Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes

Definitions

• the subject of the invention is new amino acid sequences, the nucleic acids coding for these amino acid sequences and their applications, in particular for diagnosis, in. in vitro, pathologies linked to the action of oncogenic factors.
• Protooncogenes such as H-ras, K-ras and N-ras are known (De Feo et al., (1981) Proc. Natl. Acad. Sci., USA, 78, 3328-3332; Ellis et al., ( 1981), Nature, 292, 506-511; Shimizu et al. (1983) Proc. Natl. Acad. Sci., USA, 80, 383-387) and new genes have been identified in a wide variety of organisms on the basis of their homology with the mammalian ras gene.
• the proteins encoded by these genes close to the ras genes have 50 to 30% homology with the ras proteins (Chardin et al., (1986) EMBO J., 5, 2203-2208; Madaule et al., (1985) Cell, 41, 31-40; Touchot et al., (1987) Proc. Natl. Acad. Sci., USA, 84, 8210-8214).
• Ras proteins and proteins close to ras proteins have molecular weights of about 21,000 to 24,000 d. Ras proteins are active when associated with GTP (Guanosine 5'-triphosphate) and are inactive when associated with GDP (Guanosine 5'-diphosphate) and the hydrolysis of GTP takes place by the action of their low activity.
• GTP GTP
• GDP GTP
• intrinsic GTPase Paperageorge et al., (1982) J. Virol., 44, 509-519; Mcgrath et al., (1984) Nature, 310, 644-649).
• the transforming ras proteins detected in human tumors very frequently exhibit a substitution of the amino acids located in the GTP binding domain, in position 12, 13 or 61 (Barbacid (1987) Annual Review of Biochemistry, 56, 779-827).
• This domain plays an essential role in the regulation of the biological properties of ras proteins.
• the GTP binding site consists of four non-contiguous regions which belong to all ras proteins.
• One aspect of the invention is to propose new amino acid sequences, having the ability to fix GTP and GDP and having GTPase activity.
• One of the other aspects of the invention is to propose new nucleic acids coding for amino acid sequences exhibiting interesting biological properties.
• One of the other aspects of the invention is to propose screening means, in . in vitro, certain pathologies linked either to protein contents located outside the range of values that they generally have in a healthy individual, or to mutations in these proteins.
• the subject of the invention is new amino acid sequences characterized in that they contain at least one of the following amino acid sequences: MREYKLWL (I)
• amino acid sequences are written in such a way that the leftmost end corresponds to the N-terminal end of the first amino acid delimiting the above sequences and i _ the rightmost end corresponds to the end C-terminal of the last amino acid delimiting the above-mentioned sequences.
• amino acid sequences of the invention contain at least one of the sequences (I), (II), (III), (IV) or (V) defined above.
• the invention contains at least one of the sequences (VII), (VIII), (IX) or (X) defined above.
• the invention contains at least one of the sequences (XI), (XII), (XIII), (XIV) or (XV) defined above.
• amino acid sequences of the invention comprise a threonine in position 61.
• the amino acid sequences of the invention are such that the fourth amino acid from the last C-terminal amino acid is a cysteine.
• amino acid sequences contain the 5 sequences (I), (II), (III), (IV) and (V) defined above.
• amino acid sequences according to the invention comprise the 5 sequences indicated above, these are advantageously located, from left to right, in the order (I) - (II) - (III) - (IV ) - (V), these sequences also being advantageously non-contiguous.
• amino acid sequences contain the 5 sequences (VII), (VIII), (IX) and (X) defined above.
• amino acid sequences according to the invention comprise the 5 sequences indicated above, these are advantageously located, from left to right, in the order (VII) - (VIII) - (IX) - (X ), these sequences being also advantageously non-contiguous.
• amino acid sequences contain the 5 sequences (XI), (XII), (XIII), (XIV) and (XV) defined above.
• amino acid sequences according to the invention comprise the 5 sequences indicated above, these are advantageously located, from left to right, in the order (XI) - (XII) - (XIII) - (XIV) - (X), these sequences being also advantageously non-contiguous.
• Particularly advantageous amino acid sequences according to the invention contain the chain of amino acids XVI, shown in FIG. 1.
• Particularly advantageous amino acid sequences according to the invention consist of the chain of amino acids XVI, shown in FIG. 1.
• Particularly advantageous amino acid sequences according to the invention contain the chain of amino acids XVII, shown in FIG. 2.
• Particularly advantageous amino acid sequences according to the invention consist of the chain of amino acids XVII, shown in FIG. 2.
• Particularly advantageous amino acid sequences according to the invention contain the chain of amino acids XVIII, shown in FIG. 3.
• Particularly advantageous amino acid sequences according to the invention consist of the chain of amino acids XVIII, shown in FIG. 3.
• amino acid sequences XVI, XVII and XVIII will also be designated respectively by RAP1A, RAP1B and RAP2 in the remainder of the text.
• amino acid sequences have a threonine at position 61 and a cysteine 4 amino acids from the last C-terminal amino acid.
• the position of a determined amino acid is identified with respect to the first amino acid of the sequence whereas for the nucleotide sequences (also appearing in FIGS. 1, 2 and 3) the position of a determined nucleotide is identified with respect to the first nucleotide.
• amino acid sequences have interesting biochemical and biological properties: they have the capacity to fix GTP and GDP and exhibit GTPase activity.
• amino acid sequences are also likely to bind to the intracellular membrane.
• amino acid sequences can be modified as long as they retain the biochemical and biological properties of the amino acid sequences (XVI), (XVII) and (XVIII) defined above.
• amino acid sequences falling within the scope of the invention can be distinguished from the amino acid sequences defined above:
• amino acid sequences (XVI), (XVII) and (XVIII) having mutations in position 12 (that is to say replacement of glutamine in 12 by any what other amino acid).
• amino acid sequences (XVI), (XVII) and (XVIII) having mutations at position 61 (that is to say replacement of threonine at 61 with any which other amino acid and in particular by glutamine and valine).
• the invention also relates to the amino acid sequences containing at least one of the amino acid sequences encoded by the following nucleotide sequences:
• nucleic acids containing at least one of the sequences of nucleotides coding for the amino acid sequences defined above.
• the nucleic acids belong to the human genome.
• nucleic acids of the invention code for the sequences of amino acids I to XVIII defined above. According to yet another advantageous embodiment, the nucleic acid sequences of the invention code for the amino acid sequences containing the sequences XVI, XVII and XVIII defined above. According to another advantageous embodiment of the invention, the nucleic acid sequences of the invention code for the sequences of amino acids XVI, XVII and XVIII defined above.
• GTT GAA GTA GAT GCA CAA CAG TGT ATG CTT GAA ATC TTG (7) CAA TTT ACA GCA ATG AGG GAT TTA TAC ATG AAA AAT GGA
• the nucleic acids of the invention contain the five sequences of nucleotides (1), (2), (3), (4) and (5), defined above.
• the nucleic acids of the invention contain the five sequences of nucleotides (6), (7), (8), (9) and (10), defined above.
• the nucleic acids of the invention contain the five sequences of nucleotides (11), (12), (13), (14) and (15), defined above.
• the nucleic acids of the invention contain the sequence of nucleotides (16), delimited by the end nucleotides corresponding to the nucleotides located at positions 43 and 594 in FIG. 1.
• the nucleic acids of the invention consist of the sequence of nucleotides (16), delimited by the end nucleotides corresponding to the nucleotides located at positions 43 and 594 in FIG. 1.
• the nucleic acids of the invention contain the sequence of nucleotides (19) delimited by the end nucleotides corresponding to the nucleotides located in positions 1 and 605 of FIG. 1.
• the nucleic acids of the invention consist of the sequence of nucleotides (19) delimited by the end nucleotides corresponding to the nucleotides located in positions 1 and 605 of FIG. 1.
• the nucleic acids of the invention contain the sequence of nucleotides (17), delimited by the end nucleotides corresponding to the nucleotides located at positions 54 and 605 in FIG. 2.
• nucleic acids of the invention consist of the sequence of nucleotides (17), delimited by the end nucleotides corresponding to the nucleotides located at positions 54 and 605 in FIG. 2.
• the nucleic acids of the invention contain the sequence of nucleotides (20) delimited by the end nucleotides corresponding to the nucleotides located at positions 1 and 838 in FIG. 2.
• nucleic acids of the invention consist of the sequence of nucleotides (20) delimited by the end nucleotides corresponding to the nucleotides located in positions 1 and 838 of FIG. 2.
• the nucleic acids of the invention contain the sequence of nucleotides (18), delimited by the end nucleotides corresponding to the nucleotides located at positions 4 and 552 in FIG. 3.
• nucleic acids of the invention consist of the sequence of nucleotides (18), delimited by the end nucleotides corresponding to the nucleotides located at positions 4 and 552 in Figure 3.
• the nucleic acids of the invention contain the sequence of nucleotides (21) delimited by the end nucleotides corresponding to the nucleotides located at positions 1 and 558 in FIG. 3.
• nucleic acids of the invention consist of the sequence of nucleotides (21) delimited by the end nucleotides corresponding to the nucleotides located in positions 1 and 558 of FIG. 3.
• the nucleic acids (16), (17), (18), (19), (20) and (21) of the invention all have the particularity of hybridizing with the Dras-3 gene of Drosophila as shown in FIG. 5, under the weakly stringent conditions defined below: hybridization at 60 ° C. in 5X SSC, 5X Denhardt, 0.1% SDS and 100 ⁇ g / ml of DNA from salmon sperm, last washing in 2X SSC and 0.1% SDS, at 60 ° C for about 15 min.
• nucleic acids (16), (17), (18), (19), (20) and (21) can be modified as soon as the amino acid sequences which they encode retain their biochemical and biological properties.
• nucleic acids - by addition and / or - deletion of one or more nucleotides and / or modification of one or more nucleotides, provided that the sequence thus formed is capable of hybridizing with the same RNA or DNA sequence as the corresponding sequence unmodified, and that the amino acid sequences, encoded by these nucleic acids, retain the biochemical and biological properties indicated above.
• nucleic acids (16) and (19) can also be characterized by the restriction map which is the subject of FIG. 7.
• nucleic acids (17) and (20) can also be characterized by the restriction map which is the subject of FIG. 8.
• the nucleic acids (18) and (21) can also be characterized by the restriction map which is the subject of FIG. 9.
• restriction maps should not be interpreted as having a limiting character in order to identify the nucleic acids comprising respectively the sequence indicated above.
• Equivalent nucleic acids could also be defined by a restriction map having a community with the previously defined restriction maps of at least 50% of the restriction sites, located in the same order.
• the invention also relates to the probes used to characterize the nucleic acids described above.
• probes are defined by their ability to hybridize with the above nucleic acids or their complementary sequences under the following hybridization conditions:
• the hybridization conditions are as follows: actual hybridization: 60 ° C, 5X SSC , 5X Denhardt, 0.1% SDS, 100 ⁇ g / ml of DNA 'of salmon sperm; washing: 60 ° C, 2X SSC, 0.1% SDS, 30 min;
• the hybridization conditions are as follows: actual hybridization: 45 ° C, 5X SSC, 5X Denhardt , 0.1% SDS, 100 ⁇ g / ml DNA " of salmon sperm;
• 2nd wash 45 ° C, 2X SSC, 0.1% SDS, 30mn.
• oligonucleotide probe for identifying the nucleic acid sequences of the invention it is possible, for example, to use the probe constituted by the following oligonucleotide sequence: GAA ATC CTG GAT ACT GCA GGG ACA GAG CAA TTT or the corresponding complementary sequence.
• nucleic acids (18) and (21) of the invention it is possible, for example, to use as a probe the sequence of oligonucleotides delimited by the nucleotides corresponding to the nucleotides located at positions 451 and 558 of FIG. 3, or the corresponding complementary sequence.
• the invention also relates to a recombinant nucleic acid characterized in that it contains a nucleic acid as described above, inserted into a heterologous nucleic acid with respect to the above fragment.
• the above heterologous nucleic acid may consist of a nucleic acid or a nucleic acid fragment belonging to a bacterium or even to a eukaryotic cell, said bacterium or said eukaryotic cell being chosen as the expression system of a DNA fragments described above.
• heterologous nucleic acid suitable for carrying out the invention may also consist of a plasmid or a phage.
• the invention further relates to a recombinant vector, in particular for the cloning and / or expression of a nucleic acid according to the invention, said vector being in particular of the plasmid or phage type, characterized in that it contains an acid recombinant nucleic acid as defined above, containing the above nucleic acid at one of the sites not essential for its replication.
• This recombinant vector can thus be formed by the preceding heterologous DNA insofar as this constitutes an autonomous replication system.
• the above recombinant vector is an expression vector containing, in one of its sites which are not essential for its replication, elements necessary to promote expression, in a cellular host , a nucleic acid coding for an amino acid sequence of the invention, a promoter compatible with said cellular host, in particular an inducible promoter, and optionally a signal sequence and / or an anchoring sequence.
• Another recombinant vector is characterized in that it contains the elements making it possible to insert a nucleic acid, described above, in a yeast and the expression of this nucleic acid in this yeast.
• Other eukaryotic cells may be chosen to insert the recombinant vector containing the nucleic acid of the invention, this choice being oriented by the capacity of said eukaryotic cell to organize the expression of said nucleic acid.
• the invention also relates to cellular hosts transformed by a recombinant vector such as those described above, which vector is capable of replicating in said microorganism.
• a first preferred cellular host is constituted by E.coli transformed by a recombinant vector as described previously.
• yeast transformed with a recombinant vector is Saccharo yces Cere% 'isiae.
• cellular hosts can also be used.
• the invention naturally relates to the expression product of each of the microorganisms transformed by one of the recombinant vectors described in the preceding pages.
• the invention also relates to a process for the preparation of the new peptides mentioned above by synthesis which comprises either the step-by-step addition of the chosen peptide residues, with the addition or the removal of any protective groups from the amino and carboxyl functions, or addition of selected peptide residues in order to produce fragments followed by condensation of said fragments into an appropriate amino acid sequence, with addition or elimination of the protective groups chosen.
• the invention also relates to a process for the preparation of an amino acid sequence in accordance with the description above, characterized in that: a cell host cultivated beforehand with a suitable vector containing is cultivated in an appropriate culture medium a previously defined nucleic acid,
• the process for preparing a determined amino acid sequence can be characterized in particular by culturing E.coli transformed with a recombinant vector described above and by stopping the culture at the end of the exponential growth phase of said E.coli, and recovery of the amino acid sequence determined from the culture medium free of other proteins and exocellular enzymes capable of being secreted by E.coli.
• the process for the preparation of a determined amino acid sequence can also be applied to a yeast transformed by a recombinant vector as described above, this yeast being placed in culture in an appropriate medium and said culture being stopped at the end of the exponential phase. growth of the yeast used.
• the invention also relates to antibodies directed against the above amino acid sequences.
• the invention relates to monoclonal antibodies.
• Such monoclonal antibodies can be produced by the hybridoma technique, the general principle of which is recalled below.
• one of the above amino acid sequences is inoculated into a chosen animal, the B lymphocytes of which are then capable of producing antibodies against this amino acid sequence. These antibody-producing lymphocytes are then fused with "immortal" yelomatous cells to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, a selection is then made of the cells capable of producing a particular antibody and of multiplying indefinitely. Each hybridoma is multiplied in the form of an olone, each leading to the production of a monoclonal antibody whose recognition properties with respect to the amino acid sequences of the invention can be tested, for example on an affinity column.
• the invention also relates to a method of in vitro screening of the above-mentioned amino acid sequences, from a biological sample capable of containing them.
• a screening method according to the invention can be carried out either using the above-mentioned monoclonal antibodies, or using the oligonucleotide probes described above.
• the abovementioned biological sample is taken either from fluid tissues, such as blood, or from organs, the latter type of sample making it possible in particular to obtain fine sections of tissue on which the above-mentioned antibodies are subsequently fixed.
• the screening method according to the invention proceeding by means of the abovementioned antibodies notably comprises the following steps:
• the antibodies used for the implementation of such a method are labeled in particular in an enzymatic or radioactive manner.
• Such a method according to the invention can in particular be carried out according to the ELISA method (enzyme linked sorbent assay) which comprises the following steps:
• the above-mentioned antibodies are not labeled and the detection of the immunological complexes formed between the amino acid sequences and said antibodies is carried out using a labeled immunoglobulin recognizing said complexes.
• the screening method according to the invention can also be carried out by an immunoenzymatic technique according to a mechanism of competition between the amino acid sequences likely to be contained in the biological sample, and predetermined quantities of these same sequences of amino acids, vis-à-vis the above-mentioned antibodies.
• the amino acid sequences of the invention in a predetermined amount are advantageously labeled using an enzymatic marker.
• the invention is in no way limited to the embodiments described above for the in vitro screening of the amino acid sequences of the invention, this screening can be carried out using any other immunological method, in particular immunoenzymatic currently known. .
• the invention also relates to a method of in vitro screening of the amino acid sequences of the invention carried out from a biological sample capable of containing nucleic acids coding for said amino acid sequences, characterized in that it includes: - contacting at least one of the oligonucleotide probes described above with the above-mentioned biological sample; - Detection using any appropriate means of any hybridization complexes formed between the probes and the abovementioned nucleic acids.
• the abovementioned biological sample is, prior to the implementation of the screening, treated so that the cells which it contains are lysed, and in that the genomic material contained in said cells is fragmented using restriction enzymes such as EcoRI, Ba HI etc.
• the oligonucleotide probes are labeled using an enzymatic or radioactive marker. These probes are placed on an appropriate support, in particular on a nitrocellulose or other filter, for example nylon, on which is then added the biological sample treated as indicated above.
• the subject of the invention is also a method of in vitro diagnosis of diseases correlated with amino acid sequence contents of the invention situated outside the domain delimited by the extreme values generally corresponding to the physiological state of a healthy individual, by implementing one of the screening methods in . vitro mentioned above.
• a subject of the invention is also kits or kits for implementing the above-mentioned in vitro screening methods.
• kits include in particular: a determined quantity of at least one of the above-mentioned monoclonal antibodies capable of giving rise to a specific immunological reaction with one of the amino acid sequences of the invention;
• a medium suitable for the formation of an immunological reaction between the amino acid sequences and the abovementioned antibodies advantageously reagents allowing the detection of the immunological complexes produced during the abovementioned immunological reaction.
• kits used include, for example:
• a medium suitable for the formation of a hybridization reaction between the nucleic acids and the above-mentioned probes advantageously, reagents allowing the detection of the hybridization complexes produced during the above-mentioned hybridization reaction.
• amino acid sequences according to the invention have interesting reverting properties.
• one of the normal and mutated RAPIA, RAPIB or RAP2 nucleotide sequences is inserted into a eukaryotic expression vector with a strong promoter such as LTR,
• transfections are carried out using the above vector on cells which have been previously transformed by the RAS gene,
• these cells are co-transfected with the RAPIA or RAPIB or RAP2 gene and a gene for resistance to an antibiotic, for example neomycin, as a selection marker,
• RAPIA or RAPIB or RAP2 gene on the one hand in RNA and on the other hand in proteins directly responsible for reversion is observed.
• the cDNAs corresponding to the RAPIA and RAP2 genes were inserted into eukaryotic expression vectors and transferred by transfection into different cell lines, for example fibroblastic cells in culture of murine origin N.IH3T3 in the case of RAPIA and RATl in the case of RAP2.
• the RAP2 gene cDNA does not seem to confer any particular phenotype when it is expressed in normal fibroblasts.
• its overexpression in lines transformed by the oncogenes li ⁇ ras and Ki-ras is correlated with a phenotypic reversion of these cells towards an untransformed phenotype;
• - rabbit polyclonal antibodies have been generated against the proteins RAPIA and RAP2 or peptides derived from their sequence.
• the antibodies obtained specifically recognize the RAPIA or RAP2 proteins and show no cross-reaction with the H-ras protein; the use of these antibodies to immunoprecipitate the RAP2 protein in cell lines overexpressing the has shown that c "is a membrane protein. It is synthesized as a precursor that is rapidly modified to give birth to the form mature of the protein; it is relatively stable in vivo with a metabolic half-life of around 12-24 h.
• amino acid sequences of the invention having the reversion properties indicated above are capable of being used for the therapeutic treatment of diseases linked to the RAS genes or of diseases in which the RAP gene could be involved, either by mutation, or by bad expression in certain cells.
• the gene which codes for one of the amino acid sequences of the invention is introduced into an expression vector, which must be compatible with the cells to which it is intended to direct them and whose promoters must also be compatible with said cells.
• the vector is then targeted so that it goes to be fixed in the targeted cells, so that they phagocytize it and that it is released and expressed in said cells.
• a cDNA library constructed in phage A gt10 is used from AR.m of human B lymphocyte cells (Raji line) and screened under weakly stringent hybridization conditions (defined below in the paragraph entitled " Materials and methods ”) with the complete insert of Drosophila Dras-3 DNA.
• phages are isolated from 100,000 recombinant phages.
• the EcoRI inserts are purified, subjected to cross-hybridization reactions with one another under strongly stringent conditions (defined below in the section entitled "Materials and methods") and are partially sequenced. We can thus identify two categories of clones, 14 deriving from the same gene and a fifteenth deriving from another neighboring gene.
• the first family clone is designated by RAPIA and the second family clone by RAP2.
• Figure 4 shows the hybridization profile of the cellular genes RAPIA (1) and RAP2 (2).
• the size of the fragments determined by the HindIII marker is shown on the left in kilo base pairs.
• Nucleotide sequence of the cDNAs of RAPIA and RAP2 The nucleotide sequence of the inserts of 1450 base pairs and 980 base pairs of the cDNAs of RAPIA and RAP2 is determined.
• FIGS. 1 and 3 also show the nucleic acid sequences coding for RAPIA and RAP2 respectively.
• the RAPIA and RAP2 proteins consist respectively of peptide sequences of 184 and 183 amino acids, including the initial methionine, and have a calculated molecular weight of 20,900 and 20,700d, respectively.
• the percentages are 78.5% for the Dras-3 protein of Drosophila, 59.5% for K-ras, 56 % for ral and 63% for R-ras.
• RAP2 protein A global cellular comparison for the RAP2 protein leads to a 48% homology with Dras-3 of Drosophila, 46% with K-ras, 38% with ral and 37% with R-ras.
• FIG. 5 shows the alignments of the RAPIA and RAP2 proteins with the human protein K-ras (exon 4B) and the Dras-3 protein of Drosophila.
• the reference numbering is that of the K-ras protein.
• the attachment regions of the GTP are framed.
• the region of the effector is underlined by 1 solid line and the region of attachment to the intracellular membrane is underlined in dotted lines.
• ras proteins Four domains involved in the binding site to the guanine nucleotides of ras proteins (Barbacid, M. (1987) Annual Review of Biochemistry, 56, 779-827) and corresponding to the amino acid sequences 10 to 17, 57 to 63, 113 to 120 and 143 to 147 are highly conserved among the proteins RAP, K-ras and Dras-3.
• the RAPIA and RAP2 proteins like the Dras-3 protein, have a threonine in position 61 in place of a glutamine present in all ras proteins and the proteins close to the non-oncogenic ras proteins known to date.
• the domain of residues 32 to 42, for the ras proteins - which corresponds to the domain involved in the interaction with an effector - is identical in the protein RAPIA and ras, and differs from an amino acid in the protein RAP2 compared to the ras protein.
• the C-Terminal region of ras proteins is characterized by the sequence Cys-AAX, in which A is an amino aliphatic acid and X has the ino acid C-Terminal (Powers et al., (1984) Cell, 36, 607- 612). This sequence is necessary for post-translational lipid binding and anchoring in the plasma membrane (Fujiyama et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 1266-1270). 5) Transcription of the RAPIA and RAP2 genes:
• RNA transfer of RNA to the membrane after gel electrorophoresis according to the technique designated by "Northern Blot".
• Northern Blot The conditions used for the "Northern Blot” are recalled below in the paragraph entitled “Materials and methods”.
• RNA sequences that is to say three transcripts
• 1.600, 2.700 and 5.400 nucleotides are highlighted.
• the probe originating from RAPIA is a polynucleotide delimited by the Pstl-BglII fragment of the sequence (19) and the first end of which, corresponding to the restriction by PstI, is shown in FIG. 7, and the second end of which corresponds to the restriction by BglII, is 520 base pairs to the right of the first end.
• the probe originating from RAP2 is a polynucleotide delimited by the HindII-PstI fragment of the sequence 21 and the first end of which corresponds to the restriction by HindII is shown in FIG. 9 and the second end, corresponding to the restriction by PstI, is located 340 base pairs to the right of the first end.
• FIG. 6 shows the study of the transcripts of RAPIA (column 1) and of RAP2 (column 2) after electrophoresis and transfer of polyA + mRNA extracted from human lymphocytes. The size of the different RNAs is mentioned on the left in kilobase.
• a human library constructed in the phage Agt10, was screened with an EcoRI insert of 700 base pairs from the Drosophila cDNA clone Dras-3 (Schetjer et al., (1985 ) EMBO J., 4, 407-412).
• the hybridization was carried out under weakly stringent conditions: namely hybridization at 60 ° C. in 5 x SSC, 5 x Denhardt, 0.1% SDS, 100 ⁇ g / ml of salmon sperm DNA. The last wash is done in 2 x SSC and 0.1% SDS at 60 ° C for about 15 min.
• the highly stringent conditions used for the cross-hybridization of the EcoRI inserts are as follows: the hybridization is carried out under the following conditions: 5 x SSC, 5 x Denhardt, 0.1% SDS and 100 ⁇ g / ml of sperm DNA salmon at 65 C C.
• the washing is carried out with 0.1% SSC and 0.1% SDS at 65 ° C for 30 min.
• the nucleotide sequence of the complete EcoRI insert of the RAPIA and RAP2 clones is determined using the M13 vectors by the dideoxy nucleotide chain termination method (Messing, J. (1983) Methods Enzymol., 101, 20-78 ). 7) Northern and Southern Blot analysis:
• Hybridization is carried out under the following conditions: 5 x SSC, 5 x Denhardt, 0.1% SDS and 100 ⁇ g / ml of salmon sperm DNA at 65 ° C.
• the washing is carried out using 0.1% SSC and 0.1% SDS at 65 ° C for 30 min.
• cytoplasmic RNAs are prepared from peripheral blood lymphocytes (Perry et al.,
• polyA * DNA 5 ⁇ g are fractionated on 1% formaldehyde and agarose gels, before transfer on nylon membranes (marketed under the name Pall), and the filters are hybridized with the probes originating from RAPIA and RAP2, defined above.
• the RAPIA cDNA insert is used to screen a human cDNA library prepared in the phage ⁇ gtlO.
• the RAPIA insert is marked with 32 P and makes it possible to identify a phage whose insert (of 2100 bp), after subcloning, is sequenced.
• nucleic acid sequence and the amino acid sequence encoded by this nucleic acid are shown in Figure 2.
• This amino acid sequence will subsequently be designated by RAPIB. It has a calculated molecular weight of 20,800 d.
• EXAMPLE 3 PRODUCTION OF THE RAPIA PROTEIN IN A BACTERIAL EXPRESSION VECTOR: a) The RAPIA cDNA is subcloned for the purpose of its amplification, in the vector PUC8, at the EcoRI site. After culture, the cDNA is extracted and subjected to the restriction enzyme Bgl2, then to EcoRI, and a DNA fragment is thus recovered which corresponds to the complete region coding for the protein RAPIA.
• the EcoRI / Bgl2 fragment is cloned into the phage M13Mpl1 at the BamHI and EcoRI sites, which makes it possible, after culturing the phage, to recover the single-stranded DNA.
• Site-directed mutagenesis is carried out using a synthetic oligonucleotide of the following sequence: 5 'CAGATCACATATGCGTGAGTAC 3', this oligonucleotide being previously phosphorylated by the polynucleotide kinase.
• An NDE1 site is thus introduced upstream of the ATG corresponding to the initiation of the RAPIA protein.
• Recombinant M13Mpl1 is then prepared. To do this, the double-stranded DNA of M13Mpl1 is cut with the restriction enzyme NDE1 ' and the NDE1 site is repaired using the enzyme of Kleno.
• Site-directed mutagenesis is carried out using the synthetic oligonucleotide of following sequence phosphorylated by the polynucleotide kinase: 5 'CCAGTGAATTCTATGCGTGAG 3'
• This oligonucleotide makes it possible to insert a C at the ligation site of M13Mpl1 and of RAPIA and thus reconstitute an EcoRI site.
• the phage M13Mpl1 containing the sequence RAPIA is thus obtained.
• the RAPIA insert can be isolated by cutting the phage at the EcoRI and HindIII sites.
• double-stranded DNA of Ml3Mpl1 is prepared. Cut with EcoRI and HindIII to prepare the RAPIA insert for cloning into a bacterial expression vector.
• the vector used is the vector ptac-cHras.
• the vector ptac-Hcras can be obtained from the vector pKM-tael according to the construction described in the above-mentioned article by Tucker.
• the vector pKM-tael it can be obtained according to the construction described in the article by Hermann A. De Boer et al., PNAS, vol. 80, p. 21-25, January 1983.
• the ptac-Hcras vector is subjected to the restriction enzyme HindIII, then to EcoRI, in a partial manner in order to keep a fragment of approximately 300 base pairs EcoRI-EcoRI which contains the inducible tac promoter.
• the EcoRI-HindIII fragment of RAPIA is inserted into the vector thus prepared and a ligation is carried out.
• a strain of E. coli, such as those known under the designation PRI & M15, or HB101 - PDMI vector is then transformed.
• the RAPIA protein is then expressed in the following manner.
• the RAPIA protein expressed by the E. coli strain is then recovered according to conventional techniques. This protein is in soluble form.
• Example 3 The procedure is carried out as indicated in paragraph a) of Example 3, that is to say until obtaining a phage M13Mpl1 containing the sequence RAPIA cloned at the EcoRI and HindIII sites.
• the single-stranded DNA of the phage M13Mpl1 is prepared by site-directed mutagenesis using the following synthetic oligonucleotides phosphorylated by the polynucleotide kinase:
• a glutamine is introduced in place of threonine in the context of the oligonucleotide (A) and a valine in place of threonine in the context of the oligonucleotide (B).
• the procedure is as indicated in Example 4.
• the site-directed mutagenesis is carried out using the following synthetic oligonucleotide phosphorylated by the polynucleotide kinase:
• the EcoRI-KpNI fragment of the RAPIB fragment is cloned into phage M13Mpl1 at the EcoRI and BamHI sites.
• Directed mutagenesis is carried out using a synthetic oligonucleotide following phosphorylated by the polynucleotide kinase: 10 5 ′ AAG CTT GCA TAT GCG TGA GT 3 ′ and thus an NDE1 site is introduced upstream of the ATG corresponding to the initiation of the RAPIB protein.
• Recombinant M13Mpl1 is then prepared. To do this, is cut by the restriction enzyme NdeI 15, the double-stranded DNA M13Mpll and repairs the site NdeI using Klenow enzyme.
• Site-directed mutagenesis is then performed to introduce a C to the ligation site of M13Mpl1 and RAPIB.
• the EcoRI site is thus reconstituted.
• This ⁇ site-directed mutagenesis is carried out using the synthetic oligonucleotide of following sequence phosphorylated by the polynucleotide kinase: 5 'CCA GTG AAT TCT ATG CGT GAG 3'
• Example 3 The procedure is carried out as indicated in paragraph a) of Example 3, that is to say until the phage M13Mpl1 containing the sequence RAP2 cloned at the EcoRI and HindIII sites is obtained.
• the following synthetic oligonucleotide phosphorylated by the polynucleotide kinase 5 'CGG AGG GCA TAT GCG CGA GTA 3' is used to introduce an NDE1 site, and the oligonucleotide of following sequence phosphorylated by the polynucleotide kinase: 5 'CCA GTG AAT TCT ATG CGC GAG 3 'is used to insert a C at the ligation site of M13Mpl1 and RAPIA and thus reconstitute an EcoRI site.
• the single-stranded DNA of the phage M13Mpl1 is prepared by site-directed mutagenesis using the following synthetic oligonucleotides phosphorylated by the polynucleotide kinase: 5 'GCT GGG CTC GGT CGG GGTA GGC A 3 * to transform the glycine "12" into valine.
• the double stranded DNA of the modified RAP2 sequence as indicated above is prepared, then cut with the enzymes EcoRI and HindIII, then cloned into the expression vector ptac32 described in "Biochemical Properties of Ha-ras Encoded p21 Mutants and Mechanism of the Autophosphorylation Reaction "(The Journal of Biological Chemistry, vol. 263, No. 24, p. 11792-11799, 1988), as indicated in paragraph b) of Example 3.
• the single-stranded DNA of the phage M13Mpl1 is prepared by site-directed mutagenesis using the following synthetic oligonucleotides
• EXAMPLE 12 EXPRESSION OF THE RAPIA GENE OR OF THE RAP2 GENE IN THE VETR-RAP A AND VETR-RAP2 VECTOR:
• RAPIA gene and the RAP2 gene were respectively expressed in a retrovirus-type expression vector constructed as follows:
• Vector A * is a modification of vector A, in which the EcoRI site of PBR322 has been destroyed.
• This vector was obtained by ligation of the
• the initial vector A * is represented by a circle in Figures 12a), 12b) and 12c).
• SacII / EcoRI fragment of the initial vector A * is represented by a strong line opposite which a dotted arc of circle has been drawn (inside the vector).
• the initial vector A * is cut by EcoRI and the Klenow enzyme is made to act to obtain blunt ends.
• the EcoRI site is thus destroyed and is represented below by EcoRI crossed out with a cross
• the initial vector A * is cut with PstI and SacII to recover the 3736 bp Pstl / SacII fragment.
• the initial vector A * is cut with HindIII; Klenow's enzyme is acted on to obtain blunt ends, then cut with PstI.
• the HindIII site is thus destroyed and is represented in the rest of the text by Hip_ClII.
• the Pstl / Hip-élI fragment of 5626 bp is recovered. 4) The following fragments obtained in 1), 2) and 3) above are then ligated:
• the fragment between HindIII on the one hand and Ecj_? FI / Hip-II on the other hand contains a sequence designated by "enhancer”, or stimulating sequence, represented by "E” in FIG. 12d).
• the above fragment is represented by a strong line, in FIG. 12e), opposite which points (inside the vector) have been drawn according to an arc of a circle.
• J ′ Another vector, designated by J ′, is used. This vector J ′ is obtained from the vector A * and differs from the vector A * by the following two elements:
• the SacII site present in the Harvey virus at position 940 has been replaced by an EcoRI site.
• the fragment containing the LTR5' is isolated.
• the vector J ′ is cut by Pstl / EcoRI and the Pstl / EcoRI fragment of 2305 bp is isolated, represented by a line in FIG. 12f), opposite which one has drawn (inside the vector) small solid triangles, in an arc.
• the expression vectors described above and containing RAPIA and RAP2 respectively were made to express in different normal or transformed cell lines, in particular by RAS or by other oncogenes.
• cloning into the vector PJ3 ⁇ is done from the EcoRI / Bgl2 insert obtained from the complete cDNA of RAPIA cloned directly into the vector PJ3Q at the EcoRI / Bgl2 sites of this vector.
• RAP2 As there is no Bgl2 site, the insert from the RAP2 cDNA cut at the EcoRI / PstI sites, cloned in the plasmid pUC8, is used. From this construction, the EcoRI / HindIII fragment, having 670 bp, emerges.
• the above-mentioned plasmid pUC8 containing RAP2 is cut with HindIII and repaired with the Klenow enzyme to obtain blunt ends, then cut with EcoRI.
• the EcoRI / Hi ⁇ II insert (repaired by Klenow) is isolated on agarose gel.
• the vector PJ3 ⁇ is cut at the Bgl2 site repaired by Klenow to obtain blunt ends, then cut by EcoRI.
• the Bgl2 site is destroyed and will be designated below by B ⁇ _ ⁇ .
• RAPIA and RAP2 clones are made to express in the PJ3 ⁇ vector as described above respectively in different normal or transformed cell lines, in particular by RAS or by other oncogenes.
• RAPIA the EcoRI / HindIII insert obtained from the subcloning of RAPIA into pUC8 is linked to the PJ3 ⁇ vector cut at the HindIII / EcoRI sites.
• RAPIA and RAP2 in the vector PJ3 ⁇ allows the production of antisense messenger RNA which, by hybridization with the messenger RNA endogenous RAPIA and RAP2 genes can inhibit protein synthesis.
• This vector can allow the expression of the RAP proteins either after transfection or by infection, the vector having previously been packaged using an "associated” virus ("helper").
• helper EXAMPLE 14 EXPRESSION OF THE RAPIA GENE, THE RAPIB GENE AND THE RAP2 GENE IN THE PEX V3 VECTOR:
• the vector PEX V3 is cut by the enzyme EcoRI.
• the EcoRI inserts of the complete cDNAs of RAPIA, RAPIB and RAP2 respectively are directly cloned into the PEX V3 vector cut at the EcoRI site.
• the insert is 1.4 kb, for RAPIB, the insert is 2.1 kb, and for RAP2, the insert is 950 bp.
• the correct orientation in the vector was determined by carrying out different enzymatic degradations (for example BamHI and pstl) and by the sequencing of restriction fragments including the site. Cloning EcoRI.
• RAPIA, RAPIB and RAP2 are expressed respectively in different normal or transformed cell lines.
• RAPIA and RAP2 are cloned at the BamHI site of the above vector, which was previously cut by BamHI and repaired by Klenow to obtain blunt ends.
• the BamHI site is destroyed and will be designated below by Bah ⁇ I.
• RAPIA As regards RAPIA, it is previously subcloned into the plasmid pUC8.
• the cDNA is cut by EcoRI / HindIII repaired by the Klenow (to obtain blunt ends) and will be
• the RAPIA insert is linked by a ligase making it possible to constitute the vector Zip Neo of RAPIA.
• the vector is oriented (in the direction allowing expression of the protein) by carrying out different enzymatic degradations and by carrying out sequencing at the cloning sites.
• RAPIA and RAP2 clones are made to express in P Zip Neo respectively in different normal or transformed cell lines.
• EXAMPLE 16 EXPRESSION OF THE RAPIA GENE OR OF THE RAP2 GENE IN THE YEAST VECTOR YEP51:
• Cloning is carried out as follows: The vector YEP51 is cut by Sali repaired by Klenow, then cut by HindIII.
• the Sali site is destroyed and is designated below by S l.
• the large fragment Sa ⁇ ⁇ / HindIII is purified on gel and is used for cloning.
• the insert used comes from subcloning of the RAPIA cDNA (EcoRI / Bgl2 fragment) into the vector pUC8.
• This insert is obtained by cutting RAPIA subclone in the vector pUC8 by EcoRI, followed by repair with the enzyme Klenow, then by cutting with HindIII. The EcoRI site is destroyed and
• RAPIA and RAP2 clones are made to express in the vector YEP51 in different yeast strains.
• the PD2 E2 vector is described in the literature. This vector already contains a cloned gene which is got rid of by cutting with HindIII, then by purifying on an agarose gel to isolate the vector from the insert. The vector is then repaired by the Klenow enzyme to obtain blunt ends. HindIII sites are destroyed.
• the insert from the subcloning of the cDNA of RAPIA in pUC8 (EcoRI / Bgl2 fragment) is used. This insert is obtained by cutting pUC8 RAPIA at the HindIII and EcoRI sites and is treated with the Klenow enzyme to obtain blunt ends.
• RAP2 the same type of construction is used as for RAPIA.
• RAPIA and RAP2 clones are made to express in the vector PD2E2 respectively in different strains of yeast.
• EXAMPLE 18 DETERMINATION OF THE LOCATION
• the RAP cDNAs are cloned into the plasmid pUC8. Two different fragments are used for the respective localization of each gene (cf. FIG. 10).
• the two probes used for RAPIB are either the complete cDNA (2, 1kb) or the 5 'part (0.73kb).
• the two RAP2 gene probes are either the full 0.95kb cDNA or the 0.66kb fragment in the 5 'part.
• High resolution chromosome preparations are obtained from the culture of phytohemagglutinin stimulated blood cells of two healthy men after synchronization with methothrexate according to Yunis et al.
• the probes are labeled according to the method of • random DNA primers (Kit A ersham, U.K.) 'introduced by Feinberg and Vogelstein (Feinberg, A.P., Vogelstein, B.A .: technique for radiolabeling DNA restriction endonuclease fragments to high
• These labeled probes are used at concentrations of 20 to 80 ng / l.
• the plates coated with Kodak NTB2 emulsion are exposed for one and two weeks for autoradiography: G bands are then obtained with the Wright dye.
• chromosome Ipl2-pl3 no specific deletion is associated with a particular neoplasia. Some translocations of this region are described in elanomas and various blood disorders (Bloomfield, CD., Trent, JM, Van Den Berghe, H .: Report of the committee on structural chromosome changes in neoplasia (HGM10), Cytogenet. Cell. Genet 46, 344-366, 1987).

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