CA1340868C - Nucleic acid coding for human angiotensin converting enzyme (ace), and its applications, particularly for in vitro diagnosis of high blood pressure - Google Patents

Abstract

L'invention concerne le clonage et le séquençage de l'acide nucleique codant pour l'ECA humaine ainsi que la determination de la chaîne peptidique correspondant à
l'ECA et de fragments peptidiques actifs qui en decoulent. L'invention a également pour objet l'utilisation des polypeptides sus-mentionnés pour la mise en oeuvre de méthodes de diagnostic in vitro de l'hypertension, pour l'étude conceptuelle de nouveaux inhibiteurs de l'ECA. L'invention concerne également l'utilisation des sondes nucléotidiques de l'invention pour le depistage in vitro des polymorphismes du gène codant pour l'ECA.

Description

13 ~ 086 ~

NUCLEIQIfJE ACID CO1) ANT FOR THE CONVERSION ENZYME
HUMAN ANGIOTENSIN (ACE), AND ITS
APPLICATIONS, ESPECIALLY FOR IN VITRO DIAGNOSIS
HYPERTEN. ~. ~ ION A: RTERIELLE.
The invention relates to a nucleic acid encoding for angiotensin converting enzyme (ACE) human as well as vectors containing this acid nucleic acid and the use of these for human ACE production. The invention relates to also the applications of this nucleic acid, especially in the area of new design human ACE inhibitors.
ACE, or peptidyl dipeptidase A (EC 3.4.15.1), or still kininase II, plays an important role in the regulation of blood pressure, by hydrolyzing Angiotensin I (inactive peptide released after cleavage of angiotensinogenE ~ by renin) into angiotensin II
vasopressive playing a role in the mechanisms of Arterial hypertension (SKEGGS, LT, et al (1986) J. Exp. Med., 7.03, 295-299).
Inhibition of the activity of ACE by EDTA and metal chelators, indicates that it is a metallopeptida ~; e, more particularly of a peptidase has zinc, capable of hydrolyzing, not only, Angiotensin I, also maps bradykinin (a peptide vasodilator and: natriuretique which it transforms into a heptapeptide inactive), and many other peptides has biological activity (YANG, HYT et al (1970) Biochim. Biophys. Acaa, 214, 374-376; ERDOS, EG, and al (1987) Lab. Invest:., 56, 345-348).
ACE is a widely distributed peptidase daps The organism, which we find for example in the form of membrane enzyme on cell surface 13 ~~~ 6 ~

2 vascular endothelials, and epithelial cells renales, as well as in the form of an enzyme circulating in plasma (ERDOS, et al (1987) mentioned above;
CARDWELL, PRB et al, (1976) Science, 191, 1050-1051 RYAN, US et al (1976) Tissue Cell, 8, 125-145).
Methods for purifying human ACE or animal have of: ja been described (in particular in BULL HG
et al, (1985) J. Biol. Chem., 260, 2963-2972; HOOPER, NM et al, (1! 387) Biochem. J., 247, 85-93).
However, the structure of human ACE is not not known at present. Only a few ACE peptide sequences of animal origin have have been published (BERrfSTEIN, KE et al (1988), Kidney Int., 33, 652-655: HARRIS, RB et al (1985) J. Biol.
Chem., 260, 228-2211; IWATA, K. et al (1982) Biochem.
Biophys. Res. Common, 107, 1097-1103; IWATA, K. et al (1983) Arch. Biocheam. Biophys., 227, 188-201; ST
CLAIR, DK et al (1986) Biochem. Biophys. Res. Common, 141, 968-972; SOFFE1R, RL. et al (1987) Clin. Exp. Hyp.
A9, 229-234).
Some attempts to clone the DNA coding for Animal ACEs were performed from two organs rich in EC: A, the kidneys and the lungs, but no nuc acid: complete leic coding for animal ACE
however, has not been described; only a few fragments such nuc7Leique aside have been described (DELUCA-FLAHERTY, C. et: al (: L987) Int. J. Peptide Protein Res., 29, 678-684; BERN ~~ TEIN KE et al, (1988 J. Biol.
Chem., 263, 11021-11024)). The amounts of messenger RNA
(MRNA) encoding ACE are probably too weak daps these organs to allow cloning of DNA
complementary (cDNA;) of mRNA set. No trial of ~ .34U868

3 cloning of DNA encoding human ACE has not been described so far.
Although ro: physiological ACE in extravascular tissue ~ ires is not yet known, it it is now well established that this enzyme found in the vascular endothelium and in the plasma, plays an essential role in homeostasis circulatory by its action of cleavage of the dipeptide carboxy-terminated by angiotensin I, thereby activating the latter by transforming it into angiotensin II
which is a vasoconstrictor ~~ eptide, stimulating the production of a: Ldosterone, and facilitating transmission adrenergic.
Given the essential role of angiotensin II
in the control of blood pressure, a active research has developed on the synthesis inhibitors of E; CA. Captopril was the first oral ACE inhibitor, followed by many others compounds. Currently, ACE inhibitors occupy an important ~~ lace in the treatment of Arterial hypertension. The complete structure of ACE not being known, the above-mentioned inhibitors were designed on the basis of the protease structure a zinc posseda.nt of neighboring enzyme properties those of the ECA, in particular on the basis of the structure of the active site of carboxypeptidase A
(ONDETTI, MA et al (1977) Science, 196, 441-444).
Thus, in the ab: absence of precise indication on the structure of the ACE, and more particularly on the or the sites) a ~~ tif (s) of the latter, we believe easily than the above-mentioned ACE inhibitors synthesized on the basis of analogies with the structure other enzymes may be molecules ~~~! ~ $ 6 ~

4 insufficiently active, or not completely specific of the ECA and suspects: possible to have an action insufficient therapy, or create effects secondary ires ~ irables. Among these effects undesirable, we will notably distinguish phenomena cough, t: digestive redness, rash which can be linked to the inhibition of other systems enzymatic by ACE inhibitors above tions.
One of the aims of the present invention is to allow the design of new inhibitors of The most powerful ECA: ~ and specifics of the latter than the current inhibitors mentioned above, allowing eve: ntuel7_ement a better efficiency has lower dose, and limiting the risk of effects unwanted from these: new inhibitors.
The purpose of the invention is Cloning and nucleic acid sequencing encoding ACE
human; this work was carried out from two complementary DNA banks of mRNA from human umbilical vein endothelial cells and nucleotide probes deduced from peptide sequences obtained by sequencing of purified fragments of human ACE. The techniques used for cloning and sequencing of acid nucleic sel.on 1.'invention will be more parti-especially ~ written in the detailed description of The invention.
Reference will be made in the following to the Table I and the figures in which.
- table I represents the fragments peptides obtained: enus ~ 3 from purified human ACE.

. ~ 340 ~ 68 - Figure 1 represents the comparison of acid sequences: ~ aminoterminal amines of ACE
human, and ECA dens from rabbits, beef, pork and mouse;
- Figure 2 shows the respective positions nucleic acids of the three clones used for the determination of the nucleic acid encoding ACE
human;
- Figure 3 shows the nucleic acid as well that the polypep ~ tide, deduced from the latter, corresponding human ACE;
- Figure 4 represents the homologies between certain sequences. human ACE peptides (ECAh), thermolysin (THERM), endopeptidase neutral rat or rabbit (rNEP), and collagenase human water fibroblastea (COLLh);
An in-depth study of the nucleic acid of The invention, thus included from the polypeptide deduced from of the latter cat corresponding to human ACE, leads to the following observations.
- the open reading phase of nucleic acid of figure 3 east made up of a DNA sequence delimited by the nucleotides corresponding to positions 23 and 109 of FIG. 1 coding for a signal peptide of 29 amino acids, and a sequence of DNA delimited by the nucleotides located at positions 110 to 3940 of FIG. 3, and coding for a protein containing 1277 amino acids;
- the nucleic acid of the invention is characterized by a strong internal homology (greater than 60%) between the DNA sequence delimited by nucleotides located at positions ~~ 700 and 1770, and that delimited by nucleoi ~ ides located at positions 2495 and 3565.

I34p8 ~ g The polypeptides of 257 amino acids coded res-pectively by 7 The two DNA sequences above mentioned also have strong homology between them (from o: 67.7% reduction). the strongest homology is at the amino acid level of central parts of the two above-mentioned polypeptides for example the polypeptide delimited by acids amines located at positions 361 and 404, and that delimited by amino acids located at positions 951 and 1002, pre: ~ entenit a percentage of homologies of Around 85 ~%. The two homologous polypeptides each includes a His-Glu-Met-Gly-His sequence corresponding to the amino acids located at the positions 361 to 365 and 959 .a 963 of figure 3. A study comparative (represented in figure 4) of this last sequence pe: ptidic of 5 amino amino acids, with act: ifs sites of several enzymes including thermolysin,: ~ ugger ~ e the fact that T at least one of these two sequences of 5 amino acids, which we find in the two homologous parts of the ACE, could constitute a party ~ e of the active site of the latter;
- the product of the open reading phase above-mentioned includes 17 potential sites of glycosylation, most of which are grouped in the region N-terminals of the molecule, and in the region located at the junction between the two domains of the above-mentioned homologous amino acids.
The present invention therefore concerns all acids including nucleic. the nucleotide sequence of the FIG. 3 coding: for human ACE.
The invention relates more particularly to everything nucleic acids ~ including the delimited DNA sequence by nucleoi ~ ides located at positions 23 and 3940 of 13408 ~ g figure 3, ea cod <~ nt for human pre-ECA from 1306 amino acids: the first 29 amino acids listed N-terminal rep ~ resent: ent the signal peptide, and the 1277 amino acids; remaining represent human ACE
nature.
The invention c: also relates to any acid nucleic acid understood ~ the DNA sequence delimited by the nucleotides located at positions 110 and 3940 of the Figure 3, and coding for mature human ACE.
The invention also relates to any acid nucleic acid from the DNA sequence of Figure 3 and coding for a polypeptide capable of having properties in ~: ymatic of the type of those of the ACE
human, in particular a polypeptide capable of hydrolyzing Angiotensin I and / or kinins, especially bradykinin, or whatever. no distinguishing nucleic acid of the precedent at the level of its nucleotide sequence that by nucleotide substitutions which do not cause modification of the amino acid sequence of the above polypeptide in specific conditions to make it lose the above: properties.
As such, the invention relates more particularly any nucleic acid comprising T a or the other counterpart regions of the ACE above mentioned.
Among the nucleic acids conform to The invention, particular mention will be made of any DNA fragment container.
- a DNA sequence extending between, on the one hand, a nucleotide located between positions 1 to 1177 and, on the other hand, a nuc: leotid located between the positions 3070 to 3940;

l3 ~ pg ~ g - a DNA sequence extending between, on the one hand, a nucleotide located between positions 110 to 1177, and on the other hand a nuc: leotid located between the positions 1276 ~ 1966;
- a DNA sequence extending between, on the one hand, the nucleotide is between the positions 1967 a 2971, and, on the other hand, the nucleotide located between the positions 3070 to 3940.
The invention also relates to nucleic acids whose nucleotide sequences are modified in the limits aotorized by the degsnerescence of the code genstic, as soon as the polypeptides coded by these nucleic acids either retain a structure identical primary, ie their enzymatic properties or immunologiq ~ ues.
Such non-limiting modifications lead to for example to variant nucleic acids which distinguish from; s nucleic acids above.
- by addition and / or - deletion of one or more nucleotides and or - change <~ tion ~ of one or more nucleotides.
A more particular subject of the invention is all acids nuc: leic ~ presenting the characteristic of specifically hybridize with nucleic acid represents su: r figure 3, under the conditions defined above.
- prehybridization treatment of the support (filter nitrocellulose or nylon membrane), on which is fixes the nucleic acid capable of hybridizing with that of FIG. 3, at 65 ° C. for 6 hours with a solution having the following composition. 4 x SSC, x Denhardt, ~ 3 ~ 086 ~ 3 - replacement: pre-hybridization solution in contact with the support by a buffer solution having the following composition a. 4 x SSC, 1 x Denhardt, 25 mM
NaPO4 pH 7, 2 mM EDTA, 0.5% SDS, 100, ug / ml DNA from sonic salmon sperm, comprising nucleic acid of Figure 3 while holding that probe, in particular so radioactive, ea previously denatured by a treatment at 100 ° C for 3 minutes, - incubation for 12 hours at 65 ° C, - successive washes with the following solutions.
. 2 x SSC, 1 x Denhardt, 0.5% SDS, for 45 minutes at 65 ° C four times, . 0.2 x S ~ SC, 0.1 x SSC for 45 minutes at 65 ° C a two times, . 0.1 x S; SC, 0.1% SDS for 45 minutes at 65 ° C.
I1 is ~~ remember that the composition of the Denhardt's solution. is the following . 1% ficoll, 1%
polyvinylpyrro: lidone, 1% BSA (serum albumin from beef), and that 1 x SSC consists of 0.15 M NaCl and 0.015 M sodium citrate, pH 7.
The invention also relates to any nu-cleique with the characteristic of hybridizing specifically with the nucleic acid of Figure 3 under non-stringent conditions including essential characteristics of the strin-gentes defined: ~ above ~ essus, except for what concerns temperature which is 40 ° C in non-conditions stringent, and successive washes which, in the non stringent conditions, are realized using 2 x SSC at 45 ° C for 15 minutes twice.
The invention also relates to nucleo-causes: possible to hybridize with acids nucleics dec: rits previously, or their sequences 13 ~ 08 ~~
complementary, ai: nsi that with Y messenger RNA coding for ACE and with the human gene responsible for The expression of 1 "ECA, under the hy-bridging defined c: above.
It goes without saying that the hybridization conditions stringent or not. stringent, defined above are preferred conditions for Hybridization, but are in no way limiting and can be changed without affecting the recognition and hybridization properties of probes and nucleic acids mentioned above.
Salt and temperature conditions during hybridization and washing of membranes, can be changed in the direction of a larger or a lower string ~ ence without the detection of Hybridization is affected. For example, it is possible to add formamide to lower the temperature during hybridization.
The invention also relates to the polypeptide of which 1 <~ amino acid sequence is shown in Figure 3, as well as all polypeptides: likely to have activity enzymatic type ~ that of ACE and which are coded by the above-mentioned DNA fragments derived from the acid Figure 3 nucleic acid.
Among the above-mentioned polypeptides, will distinguish us ~ ammen ~ t - po: Lypeptide extending between acids corresponding amines: in positions 1 and 619 of the Figure 3;
- po7Lypept: ide extending between acids amines corresponding to positions 620 and 1229;

~~~ (~~ 6g - the polypeptide extending between the amino acids corresponding to positions 350 and 395;
- the polypeptide: ide extending between acids corresponding amines: at positions 948 and 993.
The previous polyp ~ eptides ~ can be modified as long as they serve the biochemical properties or defined immunologic or pharmacological previously.
For example ~ ple, and in a non-limiting way, polypeptides within the scope of the invention can be distinguish from the polypeptides defined above;
- by addition and / or - suppresses; asion of one or more amino acids and or - modification of one or more acids amines, provided that the biochemical properties or immunological or p harmacological as indicated above above be ~~ reserved.
We believe that the skilled man has the possibility of identifying or even selecting those polypeptides of shorter sequences that enter in the field of _L'invention. As one of general means him. allowing to proceed with this identification, we will mention, for example, the treatment of the polypeptide of FIG. 3 with a protease cleaving the above-mentioned polypeptide into a peptidiq site ~ eu chosen, snit in a region N-terminal, exit in> a C-terminal region, followed by separation of the N-terminal fragment or of the C- fragment terminal of the remainder of said polypeptide, this "remainder"
~ so many tests for its enzymatic activity vis-a-vis angiovtensine and / or bradykinin. In case response po; ~ itive, we will have established that the 134 ~ 8 ~~

N-terminal or C-terminal fragment, did not play a role signif: icatif, if not essential for the manifestation of the enzymatic properties of said polypeptide. The ope: ration can possibly be repeated however: that we have a protease likely to recognize another specific site near an N-terminal or C-terminal end of the reatant polypeptide. Loss by the polypeptide plus small recognized of the enzymatic fragments of the fragment longer which it came from can lead to The hypothesis that the last separate fragment played a significant role in the manifestation of properties of the polypeptide of FIG. 3.
Another variant, simpler than the previous one, detection ~ of regions: ACE regions essential to manifestation of the enzyme activities of this last, pent and: re based on treatments nucleic acid enzymes encoding ACE, before incorporation of the nucleic acid as well obtained, and presumes c; oder for a polypeptide having enzymatic activities of the ACE type, in the expression vector used for setting work of a process for the production of said polypeptide in the host this: appropriate cell (process which will be more particularly ~ details in what follows). This enzymatic treatment can then consist of soft in one pruning of the ends of the initial nucleic acid (coding for example for the polypeptide of FIG. 3 in full), for example by an enzyme exonucleolytiqixe, te ~ lle que Ba131, soft by one or several restriction enzymes chosen for their recognition sites: ~ respective ance (if applicable) feeds by: directed point mutagenesis) in the 13408ti ~

initial nucleic acid sequence, either by The addition of a synthetic junction DNA fragment between the restriction enzyme cleavage site and the beginning of: The region to be expressed. Nucleic acid trunk obtained can then be tested, after incorporation into: The chosen vector and transformation of the host cs ~ llula: ire with the recombinant vector obtained, for its capacity to express a polypeptide corresponding trunk, still having the above enzymatic properties - or on the contrary do not having more: a, hence the possibility, as in the previous variant: e, to identify within the polypeptide ds ~ Figure 3, the sequences playing a important role :, if not essential in the event enzymatic properties of ACE.
The invention also relates to any acid recombinant nuclei containing any DNA fragment from the above-indicated type coding for human pre-ECA, or Mature human ACE, or for any polypeptide susceptible to: possess an enzymatic activity of type of that of the ECA, associated with a promoter and / or a transcription terminator recognized by polymerases of the host cell in which said recombinant nucleic acid is likely to be introduced.
The introduction of said recombinant nucleic acid in the ce11u7Le host, is advantageously carried out at Using vectors, in particular of the plasmid type, which are able to replicate in said host cell and to allow the expression of the sequence coding for The enzyme.
Said nuc acid: recombinant leic can also be introduced: into ~ the host cell using a viral vector (recombinant virus) capable of infecting said cells note: and allow the expression of the .code polypeptide with a nucleic acid according to 1'invention, ~~ e last being loos the control of a viral promoter acti.f daps the Note cell.
The invention therefore relates to a production process human ACE, or the above-mentioned polypeptides from EC: A, c ~ ai includes the transformation of hotel cells; using the sus-indigoes vectors, the cultivation of the transformed note cells in a appropriate medium and recovery of said polypeptides snit directly from the middle of culture, lor: that these are secret (especially in c: as or the polypeptides considered are preceded by a signal sequence when synthesis in the cell note), either after lysis of the wall of the note cell in the callus or polypeptides would not be secret outside of this last.
The above-mentioned process advantageously comprises one final purification step, for example according to chroma techniques ~ hybridophobia and affinity using an inhibitor of all or part of The fixed ECA on the column: (see detailed description following).
As such: the invention has more particularly for subject one: composition containing human ACE
pure, free of contaminants, particularly of a natural protein.
The note cells used for the mile in work of the above mentioned process can be prokaryotic cells, especially E. cells coli, or, more advantageously, cells 13 ~ 0 ~ 6 $
eukaryotes, ~ which allow, in particular, to obtain proteins on the ~ ur glycosylated and mature form (yeast, CHO cells, or insect cells infected with the bac: ulovirus).
This process ~ described above is also achievable by transfection of host cells with vectors of ~ ~ ression having integrated a modified gene The ECA. Either it is truncated parts of Complementary DNA, endothelial messenger RNA from The ECA, including for example only one of the two fields counterparts, or amputee of the 3 'end coding for the hydrophobic membrane insertion segment. The transfection of host cells is also realizable with vectors including a mutated form of ACE, or fragments thereof, the mutations carrying particularly on the bases coding for amino acids involved in the enzyme activity described above above.
I1 is p <particularly advantageous for the crystallization of the recombinant enzyme to mutate one or several potentials of N glycosylation, i.e. the codons corresponding to asparagine ~~ sparagine-X-Threonine and asparagine-X-serine.
The invention also relates to a method of preparation of the above-mentioned new polypeptides by synthesis yui includes soft 1'addition step by stage of the small residues chosen, with the addition or the removal of any protective groups from amino and carboxyl functions, or addition of residues peptides chosen to produce fragments, followed by condensation of said fragments into a ac sequence: appropriate amino acids, with addition or elimination of the protective groups chosen.
The invention also relates to antibodies.
specific di.riges against the above polypeptides.
In particular, the invention relates to antibodies monoclonal d: iriges against peptide sequences appearing to be involved in at least one of the activities of EC'.A.
Such monoclonal antibodies can be produced by the hybridoma technique, the general principle is recalled below.
First, we inoculate one of the polypeptides c: i-des: aus to a chosen animal, whose B cells are then able to produce antibody against this polypeptide. These lymphocytes antibody producers are then merged with "immortal" myeloma cells to give rise has hybridomas. From the heterogeneous mixture of cells thus obtained, we then make a selection cells capable of producing an antibody particular and. of: ~ e multiply indefinitely. Each hybridoma is multiplied in the form of clone, each leading to the production of a monoclonal antibody whose recognition properties vis-à-vis polypeptides of the invention can be tested by example on co: Lonne of affinity.
Among the ~ polypeptides used for the manufacture of the above-mentioned monoclonal antibodies, particular mention will be made of those delimited by acids amines located at positions 350 and 395, or 948 and 993 in Figure 3., The invention also relates to a methods of in vitro screening, or testing, for ACE, and more l3 ~ pg ~ g especially: human ACE, or a product derived from ACE such as the above-mentioned polypeptides having in viwo the: ~ properties of the ACE, daps a biologic ~ ue sample likely to contain them. A
such a screening method according to the invention, can be performed softly using the above monoclonal antibodies mentioned, either using the nucleotide probes of-above.
The above mentioned biological prep = vement is performs either in flowing tissues, such as the blood, either in d.es organs, this last type of permitting: in particular to obtain cuts fine of tis: aus on which the antibodies mentioned are later fixed.
The assay method according to the invention, proceeding through the above-mentioned antibodies particularly includes the following steps.
- contacting an antibody recognizing specifically ACE, or a polypeptide derived from ACE
according to the invention, with the aforementioned bio-sampling logic in conditions allowing production possible of an immunological complex formed between ACE or product derived therefrom and the above antibody mentionned ;
- detection, using any appropriate means of above complex ~~ immu: nological.
Advantage: ~ ement, the antibodies used for the implementation of such a process are particularly marked enz; ~ matiq ~ eu or radioactive.
Such a method according to the invention can in particular be carried out according to the ELISA method (enzyme linked sorbent assay) which includes the following steps.

13 ~ p868 - fixing of a predetermined amount of antibodies on a solid support, in particular to the surface of a well of a microplate;
- addition of the biological sample (in the form liquid) on said support;
- incubation for a sufficient time to allow immunological reaci: ion between said antibodies and: ACE or product derived therefrom mentionned ;
- elimi: nation of the non-fixed parts of the biologiclue sampling and washing of the solid support (in particular of the microplate wells);
- addition of an immunoglobulin marked by a enzyme capable of activating a specific substrate for this enzyme, - addition of specific activity substrate enzyme released during the immunological reaction previous ;
- detection, using any appropriate means, of degradation of the substrate by the enzyme: and - correlation of the quantity of enzymes released to the concentration of human ACE initially present in the sample: organic.
According to another embodiment of the method of the invention, the above-mentioned antibodies are not marked and the detection of complexes immunological forms between the polypeptides and said antibody is made using an immunoglobulin marked recognizing said complexes.
The dosage method according to the invention can also be rea: read by an immuno-technique enzymatic following a mechanism of competition between the polypeptides that are likely to be contained in the 13 ~ 0 ~~~

biological sample, and pre-determined amounts of these same polypeptides, with respect to antibodies above-mentioned. Dan: the latter case, the polypeptides of the invention and its predetermined amounts are advantageously marked with a marker enzymatic.
The invention is in no way limited to the modes of realization of: above criteria for in vitro assay polypeptides of the invention, this assay can be carried out with the help of any other immunological method appropriate.
The invention also relates to a method of screening, or testing:> age, in vitro of a nuclear acid coding for all or part of the ACE, realized from of a ~ snt b: iological sample likely to contain said nucleic acids, characterized in that it understand.
- the miss in contact with a nucleotide probe described below; above with the above biological sample under conditions allowing possible production of a hybridization complex formed between the acid nuclear and the so-called probe;
- detection using any appropriate means of aforementioned hy complex: bridging.
According to an embodiment of the invention, the bio7Logique sample mentioned above is, previously ~~ the mdse implementation of screening, traits so that the cells it contains are lysses and, e ~ rentue.llement, in that the material Contained in the said cells or fragments using restriction enmes: EcoRI-type, BamHI
etc ..., or that the RNAs are isolated from it.

~ 3 ~ 0 ~ 6g Advantageously, the nucleotide probes of The invention are marked with a marker enzymatic or radioactive. DNA, or RNA, from biologi.que sample are placed on a support suitable, in particular: on a nitrocellulose filter or other, e.g., single nylon membrane, on which are then added; the above-mentioned probes.
According to another advantageous embodiment of the above-mentioned process of the invention, cuts histological are carried out from the above biologic sampling and nucleotide probes of The invention feels put in direct contact with histological sections for the detection of acids nucleic acids of the invention by in situ hybridization.
The invention also relates to the application screening, or assay, methods above mentioned in the in vitro diagnosis of pathologies such as 1 "hyperitension, sarcoidosis and others granulomatous diseases, thy-ro3diens and generally all diseases correlated with levels in the organism (in the plasma or other tissues) in acid sequences amines of the im ~ ention, located outside the domain delimited by the corresponding extreme values generally in the physiological state of a healthy individual.
The in vitro assay methods of the invention using the above-mentioned antibodies also allow. follow-up in the organization of the concentration <ACE inhibitors by measuring the amount of ant: antibodies that could not be fixed on the, or the sites) act: if (s) of the ACE which are masked by The inhibitor. The assay methods of the invention are therefore part.iculiE ~ rement advantageous in the context monitoring treatment of patients with inhibitors of: ACE.
The invention was also subject to necessities or kits for the implementation of the methods of screening, or screening, in vitro mentioned above.
For example, such kits include especially:
- a determined quantity of one of the antibodies monoclonal above-mentioned: likely to give rise to a specific immunological reaction with ACE or with one of the ACE derived polypeptides according to The invention;
- and / or a specific quantity of ACE or a sleepable polypeptide gives rise to a reaction immunological with the above-mentioned antibodies - advantageously, an environment suitable for formation of an immunological reaction between the ACE or the polypeptides of the invention and the above antibodies mentioned;
advantageously, reagents allowing the detection of the immunological complexes produced during the im ~ nunological reaction mentioned above.
Within the framework of the implementation of a method in vitro screening using probes nucleotides, the kits used include example.
- a c ~ uantii: e determined from one of the probes above-mentioned nucleotides capable of sleeping place at a re ~~ ction of hybridization with one of the aids nucleic acids su: a-mentioned coding for ACE or a ACE polypeptide according to the invention;

1340 ~ 3 ~~

- advantageously, reagents allowing the detection of the hybridization complexes produced during the above-mentioned hybridization reaction.
The invention also relates to the use of the Figure 3 polypeptide or any fragment appropriate peptide derived from the latter, for the design of new inhibitors of human ACE, more powerful, and / or specific to the latter than so are the acts: the ACE inhibitors.
The invention is also intended and a method of detection or assay, of an ACE inhibitor, or quantification of its inhibitory power, which includes bringing the polypeptide of FIG. 3 into contact, or any peptide fragment derived from the latter and having an enzymatic activity of the type of that of The ACE, with said inhibitor, and the determination of the coefficient of inhibition of the enzyme by the inhibitor, in particular by measuring any activity reEaidue enzyme: It on an appropriate substrate of The ECA.
The invention also relates to the use of the polypeptide of Figure 3, or any fragment thereof the latter capable of hydrolyzing kinins, in particular bradykinin, ~ in the treatment of diseases inflammatory, or infectious, pancreatitis jitter, and more generally illnesses or release of kinineas in the body could play a pathogenic role.
As such, the invention relates more particularly pharmaceutical compositions for the treatment of the diseases indicated above, characterized by the association of all or part of the polypeptide of Figure 3, capable of hydrolyzing the 13 ~ O ~ G8 kinines, note: even bradykinin, with a vehicle pharmaceutical: reasonably acceptable.
The prese.nte invention also relates to The use of the nucleotide probes of the invention, able to hybridize: ider with the gene responsible for Expression of human ACE under the conditions described above ~~, for the determination of different forms a: lleliques of the aforementioned gene.
Arterial hypertension (HTA) is classified into two main categories according to their etiologies. the Secondary HTA is essential HTA. HTA
secondary re: grouping all forms of hypertension that the medicine can relate unequivocally to a pathology identified. It can be, in particular, a hormonal hypersecretion of tumor origin (repine or aldosterone, catecholamines, for example) or vascular (stenosis of a kidney, renal artery causing repine hypersecretion). These secondary forms of hypertension represent around 5% of the total HTA. Under the term of essential hypertension, are grouped all forms of hypertension for which no etiology exists now identifiable and which constitute 95%
remaining buses of HT.A.
The pathology of essential hypertension is not today known but many studies opt shows that genetic factors were involved in the development of hypertension. Opt family studies shows that there was a family aggregation of arterial prEassion values and that a correlation exists between the blood pressure of parents and natural children when this correlation does not exist not with adopted children. Studies conducted on mono twins or dlizygotes also opt confirmed 13 ~ 0 ~ 6 ~

Heritability: the level of blood pressure. Finally, The genetic analysis of the transmission of the level of arte pressure: riellea, in humans and in strains of genetically hypertensive rats, showed that several genEa were involved. The transmission of this characteristic, variable between individuals, calls it blood pressure level therefore goes through the transmission of allelic forznes of genes between parents and lee erifants. The genes that one hangs designer "candidate;" to the responsibility of this transmission are, primarily, those involved in the principau ~; pressure regulation systems arterial, such as: genes, renin system-angiotensin, including that coding for ACE.
The method of choice to recognize, today, the allelic forms of a gene is to identify, for this gene, the size polymorphism of the fragments of restriction of this gene (by size of the fragments of restriction we mean the number of base pairs that include the said fragments). For this type experience, the DNA of several individuals is isolates, cleave a restriction enzyme, transferred to a membrane capable of fixing it irreversible, and hybrids with a DNA probe marquee corres ~~ ondant au gene etudie.
This method makes it possible to distinguish the forms allelics of a same gene that differentiates them of an individual <~ 1 'other - by their curity: restriction, and / or - by the presence: in T one of the allelic forms dune insertic> n (additional DNA fragment in The least frequent allele), or a deletion (DNA fragment missing in the least allele 13 ~ 0 ~ 68 frequent) this insertion does not exist in other allelic forms.
In the first case mentioned above (differences based on the restriction card, or restriction site polymorphisms), under the effect digestion with a given restriction enzyme X, The allele possesses: at the level of a precise region of the genome, a restriction site recognized by this enzyme is cut by said enzyme, and the one without such a site, at the level of said region, is not cleaves. Using a susceptible nucleotide probe to hybridize to ni ~, water of said region, so we can detect two size restriction fragments different depending on whether the region concerned has been split or not. Each of these sizes corresponds to a shape allelic.
In the:> second case (presence of an insertion, or dune deletion), leis genomic DNA fragments cor-corresponding to the two allelic forms are cleaved by Restriction enzyme X at a specific site tick. We can detect, using a probe such as defined in the previous paragraph, two fragments of size restriction: the differences. To each of these sizes corresponds to an allelic shape, size la higher corresponding to the allelic form including the above-mentioned insertion.
In the two previous cases and whenever We detect size restriction fragments different (or even restriction fragments polymorphic) in a given gene and an individual has The other under the effect of the action of an enzyme of restriction X, it is: ra refers to "polymorphism X "of this gene (for example" EcoRI polymorphism "if 13 ~ 08 ~ 8 The restriction enzyme X is the EcoRI enzyme, or TaqI polymorphism if the restriction enzyme is TaqI).
The study of an X polymorphism of a chosen allele, in a given population of individuals, presumed healthy, makes it possible to note the existence of this polymorphism in a determined proportion of individuals of this population. Measuring this proportion leads to this which is called hereinafter the "allelic frequency of reference".
If the frequency: of a selected allele, next measure the same method within a population of individuals presenting a determined pathology, is different from the reference frequency, we can then send The assumption that the a7Llele in question is related to said pathology.
Similarly, we can involve an allele in the development of the disease if there is a co-segregration between the transmission of the disease, in affected families and informative for the polymorphism, and the transmission of the allele allows to involve this allele in the development of sickness. (a family is said to be informative for the polymorphism if the parent reached by the disease is helterozy ~ gote for the restriction site what which allows to follow in the allele descendants responsible for the disease).
The present invention relates to fragments of restriction po: Lymorplhes likely to hybridize with a nucleotide probe of the invention, in particular in the conditions described above, and which come from cleavage of the different allelic forms of the gene ~~~ fl ~ 6 coding for 7.'ECA using restriction enzymes determined.
The invention relates more particularly to polymorphic restriction fragments capable of hybridize with a nucleotide probe according to 1'invention, E ~ lle-mi = me likely to hybridize with The 5 'end of the nucleic acid of Figure 3 and who are following them.
- the fragments. 5.8 kb and corresponding 6 kb to the TaqI polymorphism, - the fragments. 8.8 kb and 9 kb corresponding to the Dra.I polymorphism, - fragments of 8.5 kb and corresponding 9 kb to the BglII polymorphism, - the fragments of 10.6 kb and 11 kb cor-corresponding to; KpnI polymorphism, - fragments of 8.4 kb and 8.8 kb cor-corresponding to; HindIII polymorphism, - fragments of 4, 2 kb and 3, 0 kb on the one hand, and 4 kb and 2.7 kb on the other hand, corresponding to polymorphism; BglI, - fragments of 5, 2 kb and 5, 7 kb corresponding to po: RsaI lymorphism (with presence or not a 3.0 kb fragment), - fragments of 4.3 kb on the one hand, and of 2.2 and 2.5 kb on the other hand, corresponding to the polymorphism PvuII, - fragments of 3.5 kb and 3 kb corresponding to the polymorphisms Xb; aI, - fragments of 4.3 kb and 2.7 kb cor-respond to the TaqI polymorphism.
The difference in size of the allelic fragments recognized by the probes of the invention in 13 ~ 0 ~~ 8 different individuals; s, corresponds to an insertion of a genomic fragment of approximately 400 base pairs with the margin of error due to the size analysis of these fragments by electrophoresis on agarose gel.
This insertion can be detected using any restriction enzyme cleaving nucleic acid mentioned above as long as the fragments generated be able to hybridize at least in part, with the corresponding probe.
The present invention relates to a method of screening, in. in vitro polymorphisms of the coding gene for the expression of human ACE, made from biological samples, such as blood, ceptibles of <: have genomic DNA, and have was taken from a given population of individuals not with no hypertension (or normotensive individuals), or other diseases of the vascular endothelium.
The above-mentioned samples are analyzed following the following method including.
- the processing of nucleic acids from above-mentioned biological samples using a restriction enzyme determined under conditions allowing the obtaining of restriction fragments derived from the cleavage of said nucleic acids at the level of their restriction sites recognized by said enzyme, - bringing a nucleotide probe into contact according to the invention capable of hybridizing with above-mentioned fragments: under conditions allowing event production: uelle of hybridization complexes between said probe and the restriction fragments mentioned above, in particular under the conditions of hybridization defined above, I3 ~ 0 ~ 6g - the detection of the above hybrid complexes -donation, - measuring the size of any fragments restriction polymorphs involved in complexes of above-mentioned hybridization.
Among the probes used for the implementation of such a process, mention will be made in particular of DNA
of the nucleic acid described at the figure 3, or, before: very carefully, a fraction of this CDNA likely to hybridize with different restricaion fragments corresponding to the different allelic forms of. gene for ACE.
Advantageously, the probe used in the process above mentioned is marked, in particular so radioactive or non-radioactive, in particular in the form of biotinylated probe, to allow detection of different fragment, restriction remaining hybrid with said so: nde.
Measuring the size of the different fragments of restriction p ~ olymorphs engaged Bans of complexes hybridization with: The aforementioned probe, can be carried out by mute method known per se by the man of the job ; determining the size of these fragments restrictive: Lon is realized in particular by agarose gel electrophoresis and evaluation of size of said fragments compared to those of temo fragments: ins.
The comparison of the sizes of the fragments of restriction measured in individuals of the population studied, allows to establish, within this population of individuals, the allelic frequencies of reference.

1. ~ 40 ~~~
The invention also relates to the detection of different allelic forms of the gene encoding ACE
human, likely to be linked to development in a person with hypertension, or other diseases of The vascular endothelium, especially Atherosclerosis. This ~ detection head is carried out according to the same method within a population of individuals presenting a pathology mentioned above. If the frequency of an allele measured within this population is different: rent from the reference frequency, we can then emit; be the hypothesis that this allele is probably related to hypertension.
Similarly, we can involve an allele in the development of the disease if it exists, in families affected x> ar the disease and informative for polymorphism, a cosegregation between trans-mission of certain allelic forms of the ACE gene and the transmission of HTA and / or diseases of Endothelium, especially atherosclerosis.
The invention also relates to the application results of the study of gene polymorphisms coding for human ACE in in vitro diagnosis of The possible probable genetic predisposition of a individual with hypertherapy, or other diseases of the vascular endothelium, especially 1 'atherosclero; se.

13 ~ 0 ~ 6 ~

The above-mentioned in vitro diagnostic method includes the possible detection of fragments of res-polymorphic triction: ~, derived from cleavage by an enzyme specific restriction of allies probably related to the mechanism of the HV, using probes nucleotides according to the invention, capable of hybridize with said restriction fragments polymorphs.
The conditions under which such a method in vitro diagnostics can be implemented are those described in the articles concerning techniques for detecting length polymorphisms fragments of re: atriction, still better known as The abbreviation RFL: P (Restriction Fragment-length Polymorphism). Such conditions are more particular: nt described in P article of GUSELLA
JF 'Recombinant D: NA techniques in the diagnosis of inherited disorders "published in Journal of Clinical Investigations (1986), 77, 1723-1726.
According to an aspeci ~ advantageous of the invention, in addition screening for polymorphisms in the gene encoding Human ACE, the above-mentioned in vitro diagnostic method mentioned also includes screening for polymorphisms of one or more genes coding for proteins different from ACE and involved in the mechanism of pressure regulation arterial.
As such: the in vitro diagnostic method according to the invention includes screening for polymorphisms of the ACE gene, and of the renine, and / or the kallikreine gene, and / or the gene of the ANF.

~ 3 ~ 086g Among the :: aforementioned polymorphisms of genes coding for renin, kallikrein, and ANF, we will cite in particular those who have been more particularly described in .The PCT patent application published under number W087 / 02709 dE: posed on October 23, 1987.
Using appropriate probes, the authors of this PCT application discovered the fragments of following restriction; .

- 5 kb and 9 kb corresponding to the BglI polymorphism of renin gene, - 0.9 kb corr ~ aspond ~ ant to the BglII polymorphism of the gene renin, - 20 kb and 24 kb corresponding to the RsaI polymorphism of renin gene,

- 6.2 kb and 9 kb corresponding to the HindIII polymorphism renin gene, - 9.8 kb and 1: 1 kb c: corresponding to the TaqI polymorphism of the gene for: renine, - 3.9 kb corresponding to the EcoRI polymorphism of the gene renin, - 1.4 kb and 1, ~ 8 kb corresponding to the BanII polymorphism of the ANF gene, - 4.1 and 6.2 kb corresponding to the BglI polymorphism of ANF gene, - 9.1 kb and 6,! 5 kb corresponding to the BglII polymorphism of the ANF gene, - 5.2 kb and 11.8 kb corresponding to the polymorphism EcoRI of the gene i ~ IJF, - 15 kb corresponding to the EcoRI polymorphism of the gene kallikrein ,, - 3.1 kb correspond to the PstI polymorphism of the gene for kallikrein ,, ~~~ os6s - 4.5 kb corre. Corresponding to the StuI polymorphism of the gene kallikrein.
The invention also relates to kits for the implementation of the in vitro diagnostic method defined above.
For example, such diagnostic kits understand.
- a given quantity of one or more restriction enzymes, - a determined quantity of a probe nucleotide capable of recognizing the fragments restriction resulting from the cleavage of the gene coding for ACE by the above-mentioned enzyme (s), - eventuelleme: nt a determined quantity of a probe likely to recognize fragments of restriction i: ~ above the cleavage of the gene coding for the renin with 1 '(~ or the enzymes) mentioned above, - and / or a specific quantity of a probe likely to recognize fragments from cleavage of the gene coding for kallikrein by 1 ′ (or the) enzymes) mentioned above, - and / or a specific quantity of a probe likely to recognize restriction fragments derived from the cleavage of the gene encoding ANF by 1 '(or the) enzymes) mentioned above.
Classic techniques of gene amplification, including that described in US Patent No. 4,683,202 filed on 25/10/1985, can be used to detect the polymorphism of the gene encoding ACE using primers hybridizing with this gene on both sides e insertion: about 400 base pairs above mentioned. The: ~ DNA fragments thus amplified have a 13 ~ 0 ~~~

diffe.nte se se: lon that the fragment of genomic DNA
contains or does not contain said insertion.
As such, the invention more particularly for obj and any nucleotide sequence of around 10 to 40 nucleotides from: the nucleotide sequence of the Figure 3, or its complementary sequence, or likely to hybridize with part of the sequence of the figure 3 or with the complementary sequence of this last, usable as primers for Amplification of the gene encoding ACE.
Additional characteristics of The invention appears: it will still appear during the description which follows of the cloning of the nucleic acid encoding human ACE, as well as probes used for this cloning, it being understood that this description should not be interpreted as tending to restrict the scope of claims.
I - PURIF'ICATIDN AND SEQUENCING OF THE ACE
ACE hum, groin in its complete membrane form has was purified from renal microsomes. Fraction microsomal was prepared from homogenates of kidneys of human corpses in the following way. 600 g kidney cortices were minced, suspended in a phos buffer ~~ potassium hate 20 mM, pH 8, containing 250 mM of saccharo: ~ e and a mixture of inhibitors of protease (5 mM sodium tetrathionate, N-ethylma-leimide 10 mM, phenylmethanesulfonyl fluoride 2 mM), and homogenize ~~ for 3 minutes. Debris from tissue were removed by centrifugation at 5000 g for 20 minutes and the particulate fraction was sedimented by centrifugation at 105,000 g for 1 hour. The pellet was resuspended in a buffer of potassium phosphate 150 mM; pH 8 (buffer I, 200 ml) 1 ~~~~~ 6g and treated for 18 hours with CRAPS 8 detergent mM (Serva). The supernatant, after centrifugation 5 105000 g for 1 hour, was dialyzed against buffer I for 24 hours in order to eliminate CHAPS.
Then it was placed on a column of phenyl-Sepharose * 4B (Pharmacia) and Blue has a rate of 24 ml / hour; 60% of the enzyme activity has been retained 10 on the column and elected as a single peak after applying a linear CHAPS gradient (3- (3-cholamidopropyl) dimethylammonio-1-propanesulfona-te) up to a concentration of 10 mM (800 ml).
The eluate was: enriched with ZnS04 and KC1 has 15 final concentrations of 0.1 mM and 330 mM
respectively ~ t way ~ get a connection optimal with the inhibitor, then the purification was completed on a lysinopril-Sepharose 4B column as it has been described in BULL, NG et al, (1985), J.
20 giol. Chem. ,:? 60, 2963-2972.
The protein thus isolated (1, 2 mg) was analyzed, after reduction (2 5% mercaptoethanol) and denaturation (boiling for 3 minutes), by electrophoress ~ on Bans polyacrylamide gel 6%
25 gel containing NaDodS04 (LAEMMLI, UA, (1970), Nature, 227, 680-685).
The purified enzyme hydrolyzes angiotensin I, bradykinin and: several synthetic substrates, and is inhibited by captopril and other inhibitors of 30 the ECA. the determination of enzyme activity at was carried out using the substrate furanacryloyl-L-phenylalanyl-g ~ lycyl - glycine (FAPGG) under the conditions described daps HOLMQUIST et al (1979), Anal. Biochem., 95, 540-548. hes pa: branches of the enzymatic reaction 35 determined at pH 7.5 are 136 ~ m for Km and 22100 * trademark ~. ~~ 0 ~ 68 min.'s for Kcat; treatment with bromide cyanogene a et: e used to cleave the enzyme at the level methionine residues thus generating fragments for the sequE ~ ngage. Purified ACE (0.5 mg) has been dissolved in 70% formic acid (0.2 ml) and Cyanogenic bromide (2.0 mg) was added. After 16 reaction hours at: ambient temperature in black, solution a. diluted 20 times with water and lyophilized.
The peptide fractions have been isolated by Reverse phase HPLC, using a trifluoroacetic acid (TFA) -water-acetonitrile gradient (buffer A. 0.1 M TFA in water; buffer B. 0.1%
TFA dens acet.onitrile; 1-100% B on 32 minutes, elution rate 1 ml.min. '~). Fractions correspond to different peaks have been separated and freeze-dried, and sequenced using a sequencer automatic. I ~ es peptide fragments thus obtained have been grouped in Table I. The amino-protein terminal has been determined by the Edma's method: na using an automatic sequencer using the whole ECA rotein.
The following sequence of 16 amino acids has been obtained NH2-Leu-Asp-Pro-Gly-Leu-Gln-Pro-Gly-Asp-Phe-Ser-Ala-Asp-Glu-Ala-G1: ~ -COOH
II - CLO: SWIMMING OF RNA COMPLEMENTARY DNA
MESSENGER
A) Nucleotide probe - from ~ r of the peptide sequence Met-Trp-Ala-Mei ~ -Trp-, Ale-Gln-Ser-Trp-Glu-Asn-Ile (con ~ ue according to the sequence: CNBb of table I), 64 probes HACE6 nucleotides have been synthesized at 13 ~ 08fg Using an automatic DNA synthesizer using the phosphoramidite method.
Met-Trp- ~, la-Lys-Ser-Trp-Glu-Asp-Ile- 3 ' TAC-ACC-C'.GA-TT'T-ACG-ACC-CTT-TTG-TA 5 ' GG: TG CA
- Radioactive marking. the HACE6 probe has sts marked with [yP3Z] ATP (specific activities. 5000 curies / mmole) at its 5 'end by T4 polynuclsotide kina ~ se. The specific activities of the probe is 5 x 106 cpm / pmole.
B) Cribbage of a banctue of complementary DNA
built from RNA from endothslial cells A cDNA bank of endothsial cells from umbilical veins hu: main primed with oligo (dT), built in phage agtll (Clontech Laboratories Inc.), and constitues of 1.5 x 106 recombinants independent, has been screened using the HACE6 probe marked with P32. AD: N of phages has sts transfers on nitrocellulose filter according to the Benton method and Davis (rsf. BE: NTON, WD, and DAVIS RW (1977), Science (Wash), 196, 180-182.). Hybridizations with HACE6 have sts rsalisses in. 6 x SSC, 0.1% NaDodS04, 50 mM NaPO4 buffer ~~ pH 6, 8, 5 x Denhardt, 0.1 mg / ml of DNA from salmon sperm at 50 ° C. Washing final fili: res has been done twice in 2 x SSC, 0.1% SDS, at 55 ° C for 15 minutes.
Under these conditions two different types of hybrida clones: strongly with the HACE6 probe have sts obtained. These are the clones aHECl922 and aHEC2111. The fragments of AD1V inssrss in phages have isolated sts after phage cleavage by the restriction enzyme EcoRI, and inssrss in both directions in the plasmid bluescript (St: ratagene), at the EcoRI site of this latest. From these double-stranded plasmids, single stranded matrices were obtained by reinfecting bacterial cultures carrying these plasmids using of a phage helper KC> 7.
Determination of the nucleotide sequence of these clones were assigned by the Sanger method (SANGER, F. et: al (: 1977), Proc. Natl. Acad. Sci., USA, 74, 5463-5467) using either DNA polymerase from klenow, or the modified T7 DNA polymerase (Sequenase, U: c Bioc: hemical). Some regions have been sequenced in use, ant in place of deoxyguanosine tri-phosphate (dGTF ~), deoxyinosine tri-phosphate (dITP) or deaza-deoxyguanosine tri-phosphate (7-deaza dGTP). The electrophoresis of the fragments marked by [S35] dAT: P was practiced on urea gel-polyacrylamide. The sequences were determined in synthesizing oligomers step by step all the approximately 350 base pairs that served as primers staggered along the sequence.
The nucleotide sequences inserted into these two phages so: nt overlapping over a length of 2323 base pairs :. The peptide sequence deduced from these clones contain the set of peptide sequences obtained by purification of human ACE, and which are shown in: Table I, except for the amino-terminal region.
So: re zi get the complementary DNA
correspondent ~~ the 5 'end of the messenger RNA coding for the ECA, another cDNA library was constructed in using 5 ~ g of poly (A) RNA isolated from cells endothelials of 7The umbilical vein in culture tertiary. CEas cE: cells were obtained by dissociation ~~ using collagenase as it is describes daps J ~~ FFE et al (JAFFE JM, et al (1973), J.
Clin. Invest., 52, 2745-2756), and were cultivated in presence of 20% fetal calf serum, 100 ~ g / ml heparin and 2ng / ml FGF (growth factor fibroblast ~ e) (GOSPORADOWICZ, D. et al (1983) J.
Cell. Biol. , 9 ~ 7, 16'77-1685). Total RNA was extracted from it cells ~~ near 3 subcultures by the method of Chirgwin et al (CHIRGWIN, JM et al (1979) Biochemistry, 18, 5:? 94-5299), and purifies on cellulose oligo-d (T) 7 (Pharmac: ia). The complementary DNA bank was built using the Gubler and Hoffman method (Gene (1983) 25, 263-269), using as a primer a mixture of two oligonucleotides. The first oligonucleotide is a specific primer for mRNA for ACE, determined by the sequence of clones previously obtained. It is an oligomer of 17 bases (CP5-21) complementary to a localized sequence near the 5 'end of the ACE cDNA (nucleotide 234 to 250 of Figure 3). The second primer corresponds with oligo-d (T) 12-18 seas (Pharmacia). CDNAs have been milked and inserted into the phage gGTlO cut by The EcoRI enzyme according to the method of Koenig et al (KOENIG, M. et al (1987), Cell, 50, 509-517).
Recombinant phages were screened using of a human genomic DNA fragment of 300 pairs of base isolated from a daps cloned genomic bank phage CHAR01 '~ 4A and which hybridizes with CP5-21, and with The 44-base oligonucleotide obtained from the amine-terminal sequence deduced from purified ACE
according to the method ~ 3rd described above.
This fragme: nt of restriction (SacII-SacII) of 300 base pairs includes the most 5 'terminal ACE messenger RNA; it was marked with a 13 ~ 0 ~~ 8 high activity ~ specific using the method of descriptive labeling daps FEINBERG AP et al (1983) Anal.
Biochem., 132, 6-13, and used to screen 1.5 x 106 clones under conditions of high stringency.
Among the positive clones obtained, the clone aCHDT32 has been selected and sequenced according to the same method described for previous clones. I1 has thus was determined that this clone contained an insertion of 246 base pairs cat it started 7 nucleotides in upstream of CP5-21 a ~ morce and that it stopped 25 nucleotides upstream of the ATG initiating translation.
The insertion in this clone shares 60 pairs of base with c: lone; ~ HEC2111 and contains 1 'end 5' of the sequence coding for the ACE.
The nucleotide sequence of the cDNA encoding The ACE obtained by sequencing the age of the three preceding clones therefore includes 4024 nucleotides. The 3 'end of this sequence does not contain a polyadenylation signal.
The open reading phase from the first ATG codon up to the stop codon TGA code for 1306 acids amines. La: Amino-terminal leucine determined by protein sequencing is localized after a signal peptide of 29 residues.
The clone aHEC19 ~ 22 was used as a probe for study the expression of the gene encoding ACE by the method called "the northern blot" according to the method of Thomas et al (THOMAS, PS et al (1983) Methods in Enzymology, NY Academic Press, 100, 255-266). A messenger RNA
about 4.3 kb of Ea vein endothelial cells umbilical in culture hybridizes with the above probe mentioned. In the testis, only a messenger RNA, shorter, 3 kb was detected. In the kidneys, which ~ 34 ~~ 6g are a rich source of ECA, no hybridization has was detected in the conditions used using 20 ~ g of RNA; poly (A), which suggests that the synthesis and the tau ~ ,: of turnover of the ACE are low in this organ.
Southern blot analysis of human DNA
under stringent conditions was carried out with a EcoRI-BglII fragment: from 300 base pairs locates a The 5 'end of the clone aHEC2111. Human DNA was isolates from ~ placental cell nuclei, by treatment with proteinase K followed by extraction with the phenol-c mixture: chloroform. DNA (14 ~ cg) has been digested with HindIII, separated on 0.7% agarose gel and analysis by hybridization according to the "Southern" method Blot "(SOUTHERN, IE.M. (1975) J. Mol. Biol, 98, 503-517).
The DNA fragments were labeled according to the method described in ~ FEINBERG, AP et al (1983) anal.
Biochem., 132 ,, 6-1a. This probe hybridizes with a single 9.5 kb restriction fragment.
Similar results have been obtained in using a cDNA fragment close to the 3 'end.
This result ~ at confirms the presence of a single gene.
Determination of the enzyme activity of The ECA coded by the gene thus isolated, or any active polypeptide according to the invention and derived from this last, may title determined in particular by the method described by Ho: Lmquist et al (1979) previously cited.
The dosage of this human ECA can be carried out by radioimmunological techniques (RIA) described by HIVADA K., et al (1987) Lung, 267, 27-35.
III POLYM: ORPHI ~ 51KES OF THE ACE GENE

~~ 4 ~~ ss Most DNA: Individuals Isolated From nucleated cells by lysis of red blood cells followed by lysis of white blood cells, treatment has protsinase K and: by successive extractions by the phenol and c: hloro: Eorme followed by precipitation a Isopropanol.
A quant, its determined DNA is then digsrse by a restriction enzyme and migrates on a gel agarose that separates. DNA fragments as a function of their size. The AD1V is then denature by a treatment with 0.5 N soda and transfers to a nylon membrane by blotting for 12 hours.
The DNA is then fixed to the membrane by passage through the oven for 2 hours at 80 ° C.
The member is then pre-hybridized with a solution 4 x SSC, 10 x Denhardt for at least 6 hours at 65 ° C ,. The membrane is then hybridized with a buffer solution 4 x SSC, 1 x Denhardt, 25 mM NaP04 pH 7, 2 mM ED'TA, 0.5% SDS, 100 ~ Cg / ml of sperm DNA
of sonic salmon including the marked probe denatures by: Heat at 100 ° C for 3 minutes.
The labeled probe is a mixture of cDNA fragments of the ACE contained in the clones aHEC1922 and aHEC1922.
The purified DNA fragments are labeled at high specific activities by priming technique statistic ut: using the Klenow fragment of the E. co7Li polymsrase and a dsoxynuclsotide labeled with P32 with high specific activities.
The membranes are hybridized in the presence of probe for: 12 hours: 65 ° C.
After hybridization, the membrane is washed in a solution 2 x SSC, 1 x Denhardt, 0.5% SDS at 65 ° C
for 45 minutes four times, then wash In a 0.2 x SSC solution, 0.1 x SSC for 45 minutes at 65 ° C twice, puffs in solution 0.1 x SSC, 0.1% SDS ~ for 45 minutes at 65 ° C.
The sequE ~ nces having hybridized with the probe are then visualized: Autograph readings of the membranes -80 ° C in the presence ~ of an intensifying screen on a movie.
DNA analysis of several individuals with the 1 "ECA cDNA probes or equivalent probes that is to say shortened to T one of the two ends, or extensions to the end 3 'or 5' of the part transcribed or not transcribed of the gene, or fragments of synthetic DNA: ics guff keep the property of hybridize with the restriction fragments above mentioned, pE: allows to distinguish several types of restriction fragments based on cuts DNA enzymes. These fragments are as follows:
- 5.8 kb or 6: kb by TaqI digestion, - 8.8 kb or 9 kb by DraI digestion, - 8.5 kb or 9 kb by DglII digestion, - 10.6 kb or 11 kb by KpnI digestion, - 8.4 kb or 8 .8 kb by HindIII digestion, - 4.2 kb and 3.0 kb or 4 kb and 2.7 kb by digestion BglI, - 5.2 kb or 5, '7 kb by RsaI digestion, with presence or not a 3.0 kb band.
- 3.5 kb or 3 l ~ cb by XbaI digestion, - 4.3 kb or 2, '7 by TaqI digestion.
By digestion with the enzyme PvuII, two restriction profiles or pattern including the presence or not of a ~ 4.2 kb guff band is associated with a longer band: a low of 2.2 and 2.5 kb.

Claims (23)

1. Nucleic acid characterized in that it includes all or part of the nucleic acid sequence of the Figure 3, and in that it codes for a polypeptide capable of hydrolyzing angiotensin I and / or kinins, or any nucleic acid which does not differ from the previous one at the level of its nucleotide sequence only by nucleotide substitutions that do not cause modification of the amino acid sequence of the above polypeptide under proper conditions to make it lose the above properties. 2. Nucleic acid according to claim 1, characterized in that it comprises a DNA sequence bounded by nucleotides located at positions 110 and 3940 of FIG. 3, coding for human ACE
mature. 3. Nucleic acid according to claim 2, characterized in that said DNA sequence encoding mature human ACE is preceded by a DNA sequence bounded by the nucleotides located at positions 23 and 109 of FIG. 3, the latter coding for a signal peptide of 29 amino acids. 4. Nucleic acid according to claim 1, characterized in that it comprises a DNA sequence extending between, on the one hand, the nucleotide located between positions 1 to 1177 and, on the other hand, the nucleotide located between positions 3070 to 3940 in Figure 3. 5. Nucleic acid according to claim 1, characterized in that it comprises a DNA sequence extending between, on the one hand, the nucleotide located between positions 110 to 1177 and on the other hand, the nucleotide located between positions 1276 to 1966 in Figure 3. 6. Nucleic acid according to claim 1, characterized in that it comprises a DNA sequence extending between, on the one hand, the nucleotide located between the positions 1967 to 2971 and, on the other hand, the nucleotide located between positions 3070 to 3940 of the figure 3. 7. Nucleic acid, characterized in that it includes a nucleotide sequence according to one any of claims 1 to 6 associated with a promoter under whose control the transcription of said sequence is likely to be performed and, the if necessary, a DNA sequence encoding signals transcription termination. 8. Recombinant vector chosen from the group comprising recombinant plasmid and recombinant virus, able to transform or infect a host cell appropriate, said vector containing a DNA fragment according to any one of claims 1 to 6 under the control of regulatory elements allowing expression of this DNA fragment in the host cell. 9. Process for producing human ACE, mature or not, or a polypeptide derived from human ACE
and capable of hydrolyzing angiotensin I and / or kinins, this process comprising the transformation of host cells using the vector of claim 8, culturing the transformed host cells in an appropriate culture medium and recovery of the enzyme from these cells or from the medium of culture. 10. Polypeptide encoded by nucleic acid according to claim 7, characterized in that it comprises all or part of the peptide sequence delimited by amino acids located at positions -29 to 1277 of the Figure 3, and in that it is capable of hydrolyzing Angiotensin I and / or kinins. 11. The polypeptide according to claim 10, characterized by the peptide sequence delimited by amino acids located in positions 1 and 619 of the figure 3. 12. The polypeptide according to claim 10, characterized by the peptide sequence delimited by amino acids located in positions 620 and 1229 of the figure 3. 13. The polypeptide according to claim 10, characterized by the peptide sequence delimited by amino acids located at positions 350 and 395 of the figure 3. 14. The polypeptide according to claim 10, characterized by the peptide sequence delimited by amino acids located at positions 948 and 993 of the figure 3. 15. Nucleotide probe characterized in that it consists of a nucleotide sequence complementary to all or part of the nucleic acid of claim 1, or any nucleotide probe does not distinguishing itself from the previous one in terms of its sequence nucleotide only by nucleotide substitutions not causing: the modification of the properties of hybridization of said probe with the nucleic acid of claim 1. 16. Method for hybridizing a probe nucleotide with a nucleic acid according to one any one of claims 1 to 6, characterized in that that it understands, under hybridization conditions stringentes, the following stages:
- pre-hybridization treatment of the support on which is attached nucleic acid according to any one of claims 1 to 6, at 65 ° C for 6 hours with a solution having the following composition: 4 X SSC, 10 x Denhardt;
- replacement of the pre-hybridization solution in contact with the support by a buffer solution having the following composition: 4 x SSC, 1 x Denhardt, 25mM NaPO4 pH 7, 2 mM EDTA, 0.5% SDS, 100 µg / ml of salmon sperm sonicated, comprising the labeled nucleotide probe and previously denatured by treatment at 100 ° C.
for 3 minutes;
- incubation for 12 hours at 65 ° C;

- successive washes with solutions following:
~ 2 x SSC, 1 x Denhardt, 0.5% SDS for 45 minutes at 65 ° C four times, ~ 0.2 x SSC. 0.1 x SSC for 45 minutes at 65 ° C twice, ~ 0.1 x SSC, 0.1% SDS for 45 minutes at 65 ° C, said nucleotide probe being characterized in that that it consists of a nucleotide sequence complementary to all or part of the nucleic acid of claim 1, or not distinguishable from the previous at the level of its nucleotide sequence that by nucleotide substitutions which do not cause modification of the hybridization properties of said probe with the nucleic acid of claim 1. 17. Method for hybridizing a probe nucleotide with a nucleic acid according to one any one of claims 1 to 6, characterized in that that it understands, in the hybridization conditions not stringentes, the following stages:
- pre-hybridization treatment of the support on which is attached nucleic acid, according to one any of claims 1 to 6 at 40 ° C for 6 hours with a solution having the following composition:
4 x SSC, 10 x Denhardt;
- replacement of the pre-hybridization solution in contact with the support by a buffer solution having the next composition: 4 x SSC, 1 x Denhardt, 25 mM
NaPO4 pH 7, 2 mM EDTA, 0.5 SDS, 100 µg / ml of semen sonicated salmon, including the nucleotide probe marked and previously denatured by treatment with 100 ° C for 3 minutes;
- incubation for 12 hours at 40 ° C;
- successive washes with 2 x SSC at 45 ° C
for 15 minutes twice, said nucleotide probe being characterized in that that it consists of a nucleotide sequence complementary to all or part of the nucleic acid of claim 1, or not distinguishable from the previous at the nucleotide sequence level as by nucleotide substitutions that do not cause modification of the hybridization properties of said probe with the nucleic acid of claim 1. 18. In vitro screening or assay method of a nucleic acid according to any one of claims 1 to 6 in a biological sample likely to contain it, characterized in that it includes:

- bringing a nucleotide probe into contact with the aforementioned biological sampling in conditions allowing the possible production of a hybridization complex formed between the probe and the acid above-mentioned nucleic acid, said nucleotide probe being characterized in that it is constituted by a nucleotide sequence complementary to all or part of the nucleic acid of claim 1, or not distinguishing from the previous one in terms of its sequence nucleotide only by nucleotide substitutions not causing the properties to change of hybridization of said probe with the nucleic acid of claim 1; and - the detection of the above hybridization complex. 19. In vitro screening or assay method according to claim 18, applied to in diagnosis in vitro of pathologies chosen from the group comprising hypertension, sarcoidosis and other diseases granulomatous, and thyroid dysfunctions. 20. Kit or kit for the implementation of a in vitro screening method comprising:
- a determined quantity of a probe nucleotide likely to give rise to a reaction of hybridization with a nucleic acid according to one any of claims 1 to 6;
- advantageously, an environment suitable for formation of a hybridization reaction between the acid nucleic acid and the above-mentioned probe; and advantageously, reagents allowing the detection of hybridization complexes formed between nucleic acid and probe during the above hybridization reaction, said nucleotide probe being characterized in that that it consists of a nucleotide sequence complementary to all or part of the nucleic acid of claim 1, or not distinguishable from the previous at the level of its nucleotide sequence that by nucleotide substitutions which do not cause modification. hybridization properties of said probe with the nucleic acid of claim 1. 21. Method of detection or determination of a ACE inhibitor, or quantification of its power inhibitor, which includes contacting a polypeptide according to any one of claims 10 to 14 with said inhibitor, and the determination of the coefficient of inhibition of the enzyme by the inhibitor, by measuring possible enzymatic activity residual on an appropriate ACE substrate. 22. Composition for the treatment of diseases inflammatory or infectious characterized by the association of a polypeptide according to any one from claims 10 to 14, with a vehicle pharmaceutically acceptable. 23. Method for in vitro screening of polymorphisms of the gene encoding ACE within a given population of individuals, derived from a biological sample taken from said population, characterized in that it comprises:
- the treatment of nucleic acid from the aforementioned biological sample made using of a restriction enzyme under conditions allowing restriction fragments to be obtained of the cleavage of said nucleic aside at these sites restrictions recognized by said enzyme;
- bringing a nucleotide probe into contact according to claim 15 capable of hybridizing with the above-mentioned fragments under conditions allowing the possible production of complexes of hybridization between said probe and the fragments of above-mentioned restriction, - the detection of the above complexes hybridization, and - measuring the size of any fragments restriction polymorphs involved in complexes above-mentioned hybridization.