EP0546149A1

EP0546149A1 - Nucleotide sequences hybridizable with the nov gene of chickens

Info

Publication number: EP0546149A1
Application number: EP19920913915
Authority: EP
Inventors: Bernard Perbal; Cécile MARTINERIE
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 1991-06-25
Filing date: 1992-06-25
Publication date: 1993-06-16
Also published as: WO1993000430A1; FR2678283A1

Abstract

Les séquences de nucléotides de l'invention sont caractérisées en ce qu'elles renferment un enchaînement de nucléotides capable de s'hybrider, dans des conditions stringentes (50 % de formamide, 5XSC) avec une ou plusieurs séquences du gène nov de poule dont l'ADNc comporte l'enchaînement de nucléotides représenté sur la figure. Ces séquences sont utilisables comme sondes de détection de séquences complémentaires pour l'évaluation du développement et/ou de la différentiation de tumeurs.The nucleotide sequences of the invention are characterized in that they contain a sequence of nucleotides capable of hybridizing, under stringent conditions (50% formamide, 5XSC) with one or more sequences of the hen nov gene, of which l CDNA comprises the sequence of nucleotides shown in the figure. These sequences can be used as probes for detecting complementary sequences for assessing the development and / or differentiation of tumors.

Description

NUCLEOTIDE SEQUENCES CAPABLE OF HYBRIDIZING WITH THE NOV CHICKEN GENE

The invention relates to nucleotide sequences and the corresponding amino acid sequences. It also relates to obtaining these sequences and their applications.

It has been recognized for many years that nephroblastoma induced by the avian myeloblastosis helper virus (AVM) is an animal model of Wilms' tumor in children. Although these two types of tumors have different ethiologies, no virus having been associated until now with the development of human nephroblastoma, it is conceivable that the study, at the molecular level of viral-induced nephroblastomas, can make it possible to characterize parameters that are difficult to access in the human system.

The inventors' studies concerning such avian nephroblastomas induced by MAV have enabled them to characterize in the chicken an embryonic gene called the nov gene, the expression of which proves to be stimulated at variable levels in tumors, but which is extinguished in cells. normal adult kidney.

By developing their work in this area, the inventors have developed tools allowing them to study the expression of homologous genes in human tumors and in certain cell types.

Thus, by cloning the deoxyribonucleic sequences and a complementary DNA corresponding to the nov gene of normal hen cells, the inventors established the partial nucleotide sequence of the DNAs and the complete nucleotide sequence of the cDNA. Specific molecular probes have been established based on this sequence and used to detect the presence and expression of homologous genes in various human cell types.

The invention therefore aims to provide new nucleotide sequences of a gene involved in particular in tumor cells.

It also aims to provide means for the isolation of these sequences.

The invention further relates to the corresponding coded proteins and the polyclonal and monoclonal antibodies directed against these proteins.

The invention further relates to the use of these sequences, proteins and antibodies in biological applications, in particular in detection tests.

The nucleotide sequences of the invention are characterized in that they contain a sequence of nucleotides capable of hybridizing, under stringent conditions (50% of formamide 5 XSCC) _f with one or more sequences of the hen nov gene of which the cDNA exhibits the nucleotide sequence (I), more particularly with the sequence (II).

The sequences of the nucleotide and protein sequences which are referred to in the description and the claims are given at the end of the description.

The entire nucleotide sequence of the hen nov cDNA clone is formed by 1975 bp and includes at least 5 exons. This sequence includes an open reading frame of 1.0 kb, coding for a potential protein of 32300 Da, going from nucleotide 24 to nucleotide 1076. This open reading frame is followed by 899 bp of 3 'non-coding sequences which contain two potential pattern signals AATAAA polyadenylation at position 1914 and 1932. This hen nov gene is overexpressed in avian nephroblastomas induced by MAV studied by the inventors.

Hybridization experiments carried out under stringent conditions defined above show that, unexpectedly, homologous sequences of the hen nov gene exist in the human genome.

The isolated homologous sequences, in humans or animals, can be used for screening libraries made from m-RNA, and make it possible to isolate cDNAs and thus to identify the other exons of genes of the same family. These exons and the genes which contain them, as well as the corresponding coded proteins, also form part of the invention.

It was indicated above that the hybridization experiments were carried out under stringent conditions, which makes it possible to isolate sequences having strong homologies with those of the probes.

These experiments can also be carried out under non-stringent conditions, by reducing the amount of formamide, of salt and / or the washing time, as described in "A practical guide to molecular cloning", second edition, B. Perbal, John Wiley and Sons, New York, 1988. The isolated sequences will then have a weaker homology than previously with the sequences of the probes and will lead to the identification of exons having fewer common sequences.

Nucleotide sequences of the invention are more particularly characterized in that they comprise or that they are formed by a sequence of nucleotides capable of hybridizing, under the stringent conditions mentioned above, with at least part of the second exon of the hen nov gene which comprises the nucleotide sequence (III).

The letters indicated in these sequences present the conventional meanings appearing in the work of Perbal cited above.

The invention relates in particular to the nucleotide sequences comprising the genetic information for coding for a protein having a homology of approximately 70% with the protein fragment, corresponding to the second exon of the hen nov gene, corresponding to the sequence (IV) .

The nucleotide sequences capable of hybridizing with the sequence (III) above are also characterized in that they comprise at least part of a PstI fragment of approximately 600 bp as obtained from a plasmid subclone, derived from a recombinant clone isolated from a human placenta DNA library. The enzyme restriction map of the recombinant clone, as well as that of the derived plasmid subclone containing the nucleotide sequence in question, are shown in FIG. 2A.

Such sequences are characterized in that they code for the chain of amino acids (V).

Note the presence, in these amino acid sequences encountered in humans, of a consensus sequence of binding to growth factors of insulin type (IGF). This sequence therefore appears to be conserved in humans.

The different sequences mentioned above more particularly comprise at least part of the following nucleotide sequence (VI), corresponding to the Pst I fragment mentioned above, more particularly of the sequence (VII). The sequence (VII) comprises 225 nucleotides with approximately 70% homology with exon 2 of the hen nov gene.

Other nucleotide sequences of the invention are characterized in that they are formed by or that they comprise a sequence of nucleotides capable of hybridizing, under the stringent conditions mentioned above, with at least part of the third exon of the hen nov gene, which includes the nucleotide sequence (VIII).

Sequences of the type defined above include the genetic information to code for a protein having at least about 73% homology with the potential protein fragment of the third exon of the hen nov gene corresponding to the sequence (I).

These sequences are also characterized in that they comprise at least part of a PstI fragment of approximately 800 bp and of a PstI fragment of 2 kb as obtained from a plasmid subclone derived from a recombinant clone isolated from a human placenta DNA bank. The enzymatic restriction map of the recombinant clone as well as of the derived plasmid subclone containing the nucleotide sequence in question is represented in FIG. 2A.

They are in particular sequences comprising the genetic information for coding for a protein having the amino acid sequence (X). It will be observed that this amino acid sequence can be demonstrated in humans.

These amino acid sequences more particularly comprise at least part of the nucleotide sequence (XI), more particularly of the sequence (XII). Other nucleotide sequences of the invention are characterized in that they comprise or that they are formed by a sequence of nucleotides capable of hybridizing, under the stringent conditions mentioned above, with at least part of the fourth exon of the hen nov gene, which includes the nucleotide sequence (XIII).

The invention relates to the nucleotide sequences comprising the genetic information to code for a protein having a homology of approximately 85% with the protein fragment corresponding to the fourth exon of the hen nov gene corresponding to the sequence (XIV).

Such sequences, capable of hybridizing with at least part of the above chain (XIII), are also characterized in that they comprise at least, part of a HincII fragment of approximately 400 bp, as obtained according to the methods mentioned above for the other restriction fragments (see FIG. 2B).

According to another ^" aspect, these sequences are also characterized in that they code for the chain of amino acids (XV).

The sequences mentioned above in connection with the fourth exon of the hen nov gene more particularly comprise at least part of the nucleotide sequence (XVI), corresponding to the HincII fragment mentioned above, more particularly of sequence XVII.

Still other nucleotide sequences are characterized in that they comprise or are formed by a sequence of nucleotides capable of hybridizing with at least part of the first exon of the hen nov gene which includes the nucleotide sequence XVIII.

According to another aspect, such sequences are characterized in that they contain the genetic information to code for a protein having a homology of approximately 30% with the protein fragment corresponding to the first exon of the hen nov gene responding to the sequence (XIX).

Such sequences are also characterized in that they code for the chain of amino acids (XX).

The sequences defined above in relation to the first exon of the hen nov gene more particularly comprise at least part of the nucleotide sequence (XXI).

Other nucleotide sequences of the invention are characterized in that they are formed by, or that they comprise, a sequence of nucleotides capable of hybridizing, under the stringent conditions mentioned above, with at least part third and fourth exons of the hen nov gene which comprise the nucleotide sequence (XXII).

Such sequences are further characterized in that they encode a protein fragment responding to the sequence (XXIII) following amino acids.

These sequences are also characterized in that they comprise at least part of a PstI fragment of approximately 700 bp as obtained according to the protocol mentioned above (see FIG. 2B).

Sequences of the type of those of the PstI fragment of 700 bp above are more particularly characterized in that they are formed by or that they comprise a chain of nucleotides capable of hybridizing under the stringent conditions defined above, with at least part of the third exon of the hen nov gene which comprises the sequence ( XXIV).

Other nucleotide sequences of the invention contain the genetic information to code for a protein having at least about 60% homology with the potential protein fragment of the third exon of the hen nov gene, this fragment responding to the sequence (XXV).

These are in particular sequences comprising the genetic information for coding for a protein having the amino acid sequence (XXVI).

It will be observed that this sequence can be demonstrated in humans. These sequences more particularly comprise at least part of the nucleotide sequence (XXVII), more particularly of the sequence (XXVIII).

Other nucleotide sequences of the invention are characterized in that they comprise or that they are formed by a sequence of nucleotides capable of hybridizing, under the stringent conditions mentioned above, with at least part of the fourth exon of the hen nov gene, which includes the nucleotide sequence (XXIX).

The invention relates to nucleotide sequences containing the genetic information to code for a protein having a homology of approximately 80% with the protein fragment corresponding to the fourth exon of the hen nov gene, this fragment corresponding to the sequence (XXX). . They are in particular sequences comprising the genetic information for coding for a protein having the sequence (XXXI).

These sequences are formed by or more particularly comprise the nucleotide sequence (XXXII).

According to another aspect, the invention relates to a recombinant sequence comprising one of the sequences defined above, optionally associated with a promoter capable of controlling the transcription of the sequence and a DNA sequence coding for the termination signals of the transcript.

The invention also relates to the promoter sequences of the genes comprising the nucleotide sequences defined above.

It targets in particular at least part of the promoter sequence of the human nov gene, the three and four are given in Figure 2A. This promoter sequence which corresponds to the sequence / Y IU; is located in a 2.2 kb PsTI-Hind III fragment and comprises the 283 nucleotides upstream at the start of the first exon.

The promoter sequence of the human nov gene is characterized in that it comprises several consensus sequences of different transcription factors, such as NF1 (TGGCCTTCTGCCAATC), API (TGACTAA) and Spl (GCCACTCCCC) ^•.

It also comprises a sequence of twenty repetitions of TG motifs which can constitute a polymorphism sequence, conferring an interest on this sequence as a polymorphism marker. The invention also relates to the promoter sequence of the CTGF gene identified in the EcoRI - PstI ragment of approximately 700 bp, which corresponds to the sequence (XX lA-0-

This sequence is characterized in that it includes sites for binding transcription factors such as SRF (CCTAAAAAGG), API (TGAATCA), Spl (CCCGCCC), a potential tl protein binding site (CGCCCCGGC) and a site NF kappa B (GAGAGCCCC). It also includes a basic TATA (TATAAAA).

The promoter sequence of the hen nov gene responding to the sequence. ^ X J is also part of the invention.

This sequence is contained in a Smal-Xhol fragment of approximately 1 kb which comprises consensus sequences of different transcription factors as well as a base TATA. It is characterized in particles in that it comprises the following sites for attachment of factor Spl: GGGGGCGGGG, CCCCCGCCTC, Ap2: CCGCAGGC, GGCGGGGC, GGGTCCC.

It also includes a binding site for the NF kappa E2 factor (GGCAGGTGG) and the NFKB factor (GGGAGTTTC).

It is understood that the bases of the nucleotide sequences under consideration may be in a different order from that found in the genes and / or that these bases may, where appropriate, be substituted. The corresponding sequences fall within the scope of the invention, when a fragment of these sequences used as a probe gives a characteristic and unequivocal response with regard to the ability to recognize the presence of genes coding for proteins as defined above. above expressed in tumor cells. The invention also targets, as new products, the RNAs corresponding to the different sequences defined above and the complementary sequences of the different defined nucleotide sequences.

The invention also relates to recombinant cloning and expression vectors capable of transforming an appropriate host cell, comprising at least part of a nucleotide sequence as defined above under the control of regulatory elements allowing its expression.

The strains of transformed or transfected microorganisms also fall within the scope of the invention. These strains comprise one of the nucleotide sequences defined above or also a recombinant vector as defined above.

It also targets the amino acid sequences corresponding, according to the universal genetic code, to the nucleotide sequences defined above, and the proteins expressed by the genes comprising these sequences.

Amino acid sequences homologous to those encoded by exon 2, which contain the IGF growth factor binding site are of particular interest, since the IGFII gene, which is found in humans on chromosome llpl5 , is overexpressed in certain Wilms tumors and could therefore be implicated in this pathology.

Since the consensus motif of proteins binding to IGF plays an important role in the development of nephroblastomas in conjunction with the deregulation of IGFII expression, the value of detecting an abnormal expression is measured. proteins of the invention which contain such a motif. The proteins of the invention are also characterized in that they are obtained by transformation of host cells using a recombinant vector as defined above, culturing, in an appropriate medium, of the transformed host cells or transfected and recovery of the protein from these cells or directly from the culture medium.

The production of these proteins by such a process also forms part of the invention.

The proteins of the invention and their fragments, which can also be obtained by chemical synthesis, advantageously have a high degree of purity and are used to form, according to conventional techniques, polyclonal antibodies.

Such polyclonal antibodies, as well as monoclonal antibodies capable of specifically recognizing an epitope of the above proteins, or of a fragment of these proteins, are also targeted by the invention.

The invention further relates to the biological applications of the nucleotide sequences, the corresponding proteins and monoclonal or polyclonal antibodies.

These applications include the development, from purified intragenic fragments, or from corresponding RNA, of molecular probes to search for the possible presence of nucleotide sequences related to the nov gene in various cell types.

The development of these probes includes, in particular, the denaturation of the double-stranded sequences to obtain a single-stranded sequence. The tests carried out to detect the presence of complementary sequences in various tumors and human tissues have demonstrated the great specificity of these intragenic fragments.

The use of these probes has thus made it possible to show that the gene containing the nucleotide sequences defined above is expressed in several types of human cells, including certain kidney tumors.

The invention therefore relates to detection probes characterized in that they comprise at least part of a nucleotide sequence defined above.

Any probe which differs from the previous one, in terms of its nucleotide sequence, only by nucleotide substitutions or alterations which do not result in modification of its hybridization properties with the human gene related to the hen nov gene as defined more haut falls within the scope of the invention.

The DNA fragment used as probe has a sufficient number of nucleotides to obtain the required specificity and the formation of a stable hybrid.

It is possible to use fragments reaching several kb, results of high specificity being however also obtained with shorter fragments of approximately 25 to 40 nucleotides.

Probes suitable for this type of detection are advantageously labeled with a radioactive element or any other group allowing its recognition in the hybrid state with the preparation containing the nucleotides to be studied.

According to conventional techniques, these probes are brought into contact with the biological sample to be tested or their nucleic acids, under conditions permitting possible hybridization of the nucleotide sequence of the probe with a complementary sequence, possibly contained in the product studied.

One can, for example, have recourse to the method of hybridization on spots or to the method of hybridization on replica, according to the technique of Southern. In the first method, according to the conventional technique, depositing a quantity ^v aliquot of denatured DNA on nitrocellulose membranes. The second method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the DNA with restriction enzymes, the transfer after alkaline denaturation on appropriate membranes and their hybridization with the probe under the usual conditions.

These probes constitute tumor markers by allowing the early detection of the expression of the gene containing said nucleotide sequences, which normally is not or little expressed in the corresponding normal tissues. The invention thus provides means for assessing ^" tumor development and / or di erentiation.

Detection for the specific identification of DNAs can also be carried out by DNA amplification techniques (PCR) as described in US patents 4683202 and 4683195 in the name of Cetus Corportation.

In these techniques, using two primers of about fifteen ^'nucleotides included in one of the nucleotide sequences defined above and spaced approximately 200 to 250 nucleotides. One of the sequences is capable of binding to a nucleotide sequence of one of the strands of the DNA fragment to be amplified and located at one of the ends of this fragment, for example at the 5 'end . The other sequence is capable to bind to a nucleotide sequence of the second strand of the DNA fragment to be amplified, and is located at the end of this fragment opposite to that mentioned above (at the 3 'end, when the first ends find at end 5 ').

The invention also relates to a method for detecting in vitro the presence in a biological sample of sequences complementary to those defined above. This process is characterized in that it comprises the following stages:

bringing the biological sample to be studied into contact with a nucleotide probe as defined above under conditions allowing the production of a hybridization complex formed between the probe and the desired nucleotide sequence,

- detection of the hybridization complex.

If necessary, a preliminary amplification of the quantity of nucleotide sequences capable of being contained in the sample is carried out, using primers, as described above, capable respectively of binding, of one hand at the 5 'end of one strand of said nucleotide sequence and the other hand, at the 3' end of the other strand of said nucleotide sequence.

The use of such a method represents an increase in sensitivity and a considerable saving of time compared to conventional techniques which often require a technology which can only be implemented in specialized services. It also allows rapid and highly specific detection of DNA and of the different transcription mRNA species. This process is a means of detecting a rearrangement chromosomal at the level of genes which code for RNA nov or CTGF without having to use cell cultures.

For the implementation of such an in vitro screening method, based on the use of nucleotide probes, use is advantageously made of kits or kits comprising:

a determined quantity of a nucleotide probe according to the invention,

a medium suitable for the formation of a hybridization reaction between the sequence to be detected and the probe and, advantageously,

reagents for the detection of hybridization complexes formed between the nucleotide sequence and the probe during the hybridization reaction.

- a determined quantity of a polyclonal or monoclonal antibody according to the invention,

a medium suitable for the formation of an immunological reaction between at least part of the products expressed and the antibody and, advantageously,

reagents for the detection of the immunological complexes formed during the immunological reaction.

The presence in the proteins of the invention of an insulin-like growth factor (IGF) binding sequence is advantageously used according to the invention for the determination of proteins. To this end, the proteins of the biological sample to be studied are brought into contact with an IGF comprising a labeled group, for example a radioactive group or cold probe, and the quantity of fixed product is assayed. The cloning and sequencing of the hen nov gene and of nucleotide sequences corresponding to the definitions given above are given below by way of nonlimiting examples. In these examples, reference is made to FIGS. 1 and 2,

- Figure 1 representing the cDNA sequence of the hen nov gene and that of the potential protein encoded

- Figures 2 A and 2 B the restriction maps of DNA fragments of the invention

Molecular cloning and sequencing processes reported in the examples:

purification of nucleic acids: use of dichloromethane as described in V. Maloisel et al .. Met. Mol. Cell. Biol. 1, 245-247, 1990.

Southern and Northern blots, and other cloning methods: carried out according to standard protocols published by B. Perbal in "A practical guide to molecular cloning, second edition, B. Perbal John Wiley and Sons, New York, 1988

purification of the 7 kb BamHI-HindIII and Sacl 6.6 kb DNA fragments: Geneclean method (Bio 101).

Radioactive probes: prepared by nick translation in the presence of α dCTP 32p.

Nucleotide sequencing: according to the dideoxy chain termination method in the presence of α dATP 35s, T7 polymerase or Sequenase (USB).

Example 1: Isolation of cDNA from the hen nov gene

25 ng of cDNA corresponding to poly A RNA from fibroblasts from hen embryos of 13 days are ligated with 1 μg of lambda gt10 arm to prepare a cDNA library of normal hen fibroblasts using the kit Amersha.

After screening with a cell probe derived from a tumor, 7 clones are purified, the longest insert (1.9 kb) is purified according to the Geneclean method (BIO 101) and subcloned at the Kpnl site of Bluescript KS ⁺ (Stratagene) to generate the pClK clone.

Nucleotide sequencing:

Sequencing is carried out by the method of terminating dideoxy-nucleotide chains in the presence of α 35s dATP and of T7 polymerase (Pharmacia) or of Sequenase under the conditions described by the manufacturers.

Matrices are obtained from the recombinant clones M13mpl8 and M13mpl9. The sequencing primers are from Biolabs, New England. GC compressions are resolved using deoxy-inosine (USB).

Characterization of the nov cell gene:

Northern blot analysis of RNA isolated from normal kidneys, hen embryo fibroblasts (FEP) and nephroblastomas is performed using tumor derived cell probes. The HX1024 probe makes it possible to detect, in normal FEPs, a 2.2 kb mRNA species whose expression is altered in all the other nephroblastomas. Screening of an FEP cDNA library makes it possible to isolate a 1.9 kb cDNA clone representing the 2.2 kb mRNA expressed in normal FEPs. FIG. 1 shows the entire 1975 bp nucleotide sequence of the cDNA clone of this new gene, overexpressed in the nephroblastomas studied, called gene no_3_ :. This gene appears to consist of 5 exons. A 1.0 kb open reading frame encoding a potential protein of 32300 Da has been identified from nucleotide 24 to nucleotide 1076. This open reading frame is followed by 899 bp of 3 'non-coding sequences which contain two potential motifs of polyadenylation signals (AATAAA) at positions 1914 and 1932.

The potentially coded amino acids have also been indicated in this figure. The potential nov polypeptide contains a hydrophobic nucleus characteristic of a peptide signal at its amino terminus (with 6 leucines). This nov protein is devoid of other hydrophobic regions present in the trans-membrane proteins, it is likely that it is secreted. The nov protein also contains the consensus motif GCGCCXXC of proteins binding insulin-like growth factors (IGF) and a total of 39 non-clustered cysteine residues.

F -o lP. 2: isolation in human cells of nucleotide sequences related to the hen nov gene.

A Southern blot of fragments of human DNA digested with EcoRI is carried out with the cDNA clone of the hen nov gene pClK. One operates under the stringent conditions reported by B. Perbal (see reference above).

It is found that four EcoRI fragments hybridize with sequences of the hen nov gene. These fragments contain respectively 15, 12, 8 and 5.6 kb.

Example 3: Isolation of nucleotide sequences related to the hen nov gene. From a library of human placenta DNA, two groups of recombinant lambda gtll clones are isolated using the radiolabeled pClK probe.

The partial restriction map of lambda Hu92 (which corresponds to three overlapping clones) and of lambda Hu93 (which corresponds to two overlapping clones) and those of the plasmid subclones pBH7 and p56 are shown in FIGS. 2A and 2B.

The human nucleotide sequences homologous to those of the hen nov gene are located in a 7.0 kb BamHI-HindIII DNA fragment of the clone Hu92 and those belonging to the CTGF gene in a 6.6 kb Sacl DNA fragment from the clone Hu93.

On these maps, the restriction enzymes are designated as follows: B ≈ BglII, P = PstI, K = Kpnl, H = HindIII, S = Sacl, E = EcoRI, X = Xba, B = BamHI and Hc = Hine II. The black blocks represent human exonic regions.

The subcloning of these fragments into the pUC18 and pUC19 vectors, called respectively pBH7 and pS6 clones makes it possible to more precisely locate the homologous sequences of the hen nov gene and the sequences of the CTGF gene. The first are located on the one hand in a PstI DNA fragment of 600 bp (E2), on the other hand in a PstI fragment of 800 bp (E3), and in a HincII fragment of 400 bp (E4). The pBH7 probe corresponds to the HindIII-BamHI fragment.

The location of the first, second, third, fourth and fifth human exons at GTGF are shown in Figure 2B (respective designations E1, E2, E3, E4, and E5). The use of the PstI fragments of purified DNA as probes in Southern hybridization experiments with the EcoRI fragments of Example 2 leads to the sole detection of the EcoRI DNA fragment of 12 kb with PB06 and of the EcoRI fragment of 15 kb with PSP07 demonstrating that the sequences of PBP06 and PSP07 correspond to a subset of the nov exons of chicken cDNA.

Rv-am liα A: Detection of RNA of the human genome related to the hen nov gene.

The following table reports the results of Northern hybridization experiments with different tissues and cell lines using as probes the sequences of formula VIII, XV and XVI above homologous respectively with exons E2, the hen nov gene and E3 and E4 of the CTGF gene (these codes being used in the table to designate them).

FABRICS AND PROBE CELL LINES

E2 E3-E4 kb from

(nov) (CTGF) mRNA

Bone marrow + (2,) thymus (fetal) + (2.5) Liver (fetal) (, 5; HEL

Brain (fetal)

Neuroblastoma 1 Neuroblastoma 162

Kidney (fetal) Nephroblastoma Bou

Breast tissue Breast tumor gg Breast tumor

SK-BR3 (2.5) (3.5) lung (fetal) heart (fetal) line 293

MCF7 (7, 4)

Carcinoma embry test. 8 nt (2.7) (7.4)

Teratocarcinoma test. 10 nt

Teratocarcinoma test. 11 nt

Adenocarcinoma U377 nt

HL60 nt (7.4)

nt = not tested

The results obtained show that the human gene homologous to the hen nov gene and the CTGF gene belonging to the same family are ^* expressed according to the tissues or lines in the form of different RNA species detected either by the two probes, or by a only one of them. The 7.4 kb RNA species expressed by certain tissues and lines appears to be recognized only by the PSP07 probe.

These results indicate that the regulation of genes in humans would depend on tissue specificity.

LINK I

GCGGCGGGTΛGΛCGGCCGGGΛCT ΛTG GΛG ΛCG GGC GGC GGG CΛG GGG CTG CCC GTC CTG CTG CTG CTC CTG CTC CTC CTC CGG CCG TGC GAG GTG 95

ΛGC GGG CGG GΛG GCG GCG TGC CCC CGG CCC TGC GGC GGG CGC TGC CCC GCG GAG CCG CCG CGC TGC GCC CCG GGA GTG CCC GCC GTG CTG 18

GΛC GGC TGC GGC TGC TGC CTG GTG TGC GCC CGG CΛG CGC GGC GΛG ΛGC TGC TCC CCT CTG CTG CCC TGC GAC GΛG AGC GGC GGC CTC TAC 27 TGC GΛC CGC GGC CCC GΛG GΛC GGC GGC GGC GCC GGC ATC TGC C GTC GΛC AΛC TGC GTG TTC GAT GGG ATG ATT TAC CGC 36

AΛC GGG GΛG ΛCG TTC CΛG CCC ΛGC TGC ΛΛG TΛC CAG TGC ΛCC TGC CGG GAC GGG CΛG ATC GGG TGC CTG CCC CGC TGC AAC CTG GGC CTG 45 CTG CTC CCC GGC CCC GΛC TGC CCC TTC CCG CGG AΛG ATGA GAG TG TGC GΛG AΛG TGG GTG TGC GAC CCC AGG GAT GAA 54 GTG CTC CTG GGA GGC TTT GCT ΛTG GCT GCA TΛC AGΛ CΛG GΛG GCC ACA CTT GGG ATA GAC GTG TCT GAT TCA AGT GCC AAT TGT ATT GAA 63 CAG ΛAΛ TGT TCC ΛΛΛ ΛGC TGT GGΛ ΛTG GGC TTT TCT ACC CGT GTT ΛCC AΛC AGA AAT CAG CAG TGT GΛG ATG GTG 72 AAG CAG ΛCΛ CGΛ CTT TGC ΛTG ΛTG ΛGΛ CCT TGT GAA ΛΛC GAA GAA AAA GCT GAT AAA ACA AAG AAA TCC 81

ATG ΛΛA GCT GTT CGT TTT GΛA TΛC AΛG AAC TGC ΛCC AGT GTG CAG ACT TΛC AAA CCT CGT TAC TGT GGC CTC TGC AAT GAT GGG CGA TGC 90 TGT ACC CCA CAC ΛΛG ΛCC ΛΛΛ ΛCG ATT CΛΛ GTT GTG CAG CG TTC CTA AAA AΛG CCA ATG ATG TTG ATC AAT ACC 99

TGT GTC TGT GGT CΛT ΛΛC TGT CTC AGT CΛG AΛC ΛΛT GCT TTC TTC CAG CCA TTA GAT CCC ATG TCT GCA GAA AAA ATA ΛGT TGAAATGTATA GTTTAGGTGGCCCAΛAΛGGTATGTAGTTTGTACAAAΛCTTGACCCACAATCΛGGTGAΛTGTAATAATTGCATATGTAAAATATCTGAGATTTTTTTCTAAACAGTCTGAGTGCCTTTTT 10 12 13 TTTCCTGTAGTTTACTAΛATACCTCATGACGTTTCΛCCCCTCCAΛATGTCTTTTATTCΛTTTGΛAGGAAATTTTGTACCTTGGACAGAGCCTTCTGTTGTTTCTTGACAGTGGCATAAC GΛTTACAAΛGTCAACAGCTAGTCTTTCTCTCTGAGTTTAGAGGACCTTGCCATGATTTTCAGTAGCCATAAGACTGGGCTTTTTAATAATGGATTCCTTGGGGAATGCATGATAATATG 1 TCACAΛΛΛGCTTCCΛGΛGTTTTCACTTTGAATAATGTGTACAAACACTTACACAGCCTTCTTCTTTCTGTTCAAGTTAΛATTCTTCCGGATAACTGAAAATGTTACTGATGAGAGTCTG 15 ΛATTCTTCTGGCTTΛTΛΛΛGTΛTCTTCTΛTCTGTΛCCTCTTGΛCTTTCTCTGAGGGΛTTAGTTTGCACATAGCCTCAGAAATGACATAGCTAAGATCTCGTATCTTGAAGCATAGGAGA 16

TTGATΛGÇTGATΛΛCΛΛΛTTTCTCΛTTCGTΛGCTTTΛTTAGCAGCCTΛATCCΛΛΛΛCCTΛCTGΛAGΛAAGTGTCTTACAΛGAGCTTGGTTCTAACCAGTGTCTGTCTGTAGATAAAGTA 18

GTTGTΛTGCAAΛAΛJΛ ^ ΛATTTCTGTΛΛΛTTCCTTTAAΛATACTΛACTGTΛTCΛGΛTGGTGCTTCΛCTTACTAGAAAGATGTTTATGTAAATAGAAACTGTATATATTGTAATATAACT 19

TTTΛTTΛGGTΛΛATΛΛΛCTTTΛTGTGΛTCΛΛΛATGΛΛΛAAAAAΛAAΛΛAΛΛΛAΛA -ig

LINK II

TGC GGC GGG CGC TGC CCC GCG GΛG CCG CCG CGC TGC GCC CCG GGA GTG CCC GCC GTG CTG 18 CΛC CCC TCC CGC TCC TGC CTG GTG TGC GCC CGG CAG CGC GGC GΛG Λ6C TGC TCC CCT CTG CTG CCC TGC GAC GΛG AGC GGC TAC 27

TGC CΛC CGC GGC CCC GAG GΛC GGC CGC GGC GCC GGC ATC TGC ΛTG GTG CTG GAA GGG GΛC ΛΛC TGC GTG TTC GAT GCG ATG ATT TAC CGC 36

AAC GGG CAG ACG TTC, CΛG CCC ΛGC TGC ΛΛG TAC ^' CAG TGC ΛCC TGC CGC GAC GCG CAG ATC GGG TGC CTG CCC CGC TGC AAC CTG GCC CTG 15

CTG CTC CCC GCC CCC CAC TGC CCC TTC CCG CGG AAG ATC GAA GTC CCC GGA GΛG TGC TGC GAG AAG TGG GTG TGC GAC CCC ΛGG GAT GAA 54

GTG CTC CTG GGA GGC TTT GCT ATG GCT GCA TAC AGA CAG GAG GCC ACA CTT GGG ATA GAC GTG TCT GAT TCA AGT GCC AAT TCT ATT GAA 63

CAC ACA ACA GAA TGG AGT GCT TGT TCC AAA ΛGC TGT GGA ATG GGC TTT TCT ACC CGT GTT ACC AAC AGA AAT CAG CΛG TGT GAG ATG GTG 72 AAG CλG ACA CGΛ CTT TCC ΛTG ΛTG AGA CCT TGT GAA AAC GAA GAG TAG CC AAA GGA AAA AAA TGT ATC CAA ACA AAG

LINK III

101 111 121 131 141 151 AGGTGAGCGG GCGGGAGGCG GCGTGCCCCC GGCCCTGCGG CGGGCGCTGC CCCGCGGAGC.

161 171 181 191 201 211 CGCCGCGCTG CGCCCCGGGA GTGCCCGCCG TGCTGGACGG CTGCGGCTGC TGCCTGGTGT

221 231 241 251 261 271 GCGCCCGGCA GCGCGGCGAG AGCTGCTCCC CTCTGCTGCC CTGCGACGAG AGCGGCGGCC

281 291 301 311 321 TCTACTGCGA CCGCGGCCCC GAGGACGGCG GCGGCGCCGG CATCTGCATG

LINK IV

VSGREAACPR PCGGRCPAEP PRCAPGVPAV LDGCGCCLVC ARQRGESCSP LLPCDESGGL

93 YCDRGPEDGG GAGICM

LINK V

V A A T Q R C P P Q C P G R C 756 771 786

P A T P P T C A P G V R A V L 801 816 831

D G C S C C L V C A R Q R G E 846 861 876 S C S D L E P C D E S S G L Y 891 906 921

CDRSADPSNQTGICT

355 365 375 385 395 405 CTGCAGCCAA CCGGCTTGTG CGCGTCCCAG GAGCGCGCTA TAAAACCTGT GCTGGGCGTG

415 425 435 445 455 465 ATCGGCAAGC ACCGGACCAG GGGGAAGGCG AGCAGTGCCA ATCTACAGCG AAGAAAGTCT

M

535 545 555 565 575 585 -2. TGTCTCGCGA AAGCAGTGCC TTTGCCTGAC CTTCCTGCTT CTCCATCTCC TGGGACAGTA x> r- \

595 605 615 625 635 645 M 3 3. AGTGGCACAC CCTTAAGATG CCCCCAAAGT TACTTTGCCC GCCTTGGTGG CCCCCATTTG M: 655 665 675 685 695 705 l-l GTCACCGGGC TCACTGCGTC TTCTGTCCCA GCTGAGTGGT TTCTCCTTGT

715 725 735 745 755 765 TTCAGGTCGC TGCGACTCAG CGCTGCCCTC CCCAGTGCCC GGGCCGGTGC CCTGCGACGC

775 785 795 805 815 825 CGCCGACCTG CGCCCCCGGG GTGCGCGCGG TGCTGGACGG CTGCTCATGC TGTCTGGTGT

835 845 855 865 875 885 GTGCCCGCCA GCGTGGCGAG AGCTGCTCAG ATCTGGAGCC ATGCGACGAG AGCAGTGGCC

895 905 915 925 935 945 TCTACTGTGA TCGCAGCGCG GACCCCAGCA ACCAGACTGG CATCTGCACG GGTAATCCTG

CTCCCTCTGC TGTTTGACCT CTTCTCCTGC AG

LINK VII

720 730 740 750 760 '770 GTCGCTGCGA CTCAGCGCTG CCCTCCCCAG TGCCCGGGCC GGTGCCCTGC GACGCCGCCG

780 790 800 810 820 830 ACCTGCGCCC CCGGGGTGCG CGCGGTGCTG GACGGCTGCT CATGCTGTCT GGTGTGTGCC

840 850 860 870 880 890 CGCCAGCGTG GCGAGAGCTG CTCAGATCTG GAGCCATGCG ACGAGAGCAG TGGCCTCTAC

900 910 920 930 TGTGATCGCA GCGCGGACCC CAGCAACCAG ACTGGCATCT GCACGG o

LINK VIII

331 341 351 361 371 381 GTGCTGGAAG GGGACAACTG CGTGTTCGAT GGGATGATTT ACCGCAACGG GGAGACGTTC

391 401 411 421 431 441

CAGCCCAGCT GCAAGTACCA GTGCACCTGC CGGGACGGGC AGATCGGGTG CCTGCCCCGC

451 461 471 481 491 501

TGCAACCTGG GCCTGCTGCT CCCCGGCCCC GACTGCCCCT TCCCGCGGAA GATCGAAGTC

511. 521 531 541 551 561 CCCGGAGAGT GCTGCGAGAA GTGGGTGTGC GACCCCAGGG ATGAAGTGCT CCTGGGAGGC

571 TTTGCTATGG CT

LINK IX

109 119 129 139 149 159

VLEGDNCVFD GMIYRNGETF QPSCKYQCTC RDGQIGCLPR CNLGLLLPGP DCPFPRKIEV

169 179 PGECCEKWVC DPRDEVLLGG FAMA

LINK X

116 131 146 GCG GTA GAG GGA GAT AAC TGT GTG TTC GAT GGG GTC ATC TAC CGC A V E G D N C V F D G V I Y R 161 176 191 AGT GGA GAG AAA TTT CAG CCA AGC TGC AAA TTC CAG TGC ACC TGC S G E K F Q P S C K F Q C T C

206 221 236

AGA GAT GGG CAG ATT GGC TGT GTG CCC CGC TGT CAG CTG GAT GTG R D G Q I G C V P R C Q L D V 251 266 281 CTA CTG CCT GAG CCT AAC TGC CCA GCT CCA AGA AAA GTT GAG GTG

L L P E P N C P A P R K V E V 296 311 326

CCT GGA GAG TGC TGT GAA AAG TGG ATC TGT GGC CCA GAT GAG GAG P .G E C C E K W I C G P D E E 341

GAT TCA CTG GGA GGC CTT ACC CTT GCA G

LINK XI

10 20 30 40 50 60 AAAAGGACTT GGGTTTTGGA ACATGCCCTC CAAATCTTAC ATAGCTTCTT CACTGTATTG

70 80 90 100 110 120 TGTTCTTGTT TTTCCTCTTC CTCTTTGCTT TTCACTTTGC TTCCCCAATA TTCTAGCGGT

130 140 150 160 170 180 AGAGGGAGAT AACTGTGTGT TCGATGGGGT CATCTACCGC AGTGGAGAGA AATTTCAGCC

190 200 210 220 230 240 co AAGCTGCAAA TTCCAGTGCA CCTGCAGAGA TGGGCAGATT GGCTGTGTGC CCCGCTGTCA 1-0

250 260 270 280 290 300 GCTGGATGTG CTACTGCCTG AGCCTAACTG CCCAGCTCCA AGAAAAGTTG AGGTGCCTGG

310 320 330 340 350 360 AGAGTGCTGT GAAAAGTGGA TCTGTGGCCC AGATGAGGAG GATTCACTGG GAGGCCTTAC

370 380 390 400 410 420 CCTTGCAGGT GAGAAACTCA ATATACCTAG GGCTGGTCAT AGTAGAGGGT AAATACAAAC

430 440 450 ATGAAGAATT TGCAATCTCT TGGATTTGAA AA

LINK XII

125 135 145 155 165. 175 GCGGTAGAGG GAGATAACTG TGTGTTCGAT GGGGTCATCT ACCGCAGTGG AGAGAAATTT

185 195 205 215 225 235 CAGCCAAGCT GCAAATTCCA GTGCACCTGC AGAGATGGGC AGATTGGCTG TGTGCCCCGC

245 255 265 275 285 295 TGTCAGCTGG ATGTGCTACT GCCTGAGCCT AACTGCCCAG CTCCAAGAAA AGTTGAGGTG

305 315 325 335 345 355 CCTGGAGAGT GCTGTGAAAA GTGGATCTGT GGCCCAGATG AGGAGGATTC ACTGGGAGGC J 365 CTTACCCTTG CAG

LINK XIII

583 593 603 613 623 633 GCATACAGAC AGGAGGCCAC ACTTGGGATA GACGTGTCTG ATTCAAGTGC CAATTGTATT

643 653 663 673 683 693 GAACAGACAA CAGAATGGAG TGCTTGTTCC AAAAGCTGTG GAATGGGCTT TTCTACCCGT

703. 713 723 733 743 753 GTTACCAACA GAAATCAGCA GTGTGAGATG GTGAAGCAGA CACGACTTTG CATGATGAGA

763 773 CCTTGTGAAA ACGAAGAGCC ATCTGATAA

LINK XIV

193 203 213 223 233 243

AYRQEATLGI DVSDSSANCI EQTTEWSACS KSCGMGFSTR VTNRNQQCEM VKQTRLCMMR

253 PCENEEPSDK

CHAIN XV

104 119 134 GCT TAC AGG CCA GAA GCC ACC CTA GGA GTA GAA GTC TCT GAC TCA A Y R P E A T L G V E V S D S 149 164 179

KSCGMGFSTRVTNRN

239 254 269

CGT CAA TGT GAG ATG CTG AAA CAG ACT CGG CTC TGC ATG GTG CGG R Q C E M L K Q T R L C M V R

284.

CCC TGT G

LINK XVI

10 20 30 40 50 60

ATCAGAGTCG AATGAGACCC AGTTTCTAAT AATGGCTGAA AAGGACCACT TTCCAATCCT

70 80 90 100 110 120

CACATTGATC CTAATATGGC TGTCTTTATT TATACATCCC ATAGCTTACA GGCCAGAAGC

130 140 150 160 170 180 CACCCTAGGA GTAGAAGTCT CTGACTCAAG TGTCAACTGC ATTGAACAGA CCACAGAGTG

190 200 210 220 230 240 GACAGCATGC TCCAAGAGCT GTGGTATGGG GTTCTCCACC CGGGTCACCA ATAGGAACCG

250 260 270 280 290 300 TCAATGTGAG ATGCTGAAAC AGACTCGGCT CTGCATGGTG CGGCCCTGTG AACAAGAGCC

310 320 330 340 350 360 AGAGCAGCCA ACAGATAAGG TAGGAGCCTG GAGGAAACCT CCCATCCTGA AGGTAATGGC

370 380 390 400 410 420 CTTGTGTCCT TGGAGCCTGG GCTTCAGAAA GTCACTGTTG CACTCTGTGA CGGAGAGAGC

430 AGCTATAGCG GGGAG

LINK XVII

GCTTACAGGC CAGAAGCCAC CCTAGGAGTA GAAGTCTCTG ACTCAAGTGT CAACTGCATT

173 183 193 203 213 223 GAACAGACCA CAGAGTGGAC AGCATGCTCC AAGAGCTGTG GTATGGGGTT CTCCACCCGG

233 243 253 263 273 283 GTCACCAATA GGAACCGTCA ATGTGAGATG CTGAAACAGA CTCGGCTCTG CATGGTGCGG

293 303 313 CCCTGTGAAC AAGAGCCAGA GCAGCCAACA GATAAG

CHAIN XVIII

33 43 53 63 73 83 TATGGAGACG GGCGGCGGGC AGGGGCTGCC CGTCCTGCTG CTGCTCCTGC TCCTCCTCCG

GCCGTGCGA

CHAIN XIX

10 20

METGGGQGLP VLLLLLLLLR PCE

LINK XX

285 300 315 ATG GCA ACC CCG GGG TTC GTT CCA CTT CCC CAC CCA GCC GAT CTC M A T P G F V P L P H P A D L ω 330 345 "CCC CCT CCT CCC TGC ACT GCA GCC AAC CGG CTT P P P P C T A A N R L

CHAIN XXI

294 304 314 324 334 344 ATGGCAACCC CGGGGTTCGT TCCACTTCCC CACCCAGCCG ATCTCCCCCC TCCTCCCTGC

354 ACTGCAGCCA ACCGGCTT

LINK XXII

TG CTG GΛA GGG GΛC ΛΛC TGC GTG TTC GAT GGG ΛTG ATT TAC CGC

ΛΛC GGG GΛG ΛCG TTC CΛG CCC AGC TGC AAG TAC CΛG TGC ACC TGC CGG GAC GGG CΛG ATC GGG TGC CTG CCC CGC TGC AΛC CTG GGC CTG CTG CTC CCC GGC CCC GΛC TGC CCC TTC CCG CGG AΛG ATC GAA GTC TG GGA GΛG AΛG TGG GTG TGC GAC CCC AGG GAT GAA GTG CTC CTG GGA GGC TTT GCT ΛTG GCT GCA TAC AGA CAG GΛG GCC ACA CTT GGG ΛTA GΛC GTG TCT GAT TCA AGT GCC AAT TGT ATT GAA

CAG ΛCΛ ACΛ GΛΛ TGG ΛGT GCT TGT TCC AAA AGC TGT GGA ATG GGC TTT TCT ACC CGT GTT ACC AAC AGA AAT CAG CAG TGT GAG ATG GTG

AΛG CΛG ACA CGΛ CTT TGC ΛTG ΛTG AGA CCT TGT GΛA AΛC GAA GAG CCA TCT GAT AAG

LINK XXIII

Q I P T R I P D A L D V R V P 63 78

Q C L T S A S P T P L F P S S 108 123

S P A K D G A P C I F G G T V 153 168

Y R S G Ξ S F Q S S C K Y Q C 198 213

T C L D G A V G C M P L C S M 243 258

D V R L P S P D C P F P R R V 288 303

K L P G K C C E E W V C D E P 333 346

K D Q T V G P A S R V S R V 378 393

F L * V R V V I L S Q G G S P 423 438

N C A D R T G E I P Y P G V D

468,483

H G V C V L C S R S P T G R 513 528

H V W P R P N Y D * S Q L P G 558 573

P D T E W S A C S K T C G M G

603

YSTRVTNDNA LINK XXIV

CTGCGTGTTCGATGGGATGATTTACCGCAACGGGGAGACGTTCCAGCCCAGCTGCAAGTACCAGTGCACC

350 360 370 380 390 400

190 200 210 220 230 240 250 TGCCGGGACGGGCAGATCGGGTGCCTGCCCCGCTGCAACCTGGGCCTGCTGCTCCCCGGCCCCGACTGCC 420 430 440 450 460 470

CCTTCCCGCGGAAGATCGAAG-TCCCCGGAGAGTGCTGCGAGAAGTGGGTGTGCGAC 490 500 510 520 530

XXV LINK

GECCEK

CHAIN XXVI

^{40 5} ° ^{60 70} 80 90 100

^DGAPC I ^FGG VY ^RSG E ^S F ^QSSC KY ^QC T ^C L ^DGA VGCMPLCSMDVRLP ^S PD ^C PFPRRVK PGKCCEEWVCDE

* - ^* V

LINK XXVII

1 this

3 • 18 33

CAG ATC CCA ACT CGC ATC CCT GAC GCT CTG GAT GTG AGA GTG CCC

48 63 78

CAA TGC CTG ACC TCT GCA TCC CCC ACC CCT CTC TTC CCT TCC TCT 93 108 123

TCT CCA GCC AAA GAT GGT GCT CCC TGC ATC TTC GGT GGT ACG GTG

138 153 168

TAC CGC AGC GGA GAG TCC TTC CAG AGC AGC TGC AAG TAC CAC TGC

183 198 213

ACG TGC CTG GAC GGG GCG GTG GGC TGC ATG CCC CTG TGC AGC ATG 228 243 258

GAC GTT CGT CTG CCC AGC CCT GAC TGC CCC TTC CCG AGG AGG GTC

273 288 303

AAG CTG CCC GGG AAA TGC TGC GAG GAG TGG GTG TGT GAC GAG CCC

318 333 348

AAG GAC CAA ACC GTC CTT GGG CCT GCC TCG CGG GTG AGT CGA GTC

363 378 393 _{ττc cτc TAA GTC AGG GTC GTG ATT cτc τcc CAG GGA GGG AGT ccτ}

408 423 438

AAC TGT GCC GAC CGA ACG GGG GAA ATA CCT TAT CCA GGC GTT TTA

453 468 483

CAT GGT GTT TGT GTG CTC TGC TCT CGC AGC TTA CCG ACT GGA AGA 498 513 528

CAC GTT TGG CCC AGA CCC AAC TAT GAT TAG AGC CAA CTG CCT GGT

543 558 573

CCA GAC ACA GAG TGG AGC GCC TGT TCC AAG ACC TGT GGG ATG GGC

588,603

ATC TCC ACC CGG GTT ACC AAT GAC AAC GCC TC LINK XXVIII

-. ^r 190 200 210 220 230 240

TGCCTGGTCCAGACA-CAGAGTGGAGCGCCTGTTCCAAGACCTGTGGGATGGGCATC

260 CCAA

CHAIN XXIX

^GC TTTG ^C T ^A T ^G G ^C T ^GC AT ^A CA ^GACA G ^G AGGCCACACTTGGGATAGACGTGTCT - G 570 580 590 600 610

TGT ^AT T ^GA A ^CA G ^A CAAC ^AG AAT ^G GAGTGCTTGTTCCAAAAGCTGTGGAATGGGCTT

630 640 650 660 670 680

CCAA

LINK XXX LINK

TEW _S A _CS K _SC GM _G FSTRVTNRN ^{70 80} 210 220 TEWSACSKTCGMGI

LINK XXXII

130 140 150 160 170 CTGCTC _T C _G C _A GCTTACCGACTGGAAGACACGTTTGGCCCAGACCCAACTATG

0 200 210 220 230 240

CTGGTC _C A _G ACA-CAGAGTGGAGCGCCTGTTCCAAGACCTGTGGGATGGGCAT

LINK XXXIII: fragment 1

10 20 30 40 50 60

GCTTTCTTTT TAAGGAACAG TCCTTTCTTC CCAAGAGAAC TGCTCTTTCT CTCCATTCCA

70 80 90 100 110 120 l ACCATGAGGT TCTAACTAAT CCCCATACTT CACCTTCCTT GTCCCCATTG ATTAGTCCAG

130 140 150 160 170 180 GGTGAACCCA TCCAATTTAA TTCCTGGAAC TTTTAAAGTT GGGCCTAAGA GACAGGGACA

190 200 210 220 230 240 TTCCTTCTGT GGTGATAAGG TCATAAAGTA AGAAGATTGG AAGGATCATT TTTCCCTTAT

250 260 270 280 290 300 GTGGAAGTAA TCCTGTTGGC CCTCCTCTCT CTAGATCCCA ATTGCCTCTG AGGACTCCCT

310 320 330 340 350 360 GTACCATTCC TGTGCTGTCA CTATGTGAAA CATCACAGCA TCCTTCCAGT AAAGTCCTCT

370 380 390 400 410 420 TTTCGCAAAA ACTAGTTCAA GTTTGGTTTC CATCTCTTGC AATCAAAACT GAATAGCAAT

430 440 450 460 470 480 TTTACACTTG CAGTGACTTC TTGACATGTT AATCCTTGTC TTAAAGTTAC ATTTTCCCTG

490 500 510 520 530 540 TCACCACTCC CACCCCACTC TTTCCAAGAA GAGCTAGCCC AATCTCCATG TTGCCAATTT

550 560 570 580 590 600 CTCCTTGTTC TATCTGAGTC TATTCATGCT TGGAACACTT GGCCGATGCT CTTTGCCTCC

LINK XXXIII fragment 1 (sui e)

610 620 630 640 650 660 CCATTAGCAG TGCTTCTAGT TGCTCCATTT CAAAGTACAT TAAAATGCTG TCTACCAAGA

670 680 690 700 710 720

GCCACCΛCCA GAGAATCCTA CTGAGTGGGT CAAGACTGGG GCTCAGGAAT CTGTATTTTT

•

4 ^*

730 740 750 760 770 780 AACAAAATAC ATGCTGGTTG ATTCGATCTG CAGCCAGATG GAGGCATCAT TAGGCCAAAT

GGCTTACAΛA ACCTATCAGT TTTTTTGTTT TTTGTTTTAT CTTTTTCTTA AACTTTTATT

850 860 870 880 890 900 TCAAGTTCAG GGGAAATGTG CAGGTTTGTT TACACAGGAA ATGTGTCATG GACATTTGTT

910 920 930 940 GTGCAGΛTTA TTTCATCGCC CAGGTATTAA GCCTGGTACC GAGGTACC

10 20 30 40 50 60

CΛTTAGTTAT TTTTCCCGAT CTTCTCCCTG CTCCCACCCT CCACCCTCCA AAGCCTATCA

70 80 90 100 110 120

ATTTGAΛGAG TAGGTAAATG TCCTACTCAA GAGTGCAAAT GAACTGTTTC ATCTCTAGTT

370 380 390 400 410 420 0 ^* ACATCACAGG CCTGTATAAT TTTCCTTAAA AAGTGTTTTT TGTTTTTTTC CAAAGCAACT 00

3 π> a

430 440 450 460 470 480 rt ATCCTCAAAA GAGCTGGGCA TAGTTCTCCT AGGGGCAGCA CCAGTGTTGA AGTGTGGGGG ι-o

490 500 510 520 530 540 GAΛACTGTTC TAAATCCTTC AAACAATGTC ACCTTTGGAG CAGTAAAACT GCTCCCTTTT

550 560 570 580 590 600 TCCCATGAGA GΛTGACAAGC ATGCCCCAGC AATCATTTCT TGAAAGCGGA TGCÇCGGTGA

610 620 630 640 650 660 GΛGΛAGGATT TGATTTGCTG AAGGGTCAGC CAAGTTAAGC CAGTTTCTTC CTCATTTCTT

CCCTGGCTGG AGGTTTTGAT GGTGGTGATG GTGGTTGAAC TGAACCCACT TAGAAAACTG

730,740,750,760,770,780 t ^* . TCΛAAGGTTT CTGGACTCTC AGGTGTGCCG TCTCACATTT GGTCTGCTAC AGCAGGTGCT s ^* *. o

X

790 800 810 820 830 840 H ss TCΛΛGGCTTT CTTCTGCCAA GATTTCTTTG TTTTATTTTA TGATGTTTTC TTTATGTGTG w

3

W

-_;

850 860 870 880 890 900 X TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTTTTAC TTTTATTTCT AACAAACCTG X X

910 920 930 940 950 960 TGΛCCTTGGG GTTTAAGACT GAGTGAAGCT AGAAGGATTA GAGTCAAAAG AATTTTGCCA l-h 1

970 980 990 1000 1010 1020 P ^* OQ TTTGGCCAAT AGCATTCCCC CACCTCCTGA CATATCGATT TTTTTTCTAG ATTCCCTTCC 3 π> x- tt

1030 1040 1050 1060 1070 1080 t-o CCCTGCCACT CCCCTCCCCC CAACACACAC ACACTTTTCT CTTTCTCCTC TTTCTCTCCT

1090 1100 1110 1120 1130 1140 TTCCTCCCTT GCTTCTCTCC CCTCCCTCTC AACACATTCA ATGAGTGCCC TAAACGGTGA H * r-t (0

1150 1160 1170 1180 1190 CΛΛΛCTTGCA TGTGCTTCCC TCATGACTAA ACCCCTGGCC TTCTGCCAAT CCCCTGCAG

LINK XXXIII: fragment 3

10 20 30 40 50 60

CTGCAGGCAT CCCGTAAGGA CCCCACGCTT GCAGCCCTGG TTGGAACGGT CAGGGTGGAG

70 80 90 100 110 120 GAGGATGGTG GGGAGTGGTG GTGTCTTCGT CCTGGGAGAA GGCGAAGCAA CTTCCAGGAG

130 140 150 160 170 180 GAAACGGGCG TTTCCTTCCC ACGCGCTCGA GCGAGCCCTG GGTCCTGGCC TCGGAACTCC

4>

190 200 210 220 230 240 ACCCAGCCCC TCCCCACCCT CTGGGAAAAG CCAGTCGCCA CACACAGGCA CACGCAGGCC

250 260 270 280 CCGGCGCCGC GCCCTAAGGA GAGCAGCACC CACAGCCAAT TGCC

LINK XXXIV

CGAATTTTTT AGGAATTCCT GCTGTTTGCC TCTTCAGCTA CCTACTTCCT AAAAAGGATG

70 80 90 100 110 120

TATGTCAGTG GACAGAACAG GGCAAACTTA TTCGAAAAAG AAATAAGAAA TAATTGCCAG

130 140 150 160 170 180 TGTGTTTATA AATGATATGA ATCAGGAGTG GTGCGAAGAG GATAGGGAAA AAAAAATTCT

190 200 210 220 230 240 4 ^* - ATTTGGTGCT GGAAATACTG CGCTTTTTTT TTTCCTTTTT TTTTTTTTCT GCGAGCTGGA 00

250 260 270 280 290 300

TCCATTCAGC TCATTGGCGA GCGCCGCCGC CCGGAGCGTA TAAAAGCCTC GGCCGCCCGC

430 440 450 460 470 480 CCCAAACTCA CACAACAACT CTTCCGCTGA GAGGAGACAG CCAGTGCGAC TCCACCCTCC

AGCTCGACGG CAGCCGCCCC GGCCGAGAGC CCCGA

LINK XXXV

l 10 20 30 40 50 60

GTCGAGTGCT GTGTTCAGTT TTGGGCCCCT CACTACAAGA CATCGAGGCC ATGGAGTGTG

70 80 90 100 110 120

TCCAGAGAAG GGCACGAGGT GGTGAGGAGT CTGGAGCACA TGTTTTATTG GAAGCAGCTG

130 140 150 160 170 180 4 ^* - J AGGAAGTTGG GATTGTTCAG TCCGGAGAGG CTCAGGGAAA ACATTATTGC TCTTTAAAAA vO

190 200 210 220 230 240 J TCCCTGGAAG GAGGTTGTGG TGAGGTGGAG GTCGGCCTCT GCTCCCAGGT ATCAGTGATA

250 260 270 280 290 300 GGATGAGAGG GAACTGTCTT AAATTATGCC AGGGGAGTTT CAGTTTGGAT ATCAGGAACA

310 320 330 340 350 360 ATTTTTTTTC TCCAAAAAAT TGGTGAGGTA CTGCCACAGT CTGCCCAGCG AGGTGGAATC

370 380 390 400 410 420 ACCATCCCTG GAGATGTTCA GGAAACGTGT AGATGTGGCA CTGAGGGATG TGGTTTAGTG

LINK XXXV (continued)

430 440 450 460 470 480 AGΛATGGTAG GGATGGGTTG ATGGTTGGAC TAGATTAGCT TAGCGATCTT TCCAGTCATA

490 500 510 520 530 540 ACGΛTCCTGT GATCCTACGA TCCTAAGGCG CCGGCCCCAG CGGAGCAGAC CCGCAGGCTT

550 560 570 580 590 600 CΛGCCCCGGA GCCCCGGCCG CGCGTCGGGA CGCGGGCAGG GCCGGGCACC GCCGGGCAGG

610 620 630 640 650 660 TGGCGGAGCA CAACGGGGAG CGGAGCGTAG GGCCCTGCCC GGCTCCAGCT CCCCGCCTCC

790 ACGGCCGGGA CT

Claims

1 / Nucleotide sequences, characterized in that they contain a sequence of nucleotides capable of hybridizing, under stringent conditions (50% formamide, 5XSCC) with one or more sequences of the hen nov gene of which the cDNA presents The sequence of nucleotides (I) and more especially with the sequence (II).

2 / Nucleotide sequences according to claim

1, characterized in that they are formed by, or that they comprise, a sequence of nucleotides capable of hybridizing, under the stringent conditions of claim 1, with at least part of the second exon of the hen nov gene which includes the nucleotide sequence (III).

3 / Nucleotide sequences according to claim

2, characterized in that they contain the genetic information to code for a protein containing a sequence having at least 70% homology with the protein fragment corresponding to the second exon of the hen nov gene, this fragment having the sequence amino acids (IV).

4 / Nucleotide sequences according to one of the preceding claims, characterized in that they comprise at least part of a PstI fragment of approximately 600 bp as obtained from a plasmid subclone derived from a recombinant clone isolated from a human placenta DNA library, the enzymatic restriction map of the recombinant clone as well as of the derived plasmid subclone containing the nucleotide sequence being represented in FIG. 2A. 5 / Nucleotide sequences according to one of claims 1 to 4, characterized in that they include the genetic information to code for an amino acid sequence having the V sequence.

6 / Nucleotide sequences according to one of claims 1 to 5, characterized in that they comprise at least part of the nucleotide sequence (VI), more especially of the sequence (VII).

7 / Nucleotide sequences according to claim 1, characterized in that they are capable of hybridizing, under the stringent conditions defined in claim 1, with at least part of the third exon of the hen nov gene which comprises the sequence nucleotide (VIII).

8 / Nucleotide sequences according to claim 7, characterized in that they comprise the genetic information for coding for a protein having a homology of at least 70% approximately with the potential protein fragment corresponding to the third exon of the gene nov de hen corresponding to the sequence (IX).

9 / Nucleotide sequences according to claim 7 or 8, characterized in that they comprise at least part of a PstI fragment of approximately 700 bp, as obtained from a plasmid subclone derived from a recombinant clone isolated from a DNA library of human placenta, the enzymatic restriction map of the recombinant clone as well as that of the derived plasmid subclone containing the nucleotide sequence in question being represented in FIG. 2A.

10 / Nucleotide sequences according to one of Claims 7 to 9, characterized in that they carry the genetic information to code for a protein having the amino acid sequence (X).

11 / Nucleotide sequences according to one of claims 7 to 10, characterized in that they comprise at least part of the nucleotide sequence (XI), more particularly, the nucleotide sequence (XII).

12 / Nucleotide sequences according to claim 1, characterized in that they are formed by, or that they comprise, a sequence capable of hybridizing, under the stringent conditions given in claim 1, with at least part of the fourth exon of the hen nov gene, which includes the sequence (XIII).

13 / Nucleotide sequences according to claim 12, characterized in that they are capable of coding for the protein fragment having a homology of at least 86% with the potential protein fragment corresponding to the fourth exon of the no___ gene; chicken responding to the sequence (XIV) in amino acids.

14 / Nucleotide sequences according to claim 12 or 13, characterized in that they include the genetic information to code for a protein having the sequence (XV) in amino acids.

15 / Nucleotide sequences according to one of claims 12 to 14, characterized in that they are formed by or that they comprise the nucleotide sequence (VI).

16 / Nucleotide sequences according to claim 1, characterized in that they are capable of hybridizing, under the stringent conditions defined in claim 1, with at least part of the first exon of the hen nov gene which comprises the nucleotide sequence (XVIII).

17 / Nucleotide sequences according to claim 11, characterized in that they contain the genetic information to code for a protein having a homology of at least 30% approximately with the potential protein fragment corresponding to the first exon of the nov gene. hen, this fragment presenting the sequence (XIX) in amino acids.

18 / Nucleotide sequences according to one of claims 16 or 17, characterized in that they contain the genetic information to code for a protein having the sequence (XX) in amino acids.

19 / Nucleotide sequences according to one of claims 16 to 18, characterized in that they comprise at least part of the nucleotide sequence (XXI).

20 / Nucleotide sequences according to claim 1, characterized in that they are capable of hybridizing, under the stringent conditions defined in claim 1, with at least part of the third and fourth exons of the hen nov gene which comprise the nucleotide sequence (XXII).

21 / Nucleotide sequences according to claim 20, characterized in that they include the genetic information to code for a protein having the sequence (XXIII) in amino acids.

22 / Nucleotide sequences according to one of claims 20 to 21, characterized in that they are formed by or that they comprise a sequence capable of hybridizing under the stringent conditions defined in claim 1, with at least one part of the third exon of the chicken nΩ_z gene which includes the nucleotide sequence (XXII).

23 / Nucleotide sequences according to one of claims 20 to 22, characterized in that they include the genetic information to code for a protein having a homology of at least approximately 60% with the potential protein fragment corresponding to the third exon of the hen nov gene responding to the amino acid sequence (XXIII).

24 / Nucleotide sequences according to one of claims 20 to 22, characterized in that they contain the genetic information to code for a protein having the sequence (XXIV) in amino acids.

25 / Nucleotide sequences according to one of claims 20 to 24, characterized in that they comprise at least part of the nucleotide sequence (XXV), more particularly, the nucleotide sequence (XXVI).

26 / Nucleotide sequences according to one of claims 20 to 25, characterized in that they are formed by or that they comprise a sequence capable of hybridizing under the stringent conditions given in claim 1 with at least one part the fourth exon of the chicken n_____ gene, which comprises the nucleotide chain (XXVII).

27 / Nucleotide sequences according to claim 14, characterized in that they are capable of coding for the protein fragment having a homology of at least 86% with the potential protein fragment corresponding to the fourth exon of the nov hen gene responding to the sequence (XXVIII) in amino acids. 28 / Nucleotide sequences according to claim 26 or 27, characterized in that they contain the genetic information to code for a protein having the sequence (XXIX) in amino acids.

29 / Nucleotide sequences according to one of claims 26 to 28, characterized in that they are formed by or that they comprise the nucleotide sequence (XXX).

30 / The RNAs and sequences complementary to the sequences according to any one of claims 1 to 29.

31 / Recombinant cloning and expression vectors capable of transforming an appropriate host cell comprising at least part of a nucleotide sequence according to any one of claims 1 to 30 under the control of regulatory elements allowing its expression in the host cell.

32 / Strains of transformed or transfected microorganisms, characterized in that they comprise a nucleotide sequence according to any one of claims 1 to 30 or also a recombinant vector according to claim

33 / The proteins corresponding to the nucleotide sequences according to any one of claims 1 to 30.

34 / The polyclonal and monoclonal antibodies characterized in that they specifically recognize a protein according to claim 33, or a fragment of such a protein.

35 / Detection probe, characterized in that it comprises all or part of the nucleotide sequences according to one of claims 1 to 30. 36 / A method of in vitro screening for the possible presence in a biological sample of nucleotide sequences complementary to those according to any one of claims 1 to 30, characterized in that it comprises the following steps:

bringing the biological sample into contact with a nucleotide probe according to claim 35 under conditions allowing the production of a hybridization complex formed between said probe and said nucleotide sequence,

- detection of the hybridization complex, and

- if necessary, the amplification, before the contacting step, of the nucleotide sequences according to any one of claims 1 to 30 which may be contained in the sample, using primers which may respectively to bind, on the one hand to the 5 'end of one strand of said nucleotide sequence and, on the other hand, to the 3' end of the other strand of said nucleotide sequence,

37 / Kit for implementing an in vitro screening method for the possible presence in a biological sample of sequences complementary to the sequences according to any one of claims 1 to 18, characterized in that it comprises:

a determined quantity of a nucleotide probe according to claim 35,

a medium suitable for the formation of a hybridization reaction between the sequence to be detected, and the probe, and, advantageously, reagents for the detection of hybridization complexes formed between the nucleotide sequence and the probe during the hybridization reaction.

38 / A method of in vitro screening for the presence in a biological sample of proteins according to claim 33, characterized in that it comprises:

bringing the sample into contact with an antibody according to claim 34, under conditions allowing the production of an immunological complex formed between all or part of the proteins and this antibody, and

- detection of the immunological complex.

39 / Kit for implementing an in vitro screening method for the possible presence of proteins according to claim 21 in a biological sample, characterized in that it comprises:

- a determined quantity of an antibody according to claim 33,

advantageously a medium suitable for the formation of an immunological reaction between at least part of a protein and the antibody and, advantageously,

reagents allowing the detection of the immunological complexes formed between at least part of the protein sought and the antibody during the immunological reaction.

/ A method of detection in a biological sample of proteins according to claim 33, or of their fragments, characterized by bringing the proteins of the sample, or their fragments, into contact with an IGF carrying a marker group and the determination of the quantity of product fixed. 41 / Use as primers in DNA amplification techniques, of the PCR type, of two amplimers of approximately 15 nucleotides, included in one of the sequences according to any one of Claims 1 to 18, and about 200 to 250 nucleotides apart, one of the sequences being capable of binding to the 5 'end of one strand of the sequence to be amplified and the second sequence to the 3' end of the other strand.