JPH04503054A - Novel protein, DNA encoding it and its use - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel protein, DNA encoding it and its use The present invention provides novel amino acid sequences, nucleic acids encoding these amino acid sequences. This for in vitro diagnosis of diseases related to the action of acids, as well as especially oncogenic factors. Regarding how to use them.

Already, H-ras%, -ras, and N-ras [De Feo et al., (19 1981) Proc, Natl, Acad.

Sci (Bulletin of the American Academy of Sciences, U, S, A, 78.

3328-3332: Ellis et al., (1981), Nature ( Nature), 292, 506-511; Shimizu et al. (1983), Proc. Natl, Acad, Sci, U, S, A,.

Proto-oncogenes such as [80.383-387] are known. Based on its homology with the mammalian ras gene, it has been newly discovered in various organisms. A new gene was identified.

In mammals, these genes similar to the ras gene (ral, R-ras The proteins encoded by %rho and rab) are 50 to 30 % homology [(:Hardim et al. (1986), EMBO J., 5.2203-2208; Madanle et al. (1985) C e1l (Cell), 41.31-40: Touchot et al. (1987) Proc, Natl.

Acad, Sci, U, S, A,, 84. 8210-8214].

Ras protein and proteins similar to ras protein are approximately 21,000 to 24,000 d It has a molecular weight of

When ras protein is linked to GTP (guanosine 5'-triphosphate), When activated and bound to GDP (guanosine 5'-diphosphate) GTP becomes inactive and hydrolyzed by its inherent low GTPase activity. [Papageorage et al. (1982) J, Vi rol.

(Journal of Virology), 44.509-519): McGrath et al. 1983) Nature, 310.

644-649].

Transforming ras proteins detected in human tumors very frequently Indicates amino acid substitutions located at positions 12.13 or 61 within the binding region of P. [Barbacid (1987) Animal Review of Biochemistry, 56.779-827.

This region plays a major role in regulating the biological properties of ras proteins.

The GTP binding site consists of four noncontiguous regions that belong to all ras proteins. .

Six amino acids within these regions (i.e., positions 57 to 57 of the K-ras protein) 62 (DTGAQE) is one of the ras proteins and similar ras proteins. It seems to be a characteristic [Chardi et al.

(1986), EMBOJ, 5.2203-2208: Touchot et al. (1987), Proc, Natl, Acad.

Sci, U, S, A,, 84. 8210-8214], these six residues is strictly conserved in the proteins ras, ral and rab.

In the H-ras protein, almost each residue at position 61 of the ras protein Mutations have been found to be associated with the acquisition of potential transformants [ Der et al. (1986) Cell, 44, 167-1761゜ By the way, the applicant has one threonine at position 61 among others, and even so, We have discovered a new amino acid sequence that has non-transforming properties.

One feature of the present invention is that GTPases have the ability to fix GTP and GDP. The aim is to propose new amino acid sequences that have activity.

One of the other features of the invention is for amino acid sequences that exhibit interesting biological properties. The aim is to propose new nucleic acids to encode.

Another feature of the invention is that proteins that are outside the range of values commonly found in healthy bodies. Diseases in which white matter content is or is associated with mutations in these proteins of! 15. To propose a tracking method.

The object of the present invention is that the present invention is characterized by containing at least one of the following amino acid chains: The new amino acid sequence is: MREYKLVVL (I) ALTVQFVQGIFVEKYDPTIEDSYRKQVEVDCQQCML EIL (IIIQFTAMRDLYMKNGQGFA LVYS ITAQS TFNDLQDLREQ I L RV K DTEDVPM (III) EDERVVGKEQGQNLARQWCNCAFL (IV) SKINVNE IFYDLVRQINRKTPVEKKKPKKKKSCLLL mALTVQF VQGIFVEKYDPTIEDSYRKQVEVDAQQCMLEIL (V IIIQFTAMRDLYMKNGQGFA LVYS ITAQSTFNDL QDLREQ I LRV K DTD DVPMI (VIII) EDERVVGKEQGQNLARQWNNCAFL (IXISKINVNE IFYDLVRQINRKTPVPPGKARKKSSCQLL (XIMREY KVVVL (XI) ALTVQFVTGTFIEKYDPTIEDFYRKEIEVDSSPSVL EIL (XIIIQFASMRDLY I KNGQGF I LVYSLV NQQSFQD I KPMRDQ I I RV KRYE to VPVI (X III ESEREVSSSEGRALAEEWGCPFM (XIV)SKTMVDE LFAEIVRQMNYAAQPDKDDPCC3ACNIQ (XVI and above In the following description, amino acids are referred to as terminology conventionally used in this field. It is represented by a unique character according to the table.

Amino acid sequences are defined by the left-most end of the first amino acid that defines these sequences. corresponds to the N-terminal end, and the right-most end is the last amino acid that limits these sequences. It is marked in such a way that it corresponds to the C-terminal end.

According to a preferred embodiment of the invention, the amino acid sequence of the invention is as defined above. At least one of chain (I), (III, (III), (IVI or (Vl) Contains one.

According to a preferred embodiment of the invention, the amino acid sequence of the invention is as defined above. At least one of the chains (VII), (VIII), (IXI or TX) Contains.

According to another preferred embodiment of the invention, the amino acid sequence of the invention comprises A defined chain (XII, (XIII, (X I I I I l, (XIV) or ( XV) (7)') Contains at least one of the following.

According to another preferred embodiment of the invention, the amino acid sequence of the invention at position 61 contains 1 threonine.

According to another preferred embodiment of the invention, the amino acid sequence of the invention lasts The fourth amino acid from the C-terminus of is cysteine. .

According to another embodiment according to the invention, the amino acid sequence is f11, (III, (III), (IVI and m).

When the amino acid sequence based on the present invention has the above-mentioned five chains, these chains The chains are located in the order (I) - (II) - (III - (IVI - m) from left to right. The chains are furthermore advantageously non-contiguous.

According to another embodiment according to the invention, the amino acid sequence is as defined above. Five chain formations (VIll, (VIII), (IXI and (X)) .

When the amino acid sequence based on the present invention contains the above-mentioned five chains, these chains The chains are advantageously arranged from left to right fVII)-(VIII-(IXI-(XI) (7) and these chain formations are further advantageously located adjacent to each other. Not yet.

According to another embodiment of the invention, the amino acid sequence comprises the five chains (X1 1, (XIll, (XIII, (XIVI.

and (XV).

If the amino acid sequence of the invention has the five chains shown above, these chains are , advantageously from left to right (XI)-(XIII-(XIII)-(XIV) −(Xi (7)), and these chains are also advantageously adjacent. Not yet.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Contains chain XVI.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Consists of chain XVI.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Contains chain XVII.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Consists of chain XVII.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Contains chain XVIII.

A highly advantageous amino acid sequence according to the invention is the amino acid sequence represented in FIG. Consists of chain XVIII.

Amino acid sequence XV1. XVII and XVIII are also referred to as R.A. Also called PIA, RAPIB and RAPIC.

These amino acid sequences have one threonine at position 61 and the last amino acid It has one cysteine at the fourth amino acid from the acid C-terminus.

In this regard, the amino acids represented in Figures 1, 2 and 3 respectively For sequences, the position of a given amino acid is determined by its relationship to the first amino acid in the sequence. The nucleotide sequence (also shown in Figures 1 and 2) is and shown in Figure 3), certain nucleotide positions are Marked in relation to cleotide.

These amino acid sequences exhibit advantageous biochemical and biological properties: These amino acid sequences have the ability to anchor GTP and GDP, and GTPas e activity.

Note that these amino acid sequences are non-transforming. In other words, especially the morphological characteristics of cells. and does not change the motility properties or the contact inhibition properties of the cells.

Therefore, lack of these sequences, overrepresentation (i.e., those normally suppressed in a healthy body) its expression in higher amounts) as well as some of its mutations are compatible with pathological conditions. I guess.

These amino acid sequences may also be associated with intercellular membranes. .

The preceding amino acid sequences are the aforementioned amino acid sequences (xvil, (XVIII and ( XVIII) (7) Changes may be made as long as the biochemical and biological characteristics are retained. I can do it.

For example, without limitation, amino acid sequences falling within the scope of the invention include: - addition and/or - deletion of one or more amino acids and/or - change of one or more amino acids It can be distinguished from the above-mentioned amino acid sequence by

However, in this case, the condition is that the biochemical and biological properties as mentioned above are maintained. subject.

Also within the scope of the invention are amino acid sequences exhibiting a mutation at position 12 (X VII, (XVII) and (XVIII) (i.e. gluta in 12 min is substituted with any other amino acid).

Also within the scope of the invention are amino acid sequences exhibiting a mutation at position 61. columns fXVI), (XVII) and (XVIII) (i.e. in 61 Threonine is replaced by any other amino acid, especially glutamine and valine. ).

The present invention also relates to amino acid chains encoded by the following nucleotide chains: It also relates to amino acid sequences containing at least one of the following.

ATG CGT GAG TACAAG CTA GTG GTCCTT (1 1GCT CTG ACA GTT CAG TTT GTT CAG GGA ATT TTTGTT GAA AAA TAT GACCCA A to G A TA GAA GAT TCCTACAGA AAG CAA GTT GAA GTCGAT TGCCAA GAGTGT ATG (1, TCGAA A TCCTG +21CAA Ding TT ACA GCA ATG AGG GAT TTG TAT ATG AAGAACGGC AA GGT TTT GC A CTA GTA TAT TCT ATTACA GCT CAG TC ( : ACG TTT AACGACTTA GAG GACCTG AGG G AA CAG ATT TTA CGG GTT AAG GACACGGAA GAT GTT CCA ATG ATT (31GAA GAT GAG CGA GTA GTT GGCAA GAG GAG GGCCAG AA T TTA GCA AGA CAG TGG TGT AACTGT GCC TTT TTA (41 TCA AAG ATCAAT GTT AAT GAG ATA TTT T AT GAGCTG GTCAGA CAG ATA AAT AGG AAA ACA CCA GTGGAA AAG AAG AAG CCT AAA AAG AAA T, CA, TGT CTGCTG CTC (51 ATG CGT GAG TAT AAG CTA GTCGTT CTT ( 6) G(:T TTG ACT GTA CAA TTT GTT CAA G GA ATT TTTGTA GAA AAA TA GAT CCT AC G ATA GAA GAT TCTTAT AGA AAG CAA GTT GAA GTA GAT GCA CAA CAGTGT ATG CTT GAA ATCTTG (7) CAA TTT ACA GCA ATG AG G GAT TTA TACATG AAAAAAT GGA CAA GGA TTT GCA TTA GTT TAT TCCATCACA GCA CA G TCCACA TTT AA (: GAT TTA (: AA GACCT G AGA GAA CAG ATT CTT CGA GTT AAA GA CACTGAT GAT GTT (, CA ATG ATT f8) GAA GAT GAA AGA GTT GTA GGG AAG GAA CAA GGTCAA AAT CTA GCA AGA CAA TGG AAC; AACTGT GCATTCTTA (91 TCA AAA ATA AAT GTT AAT GAG AT TTT TAT GAC (:TA GTG CGG CAA ATT AACAGA A AA ACT CCA GTGCCT GGG AAG GCT CG (: A AA AAG TCA TCA TGT CAGCTG CTT (101 ATG CGCGAG TACAAA GTG GTG GTG C, TG ( illGCCCTG ACCGTG CAG TTCGTG ACCGGCAC CTTCATCGAG AAA TACGACCCCACCATCGAG GA CTTCTACCGCAAG GAG ATCGAG GTG GAT TCG TCG (:CGTCG GTG CTG GAG ATCCTG (121 CAG TTCGCG TCCATG CGG GACCTG TACATCA AGAACGGCCAG GG(: TTCATCCTCGT(: TACAG CCTCGTCAACCAG　CAG　AGCTTCCAG　GACATCAA G CCCATG CGG GACCAG ATCATCCGCGTG AAG CGG TATGAG AAA GTG CCA GTCATC (131GA A AGT GAG AGA GAA GTA TCG TCCAGCGAA GGCAGA GCCCTT GCT GAA GAG TGG GGCTGC CCCTTTATG (141 AGT AAA ACA ATG GTG GACGAA CTCTTT GC A　GAAATT　GTG　AGG　CAG　ATG　AACTAT　GCT　 GCT CAG CCTGACAAA GAT GACCCA TGCTGT TCT GCA TGT AACATA CAA (151 Also within the scope of the invention are the amino acid sequences encoding the foregoing amino acid sequences. A nucleic acid comprising at least one chain of nucleotides.

According to one advantageous embodiment of the invention, the nucleic acid belongs to the human genome.

According to one advantageous embodiment of the invention, the nucleic acid according to the invention comprises the amino acid sequence defined above. It encodes for chains I-XVIII.

According to yet another advantageous embodiment, the nucleic acid sequence of the invention comprises the above-mentioned chain X It encodes for an amino acid sequence including VI, XVII and XVIII.

According to another advantageous embodiment of the invention, the nucleic acid sequence of the invention is as defined above. Code for the amino acid chain that is present.

Advantageous nucleic acids of the invention contain at least one of the following nucleotide sequences: It is a nucleic acid.

ATG CGT GAG TACAAG CTA GTG GTCCTT (1 1GCT CTG ACA GTT CAG TTT GTT CAG GGA ATT TTTGTT GAA AAA TAT GA (: CCA ACG ATA GAA GAT TCCTACAGA AAG CAA GTT G AA GTCGAT TGCCAA CAGTGT ATG CTCGAA A TCCTG (21CAA TTT ACA GCA ATG AGG GAT TTG TAT ATG AAGAACGGCCAA GGT TTT GC A CTA GTA TAT TCT ATTACA GCT CAG TCC ACG TTT AACGACTTA CAG GACCTG AGG GAA CAG ATT TTA CGG GTT AAG GACACGGAA G AT GTT CCA ATG ATT (31GAA GAT GAG CG A GTA GTT GGCAA GAG CAG GGCCAG AAT TTA GCA AGA CAG TGG TGT AACTGT GCCTT T TTA (4) TCA AAG ATCAAT GTT AAT GAG ATA TTT T AT GACCTG GTCAGA CAG ATA AAT AGG AAA ACA CCA GTGGAA AAG AAG AAG CCT AAA AAG AAA TCA TGT CTGCTG CTC (51 ATG CGT GAG TAT AAG CTA GTCGTT CTT ( 61GCT TTG ACT GTA CAA TTT GTT CAA GG A ATT TTTGTA GAA AAA TACGAT CCT ACG ATA GAA GAT TCTTAT AGA AAG CAA GTT G AA GTA GAT GCA CAA CAGTGT ATG CTT GA A ATCTTG (71 CAA TTT ACA GCA ATG AGG GAT TTA TACATG AAAAAAT GGA CAA GGA TT T GCA TTA GTT TAT TCCATCACA GCA CAG TCCACA TTT AACGAT TTA CAA GACCTG AGA GAA CAG ATT CTT CGA GTT AAA GACACTG AT GAT GTT CCA ATG ATT (8) GAA GAT GA A AGA GTT GTA GGG AAG GAA CAA GGTCAA AAT CTA GCA AGA CAA TGG AACAACTGT G CATTCTTA (9) TCA AAA ATA AAT GTT AAT GAG ATCTTT T AT GACCTA GTG CGG CAA ATT AACAGA AAA ACT CCA GTGCCT GGG AAG GCT CGCAAA AG TCA TCA TGT CAGCTG CTT (101 ATG CGCGAG TACAAA GTG GTG GTG CTG (1 1) GCCCTG ACCGTG CAG TTCGTG ACCGGCACC TTCATCGAG AAA TACGACCCCACCATCGAG GAC TTCTACCGCAAG GAG ATCGAG GTG GAT TCG TCG CCGTCG GTG CTG GAG ATCCTG (121CA G　TTCGCG　TCCATG　CGG　GACCTG　TACATCAAG AACGGCCAG　GGCTTCATCCTCGTCTACAGC(:TCG TCAACCAG CAG AGCTTCCAG GACATCAAG CCC ATG CGG GACCAG ATCATCCGCGTG AAG CGG TATGAG AAA GTG CCA GTCATC (131GAA AGT GAG AGA GAA GTA T(:G TCCAGCGAA GGCA GA GCCCTT GCT GAA GAG TGG GGCTGCCCCT TTATG (14) AGT AAA ACA ATG GTG GACGAA CTCTTT GC A　GAAATT　GTG　AGG　CAG　ATG　AA(:　TAT　GC T GCT CAG CCTGACAAA GAT GACCCA TGCTG T　TCT　GCA　TGT　AACAT　CAA　f15) According to another advantageous embodiment, the nucleic acid according to the invention comprises the above-mentioned nucleotides. It contains five chains (1), (2), (3), (4), and (5).

According to another advantageous embodiment of the invention, the nucleic acids according to the invention are Contains three nucleotide chains (6), (7), (8), (9), (10) .

According to another advantageous embodiment, the nucleic acid according to the invention comprises the five nucleic acids mentioned above. Contains leotide chains (11), (12), (13), (14) and (15) 6 According to another advantageous embodiment, according to the invention (the nucleic acid is located at position 43 in FIG. and by the terminal nucleotide corresponding to the nucleotide located at 594. It contains a defined nucleotide chain (16).

According to another advantageous embodiment, the nucleic acid according to the invention is located at position 43 in FIG. and by the terminal nucleotide corresponding to the nucleotide located at 594. It is composed of a defined nucleotide chain (16).

According to another advantageous embodiment, the nucleic acids of the invention are located at positions 1 and 60 in FIG. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 5 Contains a chain (19).

According to another advantageous embodiment, the nucleic acids of the invention are located at positions 1 and 60 in FIG. Nuclides delimited by terminal nucleotides corresponding to the nucleotides at 5 It is composed of leotide chain formation (19).

According to yet another embodiment, the nucleic acids of the invention are located at positions 54 and 6 in FIG. Nuclides defined by terminal nucleotides corresponding to the nucleotides at 05 Contains a leotide chain (17).

According to yet another embodiment, the nucleic acids of the invention are located at positions 54 and 6 in FIG. Nuclides defined by terminal nucleotides corresponding to the nucleotides at 05 It is composed of leotide chains (17).

According to yet another embodiment, the nucleic acids of the invention are located at positions 1 and 83 in FIG. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 8 Contains a chain (20).

According to yet another embodiment, the nucleic acids of the invention are located at positions 1 and 83 in FIG. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 8 It is composed of a chain (20).

According to yet another embodiment, the nucleic acids of the invention are located at positions 4 and 55 in FIG. Nucleotide delimited by terminal nucleotides corresponding to the nucleotides at 2 Contains a chain (18).

According to yet another embodiment, the nucleic acids of the invention are located at positions 4 and 55 in FIG. Nucleotide delimited by terminal nucleotides corresponding to the nucleotides at 2 It consists of a chain (18).

According to yet another embodiment, the nucleic acids of the invention are located at positions 1 and 55 in FIG. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 8 It contains a chain (21).

According to yet another embodiment, the nucleic acids of the invention are located at positions 1 and 55 in FIG. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 8 It is composed of a chain (21).

Nucleic acids (16), (17), (18), (19), (20) and ( 21) are all represented in Figure 5 under the less stringent conditions specified below. It has the peculiarity of hybridizing with the gene Dras-3 of the genus Drosophila, which Shown; - At 60°C, 5XSSC15X Denhardt, 0. 1% S Hybridization in PS and 100 μg salmon sperm DNA.

- Final cleaning in 2XSSC 10.1% SDS at 60°C for approximately 15 minutes.

Nucleic acids (16), (17), (18), (19).

(20) and (21) indicate that the amino acid sequences encoded by these nucleic acids are It can be modified as long as it retains its chemical and biological properties.

Such non-limiting changes may include, for example - additions and/or - deletion of one or more nucleotides and/or - single or multiple nucleotide changes; This will lead to derivative nucleic acids that are distinguished from the nucleic acids described above. However, this If the chain thus formed has the same RNA as the corresponding unaltered chain or have the ability to hybridize with DNA sequences and have the ability to hybridize with these nucleic acids. the amino acid sequence retains the above-mentioned biochemical and biological properties; subject to the following conditions.

Nucleic acids (16) and (19) are similarly characterized by the restriction map depicted in Figure 7. can be attached.

Nucleic acids (17) and (20) are also characterized by the restriction map depicted in Figure 8. be able to.

Nucleic acids (18) and (21) were similarly characterized using the restriction map shown in Figure 9. It's coming. These restriction maps each identify nucleic acids with the aforementioned linkages. Therefore, it should not be interpreted as having a restrictive meaning.

Equivalent nucleic acids are preceded by at least 50% of the restriction sites placed in the same order. It can also be defined by a single restriction map that has commonality with the defined restriction map. Ru.

The present invention also covers probes used to characterize the aforementioned nucleic acids for that purpose. shall be.

These probes may be characterized by their ability to hybridize with the nucleic acids described above or by A new nucleic acid (2) defined by its complementary sequence under hybridization conditions ), (3), (4), (5), (7), (8), (9), (io), (12), (13), (14) and (15) or their complementary sequences. For potential probes, the hybridization conditions are as follows: Hybridization: 60°C, 5XSSC15XDenhardt, 0.1% SDS, Salmon sperm DNA at 100 μs/ml: Clean: 60°C, 2X SSC 10, 1% S DS, 30 minutes - There is a possibility of hybridization with the nucleic acid or complementary sequence (1), (6) or (11). For probes that are hybridized, the hybridization conditions are as follows: : 45°C, 5XSSC15xDenhardt, 0. 1% SDS, 100u g/ml salmon sperm DNA.

1st clean two person temperature, 2XSSC10, 1% SDS, 30 minutes, Second clean = 45°C, 2XSSC10, 1% SDS, 30 minutes.

Examples of oligonucleotide probes for identifying the nucleic acid sequences of the present invention include For example, a probe constructed from the following oligonucleotide sequence can be used. Wear: GAA ATCCTG GAT ACT GCA GGG ACA GAA T TT or corresponding complementary arrangement.

In order to identify the nucleic acids (16) and (19) of the present invention, for example, as a probe, , to the nucleotides corresponding to the nucleotides at positions 493 and 605 in FIG. More limited oligonucleotide sequences or corresponding complementary sequences can be used. can.

In order to identify the nucleic acids (17) and (20) of the present invention, for example, the positions shown in FIG. limited by nucleotides corresponding to those at 504 and 88 Oligonucleotide sequences or corresponding complementary sequences can be used as probes. can.

In order to identify the nucleic acids (18) and (21) of the present invention, for example, the positions shown in FIG. limited by nucleotides corresponding to those at 451 and 558. using the oligonucleotide sequence or the corresponding complementary sequence as a probe. Can be done.

The present invention also provides for the aforementioned fragments to be inserted into a heterologous nucleic acid. It also relates to a recombinant nucleic acid characterized by containing the nucleic acid as described above. .

The above-mentioned heterologous nucleic acids consist of nucleic acids or fragments thereof belonging to bacteria or eukaryotic cells. In this case, the bacterium or eukaryotic cell contains the aforementioned DNA fragment. selected as one of the expression systems for

Another heterologous nucleic acid suitable for the practice of the invention may also be constructed as a plasmid or phage. It is good that it has been done.

The invention furthermore provides, in particular for cloning and/or expression of nucleic acids according to the invention. in recombinant vectors, especially of the plasmid or phage type, containing a recombinant nucleic acid as described above, and using this nucleic acid as one of the non-critical sites for replication. It also relates to a recombinant vector characterized in that it is accommodated in a recombinant vector.

Therefore, in this recombinant vector, the preceding heterologous DNA constitutes an autonomous replication system. Insofar as the vines formed by this DNA.

In one particular embodiment of the invention, the above-mentioned recombinant vector is In one of the non-critical sites, there is a nucleus encoding the amino acid sequence of the invention. elements necessary to promote expression of acid in the host cell, i.e., compatible with the host cell promoters, especially inducible promoters, and in some cases signal sequences. and/or one expression vector containing fixed (anchor) sequences.

The selection of promoters, signal sequences and fixed (anchor) sequences is determined by the selected expression system. Determined according to the nature of

One recombinant vector suitable for expressing the amino acid sequence according to the invention in E. coli The target is located at one site that is not important for its replication. - an element that allows insertion into E. coli; - an element that allows insertion into the vector by E. coli; elements that enable expression of nucleic acids, and - optionally a requirement allowing the export of the products resulting from the expression of the nucleic acids mentioned above; Basic, Contains.

Another recombinant vector is to insert the above-mentioned nucleic acid into yeast. It is characterized by containing elements that enable it to be expressed in yeast.

Other eukaryotic cells may also be selected for insertion of recombinant vectors containing the nucleic acids of the invention. This selection is determined by the ability of the eukaryotic cell to organize the expression of nucleic acids. Directed.

The present invention further provides host cells transformed with a recombinant vector as described above. Also related. Furthermore, such a vector can also be replicated within the above-mentioned microorganism.

A preferred first host cell is transformed with a recombinant vector as described above. ;! 5JiJi (consists of '-''''.

Another preferred host cell is yeast transformed with a recombinant vector. be. The commonly used yeast is Saccharomyces Cerevi. It is aiase.

Other host cells are also available.

It will be appreciated that the present invention also relates to The expression products of each transformed microorganism are also targeted.

The present invention also provides stepwise addition of selected peptide residues and associated amine and and the addition or removal of any protecting groups of the carboxyl functions, or alternatively fragments. Addition of selected peptide residues to generate a subsequent fragment of this Condensation in the form of one suitable amino acid sequence and concomitant addition of selected protecting groups and The present invention also relates to a method for preparing said new peptides, with configurations comprising removal and removal.

The present invention also provides a method for preparing an amino acid sequence that meets the above description, - host cells previously transformed with a suitable vector containing the aforementioned nucleic acid, culturing in a suitable culture medium; - After lysis of the cell membrane of the transformed microorganism, the amino acids produced by this microorganism recovering the amino acid sequence from the culture medium; It also relates to a method characterized by.

Methods for preparing certain amino acid sequences include, in particular, transformation with the above-mentioned recombinant vectors. The initiation of culture of λ1 and the termination of the culture at the end of the logarithmic growth phase of IJ. contains extracellular enzymes and other proteins that may be secreted by Tai II. characterized by the recovery of certain amino acid sequences from uncontained culture media.

The method for preparing certain amino acid sequences can be further improved by using recombinant vectors as described above. It can also be applied to transformed yeast. This yeast is cultured in a suitable culture medium. The culture is stopped at the end of the logarithmic growth phase of the yeast used.

The invention also relates to antibodies directed against the above-mentioned amino acid sequences. especially , the present invention relates to monoclonal antibodies.

Such monoclonal antibodies are produced using hybridoma (hybrid cell) technology. However, the overall principle of this technique will be reviewed below.

In the first step, the amino acid sequence described above is inoculated into the selected animal. child When this animal's lymphocytes B are able to produce antibodies against this amino acid sequence, can. These antibody-producing lymphocytes are then fused with "immortal" myeloma cells and Produces bleedoma.

From the heterogeneous cell mixture thus obtained, specific antibodies are produced and multiplied indefinitely. Each hybridoma produces selected cells that can reproduce. are grown in the form of lawns, each having an affinity for recognizing the amino acid sequence of the present invention. lead to the production of monoclonal antibodies that can be tested on a nitty column, etc. .

The present invention also relates to biological specimens that may contain the above-mentioned amino acid sequences. The present invention also relates to a method for tracking these amino acid sequences in blood. Based on the present invention Tracking methods such as those described above use monoclonal antibodies or oligonucleotides as described above. This can be done using a nucleotide probe.

The above-mentioned biological collections are performed in fluid tissues such as blood, or in organs; In particular, obtain thin tissue sections on which the above-mentioned antibodies will later be fixed. be able to.

The tracking method according to the invention carried out via the above-mentioned antibodies particularly comprises the following steps: Deiru ni - At least one antibody that specifically recognizes the amino acid sequence according to the present invention contacting the above-mentioned biological specimen. - The immunocomplex optionally formed between the amino acid sequence and the above-mentioned antibody is The stage of detection using all possible means.

Advantageously, the antibodies used for use in such methods are particularly enzymatic or is marked with radioactive markings.

Such a method according to the invention is in particular an ELISA (enzyme immunoassay) comprising the following steps: standard method) - Marking using an enzyme label on a solid support such as a microplate well fixing a predetermined amount of the antibody; - a step of adding a (liquid) biological specimen onto said support; - Sufficient time to allow an immune reaction between the aforementioned antibody and the aforementioned amino acid sequence. Incubation step; - addition of specific substrates whose enzymatic activity was released during a previous immune reaction; - detecting degradation of the substrate by the enzyme using any suitable means; - Correlating the amount of enzyme released to the amount of amino acid sequence present.

According to another embodiment of the tracking method of the invention, the above-mentioned antibodies are marked. detection of immune complexes formed between the amino acid sequence and the antibody This is done using a marked immunoglobulin that recognizes the complex.

The tracking method according to the invention also involves the preparation of a predetermined amount of amino acid for the above-mentioned antibody. amino acid sequences and this same amino acid sequence that may be present in biological specimens. It can also be performed by immunoenzymatic techniques following the mechanism of competition between columns. . In the former case, a predetermined amount of the amino acid sequence of the invention can be prepared using an enzymatic label. marked advantageously.

The present invention is based on the amino acid sequence based on the present invention! In the above-described embodiment for hemostasis tracking This tracking is in no way limited to other immunological methods currently known. It is also possible to perform this using an immunoenzymatic method.

The present invention may also include nucleic acids encoding for the amino acid sequences of the present invention. In the method for in vitro tracing of amino acid sequences of the present invention made from a certain biological specimen, hand, - adding at least one of said oligonucleotide probes to said biological specimen; a step of contacting with - hybridization complexes optionally formed between said nucleic acids and probes in a suitable manner; Detection using any means: It also relates to a method characterized in that it includes.

The biological specimens mentioned above must be lysed of the cells they contain prior to tracking. Moreover, the genomic material contained within these cells contains EcoRI, BamHI, etc. It has been treated with a type of restriction enzyme to be cleaved.

Advantageously, the oligonucleotide probe is labeled using an enzymatic label or a radioactive label. King has been. These probes are supported on a suitable support, especially nitrocellulose or Other (e.g. nylon) filters are then placed on this support as described above. Biological specimens processed as follows are added.

Another object of the invention is to utilize one of the above-mentioned in vitro tracking methods. , which is outside a limited range of extreme values that generally correspond to the physiological state of a healthy body. Diseases that are correlated with amino acid sequence content based on the present invention! How to diagnose hemostasis It is in.

The amino acid sequence according to the invention is present in small amounts in the biological specimen tested or not present at all, or conversely, such sequences are overrepresented within the cells of the specimen. If this is the case, this may result in the activation of anti-tumorigenic factors and oncogenic factors within these cells. It proves the imbalance, and it also proves the imbalance where there are many tumorigenic factors. in some cases, especially due to loss of contact inhibition between cells of the harvested tissue or organ, There is a risk of starting a cancerous disease.

The present invention also provides supplies or kits for implementing the above-described in vitro tracking method. is also its purpose.

As an example, such a kit may include, among other things, the following: - Possibility of causing a specific immune reaction with one of the amino acid sequences of the present invention an amount of at least one of said monoclonal antibodies, - preferably a medium suitable for the formation of an immunological reaction between the amino acid sequence and the above-mentioned antibody; - Preferably, an agent that allows the detection of the immune chains generated during the above-mentioned immune reaction. medicine.

Using oligonucleotide probes! Within the scope of implementation of the hemostasis tracking method, The kit includes, for example: - one of the above-mentioned nucleic acids encoding an amino acid sequence according to the invention The above-mentioned oligonucleotide probes that can cause hybridization reactions with an amount of at least one antibody of - preferably the amino acid sequence and a medium suitable for the formation of an immune reaction between antibodies, - Preferably, an agent that allows the detection of the immune complexes generated during the above-mentioned immune reaction. medicine.

Using oligonucleotide probes! Within the scope of implementation of the hemostasis tracking method, The kit includes, for example:

- a hybrid with one of the abovementioned nucleic acids which subsequently encodes an amino acid sequence according to the invention; Among the above oligonucleotide probes that may cause the formation reaction, at least one fixed amount, - preferably a medium suitable for the formation of a hybridization reaction between a nucleic acid and said probe; - preferably allows detection of the hybridization complex produced from said hybridization reaction; reagent.

The amino acid sequences according to the invention have advantageous revertant properties.

In order to prove these properties, the following tasks are performed: - Normal and one of the mutated nucleotide sequences RAPIA, RAP], B or RAP2 Insert.

- Cells previously transformed with the RAS gene are transformed using the vector described above. Perform transfection.

- resistance to antibiotics, e.g. neomycin, as a selectable marker; genes and genes RAPIA or RAP IB or RAP2 and these cells co-transfect.

- express RAPIA or RAPIB or RAP2 in these cells to determine the phenotype Reverse mutations can be detected.

- Cells with a normal revertant phenotype are released t1 and analyzed to identify revertant mutations. Expression of the genes RAPIA or RAPIB or RAP2 that are directly responsible for the mutation ( RNA on the one hand and protein on the other hand).

More precisely, the mechanism of proteins produced from genes RAPIA and RAP2 is For the purpose of initiating functional research, two types of tools are available: antibodies and overproducing cells. The second cell lineage was developed. - The cDNA corresponding to genes RAPIA and RAP2 is a eukaryotic expression vector. For example, in the case of RAPIA, fibrillar forms in culture taken from murine In the case of adult cells NTH3T3 and RAP2, it is referred to as RAT1. different cell lines introduced by transfection into the same strain. The cDNA of the gene RAP2 is found in normal fibers. It does not appear to confer any specific phenotype when expressed in cell lines. this against strains transformed by oncogenes H-ras and Ki-ras. Its overrepresentation in DNA that correlates with backmutation - For proteins RAPIA and RAP2 or peptides derived from their sequences , rabbit polyclonal antibodies were generated. The obtained antibody specifically binds the protein. Recognizes white matter RA, PIA or RAP2 and does not show cross-reactivity with protein H-ras stomach; - Immunoprecipitation of the protein RAP2 in cell lines overexpressing this protein By using these antibodies, we found that it is a membrane protein. It was. This protein undergoes one step before being rapidly modified to produce the mature form of the protein. It is synthesized in the form of a precursor substance. This protein is relatively stable in machining, and its metabolism is halved. The period is about 12 to 24 hours.

The amino acid sequence according to the present invention exhibiting the above-mentioned reverse mutation characteristics is the RAS gene. The RAP gene is altered due to diseases related to or sudden changes or defective expression in some cells. It is likely to be used for therapeutic treatment of diseases that are thought to be caused by children.

For this purpose, a gene encoding for one of the amino acid sequences of the invention can be combined into one Introduced into the expression vector. This expression vector guides these sequences. There must be a compatible receptor with the cell in which the desired protein is desired, and its promoter must also be compatible with the cell in which it is desired. Must be compatible with cells.

The vector is then targeted so that it colonizes within the targeted cells. These cells take up this vector, and this vector is decomposed within these cells. Allow yourself to be released and expressed.

Example 1: Ri RAPIA and RAP2's Kuroni Z Kuri-ku Saratatei 1) Human clone of cDNA homologous to Drosophila gene Dras-3 Separation: Human B lymphocyte RNAm (Raji strain) in phage λgtlo under less severe hybridization conditions (see Materials and Methods section below). (specified) Transfer the sieved cDNA bank to Drosophila D. Used with complete insert of NA Das-3.

Fifteen phages are isolated from recombinant phage i ooooo. EcoRI The certs were purified and subjected to extremely stringent conditions (specified in the Materials and Methods section below). are subjected to a cross-hybridization reaction against each other under It is determined. Thus, one derived from the same gene and another similar to identify two categories of clones, one derived from a gene of I can do it.

RAPIA is a clone of the first group, and RAP2 is a clone of the first group. This is a clone of Group 2.

Human genomic DNA taken from placenta contains BamHI, EcoRI and HindI. Columns A, B, C, D, E, and F are hydrolyzed by restriction enzymes such as II. Each corresponds to the following: Column A: BamH1 Column B: BamHI+EcoRI Column C: BamH1+Hi ndIII Column D: Hi ndIII Column E: HindIII + EcoRI Column F: EcoRI This genomic DNA was then converted into the DNAc of clones RAPIA and RAP2, respectively. is hybridized with the insert EcoRI.

Figure 4 shows the hybridization profile of cellular genes RAPIA (1) and RAP2 (2). (side view).

The size of the fragment, determined by HindIII labeling, is in kilobase pairs. The position is shown on the left.

2) Nucleotide sequences of RAPIA and RAP2 DNAc: 1450 base pairs and 980 base pairs of DNAc of RAP IA and RAP2, respectively. Determine the nucleotide sequence of the paired inserts.

The amino acid sequences encoded by these clones are shown in Figure 1 and Figure 1, respectively. This is shown in Figure 3.

In Figures 1 and 3, RAPIA and RAP2 are coded, respectively. Nucleic acid sequences are also shown.

Proteins RAP IA and RAP2 each contain 184 and 184 proteins containing initial methionine. Consists of a peptide sequence of 183 amino acids, each with a calculated molecular weight are 20900 and 20700d.

3) Proteins RAPIA and RAP2 and proteins Dras-3, K-ras, rat and the homology between R-ras On the one hand, the protein RAPI and on the other hand, the Drosophila proteins Dras-3 and The homology between R-ras, ral and R-ras is 66.5, respectively. %, 53%, 44% and 41%.

For the most conserved region of the ras protein from amino acids 1 to 84, this ratio is , 78.5% for Drosophila protein, 59.5% for K-ras %, 56% for ral and 63% for R-ras.

A global comparison of cells for the protein RAP2 shows that Drosophila Dras- 3, 48%, K-ras, 46%, ral, 38%, and R-ras, 37%. Derive homology.

If only the first 84 amino acids are considered, these proportions are 2, -63% for ras and ral, and 56% for R-ras.

Figure 5 shows the human protein -ras (exon 4B) and the Drosophila protein. The sequences of proteins RAP IA and RAP2 with white matter Dras-3 are shown.

The reference numbers are the same as for the protein -ras.

In FIG. 5, the GTP fixing area is surrounded. Actuator (effector) The area is underlined with a single solid line, and the area of attachment to the intercellular membrane is underlined with a dotted line. A line is drawn.

It is incorporated into the guanine of the ras protein at the nucleotide binding site [Ba rbacid, M. (1987) Annual Review of Biochemistry, 56, 779-827] Amino acid sequence lO-17 ．． The four areas corresponding to 57-63 degrees, 113-120, and 143-147 are: Protein RAP% has a high level of conservation among -ras and Dras-3.

The proteins RAPIA and RAP2, like the protein Dras-3, have been previously known. All ras proteins and proteins similar to ras proteins that are not oncogenes It has a threonine at position 61 instead of the glutamine present in it.

Residues for the ras protein (corresponding to the region involved in interaction with the agonist) The region 32-42 is the same in protein RAP IA and ras; The protein RAP2 differs from the ras protein by one amino acid.

The C-terminal region of the ras protein is characterized by the sequence Cys-A-AX.

In this sequence, A is a fatty acid amino acid and X is a C-terminal amino acid (Pow ers et al. (1984) (, el-1 (cell), 36°607-6123 , this sequence is required for post-translational lipid binding and anchorage within the plasma membrane. Written by Katsuyamachi (1986), Proc, Natl, Acad, Sci, 　U, S, A,.

83, 12es-+2=,o].

5) Transcription of genes RAP IA and RAP2: Transcription of genes RAPIA and RAP After electrophoresis on a gel according to a technique called ``Northern blot'' method, It is carried out by RNA transfer on the membrane.

The conditions used for the “No Sun Plot Method” are recalled in the “Materials and Methods” section. There is.

Using one probe from RAPIA, we determined that each nucleotide 600.2700 and 5400 R-N A sequences (i.e. three transcripts) )6 Using probes from RAP2, the number of nucleotides was 2200 and 250, respectively. Three R,NA sequences of 0 and 4600 are verified.

The probe from RAP, LA is a fragment PstI-B' of sequence (19) Its first end corresponding to the restriction according to gl II. is shown in FIG. Its second end, corresponding to the glII restriction, is 520 base pairs from the first end. It's on the right side.

The probe from RAP2 is a fragment of sequence 21 HindII-Ps It is a polynucleotide restricted by tI and compatible with restriction by HindII. Its corresponding first end is shown in FIG. 9 and corresponds to the restriction by PstI. The second end is 340 base pairs to the right from the first end.

Figure 6 shows electrophoresis and transfer of RNAm polyA' extracted from human lymphocytes. Later transcriptional studies of RAPIA (column 1) and RAP2 (column 2) are shown. . The sizes of the different RNAs are marked on the left in kilobases.

6) Materials and methods: Cloning and sequencing of RAPIA and RAP2 DNAc : To identify RAP DNAc, Drosophila Dras-3 DNAc The 700 base pair EcoRI insert of the clone and the phage λgt10 We sifted through a human bank constructed within [5Chetjer et al. (198 5 years) EMBOJ, 4°407-412], less stringent conditions (i.e. 5XSSC15X Denhardt, 0. 1% SDS, 1.00 pg Hybridization was performed in salmon sperm DNA at 60° C./ml.

The final wash was performed in 2X SSC and 0.1% SDS at 60°C for approximately 15 minutes. Ta.

The extremely stringent conditions used for cross-hybridization of insert ECOR■ It is as follows.

Hybridization is carried out under the following conditions: 5,X,5SC15X DenMrdt , O, 1% SDS and 10. , O’tt g l + nl salmon sperm DNA, 60℃.

Washing is done with 0.1% SSC and 0.1% SDS at 65° C. for 30 minutes.

Nucleotides of the complete EcoRI insert of clones RAPIA and RAP2 The sequence is based on the dideoxynucleotide chain termination method Messing, J. 1983) Metbods Enzymol, (Enzymological Methods) 101. 2O-78) using vector M13.

7) Northern and Southern plot analysis: A technique called the “Southern plot” method. The law is 5outhern, E, ('-1975), J, Mo1.　 Biol,,98.

503, as described in the paper. Eliminated by restriction endonuclease Electrophoresis of 10 μg of converted human placenta DNA on 0.8% agarose gel and transferred onto a nitrocellulose filter to isolate each clone RAPIA and RA. Hybridize with the EcoRI insert of P2.

This hybridization is carried out under the following conditions: 5XSSC15X Den hardt, O, 1% SDS and 100 μg/ml salmon sperm DNA, 6 5℃.

Washing is done with 0.1% SSC and 0.1% SDS for 30 minutes at 65°C. .

Regarding a technique called the "Northern blot" method, a method is used to determine the sequence of RNA. To prepare cytoplasmic RNA from peripheral blood lymphocytes [Perry et al. 72) BBA, 262, 220-226].

Before transfer onto a nylon membrane (commercially available under the name Pail) 5 μg of DNA polyA4 was separated on a 1% gel of aldehyde and agarose, and the Hybridize with probes and filters from RAP IA and RAP2.

All probes were randomly started to obtain an activity of approximately 3X10'CPM/μg. The technique marks 32p (Amershaml. Mamatis et al. Author, (1982), Molecular cloning ning).

Written in the paper of Co1d Spring Harbor Laboratory. Hybridization is carried out in 50% formaldehyde at 42°C according to the method described. .

Example 2 Cloning of two RAPIBs prepared in phage λgtlO In order to sift through the human cDNA bank, we used the RAP IA cDNA insert. Use

The insert RAPIA is marked with sap, which allows subcloning Identify phages containing inserts (2100 pb) that are sequenced after sequencing be able to.

The nucleic acid sequence and the amino acid sequence encoded by this nucleic acid are shown in Figure 2. Ru.

On the one hand, studies of the homology between this amino acid sequence and the proteins RAPIA and RAP2 According to % homology.

This amino acid sequence will then be represented by RAP IB. On that calculation The molecular weight of is 20800d.

3: RAPIA in vector 1 generation a) In the vector PUC8, at the EcoRI site, for the purpose of amplification RAPI Subcloning the cDNA of A.

After culturing, cDNA was extracted and subjected to restriction enzyme Bg12 and then EcoRI. , thus corresponding to the complete region encoding the protein RAP IA. Collect fragment A.

Fragment E c o RI / B g ] 2 has sites BamHI and E cloned into phage M12Mp11 at coRI, thus phage After culturing, single-stranded DNA can be recovered.

5′CAGATCACATATGCGTGAGTAC3′ Directed mutagenesis is performed by using oligonucleotides; Ligonucleotides are prephosphorylated by polynucleotide kinase .

Therefore, a site NDEI was introduced upstream of the ATG corresponding to the start of the protein RAPIA. do.

Next, the recombinant M13MpH is prepared. For this, a double strand of M13MpH Cut the DNA with the restriction enzyme NDEI and use Klenow enzyme to cut the NDEI site. adjust.

Next, the fragment thus obtained is cut with the restriction enzyme Hindll. The vector M13MpH was cut with EcoRI-HindIll. tied together. Note that the EcoRI site of this vector is Repaired by enzymes.

5' CCAGTGAATTC phosphorylated by polynucleotide kinase Directed mutation using synthetic nucleotides with the sequence TATGCGTGAG3' Perform mutation generation.

This oligonucleotide connects C to the ligation site of RAP IA and M13MpH. can be inserted, thus re-forming the site EcoRI. . Phage M13MpH containing the sequence RAPIA is thus obtained.

Insert RAPIA is inserted into the phage at sites EcoRI and HindIII. It can be separated by cutting.

b) Next, adjust the double-stranded DNA of M13MpH. Clocks in bacterial expression vectors EcoRI and Hi to adjust the insert RAPIA for rowing purposes. Cut with nd III.

For example, as an expression vector, Tucker et al., EMBOJ, Vol. 5, Vol. 6, p. Use the vector described in the paper 1351-1358 (1986) . More precisely, the vector used is p'tac-cHra s vector.

The ptac-Hctas vector has the structure described in the above paper by Tucker. It can be obtained from the pKM-tacI vector according to the method. pKM-tacI vector Regarding vectors, see Hermann A., De Boer et al., DNA 5, No. 8. Volume 0, p21-25. Obtained according to the structure described in the January 1983 paper. be able to.

The ptac-Hcras vector contains approximately 300 vectors containing the inducible tac promoter. To preserve the EcoRI-Ec'oRI base pair fragment, we first restricted the It is subjected to the enzyme Hindll and then to EcoRI.

Into the thus prepared vector a fragment of RAPIA, EcoRI-Hi From the resulting vector, inserting ndnl and performing ligation, PRIM15 or a large strain such as the strain known under the name HBIOI-vector PDMI. Transform the S. enterica strain.

C) Next, express the protein RAPIA using the following method.

From about 12 hours of incubation, seeding was carried out in l/l OOe culture medium, and about Grow for 3 hours and then induce with IPTG to a concentration of 50 μM for approximately 12 hours.

The protein RAPIA expressed by the E. coli strain was then extracted according to conventional techniques. to recover.

This protein is in a soluble form.

4: RAPIA's birth in 161 at Evactor As shown in section a) of Example 3, i.e. the sites EcoRI and Hin Phage M13MpH containing the sequence RAPIA cloned in dllll was obtained. Proceed with the work until it is completed.

The single-stranded DNA of phage M13MpH is activated by polynucleotide kinase. Directed mutagenesis using the following phosphorylated synthetic oligonucleotides: Therefore, it is adjusted.

5' GAT-ACT-GCA-GGG-CAA-GAG3' (A) (To convert threonine “61” to glutamine) 5”-GAT-AC, T-GCA-GGG-GTA-GAG3’ (B) (To convert threonine "61" to valine).

Thus, at position 61, threonine is substituted within the frame of oligonucleotide (A). Instead, glutamine was introduced and threonine was replaced within the framework of oligonucleotide (B). Introduce valine instead.

Respective double-stranded DNA of each of the two sequences RAPIA modified as described above is prepared here and then cleaved by the enzymes EcoRI and HindIII. and then cloned into an expression vector as shown in Example 3, section b). be logged.

The two modified proteins are obtained according to Section 03 of Example 3.

5: RAPIA's death at Evactor and sudden death at 35 Proceed as shown in Example 4. Directed mutagenesis Using the following synthetic oligonucleotide phosphorylated by cleotide kinase be done; 5' TAT-GAC-CCA-GCG-ATA-GAA-GAT3', Treo Convert nin "35" to alanine.

6: Death at Evector RAPIA's 7″ fall was released in 12 Proceed as shown in Example 4. Directed mutagenesis carried out using synthetic oligonucleotides phosphorylated by cleotide kinase. I: 5′ CTT-GGT-TCA-GTA-GGC-CTT3′, Chrysin “12 ” to valine.

h 7: RAPIA's death in Evactor 17... Proceed as shown in Example 4.

Directed mutagenesis induces the following phosphorylated by polynucleotide kinase: Performed using synthetic oligonucleotides: 5' CGT-TGG-GAA-GAA-TGC-TCT-GAC-A3', Convert phosphorus "17" to asparagine.

8: RAPIA's 1168 in King Vector suddenly passed Proceed as shown in Example 4.

Directed mutagenesis induces the following phosphorylated by polynucleotide kinase: Performed using synthetic oligonucleotides: 5' GAT-AAA-TAG-GTA-AAC-ACC-AGT3', Rigi A stop codon is introduced in place of 168.

[! 141I9: RAP IB log in vector In phage M13MpH, fragment RAP at sites EcoRI and BamHI Clone the IB fragment Ec o RI-KpN I.

The following synthetic oligonucleotides phosphorylated by polynucleotide kinase Perform directed mutagenesis using: 5' AAG-CTT-GCA-TAT-GCG-TGA-GT3', thus Introducing the site NDEI upstream of ATG corresponding to the start of protein RAP IB1 do.

Next, the recombinant M13MpH is prepared. Therefore, using the restriction enzyme NDEI, M13 Cut the MpH double-stranded DNA and use Klenow's enzyme to remove the NDEl site. to repair.

Thereafter, it is cut with restriction enzyme Hind III. after that.

Vector M13MpH cut with EcoRI-HindIII Perform ligation of the obtained fragment against. In addition, the site Eco of this vector The RI is repaired with Klenow's enzyme before ligation.

Next, in order to introduce one C into the ligation site of M13MpH and RAP IB, Perform sexual mutagenesis.

This site EcoRI is thus reformed. This directed mutagenesis Synthetic oligonucleotides of the following sequences phosphorylated by nucleotide kinases: 5' CCA-GTG-AAT-TCT-ATG-CG T-GAG3'.

Phage M13MpH containing the sequence RAPIB is thus obtained. insert RAP IB can cleave the phage at the EcoRI and Hindll sites. It can be separated by the vector ptac and inserted into an expression vector such as the vector ptac.

Protein RAP IB is carried out as indicated in Example 3, section C).

10: 112 in vector Suddenly, RAP2's As shown in section a) of Example 3, i.e. the sites EcoRI and Hin Phage M13MpH containing the sequence RAP2 cloned in dllll was obtained. Continue working until it is completed.

The following synthetic oligonucleotides phosphorylated by polynucleotide kinase : 5'CGG-AGG-GCA-TAT-GCG-CGA-GTA3" used to introduce NDEI, Oligonucleotides with the following sequences phosphorylated by polynucleotide kinase Do; 5' CCA-GTG-AAT-TCT-ATG-CGC-GAG3' is M13 Insert one C into the ligation site of MpH and RAPIA, thus making the site EcoRI Used to reshape.

The single-stranded DNA of phage M13MpH was phosphorylated by polynucleotide kinase. by directed mutagenesis using the following oxidized synthetic oligonucleotides: Arranged: 5' GCT-GGG-CTC-GGT-CGG-GGTA-GGCA3', Convert chrysin "12" to valine.

The double-stranded DNA of the sequence RAP2 modified as described above was prepared using the enzymes EcoRI and and Hi n d III and then as shown in Section b) of Example 3. Yoni r Biochemical Properties of Ha-ra s Encoded p21 Mutants and Mechanism o f Autophosphorylation Reaction (Ha-ra Biochemical properties and autophosphorylation mechanism of p21 mutant encoded by s [The Journal of Biological Chemi stry (Biochemistry Journal) Volume 263, No. 24 pi 179:2~ 11799.1988] in the expression vector ptac32. be cloned.

Something like what is known as PRI M15 or HBIOI-PDMI. The protein modified in the E. coli strain is obtained according to Section C) of Example 3. .

Italy 11: 135 in Vector Naturally, RAP2 The site obtained as shown in the previous example was clicked with EcoRI and Hindll. From phage M13MpH containing the cloned sequence RAP2, phage M1 Single-stranded DNA at 3M pH is phosphorylated by polynucleotide kinase. prepared by directed mutagenesis using synthetic oligonucleotides of: 5'TAC-GAC-CCC-GCC-ATC-GAG-GAC3',)-Le Convert onine "35" to alanine.

Modified proteins are obtained as shown in the previous examples.

Pfi12: RA in vector VETR-RAPIA and VETR-RAP2 PIA genetics is the king of RAP2i l) Retrovirus type expression constructed from initial vector A6 as follows: The RAPIA gene and RAP2 gene were respectively expressed within the vector.

Vector A0 is a version of vector A in which the EcoRI site of RBR322 has been destroyed. It is a time variation.

This vector A is a 4.5Kb HindIII of Harvey virus. -E c o RI fragment (nucleotides 3495-2540) [ 5oeda, E., and Yasuda S.

(1985), RNA1m tumor virus; molecular biology of tumor viruses, Cold Spring Harbor Laboratory Press, (:ol d Spring Harbor, H, Y, 2nd edition, p928-939) 3.3Kb EcoRI-BamHI fragment of the same virus (nucleotide 2540-378). After ligation, Lysl The fragment is located between sites HindIII and BamHI of pBR322. inserted into.

The initial Beku A9 is represented by a circle in Figures 12a), 12b) and 12c. I'm being ignored.

FIG. 12a) shows the fragments S a c II / E of the initial vector A0. c o RI is shown as a thick line, and facing this thick line (inside the vector) I drew an arc with a dotted line.

Cut the initial vector A° with EcoRI and use K1enow to obtain a free end. Let the enzyme act. In this way, the site EcoRI is destroyed, and the Ec marked with an x is shown below. It is expressed as oRI or EcoRI.

Next, cut with S　a　c　II, and create a 1600 pb fragment S　ac　II /E Collect cXXRI.

2) In Figure 12b), the fragment PstIf of the vector A0 is indicated by a thick line. /S a c II, and face it (inside the vector) and draw a circle in practice. I drew an arc.

Cut the initial vector A0 with Pstl and Sac II, and create a 3736 pb frame. Collect the fragment PstI/Sac IT.

3) In Figure 120), the thick line indicates the fragment Ps t I / of vector A0. 　Hi　　　　d　Il[, and opposite it (inside the vector) a dotted line In other words, 6 drawn by an arc with a dashed-dotted line Cut the initial vector 80 with Hind III. Kle to get the free end now enzyme and then cut with PstI. Part Hi n d III is It was destroyed in this way, and is hereinafter referred to as Section H dII■ in this book.

Recover 5626 pb fragment PstI/HKdIII .

4) Then, ligate the following fragments obtained in 1), 2), and 3) above, respectively. D - Ps t I / S a c II] 2d) Intermediate structure shown in figure Obtain structure.

In this intermediate structure, HindIII and EcχRI/Hi%d The fragment contained between Ill and Ill is indicated by rEJ in Figure 12d). It contains sequences called "enhancers", or potentiating sequences.

5) Cutting the intermediate structure using PstI/HindIII . On the one hand, LTR3' (which serves as a polyadenylation site for RNA), On the other hand, the 6345 pb fragment Pstl/Hi containing the “enhancer” region Collect nd III.

The above-mentioned fragment is represented by a thick line in Figure 12e), opposite to this thick line. A point was drawn along one arc (inside the vector).

6) Use another vector denoted J'.

This vector J' is obtained from vector A0, and has the following two elements as vector A8 and The mutation at different knee position 12 is gone: 51bp Hind Viral fragment of lll/Prull (nucleotides 1088-1139 ) was replaced by a fragment corresponding to the murine Ha r a s cell gene. It was done. [Lacal et al. (1984): Proc, Nat] Aca d, Sci, USA, Volume 81, p5305-5309].

- The site S a c II located at position 940 in the Harvey virus is the site Replaced by EcoRI.

A fragment containing LTR5' is isolated from vector J'. For this, vector The tar J'' is cut at Ps t I/EcoRI and is represented by a single line in Figure 12f). A 2305 pb fragment PstI/EcoRI is isolated.

Also, draw a small solid triangle (inside the vector) along the arc facing this line. there was.

a) RAPIA subcloned in pUC8 or subcloned in pucs The inserted RAP2 inserter hEcoRI/Hindll is then inserted (as described above). obtained as shown in Section 5 and derived from a vector called “intermediate structure J”. The coming 6345 pb fragment Hi n d III / P s t T and 2305 pb of Pstl derived from vector J′ (see Section 6 above). /EcoRI.

The above-mentioned expression vectors containing RAPIA and RAP2, respectively, can be used in normal or especially expressed in different cell lines transformed with RAS or other oncogenes. Ta.

13. RAP IA in Vector PJ30 is the king of RAP2i This vector was created by Land, H.A., Chem, C.

Morgenstern, J., P., Parada, L., F., and W. einburg.

R, A, co-author, Mol Ce1l Biol (Molecular Cell Biology) 6:1917 -1925. Written during 1986.

For RAPIA, cloning in vector PJBQ is Direct cloning in vector PJ3Q at site E c o RI / B g12 Insert E c o RI obtained from the complete cDNA of RAP IA /Bgl2.

Regarding RAP2, since site Bg12 does not exist, site Ec　R I/PstI and cloned into plasmid pUC8 An insert derived from the RAP2 CDN is used. From this structure, 670 pb Take out Fragment E c o RI / Hi n dIII.

Cut the plasmid I) UC8 containing RAP2 with Hind III, and Repair with lenow enzyme to obtain free ends, then cut with EcoRI.

Part Hi n d III was destroyed and will be referred to hereafter as Hi n d III. do.

Insert E c o RI / HV @ d III (K 1 e n o (repaired with w) are separated on an agarose gel.

Vector PJ3Q has site B repaired by Klenow to obtain a free end. p(2) and then EcoRI.

Site Bg12 was destroyed and will be referred to hereinafter as Bg12.

Insert RA P 2 (E c o RI / H1% d III) and The vector P3Q cleaved at site B g) Next, a ligation is performed.

result: RAPIA and RAP2 cloned in vector PJaQ as described above. , normal or different, specifically transformed by RAS or other oncogenes. expressed in cell lineages.

Similarly within vector PJaQ at the sites Hindlll/EcoRI, respectively. Reverse cloning of RAPIA and RAP2 was also performed.

For RAPIA, subcloning RAPIA in pUCB The insert E c o RI / Hi n d III obtained by It is ligated into the vector PJ3Q cut with indlll/EcoRI.

For RAP2, obtained from subcloning of RAP2 in pUC8 Insert EcoRI/Hind III is part Hindll/Eco It is ligated to the vector PJ3Q cut with RI.

Reverse loaning of RAPIA and RAP2 in vector PJaQ results in internal The protein is generated by hybridization with the messenger RNA of the raw genes RAP IA and RAP2. Non-transcribed messenger RNA that can suppress protein synthesis can be obtained.

This vector can be used to produce proteins after transfection or by infection. Allows representation of white matter RAP. This vector is a previously “associated” virus (“ ``Helper'').

Example 14: In Vector PEX V3 RA　P　　　　　　゛　　　　　　　PI　　　゛　　　　　　　PI　　　　゛　　゛　゛　　　　　　P　　　　　゛゛゛　】　　　　　P　　　　　゛゛　　　　　 This vector was developed by Miller, J., and Germain, R.N.

J, Exp, Med, 164: 1478-1489. Written in 1986. It is.

Cloning of RAPIA and cloning of RAP IB and RAP2 For this purpose, vector PEX V3 is cut with EcoRI enzyme.

Complete cNDA inserts of RAPIA, RAPIB and RAP2, respectively. coRI is directly cloned into the vector PEX V3 cut at the EcoRI site. -ning.

For RAPIA, the insert is 1.4kb.

For RAPIB, the insert is 2゜lkb, and for RAP2, the insert is 2゜lkb. sert is 950 pb.

Good directionality within the vector, that is, directionality that is compatible with protein expression, can be by causing extensive enzymatic degradation (e.g. BamHI and pstI) Alternatively, for sequencing a restriction fragment containing the cloning site EcoRI. Therefore, it was established.

RAPIA, RAPIB in different normal or transformed cell lines, respectively. and RAP2 are expressed.

Similarly, cloning was performed according to the opposite direction to that described above. instructor transcribed RNA as shown at the end of Example 13 can be obtained.

Example 15: RAPIA' in vector PZIP N E O is RAP2'- Con5tance, L. Cepho for this vector.

Bryan, E. Roberts and Richard, C. Mullig Anr Cells” Volume 37: 1053-1062. Described in 1984. Ru.

previously cut with BamHI and repaired by Klenow to obtain a free end. Clone RAP IA and RAP2 at the BamHI site of the vector described above. Google.

The site BamHI is destroyed and is hereinafter referred to as BamHI.

Regarding RAPIA, it was previously subcloned into plasmid pUC8. There is.

cDNA was cut with EcoRI/Hindlll repaired by Klenow. 0 site ECXRI and Hind III are destroyed (to obtain free ends) and will be expressed as EcoRI and Hi″′Ad III* Kle The insert RAPIP (E6RI/H1% d■) repaired by now is By gauze to be able to construct the RAP IA vector pZipNeo tied together. Perform sequencing at various enzymatic degradation and cloning sites. Orient the vector (in a direction that allows expression of the protein) by .

To clone RAP2, proceed as described for RAPIA. Proceed.

cut with BamHI inserted in RAP2 repaired by Klenow. vector PZipNe.

is combined with

In PZipNeo, cloned RAPIA and RAP2, respectively. Expression in normal or transformed cell lines.

16: The RAPIA word in the mother vector YEP51 is the same as the RAP2 word in the mother vector YEP51. This vector is described in Broach et al. (1983) High-level inducible expression of expressed genes, Experimental treatment of gene expression, M.

Inouye ed, fNew York: Academic Pressl It is written on pp. 83-177.

Cloning is carried out as follows: vector YEP51 is cloned into the Klenow Cut with 5alI repaired with , and then with HindIll.

Site 5alI is destroyed and is hereinafter referred to as SXI.

purified and used for cloning.

For RAPIA, the insert used was in vector pUC8. It was derived from subcloning of RAPIA cDNA.

This insert was subcloned in the vector pucS at /E c o RI. The processed RAPIA was cleaved and subsequently repaired by Klenow enzyme. , further cleaved with Hindll. The site EcoRI is destroyed and is called EcXRI.

Insert E cX RI / Hi n d II is purified and then vectorized It is coupled to iI/HindIII.

For RAP2, proceed as indicated for RAPIA.

Various RAPIA and RAP2 cloned in vector YEP51 expressed in yeast.

117: The RAPIA program in the mother vector PD2E2 is based on the RAP2' tradition. Vector PD2E2 is described in the literature.

This vector PD contains the already cloned gene, and this gene was cut with Hindllr, purified on an agarose gel and extracted from the insert. The vector is then removed by separating the vector into K1enow Repair with enzymes to obtain free ends. Site Hind III is destroyed.

For RAPIA cloning, use the RAPIA cDNA library in pUC8. Using inserts derived from book cloning (fragment EcoRI/B g12), this insert contains pU at sites HindIII and EcoRI. obtained by cleaving c8RAPIA and treated with Klenow enzyme A free end is obtained.

Sites Hi n d IH and EcoRI are destroyed, and below for RAP2, The same type of structure as RAPIA was used.

RAPIA and RAP2 cloned in vector PD2E2 expressed in different yeast strains.

18: RAPIA, RAPIB and RAP2 in humans 1) For this purpose, proceed as follows: Preparation of cDNA probe. Made by cDNARAP is cloned into plasmid pUC8. that of each gene For each localization, two different fragments are used (Fig. reference).

For localization of gene RAPIA, two probes containing the complete cDNA (Using 1.4 kbl or a portion thereof 5' (0.7 kb) including the coding region) Ta. The two probes used for RAPIB were the complete cDNA (1 2.1 kbl or a portion thereof 5' (0.73 kb+).

The two probes for gene RAP2 were either complete cNDA of 0.95kb or partial This is a 0.66 kb fragment within the 5′ region.

2) Preparation of chromosomes.

High-resolution chromosome preparation was performed by Yun et al. (Ynis).

J, J, 5avryer, J, R, Ba1l, D, W: r metaphase-pre Characterization of punting patterns of G-band chromosomes during the phase and creation of genetic maps Its use J Cytogenit, Ce1lGenet, (Cytogenetics) 2 after the same period of methotrexate according to 22,679-683゜1978) Obtained from the culture of blood cells stimulated by phytohemagglutinin from a healthy human body. It will be done.

3) Hybrid formation between specific parts The probe was manufactured by Feinberg and Vogelstein (Feinberg g.

A, P, Vogelstein, B, A; rDNA restriction endonuclea A method for radiolabeling enzyme fragments to high specific radioactivity,” Annual Report of Biochemistry The accidental DNA induction method (A. III2rsham kit, U.). 50ng Activity obtained by using 20 uCi [H”]dCTP against cNDA is 0.5 to 2×10” cps/transport.

These marked sondes are used at concentrations of 20-80 μg/-.

Harpen and 5unders (Harpen, M, E,, 5au nders.

G, F, :rA single copy of the DNA sequence on the G-band human chromosome is generated by hybridization between specific parts. Column position confirmation J Chromosoma.

83.431-439.1981), the hybridization technique described by Can ber is modified slightly according to the method of ber et al. ((:aubet J, F, its et al., [C-mos map of human transgenic trochanter against δqll, Embo , J.

42245-2248.1985).

A plate coated with Kodak NTB2 emulsion was placed on an autoradiograph. Exposure for 1-2 weeks for raffy: then G-band is exposed with Wright's dye. can get.

4) Results: Two different inserts of each of the genes RAPIA, RAPIB and RAP" , regarding hybridization between specific parts of human chromosomes to confirm the results in Figure 10. each used. Only metaphases that do not show three or more silver grains on chromosomes are Photographed and analyzed.

5) Circumferential pressure of RAPIA: Experiments performed with the 1.4 kb probe or the 0.7 kb probe 1p12-p13 revealed only one particle deposit (of the total number 6% and 5.5%) (Table 1).

No significant secondary signals are seen on other chromosomes (see Figure 11a) .

6) Confirm the location of RAPIB 2.Specific hybridization with lkb or 0.7kb probes was performed on chromosome 12g1. 4 (Table 1) shows a clear accumulation of particles (7.2% and 9.6% of the total).

There are no significant other particle deposits on other chromosomes (11b figure).

7) Confirm the position of RAP2 The two protocols used in this location confirmation were 0.950kb and 0.660kb. The curves show identical particle deposits on chromosome 13g34 in each case. (6.5% and 13.4% of the total (Table 1).

No other secondary signals are visible on other chromosomes (Figure 11c).

! 1JiI: No secondary signals or hybridization were observed on other chromosomes.

Regarding chromosome 1p12-p13, any specific deletions are associated with specific neoplasias. However, some translocations in this region have been implicated in melanoma and various blood disorders. It is explained. (Bloom-field, C, D, trent.

J, M, Van Den Berghe, H: Structural staining in r neoplasia. Committee report on body changes (HG former O1゜Cytogenet, Ce11 ．． Gent, 46.344-366゜1987).

Confirming the apparently similar proximity of the gene N-ras, which is also located on the chromosome. Can be done.

Regarding the location confirmation of gene RAPI B on chromosome 12qlJ, the following Fractures have been noted in both benign and malignant organisms; - Lipoma (Heim et al., “Reciprocal translocation in lipoma t (3; 12) (q2 7;ql3) JCancerGenet.

Cytogenet (cancer genetics, cytogenetics), 23°301-304.1 986).

- Myxolipoenteroma (Turc-Garel et al., Cytogenetic study of lipohistiomas. T I, recurrent reciprocal translocation t(12+16) (ql, 3; pHl myxolipoenteroma, cancer Genetics, Cytogenetics, 23.291-299; 1986).

- Pleomorphic adenoma (Bullerdiek et al., “Pleomorphic adenoma of the human salivary glands”) Rearrangement of chromosomal region 12q13-q15 in (PSA) J, Cytogenetics, 4 5゜1.87-190.1987 and Mark et al., “100 Human Benign Polymorphisms” Cytogenetic findings in adenoma; specificity of chromosomal abnormalities and localized tumor remains Its relationship to genes J (Anticancer Res, , 6.

299-308.1986) and -Leiomyoma () (6am et al., “Chromosomal regions 12q13-ql5 and 14q22゜ 24. The rearrangement between t (12: 14) (ql3-15: q22-24) is J (80M9, Abstract), Cytogenetics, which may be found within uterine leiomyomas. 46.628.1987).

Regarding the location of the gene RAP2 on chromosome 13q34, any specific Deletions are also not associated with neoplasia, and some deletions in this region have been linked to sporadic breast cancer. It has been reported that

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Claims (1)

[Claims] 1. An amino acid characterized by containing at least one of the following amino acid chains: Noic acid sequence: [There is an array] (I) [There is an array] (II) [There is an array] (III) [There is an array] (IV) [There is an array] (V) [There is an array] (VII) [There is an array] (VIII) [There is an array] (IX) [There is an array] (X) [There is an array] (XI) [There is an array] (XII) [There is an array] (XIII) [There is an array] (XIV) [There is an array] (XV) 2. Five chains of amino acids I, II, III, IV and Amino acid sequence according to claim 1, characterized in that it contains V and V. 3. Five chains of amino acids VII, VIII, IX and The amino acid sequence according to claim 1, characterized in that it contains X and X. 4. Five chains of amino acids XI, XII, XIII according to claim 1, The amino acid according to claim 1, characterized in that it contains XIV and XV. acid sequence. 5. Contains the amino acid chain ZVI represented in Figure 1 or alternatively this amino acid chain ZVI The amino acid according to claim 1, characterized in that it is composed of array. 6. Contains the amino acid chain XVI shown in Figure 2 or alternatively this amino acid chain The amino acid according to claim 12, characterized in that it is constituted by Acid sequence. 7. Contains amino acid chain XVIII shown in Figure 3 or is composed of this amino acid chain The amino acid sequence according to claim 1, characterized in that the amino acid sequence is 8. At least one of the amino acid chains encoded by the following nucleotide chains: An amino acid sequence characterized by containing one: [There is an array] (1) 9. Encoding the amino acid sequences set forth in claims 1 to 7 A nucleic acid comprising at least one nucleotide chain. 10. A nucleus characterized by containing at least one of the following nucleotide chains: acid: [There is an array] (I) 11. Five nucleotide chains (1), (2), ( 3), (4) and (5) according to claim 10. Nucleic acid. 12. The five nucleotide chains (6), (7), ( 8), (9) and (10). nucleic acid of 13. Five nucleotide chains (11), (12) according to claim 10 , (13), (14) and (15). Nucleic acids described in Section. 14. Corresponds to the nucleotides located at positions 43 and 594 in Figure 1 comprising a chain of nucleotides (16) defined by terminal nucleotides or limited by the terminal nucleotides corresponding to the nucleotides at positions 1 and 605 of Claim 10 characterized in that it comprises a nucleotide chain (19) Nucleic acid described in. 15. Terminal nucleotides corresponding to nucleotides at positions 54 and 605 in Figure 2. or position 1 in Figure 2. and a nucleotide defined by a terminal nucleotide corresponding to the nucleotide at 838. Claim 10, characterized in that it comprises a cleotide chain (20). Nucleic acid as described. 16. Terminal nucleotides corresponding to nucleotides at positions 4 and 552 in Figure 3 or positions 1 and 55 in Figure 3. Nucleotide defined by the terminal nucleotide corresponding to the nucleotide at 8 Nucleic acid according to claim 10, characterized in that it comprises a double chain (21). 17. inserted into a nucleic acid heterologous to said Blaument. characterized by containing the nucleic acid according to any one of paragraphs 9 to 16. recombinant nucleic acids. 18. In particular, cloning and/or expression of plasmids, cosmids or phages. a recombinant vector containing the recombinant nucleic acid according to claim 17. , characterized by housing this nucleic acid in one of the sites that are not important for replication. recombinant vector. 19. within the host cell at one of the sites not critical for replication. Elements and fields necessary to facilitate the expression of amino acid sequences according to Sections 1 to 8. In some cases, promoters, especially inducible promoters, that are compatible with the host cell are used. motors, and possibly sequential and/or fixed arrangements. The recombinant vector according to claim 18, characterized in that: 20. - an element that allows insertion into E. coli, - the size of this nucleic acid according to claims 9 to 17 inserted into a vector; elements that enable expression by enterobacteria, and - an element that allows the export of the product resulting from the expression of the nucleic acid described above; The recombinant vector according to claim 19, characterized in that it contains . 21. Any one of claims 18 to 20 that can be replicated within the host cell. A host cell transformed with the recombinant vector described in section. 22. E. coli transformed with the vector according to claim 20. Host cell according to claim 21, characterized in that. 23. For the amino acid sequence according to any one of claims 1 to 8 Antibodies, in particular monoclonal, characterized in that they are derived. 24. one of the nucleic acids according to claims 9 to 17 or its complement A nucleotide prologue characterized by hybridizing with the sequence under the following conditions: - Nucleic acids (2), (3), (4), (5), (7), (8), (9), (10), (12), (13), (14) and (15) or their complementary sequences. For probes that can be used, the hybridization conditions are as follows. So-called hybridization: 60°C, 5X, SSC, 5X Denhardt, 0.1% SDS, 100 μg/ml salmon sperm DNA; washing: 60°C, 2x SSC, 0. 1% SDS, 30 minutes; - capable of hybridizing with nucleic acids or complementary sequences (1), (6) and (11) For probes, hybridization conditions are as follows: So-called hybridization: 45°C, 5x SSC, 5x Denhardt, 0.1% SD S, 100 μg/ml salmon sperm DNA; First cleaning: ambient temperature, 2x SSC, 0.1% SDS, 30 minutes, Second clean: 45°C, 2xSSC, 0.1% SDS, 30 minutes. 25. an amino acid sequence according to any one of claims 1 to 8; , tracked in vitro in biological specimens that may contain these amino acids. In the method of - the biological specimen and at least one of the antibodies according to claim 23; Steps of contacting with: - optionally an immunological complex formed between the above-mentioned amino acid sequence and the antibody; a step of detecting using all possible means; A method characterized by comprising: 26. The amino acid sequence according to any one of claims 1 to 8 is Biology that may contain nucleic acids encoding these amino acid sequences In a method for in vitro tracking in a target specimen, - said biological material pre-treated with at least one of the oligonucleotide probes; Contacting the target specimen: - hybridization complexes optionally formed between said nucleic acids and probes in a suitable manner; detecting using any means; A method characterized by comprising: 27. A region defined by extreme values that correspond as a whole to the physiological state of a healthy body. Any amino acid sequence which is outside the scope of claims 1 to 8 and includes the amino acid sequence according to any one of claims 1 to 8. Claim 25 or The in vitro tracking method according to item 26. 28. Necessary supplies for implementing the in vitro tracking method according to claims 25 and 27 or in a kit, - an agent to be detected according to claims 1 to 8; Claim 23 that may cause an immunological reaction with the amino acid sequence - preferably an amount of at least one of the antibodies of - preferably the amino acid sequence and a medium suitable for the formation of an immune reaction between said antibodies; - an immunocomplex preferably between an amino acid sequence and an antibody produced during the above-mentioned immune reaction; reagents that allow the detection of 29. Requirements for the use of the in vitro tracking method according to claims 26 and 27 In supplies or kits, - a code for the amino acid sequence to be detected according to claims 1 to 8; A claim that may cause a hybridization reaction with one of the nucleic acids A fixed amount of at least one of the oligonucleotide probes described in Box 24: - preferably a medium suitable for the formation of a hybridization reaction between a nucleic acid and said probe; - preferably allows detection of the hybridization complex produced from said hybridization reaction; 1. An essential article or kit, characterized in that it contains a reagent. 30. - any one of claims 9 to 17 in a suitable culture medium; Culture host cells previously transformed with an appropriate vector containing the nucleic acid described in to do, - and from said culture medium, after lysis of the cell membrane of said host cell, this transformed The claimed invention is characterized in that: recovering an amino acid sequence produced by a host cell. A method for adjusting an amino acid sequence according to any one of Items 1 to 8. 31. When starting the culture of E. coli containing the recombinant vector according to claim 20, The culture is stopped at the end of the logarithmic growth phase of E. coli, and the E. coli is separated. A fixed amino acid sequence from a culture medium free of extracellular enzymes and other proteins that can be secreted. A method for adjusting a certain amino acid sequence, characterized by recovering a sequence.