genes encoding neurotransmittersand neuromodulators, particularly those which coexist withneuropeptide Y in neurons and/or influence the same phys-iological parameters as neuropeptide Y. Comparison ofneuropeptide Y mRNA levels and gene promoter regionsbetween spontaneously hypertensive and normotensive ratswill be of special interest, as differences in neuropeptide Ypeptide levels have been detected (45) and in view ofthe genepolymorphism reported here.

We are grateful to Dr. Carolyn D. Minth and Dr. Jack E. Dixon forthe human neuropeptide Y cDNA clone, Dr. James Bonner for therat genomic libraries, and Dr. Lars Rask for providing sequencing gelfacilities. We thank Briti-Marie Johansson for technical assistanceand Christina Pellettieri for secretarial help. This work was support-ed by Grants B8524-100 and B1948-103 from the Swedish NaturalScience Research Council.

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Proc. Natl. Acad Sci. USA 84 (1987)

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