CZ2000681A3 - Oligonucleotides allowing the identification of precursors of amidated polypeptide hormones - Google Patents

Abstract

Oligonucleotides and their use as probes for identifying mRNA encoding precursors of amidated polypeptide hormones, thereby identifying novel amidated polypeptide hormones. Oligonucleotides with a disclosed nucleotide sequence and a method for identifying precursors of amidated hormones.

Description

igonucleotides allowing identification of precursors amidated after 1-peptide hormones

Technical field

The present invention relates to novel oligonucleotides and their use as probes to identify mRNAs that encode amidated after 1-peptide hormone precursors and to identify novel amidated after 1-peptide hormones. Thus, the invention relates to oligonucleotides in which the nucleotide sequence is described below and to a method for identifying hormone precursors.

BACKGROUND OF THE INVENTION

Amidated after 1-peptide hormones are synthesized as precursors that undergo maturation. This maturation consists in an amidation reaction.

The C-terminal amidation reaction is a characteristic reaction of amidated 1-peptide hormones. This reaction, which occurs on precursors to one or more hormones, allows the hormone to mature and also ensures its biostability in physiological medium: the amide group formed is less vulnerable than free acid function. Thus, the hormone is more resistant to carboxypeptidases, remains active in the cell for an extended period of time, and retains optimal affinity for its receptor site.

Amidation has been widely described (Peptide amidation, Alan F. Bradbury and Derek G.Smyth, TIBS. 1.6: 112-115, March 1991)

Functional and structural characterization of peptidylamidoglycolate lyase, the enzyme catalysing of the second step in peptide amidation, AGKatopodis, DSPing, CESmith • 4 • 4 ······ · · 4 4 · 4 4 · · 4 4 4 4 « And SWMay, Biochemistry, 30 (25): 6189-6194, June 1991), and its mechanism is as follows:

~ cleavage of the precursor along the 1-peptide chain of the hormone by an endoprotease in two basic amino acids, i.e. arginine and / or lysine,

- two digestions with carboxypeptidase, resulting in an elongated glycine intermediate, the PAM enzyme (peptide 1-glycine-n-ct-monovalent monooxyganase) contains two different enzymatic activities: first, it converts the elongated the intermediate glycine to the α-hydroxyglycine derivative, the PAM subunit that performs the intervention, is PHM (peptidyl-glycine-α-α-hydroxy-monooxyganase). The obtained derivative serves as a substrate for the second PAM subunit (designated PAL:

peptidyl-α-hydroxyglycine and amidating lyase), which fixes the amino function of glycine to the amino acid, immediately fits to the flank of the W-terminal site and releases the glyoxylate.

This reaction requires the presence of a spacer site on the hormone precursor or hormones, a site that always contains the sequence: glycine and two basic amino acids (arginine or lysine) (see A.6. Catopodis et al., Biochemistry, 3.0 (25), 6189-6194, June 1991, and references cited therein).

It is known that arnidated polypeptide hormones to be secreted outside the endoplasmic reticulum contain a Ronsensual signal sequence of approximately fifteen to thirty amino acids, which sequence is present at the N-terminal end of the peptide chain. Later, it is cut by the peptidase enzyme signal so that it is no longer found in the already secreted protein (see F. Kutitta, The Anatomical Record, 236, 87-93 (1993) and references cited therein). ).

Finding a new protein is currently not easy. Proteins can be isolated and purified by various techniques: precipitation at an isoelectric point, selective extraction with certain solvents and then purification by crystallization, countercurrent distribution, adsorption, partitioning or ion exchange chromatography, electrophoresis, etc. However, these techniques assume knowledge of the properties of the protein to be isolated. In addition, if a pure sample of the new protein in question is available at the therapeutic level, there are still several steps left before a genetically modified microorganism capable of synthesizing it is available.

The method proposed by the present invention offers the advantage of using the characteristic peptide sequence of the precursor of all known amidated hormones, allowing simultaneous detection of several new hormones of this category. This screening is accomplished by direct identification of the nucleotide sequence that encodes said precursors in cDNA flasks prepared from tissues in which the precursors of these hormones can be synthesized.

Exploration using this method is much less restrictive than the aforementioned commonly used biochemical techniques because:

may lead to the isolation of several different precursors present in the same tissue on the basis of the same principle.

9 9 9 9

- allows the detection of precursors corresponding to hormones having very different biochemical and biological properties under the same technical conditions,

- allows concomitant identification of all peptide hormones that may be contained in the same precursor.

The result of the present invention is that it allows a not insignificant saving of time and money in an area where R&D costs represent a very high proportion of turnover.

The present invention also allows for a pharmacological study of active substances having a fundamental physiological role in mammalian organisms: hormones and especially amidated polypeptide neurohorrnones. Since the corresponding cDNAs are available for the first time, it will then be possible to genetically engineer the cloning vector, leading to the synthesis of hormones having therapeutic use by microorganisms.

SUMMARY OF THE INVENTION

The invention relates first to a single-stranded OX oligonucleotide which can hybridize under mild conditions to an oligonucleotide of an OY sequence Y1 - ~ Y2 - Y3 - - Y4 - Y5, wherein Y1 represents a nucleotide sequence from 1 to 12 nucleotides or Y1 is suppressed, Y2 represents the tri-nucleotide which encodes Gly, Y3 and Y4 independently represents the trisitide which encodes for Arg or Lys and Y5 represents the nucleotide sequence from 1 to 20 nucleotides or Y5 is suppressed.

The term nucleotide refers to a monomeric unit of RNA or DNA having the chemical structure of a phosphorus ester nucleoside. The nucleoside is formed by coupling a purine base (purine, adenine, guanine or analogs) or a pyrimidine base (pyrimidine, cytosine, uracil or analogs) with ribose or deoxyrose. An oligonucleotide is a nucleotide polymer indicating the sequence of a primer, probe, or RNA or DNA fragment.

These 1 igonucleotides can be obtained by synthesis, there is an automated reference method that is described in the following publications: "DNA synthesis, SANarang, Tetrahedron, 39, 3 (1983) and in Synthesis and use of synthetic igonucleotides, K.Itakura , JJ Rossi and RBWallace, Annu. Re v. Bi ochem. 53, 323 (1984).

Preferably, OX can hybridize to OY under stringent conditions.

More preferably, hybridization of OX with the γ-oligonucleotide of the YY sequence Y2-Y3-Y4-Y5 is possible.

Even more preferred is the possible hybridization of OX with an oligonucleotide of the Y1 -Y2-Y3-Y4 or Y2-Y3-Y4 OY sequence.

In a particular case, OX may hybridize to the oligonucleotide OY such that Y5 represents the nucleotide sequence Y6-Y7-Y8-Y9, wherein Y6 represents the trinucleotide that encodes for Ser, Thr, or Tyr, Y7 represents the tri nuclide that encodes for any amino acid, Y 8 represents a tri-nucleotide that encodes for Glu or Asp, and Y 9 represents a nucleotide sequence comprising 1 to 12 nucleotides. In a very special case, OX can hybridize to the oligonucleotide OY such that Y1 and Y9 are suppressed.

• · • ·

In a very special case, it may hybridize to a oligonucleotide OY in which Y2 is the tri nuclide that encodes for Oly, Y3 is the tri nuclide that encodes for Lys, Y4 is the trinucleotide that encodes for Arg, and Y5 is the 3 nucleotide sequence which code for Ser-Ala ~ 61u.

This sequence was determined using statistical study 2? known amidated sites and resulted in the definition of a given amino acid model at 6 positions: Gly-Lys-Arg-Ser-Ala-Glu.

Due to the degeneracy of the genetic code and due to the high number of codons corresponding to Gly (4 codons), Arg (6 codons) and Ser (6 codons), the sequence of the oligonucleotides was constructed using two procedures that allow this degeneration to be considered:

- use of certain inosine positions, which is a nucleotide in which the nitrogen base of hypoxanthine binds randomly to the 4 nitrogen bases that complement the DNA,

changing the nature at certain sites of the incorporated nitrogen base, thereby producing a series of oligonucleotide combinations proportional to the number of different bases introduced.

The present invention also relates to an OY oligonucleotide comprising 9 to 42 nucleotides of the sequence Y1-Y2-Y3-Y4-Y5, wherein Y1 is the nucleotide of the sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 is the trinucleotide that encodes for Gly, Y3 and Y 4 is independently a tri-nucleotide that encodes for Arg or Lys and Y 5 is a nucleotide sequence of 1 to 21 nucleotides or Y 5 is suppressed.

· 0 0 0

Preferably, the invention relates to an OY oligonucleotide such that Y1 is suppressed or that Y5 is suppressed.

In particular, the invention relates to an OY oligonucleotide such that Y5 represents the nucleotide sequence Y6-Y7-Y8-Y9, wherein Y6 represents the tri-nucleotide that encodes for Ser, Thr or Tyr, Y7 represents the tri-nucleotide that encodes for any amino acid Y8 represents a tri-nucleotide that encodes Glu or Asp, and Y9 represents a nucleotide sequence comprising 1 to 12 nucleotides.

In particular, the invention relates to an OY oligonucleotide such that Y1 and Y9 are suppressed.

In a very special case, the invention relates to an OY oligonucleotide characterized in that Y1 is suppressed, represents the tri nuclide that encodes for Gly, Y3 represents the tri nuclide that encodes for Lys, Y4 represents the tri nuclide that encodes for Arg and Y5 represents a sequence of three trinucleotides that encodes for Ser-Ala-Glu.

The present invention also relates to a single-stranded oligonucleotide 02 comprising 15 to 39 nucleotides and capable of hybridizing to a consensus signal sequence characteristic of amidated after 1-peptide hormones, said sequence having the formula Z1-22-23-24-2526 -27, wherein 21 represents the nucleotide sequence of 1 to 12 nucleotides or is 21 suppressed, 22 and 23 represent the two triad eeotids that encode for Leu, 24 and 25 represent the two tri nuclides, which encode for any two amino acids, 26 represents the trinucleotide which encodes for Leu and 27 represents the nucleotide sequence of 1 to 12 nucleotides or is a »

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Z7 suppressed.

In the present invention, the term hormone will include amidated polypeptide hormones of the endocrine system, and in particular neurohormones.

A consensus signal sequence is a sequence carried by precursors of proteins that are secreted by the cells after their maturation.

Finally, the present invention relates to a group of OX or OZ oligonucleotides that form a combinatorial library.

In the present invention, the term combinatorial library refers to a group of oligonucleotides synthesized in such a way that a nucleotide sequence that encodes for an amino acid sequence, some of which may vary, is considered as a model. Due to the degeneracy of the genetic code, a group of different oligonucleotides is obtained.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminal end, characterized by the following sequential steps:

- Obtaining a DNA bank,

2- Hybridization of one or more oligonucleotides using a given DNA bank,

3 ~ Identify the DNA sequence (s) of a given bank that hybridizes to 1 igonucleotide OX, * ftft * ftftft · ftft · ftftft ·· · ftftft * ftftftftftftft ftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftft · Ft ftft ftft

4- Identification in this sequence or in these sequences of one or more peptides with a possible amidated C-terminus.

It will be preferred that the DNA bank is a cDNA bank.

Complementary DNA (cDNA) is a nucleotide strand whose sequence is complementary to the mRNA sequence. The reaction resulting in single stranded cDNA is catalysed by inverse transcriptase. The double stranded cDNA can be obtained by the action of DNA polymerase, and is then inserted by a ligase into a plasmid or bacteriophage lambda-derived vector.

The cDNA bank contains cDNA corresponding to cytoplasmic mRNA extracted from the cell. A cell is called complete if it contains at least one bacterial clone for each initial mRNA.

Hybridization occurs when two oligonucleotides have highly complementary nucleotide sequences and can join along their length by forming hydrogen bridges between complementary bases.

Particular preference will be given to a method in which an OX oligonucleotide of OX can be detected using a labeling reagent such as ³² P or digoxigen.

The reagents most commonly used for radiolabelling are β-ray emitting elements, for example ³ H, ¹²⁰ 0, ³² P, ³³ P and ³⁵ S.

Labeling of the oligonucleotides is carried out by introducing a phosphate-bearing group (gamma- ³² P) -ATP to its 5 'end, this reaction being catalyzed by T4 polynucleotide Rinase. Digoxigenin labeling is immunoenzymatic, digoxigenin is attached to a nitrogen base and incorporated into oligonucleotides. Its presence is manifested by the use of an antibody directed against digoxigenin and attached to alkaline phosphatase. The presence is demonstrated by the use of the color induced by the substrate hydrolysed by alkaline phosphatase.

Other labeling techniques may be used: oligonucleotides modified chemically to contain a complex metal reagent (lanthanide complexes are often used), a biotin or acridine ester containing group, a fluorescent substance (fluorescein, rhodamine, Texas red) or others.

A method for identifying an amidated polypeptide hormone precursor that uses combinatorial OX oligonucleotide libraries at the hybridization stage will be particularly preferred.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminus, which comprises the following steps:

obtaining a DNA library, using a PCR technique to amplify a fragment of interest using a group of OX oligonucleotides and another group of OZ oligonucleotides, identifying the DNA sequence of the library that hybridizes to the OX oligonucleotide and which has been amplified by the PCR reaction.

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Identification in this sequence of one or more peptides with a possible amidated C-terminus.

A fragment of interest refers to a cDNA sequence that encodes for a precursor of one or more amidated after 1-peptide hormones.

DNA amplification reaction by PCR (polymerase chain reaction) requires preparation of DNA denatured by heating at 95 ° C. This preparation is then paired with an excess of two complementary oligonucleotides on opposite strands of DNA on either side of the sequence to be amplified. Each oligonucleotide then serves as a primer for DNA polymerase (extracted from a thermophilic bacterium of the type Thermus aguatis: Taq polymerase) for a copy of each of the DNA strands. This cycle can be automatically repeated by subsequent denaturations ~ renaturations.

There are many references detailing PR.C protocols: US Patent Nos. 4,683,192, 4,683,202, 4,800,159 and 4,965,188, PCR technology: principles and applications for DNA amplification on H. Erlich, edited by Stockton Press, New York (1989) and PCR. protocols: a guide to methods and applications, Innis et al., edited by Academic Press, San Diego, California (1990).

The DNA bank is preferably a cDNA bank.

More preferably, the marker can be a gene.

be said OX oligonucleotide detected by agents such as ³² P or 9

Φ Φ • • • • •

• ΦΦΦ · · ΦΦ ·

ΦΦΦΦ ΦΦ Φ ΦΦΦΦ

Φ ΦΦΦΦ • Φ · Φ φ φ ΦΦΦΦ φ «Φ ΦΦ

Particular preference will be given to a method for identifying a precursor amidated after a 1-peptide hormone, characterized in that the amplification step uses a combinatorial library of igonucleotides OX and another combinatorial library of oligonucleotides 02.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminal end comprising the following steps:

obtaining a DNA library, using a PCR technique to amplify the fragment of interest, using an OX oligonucleotide group, identifying the DNA sequence of the library which hybridizes to the OX oligonucleotide and which has been amplified by the PCR reaction, identifying in this sequence one or more peptides with possible amidated C-terminus.

The object of this method is to isolate nucleotide sequences that encode for precursors containing more than one amidated site.

The DNA bank is preferably a cDNA bank.

More preferably, the oligonucleotide OX can be detected with a labeling reagent such as ³² P or digoxigenin.

Particular preference will be given to a method for identifying a precursor amidated after a 1-peptide hormone characterized by:

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99 in that the amplification step utilizes the combinatorial library of OX oligonucleotides.

Another method of identifying a precursor polypeptide comprising an amidated C-terminal end, proposed by the present invention, is characterized by the following steps:

obtaining a DNA bank, using the PCR technique. to amplify the fragment of interest with 1 OX oligonucleotide and another single-stranded oligonucleotide capable of hybridizing under mild or stringent conditions to a universal consensus sequence contained in a plasmid vector sequence in which DNAs of a given DNA bank are cloned, such as primers T3, T7 , KS, SK, M13, Reverse, identifying the DNA sequence of a given bank that hybridizes to an oligonucleotide OX, identifying in this sequence one or more peptides with a possible amidated C-terminal end.

A universal consensus sequence is a sequence carried by a vector in which the DNA of the bank is cloned. This sequence may serve as a primer for sequencing. The nucleotide sequences of these primers are available from J. Samforook, E. F. Fritch, T. Manatis, Molecular cloning, and laboratory mannu Press.

2nd Edition, 1989, Colel Spring Harbor Laboratory

The PCR reaction requires two oligonucleotides to be

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99

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9 9 99 were fixed on cDNA cloned in the vector to amplify it. In the case where only a single sequence pertaining to the DNA fragment to be amplified is known, the solution to overcome this problem is to use a 1 oligonucleotide which can hybridize to the nucleotide sequence belonging to the vector in which the cDNA was cloned as a universal consensus sequence.

Preferably, said DNA bank is a cDNA bank.

It will be preferred by 1 igonucleotide který, which can be detected with a labeling reagent such as ³² P or digoxige η ί n.

In particular, an amplification step using a consensus library of OX oligonucleotides will be preferred.

DETAILED DESCRIPTION OF THE INVENTION

The method described in the invention has been declared valid by its application to the hormone isolated. The neurohormone selected is choececokokinin (CCK), the neuromediator that is most abundant in the brain.

1.1. Preparation of DNA matrix for PCR reactions. from the Lambda Zapp II commercial bank (Rat Brain cDNA Library Vector ref 936 501) from STRATAGENE (Lafolla, USA).

This Stratagene cDNA bank contains cDNA clones from rat brain cells.

1.1.1. Release of cloned cDNA in the form of Bluescript phagemids (Stratagene, Lajolla, USA)

4 • • • 44 • 4 • * • 4 • 444 444

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This is done according to the following protocol: 250 μΐ 2.10® PFU / ml cDNA flask, 200 μΐ XL blue bacteria (genotype: recA1 endA1 gyrA96 thi ~~ 1 hsdR.1 7 supE44 reiA1 lac / F'proAB lacPZ / X M1 5Tn 10 (Tet ·) / ^c compare Bullock, Fernandez, Short, Biotechniques, 5, 376-379 (1987) ™ optical density at 600 nm: OD - 2.5) and 1 µl of ExAssist ™ (see B. Hay, J. Short, Strategies, 5, 16-18 (1992)) at 10 ¹⁰ PFU / ml are contacted for 15 minutes at 37 ° C. The whole system is then incubated on 50 ml of LB medium (composition: a mixture of 10 g sodium chloride, 5 g yeast extract and 10 g Bactotrypton per liter sterile physiological water) for 3 hours at 37 ° C.

The culture broth is centrifuged and the supernatant is then activated by heating at 70 ° C for 20 minutes.

1.1.2 Obtaining cDNA as a double stranded plasmid bank

This step requires a incubation of 100 μΐ i per kt of the supernatant and 200 μΐ of SCO ™ bacteria at 37 ° C for 15 minutes (genotype: e14 '(McrA') ./. \ (Mcr CB-hsdSMR-mr, r) 1 7 1 SBSC RecB recJ to cast torturing :: Tn5 (Kan ^r), lac gyrA96 relA1 thi-1 endA1 lambda ^ / F 'proAB lacI ^ Z / X M15 / ^c Su' (nepot1ačující), see B.Hay, JMShort, Strategies 5 (1), 16-18 (1992) - OD ~ 1-600 nm). After adding 50 μΐ ampicillin (100 mg / ml) and 50 ml of LB medium, the whole system was incubated for one night at 37 ° C with stirring. Plasmids were prepared from a 50 ml culture using the QIAGEN Plasmid Midi Kit protocol and QIAGEN columns (QIAGEN columns contain an anion exchanger with positively charged diethylaminoethanol groups on their surface that interact with the DNA backbone phosphates). This gave a 1.37 pg / pi DNA solution.

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1.2. Amplification of a portion of the CCK precursors from the prepared bank

1.2.1. Generation of sequences of two about 1 nucleotide required for PCR. reaction

One of the two nucleotides will contain a sequence complementary to that which encodes an amidated CCK site that is known and has the sequence 61y-Arg-Arg-Ser-Ala-61u. This oligonucleotide, which will be referred to as 1 igo CCK amide, has a nucleotide sequence as its nucleus:

5'CTCAGCACTGCGCCGGCC 3 '

The second oligonucleotide, named about 1 igo CCK 5 consensus signal sequence:

he answers

5'GTGT6TCT6T6CGT6GT6 3 'is 315 pairs corresponding to these CCKs.

The size of the expected amplification product, which is the distance between the sequences that 1 igonuk1eoti house on the precursors of the base sequence, two

1.2.2. PCR. reaction

Dilute solution D1 containing 1 μΐ of Goldstar 5 U / μ 1 Taq polymerase is prepared (see P.Reynier, JFPe11 issier, JRHarle, Y.Malthiéry, Biochemical and Biophysical Research Communications, 20 May (1), 375 to 380 (1994)), 1 μΐ of buffer 10 times concentrated in standard Taq polymerase and 8 μ! water.

* 00 0000 • 0 · 0 · 0 · 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0000 000 0 «·

00

0 0 0 • 0 0 0

0 0 0

0 0 0

00

Then mix 1 μΐ of 250 ng / μΐ oligo CCK 5 ', 250 ng / μΐ of oligo CCK amide, 1 μΐ 10 mM dNTP each, 1 μΐ of 250 ng / μΐ cDNA, 5 μΐ buffer concentrated 10-fold in Taq polymerase enzyme, 2 μΐ of 25 mM magnesium chloride, μΐ of dilute solution D1 and 37 μΐ of water.

The amplification conditions are as follows: first heat treatment for 5 minutes at 95 ° C, then repeat 30 cycles. Denaturation is performed at 95 ° C for 45 seconds, hybridization at 60 ° C for 30 seconds, and extension for 1 minute at 72 ° C. Finally, an additional β-extension cycle is performed at 72 ° C for 10 minutes.

1.2.3. Results

The results are read by migrating to 0.8 X agarose gel from 1/10 PCR product. reaction. In the presence of 3.8 "diamino" 5 ~ ethyl "6"

-phenylphenanthridinium bromide (ethidium bromide) is visualized on the gel with one intense band slightly larger than a marker of molecular weight 300.

1.3 Subcloning the PCR product into a vector allowing sequencing

The vector used is pGEM T-easy Vector (supplied by PROGEMA Corporation, Madison, USA, ref. A 1380 sequence shown in Annex I). The stages are as follows:

- purification of the band corresponding to the PCR product by electroelution, ° C with 1 μΐ of vector and 1 μΐ of buffer used

- ligation for one night at pGEM T-easy at 50ng / µl at 10-fold ligation,

- extraction of 3 μΐ of product from the purified band estimated at Ong / μΙ,

- make up to 10 μΐ with water.

Bacteria JM 109 (genotype: eH- (McrA-) recAl endA1 gyrA96 thi-1 hsdR.17 (rk ~ mk +) supE44 relA1 / \ (1 ac-pr AB) / F 'traD36 proAB 1 acl® · 5 / -vi of C. Yan i sh-Perron, J.Viera,

J. Messing, Gene, 33, 103-199 (1985)) became competent by pretreatment with calcium chloride and then transformed at 45 ° C with 1/5 of the ligation mixture by thermal shock for 45 seconds. The cells were then cultured overnight in LB-ampoule medium in a Petri dish at 37 ° C.

DNA plasmids were prepared by recombination of several clones. Subcloning is then verified by enzymatic digestion with Eco RI.

1.4 Sequence

It is performed using the conventional dideoxynucleotide SANGER technique. on pGEM T-easy Vector to incorporate 315 base pairs of PCR product (prepared on a large scale using the QIAGEN tip 100 kit). The primer used for sequencing is the universal T7 igonucleotide present on plasmid pGEM T-easy Vector.

1.5 Result • · «· *> · *

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The following rough sequences were obtained

GTG GTT GTG GTG GTG GTG ATG GCA GTC GGC GCC GGC GCC GCG CTC CCG GTA GTC CCT GTA GAA GCT GTG GAC CCT ATG GAG CAG CGG GCG GCG GCC GCG GCC GCG GCC GCG TAC ATC GCG TAC ATG GCG TAC GGC CGG GCC AGT GCT GAG

The translation of the amino acid sequence results in:

VCLCVV MAVLAAGALA QPVVPVEAVD PMEQRAEEAP

R.R.QLR.AVLR.P DSEPR.AR.L6A 'LLARYI QQVR. KAPSGRMSVL

KNLQGLDPSH R.ISDR.DYMGW MDFGRR.SAE which makes it possible to easily find the nucleotide sequence of the CCK precursor (a sequence provided by the Swiss database no. P 1 13 5 5).

The abbreviations of amino acids are as follows:

A l and η i n A

Ar g i n R.

Aspartic acid D

Asparagine N

Cystein C

Glutamic acid

Leuc i n

Lys ί η K

Methi on i M

Phenylalanine F

For 1 i η P

Serin S 4 4 4 4 4 4 4

4 4 4

Glutamine G1 is histidine Isoleucine

Q Threonin T

G Tryptophan W

H Tyrosin Y

I Valin V

Claims (25)

1. A single-stranded oligonucleotide OX, characterized in that it contains 9 to 42 nucleotides and under mild conditions is capable of hybridizing with an oligonucleotide OY of the sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 2. Oligonucleotide OX according to claim 1, characterized in that it contains 9 to 42 nucleotides and is capable of hybridizing under stringent conditions with the oligonucleotide OY of the sequence Y1 “Y2-“Y 3~Y 4~YS, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y 5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 3 . 01 i gonuk1eot i d OX according to it c it's three m, that Yl 4 . 01 i gonuk1eot i d OX according to no j moving thus, that OY. 5 . 01 i gonucleot i d OX according to no j moving thus, that c recognizes22 ·· ·· the sequence Υ8-Υ7-Υ8-Υ9, in which Y 6 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp, and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides. 8. Oligonucleotide OX according to claim 5, characterized in that Y1 and Y9 are suppressed in oligonucleotide OY. 7. The oligonucleotide OX according to claim 6, characterized in that it can hybridize with a given oligonucleotide OY, in which Y2 represents a trinucleotide that codes for Gly, Y3 represents a trinucleotide that codes for Lys, Y4 represents a trinucleotide that codes for Arg and Y5 represents a sequence of three trinucleotides that codes for Ser-Ala-Glu. 8. double-stranded oligonucleotide OY, characterized in that it contains 9 to 42 nucleotides of the sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 9. The oligonucleotide OY according to claim 8, wherein Y1 is suppressed. 10. Oligonucleotide OY according to claim 8 or 9, characterized in that Y5 is suppressed. • 9 • · · · 999 ·· · 9 9 · · r · · a » ·» 4 I 9 9 4 I 9 9 4 9 9 9 9 11. The OY nucleotide according to claim 8 or 9, wherein Y5 represents the nucleotide sequence Y6~Y7~Y8~Y9, in which Y8 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides. 12. The OY igonucleotide of claim 11, wherein Y1 and Y9 are suppressed. 13. The OY nucleotide according to claim 12, characterized in that Y2 represents a trinucleotide encoding Gly, Y3 represents a trinucleotide encoding Lys, Y4 represents a trinucleotide encoding Arg and Y5 represents a sequence of 3 trinucleotides encoding C1 and C2. Ser-Al a-~G 1 u . 14. A single-stranded oligonucleotide O2, characterized in that it contains 15 to 39 nucleotides and is capable of hybridizing under mild or stringent conditions with a consensus signal sequence characteristic of amidated polypeptide hormones, said sequence having the formula 21-22-23-24-25-26-27, in which 21 represents a nucleotide sequence of 1 to 12 nucleotides or 21 is suppressed, 22 and 23 represent two trinucleotides that code for Leu, 24 and 25 represent two trinucleotides that code for any two amino acids, 28 represents a trinucleotide that codes for Leu and 27 represents a nucleotide sequence of 1 to 12 nucleotides or 27 is suppressed. 15. The OX oligonucleotide group according to any one of claims 2 4 • · 9 * 1 to 7' or oligonucleotides OZ according to claim 14, characterized in that it creates a combinatorial library. 16. A method for identifying a peptide precursor having an amidated C-terminal end, comprising the following sequential steps: - obtaining a DNA bank, - hybridizing one or more oligonucleotides according to any one of claims 1 to 7 with a given DNA library, - identifying the DNA sequence or sequences of the library that hybridizes with the oligonucleotide according to any one of claims 1 to 7 and - identification in this sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 17. The method of claim 16, wherein the hybridization step uses a combinatorial library of claim 15. 18. A method for identifying a peptide amidated C-terminal end in a network comprising the following - obtaining a DNA bank, a precursor having the following successive stages: - using the PCR technique to amplify the fragment of interest using a group of oligonucleotides according to any one of claims 1 to 7 and another group of oligonucleotides 99 9999 » 9 9 » 4 4 99 94 ► 4 9 4 ► 4 4 4 according to claim 14, - identifying the DNA sequence or sequences of the library that hybridizes with the oligonucleotide according to any one of claims 1 to 7 and - identification in this sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 19. The method of claim 18, wherein the amplification step uses the combinatorial library of claim 15. 20. A method for identifying a peptide precursor having an amidated C-terminal end, comprising the following steps: - obtaining a DNA bank, - using PCR techniques to amplify a fragment of interest using a group of oligonucleotides according to any one of claims 1 to 7, - identification of a DNA sequence or sequences of a given library which hybridizes with an oligonucleotide according to any one of claims 1 to 7 and - identification in this sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 21. The method of claim 20, wherein the amplification step uses a combinatorial library of claim 15. 22. A method for identifying a polypeptide precursor having an amidated C-terminal end, comprising the following steps; ~ obtaining a DNA library, using the PCR technique to amplify the fragment of interest using the oligonucleotide according to any one of claims 1 to 7 and another single-stranded oligonucleotide capable, under mild or stringent conditions, of hybridizing with a universal consensus sequence contained in the sequence of the plasmid vector in which the cDNA of the DNA library is cloned, such as primers T3, T7, KS, SK, M13 and Reverse, - identification of a DNA sequence of a given library which hybridizes with the oligonucleotide according to any one of claims 1 to 7 and ~ identification in this sequence of one or more peptide precursors with a possible amidated C-terminal end. 23. The method of claim 22, wherein the amplification step uses a combinatorial library of claim 15. 24. The method according to any one of claims 16 to 23, wherein the DNA library is a cDNA library. 00 00 W 0 0 * 0 0 0 · 0 0 0 · 0 0 0 0 00 00 • « · 0 · · • ·0· 0 0 0 0 0 0 0 0 00 ^{25 * * *} 25. The method of any one of claims 16 to 24, wherein the single-stranded oligonucleotide can be detected using a labeling agent such as ³² P or digoxigenin.