FR2737220A1 - NUCLEIC SEQUENCES ENCODING MELATONIN RECEPTORS AND THEIR APPLICATIONS - Google Patents

Abstract

Nucleic sequences coding for MEL1 A-type clawed toad ( DOLLAR iXenopus) melatonin receptors, also known as Mel1C, oligonucleotides included in such sequences, the uses thereof as a probe and for expressing proteins and/or fragments thereof having functional MEL1 A-type melatonin receptor activity, vectors useful for said expression, cellular hosts containing said vectors, and a melatonin receptor study model, are disclosed. A method for screening substances that are agonists or antagonists of the proteins having melatonin receptor activity, and kits for detecting the degree of affinity of various substances for said proteins having melatonin receptor activity, are also disclosed. Said nucleic sequences coding for functional MEL1 A-type melatonin receptors are selected from the following sequences: SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

Description

 The present invention relates to nucleic acid sequences coding for MEL iA type xenopal melatonin receptors, to oligonucleotides included in said sequences, to their applications as a probe and for the expression of proteins and / or fragments thereof having MEL1A A-functional melatonin receptor activity, vectors useful for said expression, cellular hosts containing said vectors and a melatonin receptor study model.

 The present invention also relates to a method for screening substances, having an agonist or antagonist effect on proteins having a melatonin receptor action, and kits or kits for detecting the degree of affinity of different substances for said melatonin receptor active proteins.

 A melatonin receptor of Xenopus laevis has been described (EBISAWA T. et al., Proc Natl Acad Sci USA, 1994, 91, 6133-6137). It is a protein of 420 amino acids.

 However, primers located in the 3 'part of the corresponding nucleic sequences did not make it possible to amplify the coding sequence for this protein.

 This is why the Applicant's goal is to search for nucleic sequences capable of encoding the MELi A melatonin receptor, which sequences are easily amplifiable in their entirety.

The present invention relates to nucleic acid sequences encoding functional receptors of melatonin, of the MELi A type and of a structure different from that of said receptor described in EBISAWA et al. and having coupling and regulatory properties different from those of the previously described receptor, which sequences are selected from the sequences SEQ ID N "1, SEQ
ID No. 3, SEQ ID No. 5 or SEQ ID No. 7, as presented in the sequence listing included in this Application.

 These sequences according to the invention have, at 3 ', a non-coding sequence more or less long (long sequences: SEQ ID NO 1 and SEQ ID NO 5 short sequences: SEQ ID NO 3 and SEQ ID No. 7), which comes from crucially on the regulation of RNA: 1/2 life of the RNA, modified compared to the sequences of the prior art.

 In particular, SEQ ID No. 1 and SEQ ID No. 3 code for a protein having 65 amino acids less, compared to the sequence described in the prior art, the 2 amino acids in the C-terminal position being, in addition. different.

This protein corresponds to the melatonin receptor called MEL1 Aa.

 SEQ ID No. 5 and SEQ ID No. 7 encode a protein also having 65 amino acids less, compared to the sequence described in the prior art, in addition, 6 amino acid residues are different from said sequence of the prior art, this protein corresponds to the melatonin receptor called MELi Ab.

 Both the MEL i Aa receptor coding sequence and the MELI Ab receptor coding sequence result in modifications in the regulation of RNA, related to the 3 'non-coding sequence (agonist ligand regulation, life of the RNA).

 The subject of the present invention is also the fragments of said sequences, useful either for a functional expression of the peptide corresponding to the corresponding melatonin receptor. or at the detection of the coding sequence for said receiver.

Among said fragments, the invention encompasses, inter alia
a sequence consisting of a 230 base pair nucleotide segment, corresponding to the nucleotides 1057-1286 of SEQ ID No. 1 or SEQ ID No. 5;
a sequence consisting of a 69 base pair nucleotide segment, corresponding to nucleotides 1057-1125 of SEQ ID No. 3 or SEQ ID No. 7.

 The present invention also relates to nucleotide probes, characterized in that they hybridize with the nucleotide sequences as defined above.

 Suitable hybridization conditions include the following: hybridization is carried out at 420C in a buffer comprising 4X 40% formamide SSC, 0.2% SDS and 5X Denhardt buffer.

Such probes have the advantage in particular of allowing the characterization of the various variants of the functional receptor of melatonin of the type
MEL1 A (MEL1 Aa or MEL1 Ab).

 The subject of the present invention is also proteins and / or protein fragments, characterized in that they are coded by a nucleotide sequence as defined above and in that they exhibit a melatonin receptor activity of the type MELi functional.

According to the invention, said protein is selected from any one of SEQ ID NO 2 (SEQ ID NO 4 being identical to SEQ
ID No. 2) or SEQ ID No. 6 (SEQ ID No. 8 being identical to SEQ ID No. 6), as presented in the sequence listing included in this Application.

 SEQ ID N 2 and N 4 correspond to the melatonin receptor designated MET 1 Aa, SEQ ID N 6 and N 8 correspond to the melatonin receptor called MELi Ab.

 These 2 proteins, the structure of which is different from that of the protein of the prior art, unexpectedly exhibit biological reactions and signals different from those previously described, in particular as regards the coupling to G proteins.

 The present invention also relates to a recombinant cloning and / or expression vector. characterized in that it comprises a nucleotide sequence according to the invention.

 Within the meaning of the present invention, the expression "recombinant vector" is understood to mean a plasmid, a cosmid, and a phage.

 According to an advantageous embodiment of said vector, it consists of a recombinant vector in which is inserted a nucleotide sequence as defined above, which vector is an expression vector of a protein having a functional receptor activity of the Melatonin type MEL i A, according to the invention.

 According to an advantageous arrangement of this embodiment, said vector consists of a plasmid pcDNA3 (Invitrogen), comprising an RSV promoter and SEQ ID No. 1.

Such a plasmid has been called X-MELla and was deposited under No. I-1583 dated June 7, 1995 in the National Collection of Cultures of Canada.
Microorganisms held by the Pasteur Institute.

 According to another advantageous arrangement of this embodiment, said vector consists of a plasmid pcDNA3 (Invitrogen), comprising an RSV promoter and SEQ ID No. 5.

Such a plasmid has been called X-MEL1b and was deposited under No. I-1584 dated June 7, 1995, with the National Collection of Cultures.
Microorganisms held by the Pasteur Institute.

 The present invention also relates to a suitable host cell, obtained by genetic transformation, characterized in that it is transformed by an expression vector according to the invention.

 Such a cell is capable of expressing a protein, having a functional melatonin receptor activity, of the MEL i A.

 According to an advantageous embodiment, the host cell is constituted in particular by the cells of the L-line.

 The subject of the present invention is also a process for expressing a protein according to the invention, characterized in that the host cell resulting from the transformation with a vector containing a nucleotide sequence according to the invention coding for a protein having functional MEL 1A melatonin functional receptor activity is cultured to produce and transport said membrane-expressed protein such that the transmembrane sequences of said receptor are exposed on the surface of the host membrane transformed cell.

 The subject of the present invention is also a model for studying melatonin receptors of the MEL1 A type, characterized in that it consists of host cells in accordance with the invention, that is to say expressing a receptor MEL 1 A type melatonin on the surface of their cell membrane.

 Such a model allows the study, from a pharmacological point of view, of melatonin receptors, in particular by allowing the identification of ligands specific for MEL1 A receptors (agonists and antagonists) and thus the development of drugs. active on the regulation of the biological clock, with particularly interesting applications, particularly in the cardiovascular (lipolysis) and oncology (phasing of cells).

The subject of the invention is also a method for detecting the capacity of a substance to behave as a ligand with respect to a protein according to the invention, which method is characterized in that it comprises
wherein said substance is brought into contact with a host cell previously transformed with an expression vector according to the invention, which host cell expresses said protein (melatonin receptor), and which contacting is carried out under conditions allowing forming a bond between at least one of the specific sites and said substance and
the detection of the possible formation of a ligand-protein complex.

The present invention further relates to a method for studying the affinity of a protein according to the invention for one or more specific ligands, which process is characterized in that it comprises
the transformation of a suitable host cell with a vector according to the invention,
the culture of the transformed host cell, under conditions permitting the expression of the melatonin receptor of the MEL1 A type, encoded by the nucleotide sequence, and the transfer of the expressed melatonin receptor towards the membrane of said cell so that the transmembrane sequences of the melatonin functional receptor are exposed on the surface of the transformed host cell,
contacting said cell with the determined ligands and
detecting an affine reaction between said transformed cell and said determined ligands.

The invention further relates to a kit for detecting the possible affinity of a ligand for a protein according to the invention, which kit is characterized in that it comprises
a culture of host cells transformed with an expression vector according to the invention,
optionally, if necessary, physical or chemical means for inducing the expression of a protein (melatonin receptor of the MEL1 A type), encoded by a nucleotide sequence according to the invention, contained in a vector whose promoter is inducible
one or more control ligands having defined affinities for said protein, and
physical or chemical means for characterizing the biological activity of the expressed protein.

Unexpectedly, the type melatonin receptors
MEL1 A according to the invention are effectively functional receptors and allow the realization of all the aforementioned applications.

In addition to the foregoing, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention, with reference to the appended drawings, in which which
Figure 1 is a schematic diagram of the technique of cloning the melatonin receptor cDNA from Xenopl®;
FIG. 2 illustrates the different cloning fragments used,
FIG. 3 illustrates the cloning and sequencing steps of the cDNA fragments of the melatonin receptor,
FIG. 4 illustrates the coding portion of the cDNA sequence coding for the functional receptor of the MEL1 A type melanoma (a or b) in comparison with the sequence EBISAWA et al. (referenced above)
- Figure 5 illustrates the melatonin-L-cell binding expressing a receptor according to the invention (MELI Aa or MEL1 Ab).

 It should be understood, however, that these examples are given solely by way of illustration of the object of the invention, of which they in no way constitute a limitation.

EXAMPLE 1 Isolation and identification of the 4 functional sequences of the melatonin receptor of MEL1 A.

 a) Amplification of the coding part of the Xenopus laevis melatonin receptor.

Total RNA from Xeziopus laevis skin is pretreated with Promega DNAse I for 20 min at 370C. The cDNA is synthesized from the total RNA of the skin of Xenopus hie screws using the MMLV reverse transcriptase
Stratagene Stratagript RNAse H- (30 min at 37 ° C., 5 min at 95 ° C.). The cDNA is amplified by PCR with different pairs of oligonucleotides specific for the melanin receptor Xeiiopzis hie screw. The PCR conditions are as follows: Boehringer Mannheim Pwo DNA polymerase with its own buffer (2 mM MgSO4) in a volume of 50 ml. The cDNA is denatured for 2 min at 94 ° C., amplified 40 times and followed by an elongation of 3 min at 72 C. One cycle corresponds to 15 sec. at 94 C; 30 sec. at the specific temperature of the oligonucleotide pair and 90 sec. at 72 C (GeneAmp PCR
System 9600 from Perkin-Elmer Cetus). The pairs of oligonucleotides are (Figure 2):
fragment 1: 28S - 241AS, specific temperature of 54 C,
fragment 2: 183 S - 462AS, specific temperature of 50 C, fragment 3 + 4: 391S - 1061AS, specific temperature of 50 ° C.

b) Amplification of the 3 'end of the melatonin receptor of
Xenopus laevis.

 The total RNA of the skin of Xenopus laevis is pretreated with Promega DNAse I for 20 min at 37 ° C. The cDNA is synthesized from the total RNA of the skin of Xenopus laevis using the kit " 3'-AmpliFINDERTMRACE "from Clonatech. The oligonucleotides specific for the Xenopiles kievis melatonin receptor used for amplification are 929S and 972S at a temperature of 58 C.

 Several primers were tested for their ability to amplify the melatonin receptor cDNA (Figure 2).

 More than 80% of the desired cDNA was amplified using 4 different primer pairs that include overlapping sequences (fragments 1-4).

 All efforts to amplify the melatonin receptor cDNA using a primer located at the 3 'end of the coding region were unsuccessful.

 The remaining portion (fragment 5) of the melatonin receptor cDNA was isolated using a modified PCR reaction, using the polyadenylation site, at the 3 'end of the cDNA as primer (principle described in Figure 3) and specific primers melatonin receptor (924 S and 972 S) located in this part of the coding region, which could be amplified by PCR.

 Two amplification fragments (estimated at 250 bp (short fragment) and 450 bp (long fragment), respectively) were obtained using this approach.

 Digestion of these fragments with two restriction enzymes shows the expected restriction profile.

 Fragments 1-5 were cloned separately into a vector and the identity verified by sequencing.

 Fragment 1 corresponds to sequence 28-241 3 clones were sequenced, which are identical with the published sequence (EBISAWA et al.).

 Fragment 2 clones were sequenced that are not identical to the nucleic acid-published sequence, but do not change the sequence to amino acids.

 Fragments 3 + 4 3 clones were sequenced, one of them is identical to the published sequence, the other two differ from the first, but are identical to each other.

 Many of the nucleic acid replacements also change the corresponding amino acid sequence (see Figure 4).

Fragment 5 (short fragment) (see SEQ ID Nos. 5 and 7): all the clones show a strong homology with the published sequence of the melatonin receptor up to the methionine in position 3s3
Beyond methionine 353, two different amino acids were detected, ie tyrosine instead of leucine and valine in place of glycine, followed by a stop codon, resulting in the loss of 65 amino acids, if compared with the published sequence of melatonin receptor XLinopus lac vis
The amino acid alignment of said receptor, from Xenopus, human and sheep, shows that the published sequence of the melatonin receptor from Xenopus has a much longer C-terminal tail than the receptor of two others. species (REPPERT et al., 1994).

 Unlike the published sequence, the melatonin receptor according to the invention obtained from Xeiiopiis laevis has the same size as the melatonin receptor obtained from humans or sheep.

 This is reinforced because it was not possible to amplify the melatonin receptor using PCR primers located in the 3 'region of the published sequence even though the same material was started.

 4 clones of the tragment C (short fragment) were sequenced. 3 clones correspond to the published sequence up to 353 methionine and a clone has a mutation that does not change the corresponding amino acid.

 The noncoding region can be isolated from the 2 clones to the polyadenylation tail.

 Fragment 5 long (see SEQ ID No. 1 and NO 3) 4 clones were sequenced, one of them corresponds to the published sequence up to 353 methionine, 3 of them show the same change as observed in the short fragment, up to 353 methionine. In all clones, amino acids 354 and 355 are modified with respect to the published sequence and have a stop codon at position 356, as seen in the short fragment.

 In addition, the 3 'non-coding region of the long fragment is identical with that of the short segment, up to the polyadenylation tail, it further comprises 160 bp, followed by its own polyadenylation tail (see sequence listing , SEQ ID NO: 1 and N 3).

 The sequence differences found throughout the sequence are highlighted in Figure 4.

 Unexpectedly, sequencing of the melatonin receptor obtained by RT-PCR reveals two different sequences (MEL1 Aa and MEL1 Ab).

EXAMPLE 2 Construction of a Vector for the Expression of the Functional Receptor of Melatonin of the Type MEL1 A.

 The Xenopus laevis melatonin receptor was cloned according to the RT-PCR method (see Figure 1).

 RNA is isolated from Xenopus skin and transcribed into cDNA using reverse transcriptase.

 Selective amplification of the melatonin receptor cDNA is performed using PCR primers specific for this receptor.

The amplified receptor is cloned into the pcDNA3-RSV expression vector and its identity verified by sequencing as follows
Successive preligations of the fragments between them, thanks to restriction enzymes common two by two, made it possible to insert the whole of the melatonin receptor in the plasmid pcDNA3 / RSV at the HindIII / Bsp120I sites with a free end, under the control of the promoter of the johns sarcoma virus (RSV). Fragments 2 and 3 + 4 are firstly linked at the level of the common "HindIII" enzyme site at position 455 of the coding sequence. The latter is then linked with fragment 1 to the common site "Bsu36I" at position 202 and with fragment 5 at the common site "XbaI" at position 1016. Each fragment is previously cut by the two enzymes essential for successive ligations. purified, consisting of 1,2,3 + 4,5 fragments, whose 5 'and 3' ends are respectively HindIII and EcoRI at a sharp end, is finally inserted into pcDNA3 / RSV.

EXAMPLE 3 Pharmacology of the Functional Receptor of Melatonin-type
MEL1 A.

 The pcDNA3-RSV vector containing the coding region and the 3 'non-coding region of the Xenopus hievis melatonin receptor according to Example 2 is transfected into mouse L cells.

The day before, 3x105 cells were passed into the 25 cm2 fiasks in DMEM medium 4.5 g / l glucose; 1 mM glutamax; 1 mM pyruvate; 10% FCS (DMEM / FCS). On the day of transfection, the medium is changed (6 ml of
DMEM / FCS / flask) and the cells are incubated for 3 hours at 37 ° C. In parallel, a coprecipitation of the DNA and CaPO4 is prepared in the following manner: in a final volume of 500 ml, 30 mg of DNA are mixed carrier (Promega pGEM3Z), 2 mg of the pcDNA3-RSV vector containing the coding region and the 3 'non-coding region of the melatonin receptor of Xenopus laevis and CaCl2 (250 mM final) (Solution
AT). 450 ml of this Solution A are added dropwise, by vortexing, to 450 ml of
HBS 2x (1.64 g NaCl, 1.19 g Hepes, 0.04 g Na2HPO4-12H20 added to 100 ml
H2O) and the pH is adjusted to 7.12. The mixture is left without stirring at room temperature for 45 minutes. 400 ml of the coprecipitate are added to a flask containing the L cells. After 4 hours of incubation at 37 ° C., the cells are washed once with 6 ml of DMEM and then incubated for 2 min in 4 ml of 15% glycerol. Then the cells are washed twice with DMEM and left in 6 ml of DMEM / FCS, supplemented with 5 mM Nabutyrate at 37 ° C overnight. The next day, the medium is removed, the cells are washed again with DMEM / FCS and incubated overnight in DMEM / FCS. On the third day the cells are distributed in 96-well plates at different dilutions (1/2, 1/4, 1/8, 1/16, 1/32) in DMEM / FCS, supplemented with 450 m / ml of Geneticin. (G-418 Sulphate) from Gibco Life
Geneticin-resistant clones are tested for expression of the melatonin receptor by binding to (125) Iodo-melatonin (200 μM) in a final volume of 250 ml (buffer: 50 mM Tris / HCl pH 7). , 4.5 mM MgCl 2). Nonspecific binding is determined in the presence of 10 mM melatonin.

Results
The following table and Figure 5 illustrate the results.

<Tb>

<SEP> KD <SEP> (pM) <SEP> fmol / mg
<tb> MELi <SEP> Aa <SEP> 226 <SEP> 537
<tb> MEL1 <SEP> Ab <SEP> 190 <SEP> 311
<Tb>
Both receptors bind melatonin with high affinity, indicating that they are MELi A subtype receptors.

 As is apparent from the foregoing, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly, it encompasses all the contrary. variants that may come to mind of the technician in the field, without departing from the scope or scope of the present invention.

SEQUENCE LIST (1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: ADIR
(B) STREET: 1 STREET. CARLE HEBERT
(C) CITY: COURBEVOIE
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 92415 CEDEX
(ii) TITLE OF THE INVENTION: MELATONIN RECEPTORS
(iii) NUMBER OF SEQUENCES: 8
(iv) COMPUTER-DEPENDABLE FORM:
(A) SUPPORT TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release 41.0, Version 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1311 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: cDNA for AwNm
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1.1065
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
ATG ATG GAG GTG AT AGC ACT TGC TTG GAT TGC AGG ACA CCT GGT ACC 48
Met Met Glu Val Asn Ser Thr Cys Leu Asp Cys Arg Thr Pro Gly Thr 5 5 L.0 15
ATA CGA ACA GAG CAG GAT GCA CAG GAC AGC GCA TCT CAG GGA CTC ACC 96
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
20 25 30
TCT GCC CTG GCG GTG GTT CTT ATA TTC ACC ATT GTT GTG GAT GTC CTG 144
Ser Ala Leu Ala Val Val Leu the Phe Thr the Val Val Asp Val Leu
35 40 45
GGC AAT ATA TTG GTC ATT TTG TCm GTC CTG AGG AAC AAG AAG CTG CAG 192
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Hrg Asn Lilies Lilies Leu Gln
50, 60 iAT GCT GGA AAT CTC TT GTT GTC AGT TTG TCT ATT GCC GAT CTG GTT 240
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
65 7C 70 75 80
GTT GCT GTG TAT CCC TAT CCG GTA ATT CTC ATA GCT ATT TTC CAG AAT 288
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
85 90 95
GGG TGG ACG CTG GGA AAT ATC CAT TGT AGC ATC AGT GGC TTC CTG ATG 336
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
100 105 110
GGA CTC AGC GTT ATT GGA TCA GTC TTC AAC ATA ACA GCC ATA GCT ATC 384
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
115 120 125
AAC AGG TAT TGC ATC TAC TGC CAC AGC CTG AGA TAT GAC AAG CTT TAT 432
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Asp Lys Leu Tyr
130 135 140
AAT CAA AGA AGC ACC TGG TGC CTG TAC GGC CTG ACA TGG CTA ATA ACT 480
Asn Gln Arg Ser Tr Tr Cys Tyr Leu Gly Leu Thr Trp Leu Leu 145 150 155 160
ATA ATT GCA ATC GTG CCA AAC TTT TTT GTT GGA TCA CTA CAG TAT GAC 528
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
165 170 175
CCC AGG ATT TTT TCT TGC ACA TTT GCG CAG ACA GTG AGT TCC TCA TAC 576
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
180 185 190
ACC ATA ACTA GTA GTG GTG GTG CAT TTT ATA GTC CCT CTT AGT GTT GTG 624
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
195 200 205
ACA TTC TGT TAC TTA AGA ATA TGG GTT TTA GTG ATC CAA GTC AAA CAC 672
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
210 215 220
AGA GTT AGA CAA GAC TTC AAG CAA AAG TTG ACA CAA ACA GAC TTG AGA 720
Arg Arg Arg Gln Asp Phe Lys Gln Lys Leu Thr Asp Thrn Arg Leu Arg 225 230 235 240
AAT TTC TTG ATG ACC TT GTG GTC TT GTA CTT TT GCA GTT TGC TGG 768
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
245 250 255
GCC CCC TTA AAC TT ATC GGC CTT GCT GTG GCC ATT AAT CCG TTT CAT 816
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Phe His
260,265,270
GTG GCA CCA AAG ATT CCA GAA TGG CTG TTT GTT TTA AGC TAT TTC ATG 864
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
275 280 285
GCC TAT TTT AAC AGT TGT CTC AAT GCT GTT ATA TAT GGT GTG CTA AAT 912
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Tire Island Gly Val Leu Asn
290,295,300
CAA AAC TTC CGC AAG GAG TAC AAA ACA ATA CTG ATG TCC TTA TTG ACT 960
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Leu Thr
305 310 305 320
CCA AGA CCG TCG TTT CTT GAG ACA TCT AGA GGA GGA ACC GAG GGA TTG 1008
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
325 330 335
AAA AGT AAG CCC TCG CCA GCT GTA ACC AAC AAC AAT CAA GCA GAT ACG 1056
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
340,345,350
TAC GTG TAA ACACAGCTTT CCACCAATAT GTACTCTGTT TCAACTATGA 1105
Tyr Val *
355
ATACAAGTTT CTTTTATGGC AGTTGCACCA TGTGCCTAAT TCTGTCCATT CATTCTAAAA 1165
TTTTTGTATA AACATACATT CTGTTGGTTT GACAGGGACA AGAGGTCTTG CTTCTCTGCA 1225
GAAAAGAAAT ATTTTGAAAT CTTGGCTGAT TTGTTATTAA TAACCATAAA TGGAATATCT 1285 TAAAAAAAAA AAAAAAAAAA GAATTC 1311
(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 355 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SLQUENCE: SEQ ID NO: 2:
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
1 5 10 15
Arg Glu Glu Asp Gln Asp Gln Asp Ser Gln Ala Ser Gly Leu Thr
20 25 30
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
35 40 45
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
50 55 60
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
65 70 75 80
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
85 90 95
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
100 105 110
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
115 120 125
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Asp Lys Leu Tyr
130 135 140
Asn Gln Arg Ser Tr Tr Cys Tyr Leu Gly Leu Thr Trp Leu Leu 145 150 155 160
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
165 170 @ 175
Pro Arg Phe Ser Cys Thr Phe Ala Thr Gln Val Sr Ser Ser Tyr
180 185 190
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
195 200 205
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gin Val Lys His
210 215 220
Arg Arg Arg Gln Asp Phe Lys Gln Lys Leu Thr Thr Thr Asp Leu Arg 225 230 235 240
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
245 250 255
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Phe His
260,265,270
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
275 280 285
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Tire Island Gly Val Leu Asn
290,295,300
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Leu Thr 305 310 315 320
Pro Arg Leu Leu Phe Leu Asp Thr Arg Ser Gly Giy Thr Glu Gly Leu
325 330 335
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
340,345,350
Tyr Val *
355 (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1147 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1065
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
ATG ATG GAG GTG AT AGC ACT TGC TTG GAT TGC AGG ACA CCT GGT ACC 48
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
ATA CGA ACA GAG CAG GAT GCA CAG GAC AGC GCA TCT CAG GGA CTC ACC 96
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
TCT GCC CTG GCG GTG GTT CTT ATA TTC ACC ATT GTT GTG GAT GTC CTG 144
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
GGC AAT ATA TTG GTC ATT TTG TCT GTC CTG AGG MC AAG AAG CTG CAG 192
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lys Leu Gln
AAT GCT GGA AAT CTC TTT GTT GTC AGT TTG TCT ATT GCC GAT CTG GTT 240
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
GTT GCT GTG TAT CCC TAT CCG GTA ATT CTC ATA GCT ATT TTC CAG AAT 288
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
GGG TGG ACG CTG GGA AAT ATC CAT TGT AGC ATC AGT GGC TTC CTG ATG 336
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
GGA CTC AGC GTT ATT GGA TCA GTC TTC AAC ATA ACA GCC ATA GCT ATC 384
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
AAC AGG TAT TGC ATC TAC TGC CAC AGC CTG AGA TAT GAC AAG CTT TAT 432
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Asp Lys Leu Tyr
AAT CAA AGA AGC ACC TGG TGC CTG TAC GGC CTG ACA TGG CTA ATA ACT 480
Asn Gln Arg Ser Tr Tr Cys Leu Leu Leu Leu Tr Tr Leu Leu
ATA ATT GCA ATC GTG CCA AAC TTT TTT GTT GGA TCA CTA CAG TAT GAC 528
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
CCC AGG ATT TTT TCT TGC ACA TTT GCG CAG ACA GTG AGT TCC TCA TAC 576
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
ACC ATA ACTA GTA GTG GTG GTG CAT TTT ATA GTC CCT CTT AGT GTT GTG 624
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
ACA TTC TGT TAC TTA AGA ATA TGG GTT TTA GTG ATC CAA GTC AAA CAC 672
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
AGA GTT AGA CAA GAC TTC AAG CAA AAG TTG ACA CAA ACA GAC TTG AGA 720
Arg Arg Arg Gln Asp Phe Lys Gln Lys Leu Thr Gln Thr Asp Leu Arg
AAT TTC TTG ATG ACC TT GTG GTC TT GTA CTT TT GCA GTT TGC TGG 768
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
GCC CCC TTA AAC TT ATC GGC CTT GCT GTG GCC ATT AAT CCG TTT CAT 816
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Phe His
GTG GCA CCA AAG ATT CCA GAA TGG CTG TTT GTT TTA AGC TAT TTC ATG 864
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
GCC TAT TTT AAC AGT TGT CTC AAT GCT GTT ATA TAT GGT GTG CTA AAT 912
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Tire Island Gly Val Leu Asn
CAA AAC TTC CGC AAG GAG TAC AGA ATA AAA CTG ATG TTA TTG ACT 960
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Leu Thr
CCA AGA CTG TTG TT CTT GAC ACA TCT AGA GGA ACT GGA GGA GGA TTG 1008
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
AAA AGT AAG CCT TCG CCA GCT GTA ACC AAC AAC AAT CAA GCA ATG GG 1056
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
TAC GTG TAA ACACAGCTTT CCACCAATAT GTACTCTGTT TCAACTATGA 1105
Tyr Val *
ATACAAGTTT CTTTTATGGC AAAAAAAAAA AAAAAAGAAT TC, 1147
(2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 355 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
1 5 10 15
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
20 25 30
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
35 40
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
50 55 60
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
65 70 75 80
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
85 90 95
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
100 105 110
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
115 120 125
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Asp Lys Leu Tyr
130 135 140
Asn Gln Arg Ser Tr Tr Cys Tyr Leu Gly Leu Thr Trp Leu Leu 145 150 155 160
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
165 170 175
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
180 185 190
Thr Island Thr Val Val Valley Val Val Val Val Phe Val Val Leu Val Val 195 200 205
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
210 215 220
Arg Arg Arg Gln Asp Phe Lys Gln Lys Leu Thr Asp Thrn Arg Leu Arg 225 230 235 240
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
245 250 255
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Phe His
260,265,270
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
275 280 285
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Tire Island Gly Val Leu Asn
290,295,300
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Leu Thr 305 310 315 320
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
325 330 335
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
340,345,350
Tyr Val *
355
(2) INFORMATION FOR SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1312 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1065
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
ATG ATG GAG GTG AT AGC ACT TGC TTG GAT TGC AGG ACA CCT GGT ACC 48
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
ATA CGA ACA GAG CAG GAT GCA CAG GAC AGC GCA TCT CAG GGA CTC ACC 96
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
TCT GCC CTG GCG GTG GTT CTT ATA TTC ACC ATT GTT GTG GAT GTC CTG 144
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
GGC AAT ATA TTG GTC ATT TTG TCT GTC CTG AGG AAC AAG AAG CTG CAG 192
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
AAT GCT GGA AAT CTC TTT GTT GTC AGT TTG TCT ATT GCC GAT CTG GTT 240
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
GTT GCT GTG TAT CCC TAT CCG GTA ATT CTC ATA GCT ATT TTC CAG AAT 288
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
GGG TGG ACG CTG GGA AAT ATC CAT TGT AGC ATC AGT GGC TTC CTG ATG 336
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
GGA CTC AGC GTT ATT GGA TCA GTC TTC AAC ATA ACA GCC ATA GCT ATC 384
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
AAC AGG TAT TGC TAC ATC TGC CAC AGC CTG AGA TAT GAC AAG CTT TT 432
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Lys Leu Phe
AAT CAA AGA AGC ACC TGG TTC CTG TAC GGC CTG ACA TGG ATA CTA ACC 480
Asn Gln Arg Ser Thr Trp Phe Tyr Leu Gly Leu Thr Trp Ile Leu Thr
ATA ATT GCC ATT GTG CCA AAC TTT TTT GTT GGA TCA CTA CAG TAT GAC 528
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
CCC AGG ATT TTC TCT TGC ACA TTT GCG AGC ACC GTA AGT TCC TCA TAC 576
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
ACC ATA ACA GTA GTG GTA GTG CAT TTT ATA GTC CCT CTT AGT GTT GTG 624
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
ACA TTC TGC TAC TTA AGA ATA TGG GTT TTA GTG ATC CAA GTC AAA CAC 672
Thr Phe Cys Tyr Leu Arg Isle Trp Val Leu Val Gln Island Val Lys His
AGA GTT AGA CAA GAC TTC AAG CAA AAG TTG ACA CCA ACA GAC TTG AGA 720
Arg Arg Arg Gln Asp Phe Lys Gln Leu Lys Thr Thr Thr Asp Arg Arg
AAT TTC TTG ACC ATG TT GTG GTC TT GTA CTT TT GCC GTT TGC TGG 768
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
GCA CCC TTG AAT TT ATC GGC CTT GCT GTG GCC ATT AAC CCA CTC CAC 816
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Leu His
GTG GCA CCA AAG ATT CCA GAG TGG TTG TTT GTG TTA AGC TAT TTC ATG 864
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
GCC TAT TTT AAC AGC TGT CTC AAT GCT GTC ATC TAC GGT CTG CTA AAT 912
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn
CAA AAC TTC CGC AAG GAA TAC AAA CGA ATA ATG TTG TCC TTA TGG ACT 960
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met * Leu Trp Thr
CCA AGA CTG TTG TT CTT GAC ACA TCT AGA GGA ACT GGA GGA GGA TTG 1008
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
AAA AGT AAG CCT TCG CCA GCT GTA ACC AAC AAC AAT CAA GCA ATG GG 1056
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
TAC GTG TAA ACACAGCTTT CCACCAATAT GTACTCTGTT TCAACTATGA 1105
Tyr Val *
ATACAAGTTT CTTTTATGGC AGTTGCACCA TGTGCCTAAT TCTTT CATTCTAAAA 1165
TTTTTGTATA AACATACATT CTGTTGGTTT GACAGGGACA AGAGGTCTTG CTTCTCTGCA 1225
GAAAAGAAAT ATTTTGAAAT CTTGGCTGAT TTGTTATTAA TAACCATAAA TGGAATATCT 1285 TTAAAAAAA AAAAAAAAAA AGAATTC 1312
(2) INFORMATION FOR SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 355 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF NOLECULE: Protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
1 5 10 15
Arg Glu Glu Asp Gln Al Gln Asp Ser Ala Ser Gln Gly Leu Tyr
20 25 30
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Island Val Val Asp Val Leu
35 40 45
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
50 55 60
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
65 70 75 80
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
85 90 95
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
100 105 110
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
115 120 125
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Lys Leu Phe
130 135 140
Asn Gln Arg Ser Thr Trp Phe Tyr Leu Gly Leu Thr Trp Ile Leu Thr 145 150 155 160
Ile Ile Ala Ile Val Pro Asn Phe Phe Val Gly Ser Leu Gin Tyr Asp
165 170 175
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
180 185 190
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
195 200 205
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
210 215 220
Arg Arg Arg Gln Asp Phe Lys Gln Leu Lys Thr Pro Thr Asp Leu Arg 225 230 235 240
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
245 250 255
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Leu His
260,265,270
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
275 280 285
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn
290,295,300
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Trp Thr 305 310 315 320
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
325 330 335
Lys Ser Lys Ser Ser Pro Ala Val Thr Asn Asn Asin Gin Ala Asp Met
340,345,350
Tyr Val *
355
(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1147 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1065
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
ATG ATG GAG GTG AT AGC ACT TGC TTG GAT TGC AGG ACA CCT GGT ACC 48
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
ATA CGA ACA GAG CAG GAT GCA CAG GAC AGC GCA TCT CM GGA CTC ACC 96
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
TCT GCC CTG GCG GTG GTT CTT ATA TTC ACC ATT GTT GTG GAT GTC CTG 144
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
GGC AAT ATA TTG GTC ATT TTG TCT GTC CTG AGG AAC AAG AAG CTG CAG 192
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
AAT GCT GGA AAT CTC TTT GTT GTC AGT TTG TCT ATT GCC GAT CTG GTT 240
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
GTT GCT GTG TAT CCC TAT CCG GTA ATT CTC ATA GCT ATT TTC CAG AAT 288
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
GGG TGG ACG CTG GGA AAT ATC CAT TGT AGC ATC AGT GGC TTC CTG ATG 336
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
GGA CTC AGC GTT ATT GGA TCA GTC TTC AAC ATA ACA GCC ATA GCT ATC 384
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
AAC AGG TAT TGC TAC ATC TGC CAC AGC CTG AGA TAT GAC AAC CTT TTT 432
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Lys Leu Phe
AAT CAA AGA AGC ACC TGG TTC CTG TAC GGC CTG ACA TGG ATA CTA ACC 480
Asn Gln Arg Ser Thr Trp Phe Tyr Leu Gly Leu Thr Trp Ile Leu Thr
ATA ATT GCC ATT GTG CCA AAC TTT TTT GTT GGA TCA CTA CAG TAT GAC 528
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
CCC AGG ATT TTC TCT TGC ACA TTT GCG AGC ACC GTA AGT TCC TCA TAC 576
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
ACC ATA ACA GTA GTG GTA GTG CAT TTT ATA GTC CCT CTT AGT GTT GTG 624
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
ACA TTC TGC TAC TTA AGA ATA TGG GTT TTA GTG ATC CAA GTC AAA CAC 672
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
AGA GTT AGA CAA GAC TTC AAG CAA AAG TTG ACA CCA ACA GAC TTG AGA 720
Arg Arg Arg Gln Asp Phe Lys Gln Lys Leu Thr Pro * Asp Leu Arg
AAT TTC TTG ACC ATG TT GTG GTC TT GTA CTT TT GCC GTT TGC TGG 768
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Als Val Cys Trp
GCA CCC TTG AAT TT ATC GGC CTT GCT GTG GCC ATT AAC CCA CTC CAC 816
Ala Pro Leu Asn Phe Island Gly Leu Ala Val Ala Island Asn Pro Leu His
GTG GCA CCA AAG ATT CCA GAG TGG TTG TTT GTG TTA AGC TAT TTC ATG 864
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
GCC TAT TTT AAC AGC TGT CTC AAT GCT GTC ATC TAC GGT CTG CTA AAT 912
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn
CAA AAC TTC CGC AAG GAA TAC AAA CGA ATA ATG TTG TCC TTA TGG ACT 960
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Trp Thr
CCA AGA CTG TTG TT CTT GAC ACA TCT AGA GGA ACT GGA GGA GGA TTG 1008
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
AAA AGT AAG CCT TCG CCA GCT GTA ACC AAC AAC AAT CAA GCA ATG GG 1056
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
TAC GTG TAA ACACAGCTTT CCACCAATAT GTACTCTGTT TCAACTATGA 1105
Tyr Val *
ATACAAGTTT CTTTTATGGC AAAAAAAAAA AAAAAAGAAT TC 1147
(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 355 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Met Glu Val Asn Thr Ser Cys Leu Asp Cys Arg Thr Pro Gly Thr
1 5 10 15
Arg Thr Glu Gln Gln Asp Ala Gln Asp Ser Ala Ser Gln Gly Leu Thr
20 25 30
Ser Ala Leu Ala Val Val Leu Ile Phe Thr Val Val Asp Asp Val Leu
35 40
Gly Asn Ile Leu Val Ile Leu Ser Val Leu Arg Asn Lily Lys Leu Gln
50 55 60
Asn Ala Gly Asn Leu Phe Val Ser Val Ser Leu Ala Island Asp Leu Val
65 70 75 80
Val Ala Val Tyr Pro Tyr Pro Val Ile Leu Ile Ala Ile Phe Gln Asn
85 90 95
Gly Trp Thr Leu Gly Asn Ile His Cys Gln Ile Ser Gly Phe Leu Met
100 105 110
Gly Leu Ser Val Gly Ser Island Val Phe Asn Island Thr Ala Island Ala Ile
115 120 125
Asn Arg Tyr Cys Tyr Isle Cys His Ser Leu Arg Tyr Asp Lys Leu Phe
130 135 140
Asn Gln Arg Ser Thr Trp Phe Tyr Leu Gly Leu Thr Trp Ile Leu Thr 145 150 155 160
Ile Ala Island Val Pro Island Asn Phe Phe Valley Val Gly Ser Leu Gln Tyr Asp
165 170 175
Pro Arg Phe Ser Cys Thr Phe Ala Gln Thr Ser Ser Ser Ser Tyr
180 185 190
Thr Island Thr Val Val Val Val Val Val Val Val Val Val
195,200
Thr Phe Cys Tyr Leu Arg Ile Trp Val Leu Val Ile Gln Val Lys His
210 215 220
Arg Arg Arg Gln Asp Phe Lys Gln Leu Lys Thr Pro Thr Asp Leu Arg 225 230 235 240
Asn Phe Leu Thr Phe Val Val Phe Val Leu Phe Ala Val Cys Trp
245 25G 255
Ala Pro Leu Asn Phe Island Gly re Val Ala Island Asn Pro Leu His
260 ^ 55 270
Val Ala Pro Lys Pro Island Glu Trp Leu Phe Leu Val Ser Tyr Phe Met
275 230 285
Ala Tyr Phe Asn Ser Cys Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn
290,295,300
Gln Asn Phe Arg Lys Glu Tyr Lys Arg Ile Leu Met Ser Leu Trp Thr 305 310 315 320
Pro Arg Leu Leu Phe Leu Asp Ser Ser Arg Gly Gly Thr Glu Gly Leu
325 330 335
Lys Ser Lys Ser Ser Pro Ala Val Asn Thr Asn Asn Gln Ala Asp Met
340,345,350
Tyr Val *
355

Claims (4)

claims  1) Nucleic sequences coding for functional receptors of melatonin of MET 1 type. and characterized in that they are selected from the following sequences: SEQ ID N 1 SEQ ID N 3, SEQ ID N 5 or SEQ ID N 7.  2) Fragments of the sequences according to claim 1, characterized in that they are selected from the group which comprises  a sequence consisting of a segment of 230 nucleotide base pairs, corresponding to the nucleotides 1057-1286 of SEQ ID N I or SEQ ID NO 5  a sequence consisting of a 69 base pair nucleotide segment corresponding to nucleotides 1057-1125 of SEQ ID No. 3 or SEQ ID N 7.  3) nucleotide probes, characterized in that they hybridize with the nucleotide sequences according to claim 1 or claim 2.  4) Protein and / or protein fragments, characterized in that they are encoded by a nucleotide sequence according to claim 1 or claim 2 and in that they exhibit a melanin receptor activity of the functional MEL1A type.  z) Protein according to claim 4, characterized in that it has any of the following sequences. SEQ ID N 2 or SEQ ID N 6.  6) recombinant cloning and / or expression vector, characterized in that it comprises a nucleotide sequence according to claim 1 or claim 2.  7) Vector according to claim 6. characterized in that it consists of a plasmid comprising an RSV promoter and SEQ ID N 1. Cultures of Microorganisms held by the Pasteur Institute.  8) Vector according to claim 7, characterized in that it was deposited under No. I-1583 dated June 7, 1995 from the National Collection of  9) Vector according to claim 6, characterized in that it consists of a plasmid comprising an RSV promoter and SEQ ID No. 5. Cultures of Microorganisms held by the Institut Pasteur.  10) Vector according to claim 9, characterized in that it was deposited under No. I-1584 dated June 7, 1995 with the National Collection of  11 ") Suitable host cell, obtained by genetic transformation, characterized in that it is transiormee by an expression vector according to any one of claims 6 to 10.  12 ") Cell host according to claim 11, characterized in that it consists of the cells of the L line.  13) The expression process of a protein according to claim 4 or claim 5, characterized in that the host cell resulting from the transformation with a vector containing a nucleotide sequence according to claim 1 or claim 2 coding for a protein having functional MET1 melatonin functional receptor activity, is cultured to produce and transport said membrane-expressed protein such that the transmembrane sequences of said receptor are exposed on the membrane surface of the membrane. transformed cellular host. MEL1A, characterized in that it consists of host cells according to claim 11 or claim 12 that is to say expressing a functional receptor of melatonin type MELi A on the surface of their cell membrane.  14 ") Model study of melatonin receptors of type  the detection of the possible formation of a ligand-protein complex.  contacting said substance with a host cell previously transformed with an expression vector according to any one of claims 6 to 10, which host cell expresses said protein (melatonin receptor), and which contacting is performed under conditions allowing the formation of a bond between at least one of the specific sites and said substance and  A method of detecting the ability of a substance to behave as a ligand to a protein according to claim 4 or claim 5, which process is characterized in that it comprises  detecting an affine reaction between said transformed cell and said determined ligands.  contacting said cell with the determined ligands and  culturing the transformed host cell under conditions permitting the expression of the MEL1 A-type melatonin receptor, encoded by the nucleotide sequence, and the transfer of the expressed melatonin receptor to the membrane of said cell so that the transmembrane sequences of the functional melatonin receptor are exposed on the surface of the transformed host cell;  transforming a suitable host cell with a vector according to any one of claims 6 to 10,  16) Process for the study of the affinity of a protein according to claim 4 or claim 5 for one or more specific ligands, which process is characterized in that it comprises  physical or chemical means for characterizing the biological activity of the expressed protein.  one or more control ligands having defined affinities for said protein, and  - eventually. if necessary, physical or chemical means for inducing the expression of a protein (melatonin receptor of the MELi A type), encoded by a nucleotide sequence according to claim 1 or claim 2, contained in a vector whose promoter is inducible,  a host cell culture transformed with an expression vector according to any one of claims 6 to 10,  17) Kit for detecting the possible affinity of a ligand for a protein according to claim 4 or claim 5, which kit is characterized in that it comprises: