Gene involved in cadasil, method of diagnosis and therapeutic application

The present invention relates to the demonstration of the involvement of the Notch3 protein in CADASIL thus allowing in particular a diagnosis of a predisposition to certain neurological disorders, in particular CADASIL, and models which make it possible to test the therapies possible for this type of pathology.

CADASIL or “Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy” has recently been identified as a cause of cerebral attacks and of dementia whose main characteristics include recidivous subcortical infarcts, migraines and a vascular dementia, in association with MRI images objectivizing diffuse abnormalities of the cerebral white substance.

An anatomicopathological examination shows multiple small deep cerebral infarcts, a leukoencephalopathy and a nonatherosclerotic and nonamyloid angiopathy involving essentially the small cerebral arteries.

As its name indicates, CADASIL is a hereditary disease with a dominant autosomal character. For more information, there may be found in particular a study of the clinical spectrum of CADASIL in H. Chabriat et al., The Lancet, Vol. 346, Oct. 7, 1995.

This highly incapacitating and very often lethal disease has probably remained so far largely undiagnosed as such; the study of about one hundred families since 1993 shows that erroneous diagnoses were most often given to the patient (multiple sclerosis, Alzheimer's disease and the like). Current studies would tend to demonstrate that it is a condition which is much more widespread than what was thought during its discovery.

The research studies currently pursued have the objective of identifying diagnostic tools for the disease and, by virtue, in particular of the models and the possibilities offered by genetic engineering, of developing a possible substitute therapy.

The gene involved in CADASIL has been localized on chromosome 19 and a finer localization is in particular mentioned in two patent applications with the same inventors.

It has now been possible to identify the gene involved in CADASIL which is the Notch3 gene.

The demonstration of the involvement of Notch3 in CADASIL has been possible given the previous limits which had been mentioned especially in the patent applications in question, the first interval (size 14 cM) was D19S221-D19S215 (first patent application), and then the second interval (size 2 cM) was D19S226-D19S199 (second patent application). The region of interest was cloned into a BAC and YAC contig (continuous nucleotide sequence) and its size was estimated at 800 kb. Analysis of this region with the aid of restriction enzymes showed a very high density of NotI, EagI and SacII sites which suggested the presence of numerous genes. Among the numerous transcripts identified by cDNA selection, one transcript showed a very strong homology with a sequence situated at the coding 5′ end of the mouse gene Notch3. Since other analytical factors seemed to corroborate this presence of the Notch3 gene in this situation, the latter was considered to be a good candidate gene by its position in the interval of interest.

The comparative studies carried out on known CADASIL families in comparison with healthy subjects have made it possible to identify mutations on this Notch3 gene in a large number of CADASIL subjects whereas such mutations were not observed on the healthy subjects analyzed. Since, finally, it has been possible to demonstrate the cosegregation of these mutations with the disease phenotype within effected families, the involvement of the Notch3 gene in CADASIL became incontestable.

All the point mutations observed lead to the creation or to the disappearance of a cysteine in one of the EGF domains of this protein. These mutations are clustered for a large part of them into the first six EGFs. The clustering of the mutations is certainly important in diagnostic terms especially for the “sequential” search for these mutations.

Moreover, all these mutations lead to the presence of an odd number of cysteines in one of the EGFs (either seven, or five cysteines) instead of the six cysteines normally present. These mutations could thus result in the formation of either intra- or intermolecular (and in this case in the formation of homo- or heterodimers) aberrant disulfide bridges.

The role of a normal or abnormal dimerization in the functioning of receptors, in particular their activation, is well known.

The Notch genes have been known for a very long time, especially in drosophila and their equivalent is known in vertebrates, in particular in mice. Its English name “notch” comes from the fact that some mutations of this gene produce a notch in the wings of flies. The article by Spyros Artavanis-Tskanas et al., Science 268, 225 (1995) as well as the references which it contains indicate that the Notch proteins are essentially involved, especially in drosophila, in the specification of the cellular destiny during development, and although the protein is always expressed in adult organisms, its functions in the latter remain unknown. More precisely, it appears that the product of the Notch3 gene, hereinafter “Notch3 protein”, is a cell receptor which controls a cascade of cellular events and whose mutation necessarily leads to greater or lesser disruptions in this cascade which may lead to many other neurological, especially cerebrovascular, disorders.

It should be recalled that, while in the text which follows there is interest more particularly in neurological disorders, in particular cerebrovascular-type disorders and most particularly CADASIL, it is probable, given the function of the cell receptor for the product of the Notch3 gene, that impairment of this receptor can lead to a disorganization of its interaction with various ligands but also with the various partners involved in the transduction cascade. Account should be taken, in addition, of the fact that the Notch3 protein might have other functions which have not yet been demonstrated. Under these conditions, it is highly probable that conditions exhibiting similarities with CADASIL may also be involved in the case of a mutation in the Notch3 gene.

Among the relevant diseases, there may be mentioned the sporadic forms of CADASIL, that is to say which occur without a family history but following a neomutation. Notch3 might moreover be involved in other conditions which may be classified into different groups:

Migraine and Hemiplegic Migraine

It was shown that at least one of the genes involved in familial hemiplegic migraine (FHM), the dominant autosomal form of migraine with aura, was located in the same region of chromosome 19 as the CADASIL gene. It should be noted that more than 30% of patients suffering from CADASIL, a condition characterized by the repeated onset of cerebrovascular accidents and of a vascular dementia, suffered from migraine with aura. However, the latter is observed in only about 5% of the population; it is this observation which led to testing the involvement of the CADASIL gene in the mechanisms of this condition. The involvement of this gene in a form of migraine with or without aura was of considerable diagnostic and therapeutic interest because of the frequency of migraine with aura and of migraine without aura in the general population.

Other Vascular (Cerebral Infarct) and/or Dementia Pathologies of Unknown Etiology

This group corresponds to a very large number of patients in neurology, psychiatric and internal medicine departments and it is everything to do reasonable to think that Notch3 or a partner in this signaling pathway may be involved in these conditions for the reasons stated above.

Familial Paroxytic Ataxia

The situation is the same as for FHM. A gene responsible for this condition has been located in the same region of chromosome 19 and Notch3 could be implicated in this condition.

Moreover, the mutations of this gene are responsible for developmental abnormalities which are well known in other species as well as for neoplastic-type pathologies. Malformative and/or neoplastic syndromes in which there may be demonstrated a rearrangement of the region which contains this gene might be major candidates for the search for an involvement of this gene in their physiopathology.

These disorders may be grouped under the name of “disorders linked to the Notch3 receptor”.

In some cases, this may involve disorders having a multigenic origin but in which the modifications of Notch3 might contribute to the onset of the pathology or to its worsening.

The present invention relates, first of all, to an isolated nucleotide sequence, characterized in that it is chosen from:

a) the sequences encoding the human Notch3 protein and its allelic variants,

b) the sequences encoding a fragment of these proteins and having at least 10 bases,

c) the human Notch3 genomic sequences and its alleles,

d) the sequences exhibiting at least 80%, and preferably at least 90%, homology with the sequences (a) and (c),

e) the fragments of the sequences (c) or (d) having at least 10 bases,

f) the sequences which hybridize with a sequence of (a) to (e).

It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, that is to say in the natural state; they are sequences which have been isolated, that is to say that they were collected directly or indirectly, for example by copying (cDNA), their environment having been at least partially modified.

Thus, this may also involve both cDNA and genomic DNA which is partially modified or carried by sequences which are at least partially different from the sequences carrying them naturally.

These sequences may also be described as being “nonnatural”.

“Nucleic sequence” is understood to mean a natural isolated, or synthetic, fragment of DNA and/or RNA designating a precise linkage of nucleotides, modified or otherwise, making it possible to define a fragment, a segment or a region of a nucleic acid.

“Allelic variant” of the protein is understood to mean all the mutated proteins and the polymorphisms which may exist in a human being, which are obtained in particular by truncation, substitution, deletion or addition of amino acid residues, as well as the artificial variants.

According to the invention, the nucleic sequence fragments may in particular encode domains of the protein or may be used as probe or as primer in methods of detection, identification or amplification. These fragments have a minimum size of 10 bases and fragments of 20 bases, preferably 30 bases, will be preferred.

According to the invention, the homology is solely of the statistical type; it means that the sequences exhibit at least 80%, and preferably 90%, of nucleotides in common.

The hybridization conditions should make it possible, according to the invention, to ensure at least 95% homology.

More particularly, the present invention relates to a nucleotide sequence chosen from:

a) the sequences encoding a polypeptide comprising the amino acids according to the sequence in

FIG. 1

,

b) the nucleic sequences corresponding to

FIG. 1

,

c) a fragment of a sequence according to (a) or (b) containing at least 10 bases, and

d) a sequence which contains, relative to the sequences (a), (b) or (c), at most 20 partial mutations.

FIG. 1

represents the sequences of Notch3 as were sequenced on a normal genome.

The sequences are identified by references which make it possible to position them relative to each other using FIG.

3

.

As regards the special remarks on (a), (b), (c) and (d), the previous remarks apply.

The invention also relates to the fragments of these sequences, in particular sequences encoding polypeptides which have retained all or part of the activity of the Notch3 protein.

Among the particularly advantageous sequences, there may be mentioned those encoding domains or combinations of domains of the Notch3 protein, that is to say the sequences:

“EGF” repeats

“Notch/lin12” repeats

“cdc10/SW16” repeats

or the transmembrane sequence.

Among the advantageous sequences are in particular the sequence encoded by the second transcript which will be described in the text which follows, the said transcript having an estimated size of between 1.3 and 2.4 kb.

These sequences may be identified with reference in particular to

FIG. 2

which schematically represents the organization of Notch3.

These partial sequences can be used for numerous applications, as described below, especially for preparing Notch-type or different types of protein constructs, but also for preparing, for example, truncated Notch-like proteins which will serve as lure for the Notch3 ligand or as agonist for the protein.

It is also possible to envisage using these protein sequences for their intrinsic effects; thus, the EGF domains are present in other proteins, especially other receptors; reference may be made for example to Iain D. Campbell, Current Biology, 3: 385-392 (1993) for other applications of the EGF sequences in question.

While the sequences described are in general normal sequences, the invention also relates to the mutated sequences insofar as they contain at least one point mutation and preferably less than 20 mutations in total.

Preferably, the present invention relates to the nucleotide sequences in which the point mutations are not silent, that is to say that they lead to a modification of the amino acid encoded relative to the normal sequence. Still more preferably, these mutations affect amino acids which structure the Notch3 protein or the corresponding fragments thereof, that is to say in particular the mutations which suppress the cysteines or, on the contrary, which make them appear, but also the mutations which change the character of the protein, either from the charge point of view, or from the hydrophobicity point of view.

The present invention also relates to the mutations which may occur in the promoter and/or regulatory sequences of the human Notch3 gene, which may have effects on the expression of the protein.

Examples of such mutations will be described in the text which follows.

In general, the present invention relates to both the normal Notch3 protein and the mutated Notch3 proteins, as well as to their fragments and to the corresponding DNA and RNA sequences, that is to say the alleles.

It should be noted that the Northern blot study of the expression of this gene in human tissues reveals two transcripts. One having a size estimated at 7.5-9.5 kb is present in all the tissues tested; the other, whose size is between 1.3 and 2.4 kb, is detected only in some parts of the central nervous system. The present invention relates to these two transcripts.

Among the nucleotide fragments, there may be mentioned the intron genomic sequences of the Notch3 gene and more particularly the joining sequences between the introns and the exons, especially as are represented in Table A; and finally, the present invention relates to all the primers which may be deduced from the preceding nucleotide sequences and which may make it possible to detect them using an amplification method such as the PCR method, especially those presented in Table B.

The present invention also relates to the nucleotide sequences which may contain nonnatural nucleotides, especially sulfur-containing nucleotides for example or having an α or β structure.

Finally, the present invention of course relates to both the DNA and RNA sequences, as well as the corresponding double-stranded DNAs.

As will be described below for some applications, it may be necessary to provide for mixed constructs, protein/DNA/chemical compound, especially the use of intercalating agents for example; it should be understood that such compounds are covered by the patent as containing a sequence according to the invention.

The present invention also relates to the polypeptide or peptide proteins corresponding to the abovementioned sequences, in a nonnatural form, that is to say that they are not taken in their natural environment but obtained by purification from natural sources or obtained by genetic recombination, as will be described below.

The invention also relates to the same polypeptides or proteins obtained by chemical synthesis and capable of containing nonnatural amino acids.

The present invention relates to the recombinant proteins thus obtained both in a glycosylated and nonglycosylated form and capable of having or otherwise the natural tertiary structure.

In particular, the present invention relates to the Notch3 fragments which exhibit an activity similar to the total receptor, especially the soluble part(s) of said receptor corresponding in particular to their extracellular domain. These may be used especially as a lure in a therapy, as will be described below.

The present invention also relates to the cloning and expression vectors containing a nucleotide sequence as described above.

These cloning and expression vectors may contain elements ensuring the expression of the sequence in a host cell, especially promoter sequences and regulatory sequences which are efficient in said cell (see reference below).

The vector in question may be autonomously replicating or intended to ensure the integration of the sequence into the chromosomes of the host cell.

In the case of autonomously replicating systems, depending on the prokaryotic or eukaryotic host cell, plasmid-type systems or viral systems will preferably be used, it being possible for the viral vectors to be especially adenoviruses, poxviruses or herpesviruses. Persons skilled in the art know the technologies which can be used for each of these viruses (see reference below).

When the integration of the sequence into the chromosomes of the host cell is desired, it will be necessary to provide for, on either side of the nucleotide sequence to be integrated, one or more sequences obtained from the host cell in order to bring about the recombination. These are also methods which are widely described in the prior art. It will be possible, for example, to use plasmid or viral type systems; such viruses will be, for example, retroviruses or AAVs (Adeno-Associated Viruses).

The invention also relates to the prokaryotic or eukaryotic cells transformed by a vector as described above, and this being in order to bring about the expression of a natural or mutated Notch3 protein or, for example, of one of its subunits.

As indicated above, the present invention also relates to the proteins, peptides or polypeptides obtained by culturing the cells thus transformed and recovering the protein expressed, it being possible for said recovery to be carried out intracellularly or extracellularly from the culture medium when the vector has been designed to bring about the excretion of the protein via for example a “leader” sequence, the protein being in a pre-protein or prepro-protein form. The constructs allowing the secretion of the proteins are known both for prokaryotic systems and eukaryotic systems.

Among the cells which can be used for the production of these proteins, there may of course be mentioned bacterial cells, but also yeast cells, as well as animal cells, in particular mammalian cell cultures, but also insect cells in which methods using baculoviruses for example may be used (see reference below).

The cells thus obtained can make it possible to prepare natural or mutated Notch3 proteins, but also fragments of these proteins, especially polypeptides which may correspond to the different domains in question.

However, the cells transformed as described above may also be used as a model to study the interactions between the Notch gene and its various ligands as well as its influence on the products downstream of the receptor, but in particular they may be used in an application for the selection of products interacting with the natural or mutated Notch3 receptor, as an agonist or an antagonist of this receptor.

This type of cellular model may be produced using genetic engineering techniques. It involves, depending on the type of cells which it is desired to use, cloning the gene in question in its normal form or in its mutated form into an expression vector, whether it is an autonomously replicating vector or an integration vector, said vector containing all the elements allowing the expression of the gene in the cell in question, or the latter having all the elements allowing the expression of the sequence in question.

There are thus obtained eukaryotic or prokaryotic cells expressing the Notch3 protein(s) which, given its characteristics, will be situated like a transmembrane protein whose fine structure will be described in the text which follows, it being possible for said cells to then constitute models which make it possible to test at the same time the interactions of various ligands with the product of the Notch3 protein or to test synthetic chemical products capable of interacting with the product of the Notch3 gene, and this by adding them to the culture medium for said cells.

It should in particular be noted that the products in question may also be products with either antagonist or agonist activity.

The use of cellular models to test pharmaceutical products is well known; here again, there is no need to present this type of model in detail.

Another potential application of the characterization of this gene is the possibility of identifying potential ligands for this protein, either because they have a conserved sequence with human Notch3, or because they interact with Notch3 (affinity methods) or partners for this signaling pathway.

These models may be of the in vitro type, for example cultures of human cells, either in a normal culture, or possibly in the form of an isolated organ, such as for example certain types of vessels which may be transformed in order to cause them to express the desired phenotypes.

The present invention also relates to the organisms, such as animals, in particular mice, expressing the phenotype corresponding to the normal or mutated Notch3 of human origin. Here again, these animals may be used as model animals to test the efficacy of certain pharmaceutical products.

The present invention also relates to the products obtained using the preceding cellular models.

There will thus be obtained, depending on the type of interaction determined, therapeutic compositions characterized in that they contain, as active ingredient, a compound with a pro-Notch3 activity; this may be in particular all or part of a polypeptide as were described above or a vector expressing these same polypeptides, or else chemical or biological compounds having a pro-Notch3 activity, a Notch3-like activity or inducing the production of natural Notch3.

It will also be possible to demonstrate therapeutic compositions in which the active ingredient will have an anti-Notch3 action.

This may involve, here again, modified proteins described above which may play the role of a lure, or anti-Notch3 antibodies, in particular when these antibodies recognize the mutated receptors and will, under these conditions, be able to block the activity of the normal receptor.

This may also involve chemical products having an anti-Notch3 activity, or Notch3 antagonists.

In some cases, the use of some of the Notch3 domains may allow a therapeutic approach blocking the activity of the Notch3 receptor when the latter is mutated using soluble receptors which will serve as lure to the natural ligands; in other cases, it will be possible, by expressing the entire receptor, to provide a replacement therapy using either directly the proteins or fragments thereof, or directly expressing the protein, especially via gene therapy and using the vectors which were described above.

In the context of gene therapy, it is also possible to provide for the use of the sequences of the genes or cDNAs described above as “naked”; this technique was in particular developed by the company Vical; it has shown that it was possible, under these conditions, to express the protein in certain tissues without requiring the use of the support for a viral vector in particular.

Still in the context of gene therapy, it is also possible to provide for the use of cells transformed ex vivo, which cells may then be reimplanted either as such or in systems of the organoid type, as is also known in the state of the art. It is also possible to envisage the use of an agent facilitating targeting of the determined cell type, penetration into the cells or transport to the nucleus.

Among the numerous pharmaceutical compounds which can be used, there should be mentioned more particularly, in addition to the ligands for the Notch3 product, the sense or anti-sense sequences interacting with the normal or mutated Notch3 gene, or interacting on the regulation or expression of these genes, it being also possible for said products to interact downstream of the expression products induced by the Notch3 receptors. The soluble sequences corresponding to Notch3 should furthermore be cited.

There should also be mentioned the monoclonal antibodies blocking the Notch3 receptors, in particular the mutated Notch3 receptors, and/or blocking the corresponding ligands and/or the products induced by said receptors which may therefore have pro or anti activities.

It should be recalled that the monoclonal antibodies directed against the Notch3 receptor may, depending on the epitope recognized, have a pro or anti-Notch3 activity which makes them useable in therapeutic compositions.

Finally, the present invention relates, as was said above, more particularly to the methods of diagnosing a predisposition to neurological conditions, especially of the CADASIL type, or of diseases linked to the Notch3 receptor in a patient, characterized in that the presence of a mutation in Notch3 is determined using a biological sample from said patient by analysis of all or part of a nucleic sequence corresponding to said gene, the presence of at least one such mutation being indicative of a predisposition of said patient to neurological conditions or diseases linked to the Notch3 receptor.

Other diagnostic methods can make it possible to characterize, by means of antibodies, the deposit expected in the basal membrane of the vascular smooth muscle cells, a deposit which might consist of the Notch3 protein itself or one of its cleavage products.

Among the desired mutations, there may be mentioned more particularly the mutations referenced in Table C and FIG.

3

.

The nucleic acid sequences may be either genomic DNA, a cDNA or an mRNA.

As was said above, among the neurological disorders which may be demonstrated, there is understood more particularly disorders of the cerebrovascular type and especially CADASIL, but the list of certain disorders which might be linked to an abnormality in the Notch3 receptor has been previously given; among these conditions, there may be mentioned most particularly the potential involvement of Notch3 in migraines with or without aura and dementias of currently unknown etiology.

The diagnostic tools based on the present invention may allow a positive and differential diagnosis in a patient taken in isolation or alternatively a presymptomatic diagnosis in an at-risk subject (family history for example), it is also possible to envisage an antenatal diagnosis.

In addition, the detection of a specific mutation may allow an evolutive diagnosis.

The methods which make it possible to demonstrate the mutation in a gene relative to the natural gene are of course very numerous; they may be carried out by studying the genomic DNA, the cDNA and/or the protein. They can be essentially divided into two large categories, the first type of method is that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding nonmutated natural sequence, and the second type in which the presence of the mutation is detected indirectly, for example, by detecting the mismatches due to the presence of the mutation.

In both cases, the methods in which all or part of the sequence corresponding to Notch3 is amplified prior to the detection of the mutation will be preferred in general; these amplification methods may be carried out by the so-called PCR (see reference below) or PCR-like methods. PCR-like will be understood to designate all the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified; these techniques are well known, in general they relate to the amplification of DNA by polymerase; when the original samples is an RNA, it is advisable to carry out a reverse transcription beforehand; a great number of methods allowing this amplification currently exists, for example the so-called NASBA and TMA methods which are well known to persons skilled in the art.

Table B gives the sequences of the PCR primers which make it possible to amplify the exons as well as the temperatures for the PCR reactions.

A general methodology for amplification of the sequences will be described in the examples.

Test for Point Mutations

In addition to the direct sequencing of the mutation, various methods may be used. The techniques will be briefly cited:

1) test for “Single Strand Conformation Polymorphisms” (SSCP) (see reference below) or denaturing gradient gel electrophoresis (DGGE).

2) the methods based on a cleavage of the mismatched regions (enzymatic cleavage by S1 nuclease, chemical cleavage by various compounds such as piperidine or osmium tetroxide, and the like.

3) heteroduplex detection by electrophoresis,

4) methods based on the use in hybrication of allele-specific oligonucleotide (ASO) probes.

Other well known methods based on hybrication techniques can be used.

Test for Deletion, Inversion or Duplication Type Rearrangements

Other well known methods based on the techniques of hybridization with the aid of genomic probes, of cDNA probes, of oligonucleotide probes, of riboprobes, of so-called capture probes or of so-called detection probes, may be used for the test for this type of rearrangement.

Another diagnostic approach which can be used when DNA from several subjects of the same family is available is based on the method of linkage analysis which makes it possible to calculate the risk which a subject belonging to a linked family has of being a carrier or otherwise of the diseased gene. This analysis may be carried out with polymorphic markers situated in the immediate vicinity of the gene, or intragenic polymorphic markers.

It is important to recall that in the CADASIL families, the existence of mutations in the Notch3 gene corresponds to mutations which change amino acids which are essential for the function of the protein for which it encodes.

Moreover, in the examples, the situations of the mutations currently detected are indicated, but it is possible that other mutations exist in the Notch3 gene which have not yet been detected but which should lead to the same types of risks from the pathological point of view.

In any case, the mutated Notch3 proteins may exhibit an antigenicity which is different from that of the natural protein.

It is therefore possible to carry out a diagnosis or a prognosis of a susceptibility to neurological, in particular cerebrovascular, disorders of the CADASIL type and disorders linked to the Notch3 receptor, by detecting the product of the mutated gene for Notch3; this type of detection can be carried out, for example, with the aid of monoclonal or polyclonal antibodies. Under these conditions, it is possible to detect and assay the abnormal product of the Notch3 gene by well known methods, RIA or ELISA for example; these technologies being known, they will not be further developed beforehand in the text which follows. Antibodies directed against the normal protein could also be used if the deposit present in the arteries of the skin corresponded to the Notch3 protein or to one of its cleavage products.

The present invention also relates to the labeled monoclonal or polyclonal antibodies corresponding to all or part of the mutated proteins so as to serve as imaging agent in vivo or ex vivo on biological samples.

Thus, it appears that the granular masses present in the basals of the vascular smooth muscle cells are due to an accumulation of the abnormal protein and the test for this protein with the aid of antibodies, either in biopsies or in vivo, is of a diagnostic interest.

Methods Based on the Detection of the Product of the Gene

The mutations of the Notch3 gene may be responsible for various modifications of the product of this gene, modifications which can be used for a diagnostic approach. Briefly, the protein may be truncated, reduced in size or absent; its properties, in particular its antigenicity, may be modified. All these modifications may be used in a diagnostic approach using several well known methods based on the use of mono- or polyclonal antibodies which recognize the normal protein or mutated variants, and this using the study of protein extracts or of tissue sections (for example skin biopsies), or studies carried out in vivo (imaging with the aid of antibodies coupled to a molecule which is detectable in PET-scan type imaging, and the like).

The polyclonal or monoclonal antibodies may be obtained by immunological reaction of a human or animal organism with an immunogenic agent consisting of a protein or a polypeptide capable of being obtained from prokaryotic or eukaryotic cells transformed by a vector as described above. Preferably, the immunogenic agent consists of a specific polypeptide of the mutated form of the Notch protein whose sequence is chosen from the polypeptide sequences comprising at least one mutation chosen from the mutations corresponding to

FIG. 3

or to Table C.

The present invention finally relates to therapeutic compositions containing, as active ingredient, a compound with a pro-Notch3 activity, especially as described above, as well as therapeutic compositions containing, as active ingredient, a compound with an anti-Notch3 activity.

Other characteristics and advantages of the present invention will appear on reading the examples below, with reference to the accompanying drawings in which:

FIG. 1

reproduces the cDNA sequence (SEQ ID NO:1) of human Notch3 as well as the corresponding protein sequence (SEQ ID NO:2). Panels 1A-1H present the sequences in consecutive order, with the sequences on each panel following directly from the preceding panel;

FIG. 2

represents the general structure of the product of the Notch3 gene as well as the mutations which were detected by aligning the human cDNA clones with mouse Notch3,

A—mouse Notch3 gene with its 34 EGF domains, 3 Notch/Lin12 repeats and 6 cdc10 repeats, as well as the transmembrane domain,

B—at the bottom, 8 of the human cDNAs with the identifications corresponding to

FIG. 1

, at the top, the alignment of some genomic sequences with the cDNA of at least 29 exons, the origins of the various fragments appear at the bottom of

FIG. 4

; the various clones are available commercially or through libraries;

the clones 261623; 153875; 149693 are available from the IMAGE consortium;

the clone C-32b03 is available from GENEXPRESS (Généthon, Evry, France);

the clones p28-20; CNA-20 are available from CLONTECH;

FIG. 3

schematically represents the situation and the nature of the mutations involved in CADASIL;

FIG. 4

represents a “Northern blot” analysis, the Northern blots containing 2 μg per line of human poly (A

+

) DNA, from left to right:

of various brain tissues,

of various adult organs,

of various fetal organs;

they are hybridized with p28-20, a Notch3 human 1.45 kb cDNA probe; a 7.5 to 9.5 kb transcript is detected in all the tissues, both adult and fetal, with the exception of the liver, the transcript is weakly expressed in the brain tissue in the middle and on the right; on the contrary, on the left, not only are transcripts of all the tissues observed, but also the presence of a transcript of between 2.4 and 1.3 kb whose presence has never been mentioned and which may be of a very high importance is observed.

EXAMPLE 1

Scheme for the Isolation and Analysis of the Notch3 Gene

Following the remarks and the analysis which were summarized at the beginning of the description as regards the location of the gene, murine cDNA probes were used to isolate the cDNA for the human Notch3 gene, and then genomic clones whose sequences could be aligned with it and with the murine cDNA sequences.

Additional information on the sequences were obtained from a cDNA fragment (884Na4) obtained by cDNA selection on YAC (884g1) and from two genomic fragments (J431NH and J432NH) which were obtained by subcloning of BAC 13J4 NotI-HindIII fragments.

In the screening of the (dBest) data bank with all the sequences, it has been possible to identify additional clones (IMAGE clones, Genexpress).

The coding sequence of the human Notch3 gene, which is highly homologous to the corresponding murine gene, is represented in FIG.

1

.

Table A schematically represents the structure of the gene, specifying the sequence and the position of the exon-intron junctions.

In this table, the first exon corresponds to a sequence whose 5′ end was not completely cloned, likewise for exon 33.

It should be noted that alternative cDNAs may exist which correspond to the known phenomenon of alternative splicing.

This sequence contains 34 EGF domains, 3 Notch/lin12 repeats, as well as 3 cdc10 ankyrin-like repeats. The human and murine proteins exhibit 90.5% identity on the sequence currently available. A 1.45 kb partial human cDNA probe containing the EGF-like domains reveals a ubiquitous transcript in the fetal tissues, as well as in the human adult tissues whose size of between 7.5 and 9.5 kb is similar to the murine transcript (FIG.

4

).

This probe reveals another transcript in certain subregions of the brain whose size is estimated at between 1.3 and 2.4 kb (FIG.

4

).

EXAMPLE 2

Study of the Mutations

In order to study the extent of the mutations in the Notch3 gene on CADASIL, the possible presence of a substantial genomic DNA rearrangement was first studied using various combinations of enzymes and of Notch3 probes.

No drastic rearrangement could be detected in the CADASIL patients, that is why point mutations were then tested for.

Thus, the mutations of the total coding sequence of the Notch3 gene in the genomic DNA were studied using a combination of SSCP method and heteroduplex analysis in 51 CADASIL patients with no family relationship. 28 of them belong to families for which the evidence for a relationship with chromosome 19 has been demonstrated and 33 exhibit ultrastructural lesions of the wall of the arterioles of the skin (presence of osmiophilic granular deposits in the basal membrane of the vascular smooth muscle cells).

All the splicing junctions, except 3, were analyzed. In addition, direct sequencing of the PCR products of exon 4 and its splicing sites was carried out on all the patients.

Impairments which were compatible with corresponding mutations were found in 42 patients (82%), said mutations not being observed in any of the 200 control chromosomes. For 26 patients, it was possible to analyze one or more which were related, affected or otherwise, and in each case it was established that the mutation segregated with the CADASIL phenotype. There are 29 different mutations, of which 20 are described for the first time. They include 24 missense mutations which appear in 40 patients, which mutations should replace (16) one amino acid with an additional or mutated cysteine (8) one of the 6 cysteine residues, which are the key elements of the EGF domains.

Two of the mutations at the 5′ splicing site in the latter two patients should normally affect the splicing of exon 4. The last three mutations are missense mutations which appear in 3 patients simultaneously with the mutations described above.

Patient 21 carries 2 distinct mutations which change an arginine to cysteine at codon 141 in EGF 3 and which changes a conserved glycine to alanine at codon 288 in EGF 7. This patient's pedigree was not available; it was not therefore possible to study the cosegregation of these two mutations.

Patient 29 carries a first mutation in EGF 4 which changes an arginine 182 to cysteine and a second which changes a highly conserved alanine 1852 to threonine in the cdc10 domain. These two mutations segregated with the disease.

The last patient 55 is a carrier of two distinct mutations in the EGF domains, which change a cysteine (224) to a tryptophan and a nonconserved leucine (497) to a serine residue.

Although the latter three missense mutations are not detected in the 200 control chromosomes, they may involve rare polymorphisms given the presence also of missense mutations which mutate or create cysteine residues.

It should be noted that most of these 26 mutations having a pathogenic effect lie exclusively in the EGF parts. 41% of these mutations (11 out of 26) appearing in 25 patients are situated in exon 4 and 65% (17 out of 26) lie in the first 6 EGF domains (see in particular FIG.

3

.

FIG. 3

allows the detection to characterize the main mutations detected, the nomenclature chosen indicates the position of the mutation as well as the corresponding modifications of the protein.

As was indicated above, the fact that a Notch gene is involved in neurological disorders in adults appears completely surprising since Notch is mainly known to be involved during development in drosophila. None of the CADASIL families studied up until now exhibits developmental abnormalities.

EXAMPLE 3

Detection of the Mutations in Patients by the SSCP Method

The oligonucleotides used as primers were synthesized from intron-exon joining sequences (Table B) so as to amplify genomic fragments of about 200 bp. The sequences of the PCR primers are given below (Table A).

The analyses can be carried out using DNA extracted from blood samples or any other tissue.

The amplification reactions are carried out in a final volume of 25 μl containing 100 ng of genomic DNA, 0.5 μm of each primer, 150 μg of a mixture of 4dNTPs, Taq polymerase 1XPCR from Cetus, 1 U Taq polymerase (BRL), 1.5 μCi αdCTP labeled with P33 according to a protocol comprising 30 identical cycles (94° C., 15 s; 65° C., 15 s; 72° C., 15 s).

For some of the primers, an “annealing” temperature of 70° C. should be used, as indicated in Table A.

The PCR products are denatured in 50% formamide and separated by electrophoresis in a 6% nondenaturing polyarylamide gel.

After autoradiography, the SSCP bands obtained in the patients are compared with those of healthy controls in search of abnormal variants. Their analysis can be used as a diagnostic approach. These variants can then be sequenced if necessary.

TABLE A

Exon-intron structure of the Notch3 gene (sequences and positions of the exon-

intron junctions)

Splice Acceptor Site

Exon

Splice Donor Site

Intron/Exon

(Size)

Position

Exon/Intron

1

1-196

CTGCAG/gtgaggggc

(SEQ ID NO:3)

laAlaAl

cccacacag/CCCCCC

(SEQ ID NO:4)

2 (79)

197-275

CTGCCT/gtgagtgcc

(SEQ ID NO:5)

aProPr

aCysLe

gcccacag/GTGCCC

(SEQ ID NO:6)

3 (143)

276-418

TCCGAG/gtgagagg

(SEQ ID NO:7)

uCysPr

heArgG

ccctccag/GCCCTG

(SEQ ID NO:8)

4 (339)

419-757

TTCCTG/gtgagtga

(SEQ ID NO:9)

lyProA

euProG

cttgttag/GGTTTG

(SEQ ID NO:10)

5 (123)

758-880

GGACAG/gtgggcac

(SEQ ID NO:11)

lyPheG

rpThrG

tgccacag/GCCAGT

(SEQ ID NO:12)

6 (234)

881-1114

AGACTG/gtgagtgg

(SEQ ID NO:13)

lyGlnP

ysThrG

cttcccag/GCCTCC

(SEQ ID NO:14)

7 (156)

1115-1270

CTATCG/gtgagggg

(SEQ ID NO:15)

lyLeuL

erIleG

tccggcag/GCGCCA

(SEQ ID NO:16)

8 (187)

1271-1456

TGGCAG/gtgggtgg

(SEQ ID NO:17)

lyAlaA

etAlaG

tgccccag/GCTTCA

(SEQ ID NO:18)

9 (114)

1457-1570

CCTCGG/gtgaggac

(SEQ ID NO:19)

lyPheT

roSerG

caccccag/GCTTCA

(SEQ ID NO:20)

10 (114)

1571-1684

CCGAGG/gtgaggcg

(SEQ ID NO:21)

lyPheS

laGluG

ccccacag/GCTTTG

(SEQ ID NO:22)

11 (234)

1685-1918

CCACAG/gtgggacc

(SEQ ID NO:23)

lyPheG

hrThrG

gcccctag/GTGTGA

(SEQ ID NO:24)

12 (111)

1919-2029

TCACAG/gtgggcaa

(SEQ ID NO:25)

lyValA

heThrG

ctccccag/GGCCCC

(SEQ ID NO:26)

13 (193)

2030-2222

TGGCGG/gtgagggc

(SEQ ID NO:27)

lyProL

oGlyGl

cctgccag/GTTCCG

(SEQ ID NO:28)

14 (152)

2223-2374

TCCAGG/gtgtgtac

(SEQ ID NO:29)

yPheAr

alGlnG

cccaacag/GACGTC

(SEQ ID NO:30)

15 (114)

2375-2488

GGCAAG/gtatgccac

(SEQ ID NO:31)

lyArgG

rpGlnG

tacccccag/GCCCAC

(SEQ ID NO:32)

16 (156)

2489-2644

ACCCCA/gtgagtgca

(SEQ ID NO:33)

lyProA

spProA

gtccgcag/ACCCAT

(SEQ ID NO:34)

17 (226)

2645-2870

CCCCAG/gtgggcgg

(SEQ ID NO:35)

snProC

rProSe

cgctccag/CTCCTG

(SEQ ID NO:36)

18 (202)

2871-3072

TGCCAG/gtgggtgg

(SEQ ID NO:37)

rSerCy

CysGln

ccctccag/ACGCTG

(SEQ ID NO:38)

19 (148)

3073-3220

AGATCG/gtgagtgg

(SEQ ID NO:39)

ThrLeu

lnIleG

ctttgcag/GGGTGC

(SEQ ID NO:40)

20 (185)

3221-3405

TGTGAG/gtaagggg

(SEQ ID NO:41)

lyValA

CysGlu

cactgaag/TGTCTT

(SEQ ID NO:42)

21 (133)

3406-3538

CGCTGG/gtatgcca

(SEQ ID NO:43)

CysLeu

hrLeuG

tcccccag/GGGTGC

(SEQ ID NO:44)

22 (258)

3539-3796

TCTCAG/gttaacct

(SEQ ID NO:45)

lyValL

heSerG

tcgctcag/GTCCTC

(SEQ ID NO:46)

23 (119)

3797-3915

GCCCAG/gtaggtgtg

(SEQ ID NO:47)

lyProA

AlaGln

gacccccag/CCGTTC

(SEQ ID NO:48)

24 (566)

3916-4481

TTGCAA/gtgagccc

(SEQ ID NO:49)

ProPhe

rCysAs

cccaccag/CCCGGT

(SEQ ID NO:50)

25 (333)

4482-4814

GATCGG/gtgagtgac

(SEQ ID NO:51)

nProVa

lIleG

tccctgcag/CTCGGT

(SEQ ID NO:52)

26 (155)

4815-4969

TGCGGG/gtgcggcc

(SEQ ID NO:53)

ySerVal

alArgG

tgctcttag/GGGGAGC

(SEQ ID NO:54)

27 (223)

4970-5192

CATGAA/gtgagaac

(SEQ ID NO:55)

lyGluP

yMetLy

tccgccag/GAACAT

(SEQ ID NO:56)

28 (85)

5193-5277

CTAAAG/gtactgcc

(SEQ ID NO:57)

sAsnMe

LeuLys

cccctccag/GTAGAG

(SEQ ID NO:58)

29 (162)

5278-5440

GCCCAG/gtcagtgac

(SEQ ID NO:59)

ValGlu

lyProA

ccctgcag/ATGGCT

(SEQ ID NO:60)

30 (305)

5441-5745

TTCCAG/gtgagata

(SEQ ID NO:61)

spGlyP

PheGln

tgtcctag/ATTCTC

(SEQ ID NO:62)

31 (148)

5746-5893

AGCTTG/gtaggttg

(SEQ ID NO:63)

IleLeu

luLeuG

ccctccag/GGAAAT

(SEQ ID NO:64)

32 (99)

5894-5992

AGCAAG/gtgagccc

(SEQ ID NO:65)

lyLysS

SerLys

ccccccag/GAGGAG

(SEQ ID NO:66)

33

5993-

GluGlu

TABLE B

Sequences of the primers used for the screening of the mutations of

the Notch3 gene

PCR

product

Exon

Size

Domain

Primers

size

1

Signal

EOF

AAGGAGGGAGGAGGGGAG

125

(SEQ ID NO:66)

peptide

E0R

TGGGGGTTCTTGCACTCC*

(SEQ ID NO:67)

EOF

AAGGAGGGAGGAGGGGAG

163

(SEQ ID NO:68)

E0RBIS

GGTTCCTGCCTCCCATGA*

(SEQ ID NO:69)

2

79

EGF1

E1F

TCCTCCACCTTCCTTCAC*

148

(SEQ ID NO:70)

E1R

ACACACAGGGCCCACTGGT*

(SEQ ID NO:71)

3

143

EGF 1-2

N1 F

TGTGCTGCCCAACCAAGCCA*

224

(SEQ ID NO:72)

N1 R

ACTGACCACACCCCCGACTA*

(SEQ ID NO:73)

4

339

EGF 2-5

N2A F

TAGTCGGGGGTGTGGTCAGT*

192

(SEQ ID NO:74)

N2A R

TCATCCACGTCGCTTCGGCA

(SEQ ID NO:75)

CNA F

ATGGACGCTTCCTCTGCTC

167

(SEQ ID NO:76)

CNA R

ACATAGTGGCCCTGTGTAGC

(SEQ ID NO:77)

CNA F

ATGGACGCTTCCTCTGCTCC

295

(SEQ ID NO:78)

N3AR

CCTCTGACTCTCCTGAGTAG*

(SEQ ID NO:79)

5

123

EGF 5-6

N23Fbis

TGACCATCCTTGCCCCCTT*

241

(SEQ ID NO:80)

N23 R

CTGGCCTGTGGCACACAGAT *

(SEQ ID NO:81)

6

234

EGF6-8

N13A F

TGGACTGCTGCATCTGTGTG*

191

(SEQ ID NO:82)

N13A R

ACACGCCTGTGGCACAGTCA

(SEQ ID NO:83)

N13B F

GAGCTGCAGTCAGAATATCG

145

(SEQ ID NO:84)

N13B R

ATCCATGGCTCCCTGCAGAG*

(SEQ ID NO:85)

7

156

EGF 8-10

N24 F

CAGAGCAGGAAGATCTGCCT*

229

(SEQ ID NO:86)

N24 R

CATTCACAGACGACGGAGCT*

(SEQ ID NO:87)

8

187

EGF 10-11

N3 F

ATCGCACTCCATCCGGCA*

212

(SEQ ID NO:88)

N3 R

ACCCACCTGCCATACAGA*

(SEQ ID NO:89)

9

114

EGF 11-12

N25A F

CGTTCACACCATAGGGTAGC*

215

(SEQ ID NO:90)

N25A R

CCCCTTCCCAGACATGTCTT

(SEQ ID NO:91)

10

114

EGF 12-13

N25BF

CTTGTCGGACTGTCATTGG

195

(SEQ ID NO:92)

N25BR

GTGTACTGCTCTCACCCTT*

(SEQ ID NO:93)

11

234

EGF 13-15

N4AF

ATTGGTCCGAGGCCTCACTT*

213

(SEQ ID NO:94)

N4AR

ACCTGGCTCTCGCAGCGTGT

(SEQ ID NO:95)

N4B R

CCATTCCCAACCCCTCTGTG

199

(SEQ ID NO:96)

N4B F

TGCCTGTGCTCCTGGCTACA*

(SEQ ID NO:97)

12

111

EGF 15-16

N5 F

TGGCCACTCCATGCCATGTT*

166

(SEQ ID NO:98)

N5 R

TCTCATGGCAGCCACTTGCC*

(SEQ ID NO:99)

13

193

EGF 16-18

N14 F

ATGAGTGTGCTTCCAGCCCA*

258

(SEQ ID NO:100)

N14 R

GCAGTGTCTGAGGCTGAGAA*

(SEQ ID NO:101)

14

152

EGF 18-19

N6 F

TCCCTGGCCTGACTACCTTC*

207

(SEQ ID NO:102)

N6 R

CTGCAGAGGGAAGGTGAGGT*

(SEQ ID NO:103)

15

114

EGF 19-20

N26BF

AAGGCTATCCTGCTTCC*

183

(SEQ ID NO:104)

N26BR

GAGGAGGAGGGAAGAGAA*

(SEQ ID NO:105)

16

156

EGF 20 22

S13FBIS

AGGATGTGGACGAGTGTGCT

195

(SEQ ID NO:106)

N26CR

GCTTAATGACTGTGTTCC*

(SEQ ID NO:107)

17

226

EGF 22-24

N15A F

TCAGACTGGGCTAATGGGGG*

257

(SEQ ID NO:108)

N15A R

TCGCAGTGGAAGCCTCCGTA

(SEQ ID NO:109)

N15B F

GATGTGGATGAGTGCCTGAG

166

(SEQ ID NO:110)

N15B R

GTCCTGCTCTTCAAGCAGA*

(SEQ ID NO:111)

18

202

EGF 24-25

N27F

GATCCTCCCTCCCACTCCTT*

256

(SEQ ID NO:112)

N27R

AGGTCCCCAGTAACTCCA*

(SEQ ID NO:113)

19

148

EGF 25-27

N22F

ACTGACTCTAAGTGCTTCCC*

240

(SEQ ID NO:114)

N22R

AGCAGGAGGTACGTGCATGA*

(SEQ ID NO:115)

20

185

EGF 27-28

N7 F

TGTTCCTGTGCCACTCTCCT*

249

(SEQ ID NO:116)

N7 R

ACCTCCTCTTCCCTCTCCT*

(SEQ ID NO:117)

21

133

EGF 28-29

N8 F

TCTGTGTCCCACTAAGCTGA*

237

(SEQ ID NO:118)

N8 R

CAAGAGGAAATGAAGACAGC*

(SEQ ID NO:119)

22

258

EGF 29-31

N9A F

TTCCTCTTGACCACCCCTCG*

217

(SEQ ID NO:120)

N9A R

TGGCAGGCACCTGAGCGACA

(SEQ ID NO:121)

N9B F

CAGGATACACTGGTTTGCGC

209

(SEQ ID NO:122)

N9B R

TGCCACGTTATGGATCAGCC*

(SEQ ID NO:123)

23

119

EGF 31-32

N10 F

GATCTACATGCTCCCGCTCG*

178

(SEQ ID NO:124)

N10 R

TACTCCTCCTCCATAGGCCG*

(SEQ ID NO:125)

24

566

EGF 32-34

N16AFTR

CGTTCTGGGGTCCGCGTT

249

(SEQ ID NO:126)

Lin12 N1-3

N16DR

AAGCGCAGCGGAAGAAGGGC

(SEQ ID NO:127)

N16FF

GCCCTTCTTCCGCTGCGCTT

230

(SEQ ID NO:128)

N16FR

ACTGCAGCGCCTCGCATTGC

(SEQ ID NO:129)

N16GF

CTGCGACCGCGAGTGCAACA

239

(SEQ ID NO:130)

N16HR

ATAGACAGACGGATCGAT*

(SEQ ID NO:131)

25

331

Lin12 N3

N21CF

CTCTCTGCCTCACCCTT*

207

(SEQ ID NO:132)

N21CR

GCTGGAACGCAGTAGCT

(SEQ ID NO:133)

N21DF

TGCTCACAGTGCTGCTG

223

(SEQ ID NO:134)

N21DR

CACGGCTTTTCCAGGTG*

(SEQ ID NO:135)

26

155

N34F

TTTGAGCCCTCTGGTCC*

306

(SEQ ID NO:136)

N34R

AAGAGCAGGAAGCAGAG*

(SEQ ID NO:137)

27

222

TM

N28Fbis

TCCCTCTGCTTCCTGCTCTT*

291

(SEQ ID NO:138)

N28R

TCACAAGGTCCCCGTAGTCA*

(SEQ ID NO:139)

28

85

J5N3 F

CTCACATCCCCTCTTCCCAT*

203

(SEQ ID NO:140)

J5N3 R

ATCACGCCCATCATCCACTG*

(SEQ ID NO:141)

29

163

Cdc10 N1

L24bisf

CAGCACCAAAGGGTG*

241

(SEQ ID NO:142)

L24bisR

CATCCCTTTGGGAGG*

(SEQ ID NO:143)

30

305

Cdc10 N1-3

N17AF

ATGGCTTCACCCCGCTAATG

176

(SEQ ID NO:144)

N17AR

AGCCAGGTGCAAAGCAGTCT

(SEQ ID NO:145)

N17BF

TCAGCTTGGGGCACGGACTG

165

(SEQ ID NO:146)

N17BR

GCATCGGCTGTGACAGCTGT

(SEQ ID NO:147)

31

148

Cdc10 N4-5

N26FBIS

TGTTCCTGCCATGACCCCT*

239

(SEQ ID NO:148)

N26RBIS

CAGGTGACACTAACCCAGTC*

(SEQ ID NO:149)

32

98

Cdc10 N5-6

N31F

TCCTGACCTCTCTCCCCTTC*

178

(SEQ ID NO:150)

N31R

AATGGCGCTGTGCCACTGCT*

(SEQ ID NO:151)

33

Cdc10 N6

N32AF

GCTACTGTTAGCTGGGGTTT*

214

(SEQ ID NO:152)

NLS

N32AR

TGATCCAGCAAGCGCACGAT

(SEQ ID NO:153)

PEST

N32EFTER

TCACCGACCACCTGGACA

425

(SEQ ID NO:154)

N32DR

ACCAAGCTGTGCCAGAGA

(SEQ ID NO:155)

N32DF

TCCAAGAAGAGCAGGAGG

246

(SEQ ID NO:156)

N32DR

ACCAAGCTGTGCCAGAGA

(SEQ ID NO:157)

N32B F

CAGTGTCTCTGGCACAGCTT

248

(SEQ ID NO:158)

N32BR

TCCTGGGACTGCCAGGTAA

(SEQ ID NO:159)

N32CF

AGCTGCTCAACCCAGGGA

229

(SEQ ID NO:160)

N32CR

GTGGATTCGGACCAGTCT

(SEQ ID NO:161)

N32GF

GAATCCCCTGAGCACT

235

(SEQ ID NO:162)

N32GR

CTAAGAACTGACGAGC

(SEQ ID NO:163)

*intronic primers

TABLE C

Notch3 mutations in CADASIL patients

Evidence of

SMC

Notch3

Patient

linkage

lesions

nt

a

Notch3 mutation

Effect

Exon

Domain

Segregation

52*

nd

nd

224

T

G

T-->T

A

T

C

49

-->Y*

N2

EGF1

nd

56

nd

+

291

TG

G

-->TG

T

W

71

-->C

N3

EGF1

nd

11

+

nd

406

C

GT-->

T

GT

R

110

-->C

N3

EGF2

+

3

+

+

419(-2)

A

G-->

G

G

abnormal splicing

N4

+

of exon 4?

39

nd

+

419(-2)

A

G-->

C

G

abnormal splicing

N4

nd

of exon 4?

10

+

+

475

C

GC-->

T

GC

R

133

-->C

N4

EGF3

+

20

nd

+

475

C

GC-->

T

GC

R

133

-->C

N4

EGF3

nd

46

+

nd

475

C

GC-->

T

GC

R

133

-->C

N4

EGF3

+

6

+

nd

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

+

12

+

+

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

+

19

+

nd

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

+

21*

nd

+

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

nd

941

G

G

C-->G

C

G

G

288

-->A*

N5

EGF7

nd

38

+

nd

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

+

49

+

+

499

C

GC-->

T

GC

R

141

-->C

N4

EGF3

+

26

+

+

514

T

GC-->

C

GC

C

146

-->R

N4

EGF3

+

4

+

+

535

C

GC-->

T

GC

R

153

-->C

N4

EGF3

+

50

nd

+

535

C

GC-->

T

GC

R

153

-->C

N4

EGF3

+

9*

+

+

583

C

GC-->

T

GC

R

169

-->C*

N4

EGF4

+

15*

+

nd

583

C

GC-->

T

GC

R

169

-->C*

N4

EGF4

+

24

+

nd

583

C

GC-->

T

GC

R

169

-->C

N4

EGF4

+

36*

nd

+

583

C

GC-->

T

GC

R

169

-->C*

N4

EGF4

nd

48

nd

+

583

C

GC-->

T

GC

R

169

-->C

N4

EGF4

nd

1

+

nd

589

G

GT-->

T

GT

G

171

-->C

N4

EGF4

+

45*

+

+

622

C

GC-->

T

GC

R

182

-->C*

N4

EGF4

+

47*

nd

+

622

C

GC-->

T

GC

R

182

-->C*

N4

EGF4

nd

29*

+

+

622

C

GC-->

T

GC

R

182

-->C

N4

EGF4

+

5632

G

CT-->

A

CT

A

1852

-->T*

N30

cdc10

+

41

nd

+

631

T

GT-->

C

GT

C

185

-->R

N4

EGF4

nd

57

nd

+

712

T

GC-->

A

GC

C

212

-->S

N4

EGF5

nd

8

+

nd

742

T

GT-->

G

GT

C

222

-->G

N4

EGF5

+

55

nd

nd

749

T

G

T-->T

A

T

C

224

-->Y

N4

EGF5

+

1568

T

C

G-->T

T

G

S

497

-->L

N9

EGF12

−

14

+

+

851

T

A

T-->T

G

T

Y

258

-->C

N5

EGF6

+

54*

nd

+

1703

T

G

T-->T

A

T

C

542

-->Y*

N11

EGF13

nd

17*

+

+

1750

C

GC-->

T

GC

R

558

-->C*

N11

EGF14

+

18*

+

+

1750

C

GC-->

T

GC

R

558

-->C*

N11

EGF14

+

31*

nd

+

1810

C

GC-->

T

GC

R

578

-->C*

N11

EGF14

+

43

nd

nd

2260

C

GC-->

T

GC

R

728

-->C

N14

EGF18

nd

25

+

+

3031

C

GC-->

T

GC

R

985

-->C

N18

EGF25

+

42

nd

+

3031

C

GC-->

T

GC

R

985

-->C

N18

EGF25

nd

7

+

nd

3094

C

GC-->

T

GC

R

1006

-->C

N19

EGF26

+

35

nd

nd

3169

C

GC-->

T

GC

R

1031

-->C

N19

EGF26

+

33

nd

nd

3769

C

GC-->

T

GT

R

1231

-->C

N22

EGF31

nd

58*

nd

+

3859

T

GC-->

C

GC

C

1261

-->R*

N23

EGF32

nd

*patient and mutation previously reported

7

SMC: smooth muscle cell

REFERENCES FOR THE VARIOUS METHODS CITED ABOVE

Polymerase Chain Reaction (PCR)

Saiki et al., Science 239, p. 487, 1988+reference manual.

SSCP

Orita et al., Proc. Natl. Acad. Sci. USA, 86, p. 2766-2770, 1989.

Techniques for Detection of Mutations Based on the Demonstration of Mismatches

chemical cleavage

enzymatic cleavage (S1 nuclease)

heteroduplex

Allele Specific Oligonucleotide probes (ASO)

References

Cotton et al., Proc. Natl. Acad. Sci. USA, 85, 4397, 1988

Sherk et al., Proc. Natl. Acad. Sci. USA, 72, 989, 1975 Cariello, Hum. Genet., 42, 726, 1988

Cloning Vectors and Basic Molecular Biology Techniques

Current protocols in molecular biology, Eds F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, S. G. Seldman, J. A. Smith and K. Struhl, Published by Green Publishing Associates and Wiley Interscience, 1st edition 1987, John Wiley and sons

Molecular cloning. A laboratory manual, J. Sambrook, EF Fritsch and T. Mariatis, 2nd edition, 1989, Cold Spring Harbor Laboratory Press

163

1

8091

DNA

Homo sapiens

CDS

(79)..(7041)

human ADNc Notch 3

1
acgcggcgcg gaggctggcc cgggacgcgc ccggagccca gggaaggagg gaggagggga 60
gggtcgcggc cggccgcc atg ggg ccg ggg gcc cgt ggc cgc cgc cgc cgc 111
Met Gly Pro Gly Ala Arg Gly Arg Arg Arg Arg
1 5 10
cgt cgc ccg atg tcg ccg cca ccg cca ccg cca ccc gtg cgg gcg ctg 159
Arg Arg Pro Met Ser Pro Pro Pro Pro Pro Pro Pro Val Arg Ala Leu
15 20 25
ccc ctg ctg ctg ctg cta gcg ggg ccg ggg gct gca gcc ccc cct tgc 207
Pro Leu Leu Leu Leu Leu Ala Gly Pro Gly Ala Ala Ala Pro Pro Cys
30 35 40
ctg gac gga agc ccg tgt gca aat gga ggt cgt tgc acc cag ctg ccc 255
Leu Asp Gly Ser Pro Cys Ala Asn Gly Gly Arg Cys Thr Gln Leu Pro
45 50 55
tcc cgg gag gct gcc tgc ctg tgc ccg cct ggc tgg gtg ggt gag cgg 303
Ser Arg Glu Ala Ala Cys Leu Cys Pro Pro Gly Trp Val Gly Glu Arg
60 65 70 75
tgt cag ctg gag gac ccc tgt cac tca ggc ccc tgt gct ggc cgt ggt 351
Cys Gln Leu Glu Asp Pro Cys His Ser Gly Pro Cys Ala Gly Arg Gly
80 85 90
gtc tgc cag agt tca gtg gtg gct ggc acc gcc cga ttc tca tgc cgg 399
Val Cys Gln Ser Ser Val Val Ala Gly Thr Ala Arg Phe Ser Cys Arg
95 100 105
tgc ccc cgt ggc ttc cga ggc cct gac tgc tcc ctg cca gat ccc tgc 447
Cys Pro Arg Gly Phe Arg Gly Pro Asp Cys Ser Leu Pro Asp Pro Cys
110 115 120
ctc agc agc cct tgt gcc cac ggt gcc cgc tgc tca gtg ggg ccc gat 495
Leu Ser Ser Pro Cys Ala His Gly Ala Arg Cys Ser Val Gly Pro Asp
125 130 135
gga cgc ttc ctc tgc tcc tgc cca cct ggc tac cag ggc cgc agc tgc 543
Gly Arg Phe Leu Cys Ser Cys Pro Pro Gly Tyr Gln Gly Arg Ser Cys
140 145 150 155
cga agc gac gtg gat gag tgc cgg gtg ggt gag ccc tgc cgc cat ggt 591
Arg Ser Asp Val Asp Glu Cys Arg Val Gly Glu Pro Cys Arg His Gly
160 165 170
ggc acc tgc ctc aac aca cct ggc tcc ttc cgc tgc cag tgt cca gct 639
Gly Thr Cys Leu Asn Thr Pro Gly Ser Phe Arg Cys Gln Cys Pro Ala
175 180 185
ggc tac aca ggg cca cta tgt gag aac ccc gcg gtg ccc tgt gcg ccc 687
Gly Tyr Thr Gly Pro Leu Cys Glu Asn Pro Ala Val Pro Cys Ala Pro
190 195 200
tca cca tgc cgt aac ggg ggc acc tgc agg cag agt ggc gac ctc act 735
Ser Pro Cys Arg Asn Gly Gly Thr Cys Arg Gln Ser Gly Asp Leu Thr
205 210 215
tac gac tgt gcc tgt ctt cct ggg ttt gag ggt cag aat tgt gaa gtg 783
Tyr Asp Cys Ala Cys Leu Pro Gly Phe Glu Gly Gln Asn Cys Glu Val
220 225 230 235
aac gtg gac gac tgt cca gga cac cga tgt ctc aat ggg ggg aca tgc 831
Asn Val Asp Asp Cys Pro Gly His Arg Cys Leu Asn Gly Gly Thr Cys
240 245 250
gtg gat ggc gtc aac acc tat aac tgc cag tgc cct cct gag tgg aca 879
Val Asp Gly Val Asn Thr Tyr Asn Cys Gln Cys Pro Pro Glu Trp Thr
255 260 265
ggc cag ttc tgc acg gag gac gtg gat gag tgt cag ctg cag ccc aac 927
Gly Gln Phe Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Gln Pro Asn
270 275 280
gcc tgc cac aat ggg ggt acc tgc ttc aac acg ctg ggt ggc cac agc 975
Ala Cys His Asn Gly Gly Thr Cys Phe Asn Thr Leu Gly Gly His Ser
285 290 295
tgc gtg tgt gtc aat ggc tgg aca ggt gag agc tgc agt cag aat atc 1023
Cys Val Cys Val Asn Gly Trp Thr Gly Glu Ser Cys Ser Gln Asn Ile
300 305 310 315
gat gac tgt gcc aca gcc gtg tgc ttc cat ggg gcc acc tgc cat gac 1071
Asp Asp Cys Ala Thr Ala Val Cys Phe His Gly Ala Thr Cys His Asp
320 325 330
cgc gtg gct tct ttc tac tgt gcc tgc ccc atg ggc aag act ggc ctc 1119
Arg Val Ala Ser Phe Tyr Cys Ala Cys Pro Met Gly Lys Thr Gly Leu
335 340 345
ctg tgt cac ctg gat gac gcc tgt gtc agc aac ccc tgc cac gag gat 1167
Leu Cys His Leu Asp Asp Ala Cys Val Ser Asn Pro Cys His Glu Asp
350 355 360
gct atc tgt gac aca aat ccg gtg aac ggc cgg gcc att tgc acc tgt 1215
Ala Ile Cys Asp Thr Asn Pro Val Asn Gly Arg Ala Ile Cys Thr Cys
365 370 375
cct ccc ggc ttc acg ggt ggg gca tgt gac cag gat gtg gac gag tgc 1263
Pro Pro Gly Phe Thr Gly Gly Ala Cys Asp Gln Asp Val Asp Glu Cys
380 385 390 395
tct atc ggc gcc aac ccc tgc gag cac ttg ggc agg tgc gtg aac acg 1311
Ser Ile Gly Ala Asn Pro Cys Glu His Leu Gly Arg Cys Val Asn Thr
400 405 410
cag ggc tcc ttc ctg tgc cag tgc ggt cgt ggc tac act gga cct cgc 1359
Gln Gly Ser Phe Leu Cys Gln Cys Gly Arg Gly Tyr Thr Gly Pro Arg
415 420 425
tgt gag acc gat gtc aac gag tgt ctg tcg ggg ccc tgc cga aac cag 1407
Cys Glu Thr Asp Val Asn Glu Cys Leu Ser Gly Pro Cys Arg Asn Gln
430 435 440
gcc acg tgc ctc gac cgc ata ggc cag ttc acc tgt atc tgt atg gca 1455
Ala Thr Cys Leu Asp Arg Ile Gly Gln Phe Thr Cys Ile Cys Met Ala
445 450 455
ggc ttc aca gga acc tat tgc gag gtg gac att gac gag tgt cag agt 1503
Gly Phe Thr Gly Thr Tyr Cys Glu Val Asp Ile Asp Glu Cys Gln Ser
460 465 470 475
agc ccc tgt gtc aac ggt ggg gtc tgc aag gac cga gtc aat ggc ttc 1551
Ser Pro Cys Val Asn Gly Gly Val Cys Lys Asp Arg Val Asn Gly Phe
480 485 490
agc tgc acc tgc ccc tcg ggc ttc agc ggc tcc acg tgt cag ctg gac 1599
Ser Cys Thr Cys Pro Ser Gly Phe Ser Gly Ser Thr Cys Gln Leu Asp
495 500 505
gtg gac gaa tgc gcc agc acg ccc tgc agg aat ggc gcc aaa tgc gtg 1647
Val Asp Glu Cys Ala Ser Thr Pro Cys Arg Asn Gly Ala Lys Cys Val
510 515 520
gac cag ccc gat ggc tac gag tgc cgc tgt gcc gag ggc ttt gag ggc 1695
Asp Gln Pro Asp Gly Tyr Glu Cys Arg Cys Ala Glu Gly Phe Glu Gly
525 530 535
acg ctg tgt gat cgc aac gtg gac gac tgc tcc cct gac cca tgc cac 1743
Thr Leu Cys Asp Arg Asn Val Asp Asp Cys Ser Pro Asp Pro Cys His
540 545 550 555
cat ggt cgc tgc gtg gat ggc atc gcc agc ttc tca tgt gcc tgt gct 1791
His Gly Arg Cys Val Asp Gly Ile Ala Ser Phe Ser Cys Ala Cys Ala
560 565 570
cct ggc tac acg ggc aca cgc tgc gag agc cag gtg gac gaa tgc cgc 1839
Pro Gly Tyr Thr Gly Thr Arg Cys Glu Ser Gln Val Asp Glu Cys Arg
575 580 585
agc cag ccc tgc cgc cat ggc ggc aaa tgc cta gac ctg gtg gac aag 1887
Ser Gln Pro Cys Arg His Gly Gly Lys Cys Leu Asp Leu Val Asp Lys
590 595 600
tac ctc tgc cgc tgc cct tct ggg acc aca ggt gtg aac tgc gaa gtg 1935
Tyr Leu Cys Arg Cys Pro Ser Gly Thr Thr Gly Val Asn Cys Glu Val
605 610 615
aac att gac gac tgt gcc agc aac ccc tgc acc ttt gga gtc tgc cgt 1983
Asn Ile Asp Asp Cys Ala Ser Asn Pro Cys Thr Phe Gly Val Cys Arg
620 625 630 635
gat ggc atc aac cgc tac gac tgt gtc tgc caa cct ggc ttc aca ggg 2031
Asp Gly Ile Asn Arg Tyr Asp Cys Val Cys Gln Pro Gly Phe Thr Gly
640 645 650
ccc ctt tgt aac gtg gag atc aat gag tgt gct tcc agc cca tgc ggc 2079
Pro Leu Cys Asn Val Glu Ile Asn Glu Cys Ala Ser Ser Pro Cys Gly
655 660 665
gag gga ggt tcc tgt gtg gat ggg gaa aat ggc ttc cgc tgc ctc tgc 2127
Glu Gly Gly Ser Cys Val Asp Gly Glu Asn Gly Phe Arg Cys Leu Cys
670 675 680
ccg cct ggc tcc ttg ccc cca ctc tgc ctc ccc ccg agc cat ccc tgt 2175
Pro Pro Gly Ser Leu Pro Pro Leu Cys Leu Pro Pro Ser His Pro Cys
685 690 695
gcc cat gag ccc tgc agt cac ggc atc tgc tat gat gca cct ggc ggg 2223
Ala His Glu Pro Cys Ser His Gly Ile Cys Tyr Asp Ala Pro Gly Gly
700 705 710 715
ttc cgc tgt gtg tgt gag cct ggc tgg agt ggc ccc cgc tgc agc cag 2271
Phe Arg Cys Val Cys Glu Pro Gly Trp Ser Gly Pro Arg Cys Ser Gln
720 725 730
agc ctg gcc cga gac gcc tgt gag tcc cag ccg tgc agg gcc ggt ggg 2319
Ser Leu Ala Arg Asp Ala Cys Glu Ser Gln Pro Cys Arg Ala Gly Gly
735 740 745
aca tgc agc agc gat gga atg ggt ttc cac tgc acc tgc ccg cct ggt 2367
Thr Cys Ser Ser Asp Gly Met Gly Phe His Cys Thr Cys Pro Pro Gly
750 755 760
gtc cag gga cgt cag tgt gaa ctc ctc tcc ccc tgc acc ccg aac ccc 2415
Val Gln Gly Arg Gln Cys Glu Leu Leu Ser Pro Cys Thr Pro Asn Pro
765 770 775
tgt gag cat ggg ggc cgc tgc gag tct gcc cct ggc cag ctg cct gtc 2463
Cys Glu His Gly Gly Arg Cys Glu Ser Ala Pro Gly Gln Leu Pro Val
780 785 790 795
tgc tcc tgc ccc cag ggc tgg caa ggc cca cga tgc cag cag gat gtg 2511
Cys Ser Cys Pro Gln Gly Trp Gln Gly Pro Arg Cys Gln Gln Asp Val
800 805 810
gac gag tgt gct ggc ccc gca ccc tgt ggc cct cat ggt atc tgc acc 2559
Asp Glu Cys Ala Gly Pro Ala Pro Cys Gly Pro His Gly Ile Cys Thr
815 820 825
aac ctg gca ggg agt ttc agc tgc acc tgc cat gga ggg tac act ggc 2607
Asn Leu Ala Gly Ser Phe Ser Cys Thr Cys His Gly Gly Tyr Thr Gly
830 835 840
cct tcc tgt gat cag gac atc aat gac tgt gac ccc aac cca tgc ctg 2655
Pro Ser Cys Asp Gln Asp Ile Asn Asp Cys Asp Pro Asn Pro Cys Leu
845 850 855
aac ggt ggc tcg tgc caa gac ggc gtg ggc tcc ttt tcc tgc tcc tgc 2703
Asn Gly Gly Ser Cys Gln Asp Gly Val Gly Ser Phe Ser Cys Ser Cys
860 865 870 875
ctc cct ggt ttc gcc ggc cca cga tgc gcc cgc gat gtg gat gag tgc 2751
Leu Pro Gly Phe Ala Gly Pro Arg Cys Ala Arg Asp Val Asp Glu Cys
880 885 890
ctg agc aac ccc tgc ggc ccg ggc acc tgt acc gac cac gtg gcc tcc 2799
Leu Ser Asn Pro Cys Gly Pro Gly Thr Cys Thr Asp His Val Ala Ser
895 900 905
ttc acc tgc acc tgc ccg ccg ggc tac gga ggc ttc cac tgc gaa cag 2847
Phe Thr Cys Thr Cys Pro Pro Gly Tyr Gly Gly Phe His Cys Glu Gln
910 915 920
gac ctg ccc gac tgc agc ccc agc tcc tgc ttc aat ggc ggg acc tgt 2895
Asp Leu Pro Asp Cys Ser Pro Ser Ser Cys Phe Asn Gly Gly Thr Cys
925 930 935
gtg gac ggc gtg aac tcg ttc agc tgc ctg tgc cgt ccc ggc tac aca 2943
Val Asp Gly Val Asn Ser Phe Ser Cys Leu Cys Arg Pro Gly Tyr Thr
940 945 950 955
gga gcc cac tgc caa cat gag gca gac ccc tgc ctc tcg cgg ccc tgc 2991
Gly Ala His Cys Gln His Glu Ala Asp Pro Cys Leu Ser Arg Pro Cys
960 965 970
cta cac ggg ggc gtc tgc agc gcc gcc cac cct ggc ttc cgc tgc acc 3039
Leu His Gly Gly Val Cys Ser Ala Ala His Pro Gly Phe Arg Cys Thr
975 980 985
tgc ctc gag agc ttc acg ggc ccg cag tgc cag acg ctg gtg gat tgg 3087
Cys Leu Glu Ser Phe Thr Gly Pro Gln Cys Gln Thr Leu Val Asp Trp
990 995 1000
tgc agc cgc cag cct tgt caa aac ggg ggt cgc tgc gtc cag act ggg 3135
Cys Ser Arg Gln Pro Cys Gln Asn Gly Gly Arg Cys Val Gln Thr Gly
1005 1010 1015
gcc tat tgc ctt tgt ccc cct gga tgg agc gga cgc ctc tgt gac atc 3183
Ala Tyr Cys Leu Cys Pro Pro Gly Trp Ser Gly Arg Leu Cys Asp Ile
1020 1025 1030 1035
cga agc ttg ccc tgc agg gag gcc gca gcc cag atc ggg gtg cgg ctg 3231
Arg Ser Leu Pro Cys Arg Glu Ala Ala Ala Gln Ile Gly Val Arg Leu
1040 1045 1050
gag cag ctg tgt cag gcg ggt ggg cag tgt gtg gat gaa gac agc tcc 3279
Glu Gln Leu Cys Gln Ala Gly Gly Gln Cys Val Asp Glu Asp Ser Ser
1055 1060 1065
cac tac tgc gtg tgc cca gag ggc cgt act ggt agc cac tgt gag cag 3327
His Tyr Cys Val Cys Pro Glu Gly Arg Thr Gly Ser His Cys Glu Gln
1070 1075 1080
gag gtg gac ccc tgc ttg gcc cag ccc tgc cag cat ggg ggg acc tgc 3375
Glu Val Asp Pro Cys Leu Ala Gln Pro Cys Gln His Gly Gly Thr Cys
1085 1090 1095
cgt ggc tat atg ggg ggc tac atg tgt gag tgt ctt cct ggc tac aat 3423
Arg Gly Tyr Met Gly Gly Tyr Met Cys Glu Cys Leu Pro Gly Tyr Asn
1100 1105 1110 1115
ggt gat aac tgt gag gac gac gtg gac gag tgt gcc tcc cag ccc tgc 3471
Gly Asp Asn Cys Glu Asp Asp Val Asp Glu Cys Ala Ser Gln Pro Cys
1120 1125 1130
cag cac ggg ggt tca tgc att gac ctc gtg gcc cgc tat ctc tgc tcc 3519
Gln His Gly Gly Ser Cys Ile Asp Leu Val Ala Arg Tyr Leu Cys Ser
1135 1140 1145
tgt ccc cca gga acg ctg ggg gtg ctc tgc gag att aat gag gat gac 3567
Cys Pro Pro Gly Thr Leu Gly Val Leu Cys Glu Ile Asn Glu Asp Asp
1150 1155 1160
tgc ggc cca ggc cca ccg ctg gac tca ggg ccc cgg tgc cta cac aat 3615
Cys Gly Pro Gly Pro Pro Leu Asp Ser Gly Pro Arg Cys Leu His Asn
1165 1170 1175
ggc acc tgc gtg gac ctg gtg ggt ggt ttc cgc tgc acc tgt ccc cca 3663
Gly Thr Cys Val Asp Leu Val Gly Gly Phe Arg Cys Thr Cys Pro Pro
1180 1185 1190 1195
gga tac act ggt ttg cgc tgc gag gca gac atc aat gag tgt cgc tca 3711
Gly Tyr Thr Gly Leu Arg Cys Glu Ala Asp Ile Asn Glu Cys Arg Ser
1200 1205 1210
ggt gcc tgc cac gcg gca cac acc cgg gac tgc ctg cag gac cca ggc 3759
Gly Ala Cys His Ala Ala His Thr Arg Asp Cys Leu Gln Asp Pro Gly
1215 1220 1225
gga ggt ttc cgt tgc ctt tgt cat gct ggc ttc tca ggt cct cgc tgt 3807
Gly Gly Phe Arg Cys Leu Cys His Ala Gly Phe Ser Gly Pro Arg Cys
1230 1235 1240
cag act gtc ctg tct ccc tgc gag tcc cag cca tgc cag cat gga ggc 3855
Gln Thr Val Leu Ser Pro Cys Glu Ser Gln Pro Cys Gln His Gly Gly
1245 1250 1255
cag tgc cgt cct agc ccg ggt cct ggg ggt ggg ctg acc ttc acc tgt 3903
Gln Cys Arg Pro Ser Pro Gly Pro Gly Gly Gly Leu Thr Phe Thr Cys
1260 1265 1270 1275
cac tgt gcc cag ccg ttc tgg ggt ccg cgt tgc gag cgg gtg gcg cgc 3951
His Cys Ala Gln Pro Phe Trp Gly Pro Arg Cys Glu Arg Val Ala Arg
1280 1285 1290
tcc tgc cgg gag ctg cag tgc ccg gtg ggc gtc cca tgc cag cag acg 3999
Ser Cys Arg Glu Leu Gln Cys Pro Val Gly Val Pro Cys Gln Gln Thr
1295 1300 1305
ccc cgc ggg ccg cgc tgc gcc tgc ccc cca ggg ttg tcg gga ccc tcc 4047
Pro Arg Gly Pro Arg Cys Ala Cys Pro Pro Gly Leu Ser Gly Pro Ser
1310 1315 1320
tgc cgc agc ttc ccg ggg tcg ccg ccg ggg gcc agc aac gcc agc tgc 4095
Cys Arg Ser Phe Pro Gly Ser Pro Pro Gly Ala Ser Asn Ala Ser Cys
1325 1330 1335
gcg gcc gcc ccc tgt ctc cac ggg ggc tcc tgc cgc ccc gcg ccg ctc 4143
Ala Ala Ala Pro Cys Leu His Gly Gly Ser Cys Arg Pro Ala Pro Leu
1340 1345 1350 1355
gcg ccc ttc ttc cgc tgc gct tgc gcg cag ggc tgg acc ggg ccg cgc 4191
Ala Pro Phe Phe Arg Cys Ala Cys Ala Gln Gly Trp Thr Gly Pro Arg
1360 1365 1370
tgc gag gcg ccc gcc gcg gca ccc gag gtc tcg gag gag ccg cgg tgc 4239
Cys Glu Ala Pro Ala Ala Ala Pro Glu Val Ser Glu Glu Pro Arg Cys
1375 1380 1385
ccg cgc gcc gcc tgc cag gcc aag cgc ggg gac cag cgc tgc gac cgc 4287
Pro Arg Ala Ala Cys Gln Ala Lys Arg Gly Asp Gln Arg Cys Asp Arg
1390 1395 1400
gag tgc aac agc cca ggc tgc ggc tgg gac ggc ggc gac tgc tcg ctg 4335
Glu Cys Asn Ser Pro Gly Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu
1405 1410 1415
agc gtg ggc gac ccc tgg cgg caa tgc gag gcg ctg cag tgc tgg cgc 4383
Ser Val Gly Asp Pro Trp Arg Gln Cys Glu Ala Leu Gln Cys Trp Arg
1420 1425 1430 1435
ctc ttc aac aac agc cgc tgc gac ccc gcc tgc agc tcg ccc gcc tgc 4431
Leu Phe Asn Asn Ser Arg Cys Asp Pro Ala Cys Ser Ser Pro Ala Cys
1440 1445 1450
ctc tac gac aac ttc gac tgc cac gcc ggt ggc cgc gag cgc act tgc 4479
Leu Tyr Asp Asn Phe Asp Cys His Ala Gly Gly Arg Glu Arg Thr Cys
1455 1460 1465
aac ccg gtg tac gag aag tac tgc gcc gac cac ttt gcc gac ggc cgc 4527
Asn Pro Val Tyr Glu Lys Tyr Cys Ala Asp His Phe Ala Asp Gly Arg
1470 1475 1480
tgc gac cag ggc tgc aac acg gag gag tgc ggc tgg gat ggg ctg gat 4575
Cys Asp Gln Gly Cys Asn Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp
1485 1490 1495
tgt gcc agc gag gtg ccg gcc ctg ctg gcc cgc ggc gtg ctg gtg ctc 4623
Cys Ala Ser Glu Val Pro Ala Leu Leu Ala Arg Gly Val Leu Val Leu
1500 1505 1510 1515
aca gtg ctg ctg ccg ccg gag gag cta ctg cgt tcc agc gcc gac ttt 4671
Thr Val Leu Leu Pro Pro Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe
1520 1525 1530
ctg cag cgg ctc agc gcc atc ctg cgc acc tcg ctg cgc ttc cgc ctg 4719
Leu Gln Arg Leu Ser Ala Ile Leu Arg Thr Ser Leu Arg Phe Arg Leu
1535 1540 1545
gac gcg cac ggc cag gcc atg gtc ttc cct tac cac cgg cct agt cct 4767
Asp Ala His Gly Gln Ala Met Val Phe Pro Tyr His Arg Pro Ser Pro
1550 1555 1560
ggc tcc gaa ccc cgg gcc cgt cgg gag ctg gcc ccc gag gtg atc ggc 4815
Gly Ser Glu Pro Arg Ala Arg Arg Glu Leu Ala Pro Glu Val Ile Gly
1565 1570 1575
tcg gta gta atg ctg gag att gac aac cgg ctc tgc ctg cag tcg cct 4863
Ser Val Val Met Leu Glu Ile Asp Asn Arg Leu Cys Leu Gln Ser Pro
1580 1585 1590 1595
gag aat gat cac tgc ttc ccc gat gcc cag agc gcc gct gac tac ctg 4911
Glu Asn Asp His Cys Phe Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu
1600 1605 1610
gga gcg ttg tca gcg gtg gag cgc ctg gac ttc ccg tac cca ctg cgg 4959
Gly Ala Leu Ser Ala Val Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg
1615 1620 1625
gac gtg cgg ggg gag ccg ctg gag cct cca gaa ccc agc gtc ccg ctg 5007
Asp Val Arg Gly Glu Pro Leu Glu Pro Pro Glu Pro Ser Val Pro Leu
1630 1635 1640
ctg cca ctg cta gtg gcg ggc gct gtc ttg ctg ctg gtc att ctc gtc 5055
Leu Pro Leu Leu Val Ala Gly Ala Val Leu Leu Leu Val Ile Leu Val
1645 1650 1655
ctg ggt gtc atg gtg gcc cgg cgc aag cgc gag cac agc acc ctc tgg 5103
Leu Gly Val Met Val Ala Arg Arg Lys Arg Glu His Ser Thr Leu Trp
1660 1665 1670 1675
ttc cct gag ggc ttc tca ctg cac aag gac gtg gcc tct ggt cac aag 5151
Phe Pro Glu Gly Phe Ser Leu His Lys Asp Val Ala Ser Gly His Lys
1680 1685 1690
ggc cgg cgg gaa ccc gtg ggc cag gac gcg ctg ggc atg aag aac atg 5199
Gly Arg Arg Glu Pro Val Gly Gln Asp Ala Leu Gly Met Lys Asn Met
1695 1700 1705
gcc aag ggt gag agc ctg atg ggg gag gtg gcc aca gac tgg atg gac 5247
Ala Lys Gly Glu Ser Leu Met Gly Glu Val Ala Thr Asp Trp Met Asp
1710 1715 1720
aca gag tgc cca gag gcc aag cgg cta aag gta gag gag cca ggc atg 5295
Thr Glu Cys Pro Glu Ala Lys Arg Leu Lys Val Glu Glu Pro Gly Met
1725 1730 1735
ggg gct gag gag gct gtg gat tgc cgt cag tgg act caa cac cat ctg 5343
Gly Ala Glu Glu Ala Val Asp Cys Arg Gln Trp Thr Gln His His Leu
1740 1745 1750 1755
gtt gct gct gac atc cgc gtg gca cca gcc atg gca ctg aca cca cca 5391
Val Ala Ala Asp Ile Arg Val Ala Pro Ala Met Ala Leu Thr Pro Pro
1760 1765 1770
cag ggc gac gca gat gct gat ggc atg gat gtc aat gtg cgt ggc cca 5439
Gln Gly Asp Ala Asp Ala Asp Gly Met Asp Val Asn Val Arg Gly Pro
1775 1780 1785
gat ggc ttc acc ccg cta atg ctg gct tcc ttc tgt ggg ggg gct ctg 5487
Asp Gly Phe Thr Pro Leu Met Leu Ala Ser Phe Cys Gly Gly Ala Leu
1790 1795 1800
gag cca atg cca act gaa gag gat gag gca gat gac aca tca gct agc 5535
Glu Pro Met Pro Thr Glu Glu Asp Glu Ala Asp Asp Thr Ser Ala Ser
1805 1810 1815
atc atc tcc gac ctg atc tgc cag ggg gct cag ctt ggg gca cgg act 5583
Ile Ile Ser Asp Leu Ile Cys Gln Gly Ala Gln Leu Gly Ala Arg Thr
1820 1825 1830 1835
gac cgt act ggc gag act gct ttg cac ctg gct gcc cgt tat gcc cgt 5631
Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala Ala Arg Tyr Ala Arg
1840 1845 1850
gct gat gca gcc aag cgg ctg ctg gat gct ggg gca gac acc aat gcc 5679
Ala Asp Ala Ala Lys Arg Leu Leu Asp Ala Gly Ala Asp Thr Asn Ala
1855 1860 1865
cag gac cac tca ggc cgc act ccc ctg cac aca gct gtc aca gcc gat 5727
Gln Asp His Ser Gly Arg Thr Pro Leu His Thr Ala Val Thr Ala Asp
1870 1875 1880
gcc cag ggt gtc ttc cag att ctc atc cga aac cgc tct aca gac ttg 5775
Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn Arg Ser Thr Asp Leu
1885 1890 1895
gat gcc cgc atg gca gat ggc tca acg gca ctg atc ctg gcg gcc cgc 5823
Asp Ala Arg Met Ala Asp Gly Ser Thr Ala Leu Ile Leu Ala Ala Arg
1900 1905 1910 1915
ctg gca gta gag ggc atg gtg gaa gag ctc atc gcc agc cat gct gat 5871
Leu Ala Val Glu Gly Met Val Glu Glu Leu Ile Ala Ser His Ala Asp
1920 1925 1930
gtc aat gct gtg gat gag ctt ggg aaa tca gcc tta cac tgg gct gcg 5919
Val Asn Ala Val Asp Glu Leu Gly Lys Ser Ala Leu His Trp Ala Ala
1935 1940 1945
gct gtg aac aac gtg gaa gcc act ttg gcc ctg ctc aaa aat gga gcc 5967
Ala Val Asn Asn Val Glu Ala Thr Leu Ala Leu Leu Lys Asn Gly Ala
1950 1955 1960
aat aag gac atg cag gat agc aag gag gag acc ccc cta ttc ctg gcc 6015
Asn Lys Asp Met Gln Asp Ser Lys Glu Glu Thr Pro Leu Phe Leu Ala
1965 1970 1975
gcc cgc gag ggc agc tat gag gct gcc aag ctg ctg ttg gac cac ttt 6063
Ala Arg Glu Gly Ser Tyr Glu Ala Ala Lys Leu Leu Leu Asp His Phe
1980 1985 1990 1995
gcc aac cgt gag atc acc gac cac ctg gac agg ctg ccg cgg gac gta 6111
Ala Asn Arg Glu Ile Thr Asp His Leu Asp Arg Leu Pro Arg Asp Val
2000 2005 2010
gcc cag gag aga ctg cac cag gac atc gtg cgc ttg ctg gat caa ccc 6159
Ala Gln Glu Arg Leu His Gln Asp Ile Val Arg Leu Leu Asp Gln Pro
2015 2020 2025
agt ggg ccc cgc agc ccc ccc ggt ccc cac ggc ctg ggg cct ctg ctc 6207
Ser Gly Pro Arg Ser Pro Pro Gly Pro His Gly Leu Gly Pro Leu Leu
2030 2035 2040
tgt cct cca ggg gcc ttc ctc cct ggc ctc aaa gcg gca cag tcg ggg 6255
Cys Pro Pro Gly Ala Phe Leu Pro Gly Leu Lys Ala Ala Gln Ser Gly
2045 2050 2055
tcc aag aag agc agg agg ccc ccc ggg aag gcg ggg ctg ggg ccg cag 6303
Ser Lys Lys Ser Arg Arg Pro Pro Gly Lys Ala Gly Leu Gly Pro Gln
2060 2065 2070 2075
ggg ccc cgg ggg cgg ggc aag aag ctg acg ctg gcc tgc ccg ggc ccc 6351
Gly Pro Arg Gly Arg Gly Lys Lys Leu Thr Leu Ala Cys Pro Gly Pro
2080 2085 2090
ctg gct gac agc tcg gtc acg ctg tcg ccc gtg gac tcg ctg gac tcc 6399
Leu Ala Asp Ser Ser Val Thr Leu Ser Pro Val Asp Ser Leu Asp Ser
2095 2100 2105
ccg cgg cct ttc ggt ggg ccc cct gct tcc cct ggt ggc ttc ccc ctt 6447
Pro Arg Pro Phe Gly Gly Pro Pro Ala Ser Pro Gly Gly Phe Pro Leu
2110 2115 2120
gag ggg ccc tat gca gct gcc act gcc act gca gtg tct ctg gca cag 6495
Glu Gly Pro Tyr Ala Ala Ala Thr Ala Thr Ala Val Ser Leu Ala Gln
2125 2130 2135
ctt ggt ggc cca ggc cgg gca ggt cta ggg cgc cag ccc cct gga gga 6543
Leu Gly Gly Pro Gly Arg Ala Gly Leu Gly Arg Gln Pro Pro Gly Gly
2140 2145 2150 2155
tgt gta ctc agc ctg ggc ctg ctg aac cct gtg gct gtg ccc ctc gat 6591
Cys Val Leu Ser Leu Gly Leu Leu Asn Pro Val Ala Val Pro Leu Asp
2160 2165 2170
tgg gcc cgg ctg ccc cca cct gcc cct cca ggc ccc tcg ttc ctg ctg 6639
Trp Ala Arg Leu Pro Pro Pro Ala Pro Pro Gly Pro Ser Phe Leu Leu
2175 2180 2185
cca ctg gcg ccg gga ccc cag ctg ctc aac cca ggg acc ccc gtc tcc 6687
Pro Leu Ala Pro Gly Pro Gln Leu Leu Asn Pro Gly Thr Pro Val Ser
2190 2195 2200
ccg cag gag cgg ccc ccg cct tac ctg gca gtc cca gga cat ggc gag 6735
Pro Gln Glu Arg Pro Pro Pro Tyr Leu Ala Val Pro Gly His Gly Glu
2205 2210 2215
gag tac ccg gtg gct ggg gca cac agc agc ccc cca aag gcc cgc ttc 6783
Glu Tyr Pro Val Ala Gly Ala His Ser Ser Pro Pro Lys Ala Arg Phe
2220 2225 2230 2235
ctg cgg gtt ccc agt gag cac cct tac ctg acc cca tcc ccc gaa tcc 6831
Leu Arg Val Pro Ser Glu His Pro Tyr Leu Thr Pro Ser Pro Glu Ser
2240 2245 2250
cct gag cac tgg gcc agc ccc tca cct ccc tcc ctc tca gac tgg tcc 6879
Pro Glu His Trp Ala Ser Pro Ser Pro Pro Ser Leu Ser Asp Trp Ser
2255 2260 2265
gaa tcc acg cct agc cca gcc act gcc act ggg gcc atg gcc acc acc 6927
Glu Ser Thr Pro Ser Pro Ala Thr Ala Thr Gly Ala Met Ala Thr Thr
2270 2275 2280
act ggg gca ctg cct gcc cag cca ctt ccc ttg tct gtt ccc agc tcc 6975
Thr Gly Ala Leu Pro Ala Gln Pro Leu Pro Leu Ser Val Pro Ser Ser
2285 2290 2295
ctt gct cag gcc cag acc cag ctg ggg ccc cag ccg gaa gtt acc ccc 7023
Leu Ala Gln Ala Gln Thr Gln Leu Gly Pro Gln Pro Glu Val Thr Pro
2300 2305 2310 2315
aag agg caa gtg ttg gcc tgagacgctc gtcagttctt agatcttggg 7071
Lys Arg Gln Val Leu Ala
2320
ggcctaaaga gacccccgtc ctgcctcctt tctttctctg tctcttcctt ccttttagtc 7131
tttttcatcc tcttctcttt ccaccaaccc tcctgcatcc ttgccttgca gcgtgaccga 7191
gataggtcat cagcccaggg cttcagtctt cctttattta taatgggtgg gggctaccac 7251
ccaccctctc agtcttgtga agagtctggg acctccttct tccccacttc tctcttccct 7311
cattcctttc tctctccttc tggcctctca tttccttaca ctctgacatg aatgaattat 7371
tattattttt ctttttcttt ttttttttac attttgtata gaaacaaatt catttaaaca 7431
aacttattat tattattttt tacaaaatat atatatggag atgctccctc cccctgtgaa 7491
ccccccagtg cccccgtggg gctgagtctg tgggcccatt cggccaagct ggattctgtg 7551
tacctagtac acaggcatga ctgggatccc gtgtaccgag tacacgaccc aggtatgtac 7611
caagtaggca cccttgggcg cacccactgg ggccaggggt cgggggagtg ttgggagcct 7671
cctccccacc ccacctccct cacttcactg cattccagat tggacatgtt ccatagcctt 7731
gctggggaag ggcccactgc caactccctc tgccccagcc ccacccttgg ccatctccct 7791
ttgggaacta gggggctgct ggtgggaaat gggagccagg gcagatgtat gcattccttt 7851
atgtccctgt aaatgtggga ctacaagaag aggagctgcc tgagtggtac tttctcttcc 7911
tggtaatcct ctggcccagc cttatggcag aatagaggta tttttaggct atttttgtaa 7971
tatggcttct ggtcaaaatc cctgtgtagc tgaattccca agccctgcat tgtacagccc 8031
cccactcccc tcaccaccta ataaaggaat agttaacact caaaaaaaaa aaaaaaaaaa 8091

2

2321

PRT

Homo sapiens

human ADNc Notch 3

2
Met Gly Pro Gly Ala Arg Gly Arg Arg Arg Arg Arg Arg Pro Met Ser
1 5 10 15
Pro Pro Pro Pro Pro Pro Pro Val Arg Ala Leu Pro Leu Leu Leu Leu
20 25 30
Leu Ala Gly Pro Gly Ala Ala Ala Pro Pro Cys Leu Asp Gly Ser Pro
35 40 45
Cys Ala Asn Gly Gly Arg Cys Thr Gln Leu Pro Ser Arg Glu Ala Ala
50 55 60
Cys Leu Cys Pro Pro Gly Trp Val Gly Glu Arg Cys Gln Leu Glu Asp
65 70 75 80
Pro Cys His Ser Gly Pro Cys Ala Gly Arg Gly Val Cys Gln Ser Ser
85 90 95
Val Val Ala Gly Thr Ala Arg Phe Ser Cys Arg Cys Pro Arg Gly Phe
100 105 110
Arg Gly Pro Asp Cys Ser Leu Pro Asp Pro Cys Leu Ser Ser Pro Cys
115 120 125
Ala His Gly Ala Arg Cys Ser Val Gly Pro Asp Gly Arg Phe Leu Cys
130 135 140
Ser Cys Pro Pro Gly Tyr Gln Gly Arg Ser Cys Arg Ser Asp Val Asp
145 150 155 160
Glu Cys Arg Val Gly Glu Pro Cys Arg His Gly Gly Thr Cys Leu Asn
165 170 175
Thr Pro Gly Ser Phe Arg Cys Gln Cys Pro Ala Gly Tyr Thr Gly Pro
180 185 190
Leu Cys Glu Asn Pro Ala Val Pro Cys Ala Pro Ser Pro Cys Arg Asn
195 200 205
Gly Gly Thr Cys Arg Gln Ser Gly Asp Leu Thr Tyr Asp Cys Ala Cys
210 215 220
Leu Pro Gly Phe Glu Gly Gln Asn Cys Glu Val Asn Val Asp Asp Cys
225 230 235 240
Pro Gly His Arg Cys Leu Asn Gly Gly Thr Cys Val Asp Gly Val Asn
245 250 255
Thr Tyr Asn Cys Gln Cys Pro Pro Glu Trp Thr Gly Gln Phe Cys Thr
260 265 270
Glu Asp Val Asp Glu Cys Gln Leu Gln Pro Asn Ala Cys His Asn Gly
275 280 285
Gly Thr Cys Phe Asn Thr Leu Gly Gly His Ser Cys Val Cys Val Asn
290 295 300
Gly Trp Thr Gly Glu Ser Cys Ser Gln Asn Ile Asp Asp Cys Ala Thr
305 310 315 320
Ala Val Cys Phe His Gly Ala Thr Cys His Asp Arg Val Ala Ser Phe
325 330 335
Tyr Cys Ala Cys Pro Met Gly Lys Thr Gly Leu Leu Cys His Leu Asp
340 345 350
Asp Ala Cys Val Ser Asn Pro Cys His Glu Asp Ala Ile Cys Asp Thr
355 360 365
Asn Pro Val Asn Gly Arg Ala Ile Cys Thr Cys Pro Pro Gly Phe Thr
370 375 380
Gly Gly Ala Cys Asp Gln Asp Val Asp Glu Cys Ser Ile Gly Ala Asn
385 390 395 400
Pro Cys Glu His Leu Gly Arg Cys Val Asn Thr Gln Gly Ser Phe Leu
405 410 415
Cys Gln Cys Gly Arg Gly Tyr Thr Gly Pro Arg Cys Glu Thr Asp Val
420 425 430
Asn Glu Cys Leu Ser Gly Pro Cys Arg Asn Gln Ala Thr Cys Leu Asp
435 440 445
Arg Ile Gly Gln Phe Thr Cys Ile Cys Met Ala Gly Phe Thr Gly Thr
450 455 460
Tyr Cys Glu Val Asp Ile Asp Glu Cys Gln Ser Ser Pro Cys Val Asn
465 470 475 480
Gly Gly Val Cys Lys Asp Arg Val Asn Gly Phe Ser Cys Thr Cys Pro
485 490 495
Ser Gly Phe Ser Gly Ser Thr Cys Gln Leu Asp Val Asp Glu Cys Ala
500 505 510
Ser Thr Pro Cys Arg Asn Gly Ala Lys Cys Val Asp Gln Pro Asp Gly
515 520 525
Tyr Glu Cys Arg Cys Ala Glu Gly Phe Glu Gly Thr Leu Cys Asp Arg
530 535 540
Asn Val Asp Asp Cys Ser Pro Asp Pro Cys His His Gly Arg Cys Val
545 550 555 560
Asp Gly Ile Ala Ser Phe Ser Cys Ala Cys Ala Pro Gly Tyr Thr Gly
565 570 575
Thr Arg Cys Glu Ser Gln Val Asp Glu Cys Arg Ser Gln Pro Cys Arg
580 585 590
His Gly Gly Lys Cys Leu Asp Leu Val Asp Lys Tyr Leu Cys Arg Cys
595 600 605
Pro Ser Gly Thr Thr Gly Val Asn Cys Glu Val Asn Ile Asp Asp Cys
610 615 620
Ala Ser Asn Pro Cys Thr Phe Gly Val Cys Arg Asp Gly Ile Asn Arg
625 630 635 640
Tyr Asp Cys Val Cys Gln Pro Gly Phe Thr Gly Pro Leu Cys Asn Val
645 650 655
Glu Ile Asn Glu Cys Ala Ser Ser Pro Cys Gly Glu Gly Gly Ser Cys
660 665 670
Val Asp Gly Glu Asn Gly Phe Arg Cys Leu Cys Pro Pro Gly Ser Leu
675 680 685
Pro Pro Leu Cys Leu Pro Pro Ser His Pro Cys Ala His Glu Pro Cys
690 695 700
Ser His Gly Ile Cys Tyr Asp Ala Pro Gly Gly Phe Arg Cys Val Cys
705 710 715 720
Glu Pro Gly Trp Ser Gly Pro Arg Cys Ser Gln Ser Leu Ala Arg Asp
725 730 735
Ala Cys Glu Ser Gln Pro Cys Arg Ala Gly Gly Thr Cys Ser Ser Asp
740 745 750
Gly Met Gly Phe His Cys Thr Cys Pro Pro Gly Val Gln Gly Arg Gln
755 760 765
Cys Glu Leu Leu Ser Pro Cys Thr Pro Asn Pro Cys Glu His Gly Gly
770 775 780
Arg Cys Glu Ser Ala Pro Gly Gln Leu Pro Val Cys Ser Cys Pro Gln
785 790 795 800
Gly Trp Gln Gly Pro Arg Cys Gln Gln Asp Val Asp Glu Cys Ala Gly
805 810 815
Pro Ala Pro Cys Gly Pro His Gly Ile Cys Thr Asn Leu Ala Gly Ser
820 825 830
Phe Ser Cys Thr Cys His Gly Gly Tyr Thr Gly Pro Ser Cys Asp Gln
835 840 845
Asp Ile Asn Asp Cys Asp Pro Asn Pro Cys Leu Asn Gly Gly Ser Cys
850 855 860
Gln Asp Gly Val Gly Ser Phe Ser Cys Ser Cys Leu Pro Gly Phe Ala
865 870 875 880
Gly Pro Arg Cys Ala Arg Asp Val Asp Glu Cys Leu Ser Asn Pro Cys
885 890 895
Gly Pro Gly Thr Cys Thr Asp His Val Ala Ser Phe Thr Cys Thr Cys
900 905 910
Pro Pro Gly Tyr Gly Gly Phe His Cys Glu Gln Asp Leu Pro Asp Cys
915 920 925
Ser Pro Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Val Asn
930 935 940
Ser Phe Ser Cys Leu Cys Arg Pro Gly Tyr Thr Gly Ala His Cys Gln
945 950 955 960
His Glu Ala Asp Pro Cys Leu Ser Arg Pro Cys Leu His Gly Gly Val
965 970 975
Cys Ser Ala Ala His Pro Gly Phe Arg Cys Thr Cys Leu Glu Ser Phe
980 985 990
Thr Gly Pro Gln Cys Gln Thr Leu Val Asp Trp Cys Ser Arg Gln Pro
995 1000 1005
Cys Gln Asn Gly Gly Arg Cys Val Gln Thr Gly Ala Tyr Cys Leu Cys
1010 1015 1020
Pro Pro Gly Trp Ser Gly Arg Leu Cys Asp Ile Arg Ser Leu Pro Cys
1025 1030 1035 1040
Arg Glu Ala Ala Ala Gln Ile Gly Val Arg Leu Glu Gln Leu Cys Gln
1045 1050 1055
Ala Gly Gly Gln Cys Val Asp Glu Asp Ser Ser His Tyr Cys Val Cys
1060 1065 1070
Pro Glu Gly Arg Thr Gly Ser His Cys Glu Gln Glu Val Asp Pro Cys
1075 1080 1085
Leu Ala Gln Pro Cys Gln His Gly Gly Thr Cys Arg Gly Tyr Met Gly
1090 1095 1100
Gly Tyr Met Cys Glu Cys Leu Pro Gly Tyr Asn Gly Asp Asn Cys Glu
1105 1110 1115 1120
Asp Asp Val Asp Glu Cys Ala Ser Gln Pro Cys Gln His Gly Gly Ser
1125 1130 1135
Cys Ile Asp Leu Val Ala Arg Tyr Leu Cys Ser Cys Pro Pro Gly Thr
1140 1145 1150
Leu Gly Val Leu Cys Glu Ile Asn Glu Asp Asp Cys Gly Pro Gly Pro
1155 1160 1165
Pro Leu Asp Ser Gly Pro Arg Cys Leu His Asn Gly Thr Cys Val Asp
1170 1175 1180
Leu Val Gly Gly Phe Arg Cys Thr Cys Pro Pro Gly Tyr Thr Gly Leu
1185 1190 1195 1200
Arg Cys Glu Ala Asp Ile Asn Glu Cys Arg Ser Gly Ala Cys His Ala
1205 1210 1215
Ala His Thr Arg Asp Cys Leu Gln Asp Pro Gly Gly Gly Phe Arg Cys
1220 1225 1230
Leu Cys His Ala Gly Phe Ser Gly Pro Arg Cys Gln Thr Val Leu Ser
1235 1240 1245
Pro Cys Glu Ser Gln Pro Cys Gln His Gly Gly Gln Cys Arg Pro Ser
1250 1255 1260
Pro Gly Pro Gly Gly Gly Leu Thr Phe Thr Cys His Cys Ala Gln Pro
1265 1270 1275 1280
Phe Trp Gly Pro Arg Cys Glu Arg Val Ala Arg Ser Cys Arg Glu Leu
1285 1290 1295
Gln Cys Pro Val Gly Val Pro Cys Gln Gln Thr Pro Arg Gly Pro Arg
1300 1305 1310
Cys Ala Cys Pro Pro Gly Leu Ser Gly Pro Ser Cys Arg Ser Phe Pro
1315 1320 1325
Gly Ser Pro Pro Gly Ala Ser Asn Ala Ser Cys Ala Ala Ala Pro Cys
1330 1335 1340
Leu His Gly Gly Ser Cys Arg Pro Ala Pro Leu Ala Pro Phe Phe Arg
1345 1350 1355 1360
Cys Ala Cys Ala Gln Gly Trp Thr Gly Pro Arg Cys Glu Ala Pro Ala
1365 1370 1375
Ala Ala Pro Glu Val Ser Glu Glu Pro Arg Cys Pro Arg Ala Ala Cys
1380 1385 1390
Gln Ala Lys Arg Gly Asp Gln Arg Cys Asp Arg Glu Cys Asn Ser Pro
1395 1400 1405
Gly Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Ser Val Gly Asp Pro
1410 1415 1420
Trp Arg Gln Cys Glu Ala Leu Gln Cys Trp Arg Leu Phe Asn Asn Ser
1425 1430 1435 1440
Arg Cys Asp Pro Ala Cys Ser Ser Pro Ala Cys Leu Tyr Asp Asn Phe
1445 1450 1455
Asp Cys His Ala Gly Gly Arg Glu Arg Thr Cys Asn Pro Val Tyr Glu
1460 1465 1470
Lys Tyr Cys Ala Asp His Phe Ala Asp Gly Arg Cys Asp Gln Gly Cys
1475 1480 1485
Asn Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys Ala Ser Glu Val
1490 1495 1500
Pro Ala Leu Leu Ala Arg Gly Val Leu Val Leu Thr Val Leu Leu Pro
1505 1510 1515 1520
Pro Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe Leu Gln Arg Leu Ser
1525 1530 1535
Ala Ile Leu Arg Thr Ser Leu Arg Phe Arg Leu Asp Ala His Gly Gln
1540 1545 1550
Ala Met Val Phe Pro Tyr His Arg Pro Ser Pro Gly Ser Glu Pro Arg
1555 1560 1565
Ala Arg Arg Glu Leu Ala Pro Glu Val Ile Gly Ser Val Val Met Leu
1570 1575 1580
Glu Ile Asp Asn Arg Leu Cys Leu Gln Ser Pro Glu Asn Asp His Cys
1585 1590 1595 1600
Phe Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu Gly Ala Leu Ser Ala
1605 1610 1615
Val Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg Asp Val Arg Gly Glu
1620 1625 1630
Pro Leu Glu Pro Pro Glu Pro Ser Val Pro Leu Leu Pro Leu Leu Val
1635 1640 1645
Ala Gly Ala Val Leu Leu Leu Val Ile Leu Val Leu Gly Val Met Val
1650 1655 1660
Ala Arg Arg Lys Arg Glu His Ser Thr Leu Trp Phe Pro Glu Gly Phe
1665 1670 1675 1680
Ser Leu His Lys Asp Val Ala Ser Gly His Lys Gly Arg Arg Glu Pro
1685 1690 1695
Val Gly Gln Asp Ala Leu Gly Met Lys Asn Met Ala Lys Gly Glu Ser
1700 1705 1710
Leu Met Gly Glu Val Ala Thr Asp Trp Met Asp Thr Glu Cys Pro Glu
1715 1720 1725
Ala Lys Arg Leu Lys Val Glu Glu Pro Gly Met Gly Ala Glu Glu Ala
1730 1735 1740
Val Asp Cys Arg Gln Trp Thr Gln His His Leu Val Ala Ala Asp Ile
1745 1750 1755 1760
Arg Val Ala Pro Ala Met Ala Leu Thr Pro Pro Gln Gly Asp Ala Asp
1765 1770 1775
Ala Asp Gly Met Asp Val Asn Val Arg Gly Pro Asp Gly Phe Thr Pro
1780 1785 1790
Leu Met Leu Ala Ser Phe Cys Gly Gly Ala Leu Glu Pro Met Pro Thr
1795 1800 1805
Glu Glu Asp Glu Ala Asp Asp Thr Ser Ala Ser Ile Ile Ser Asp Leu
1810 1815 1820
Ile Cys Gln Gly Ala Gln Leu Gly Ala Arg Thr Asp Arg Thr Gly Glu
1825 1830 1835 1840
Thr Ala Leu His Leu Ala Ala Arg Tyr Ala Arg Ala Asp Ala Ala Lys
1845 1850 1855
Arg Leu Leu Asp Ala Gly Ala Asp Thr Asn Ala Gln Asp His Ser Gly
1860 1865 1870
Arg Thr Pro Leu His Thr Ala Val Thr Ala Asp Ala Gln Gly Val Phe
1875 1880 1885
Gln Ile Leu Ile Arg Asn Arg Ser Thr Asp Leu Asp Ala Arg Met Ala
1890 1895 1900
Asp Gly Ser Thr Ala Leu Ile Leu Ala Ala Arg Leu Ala Val Glu Gly
1905 1910 1915 1920
Met Val Glu Glu Leu Ile Ala Ser His Ala Asp Val Asn Ala Val Asp
1925 1930 1935
Glu Leu Gly Lys Ser Ala Leu His Trp Ala Ala Ala Val Asn Asn Val
1940 1945 1950
Glu Ala Thr Leu Ala Leu Leu Lys Asn Gly Ala Asn Lys Asp Met Gln
1955 1960 1965
Asp Ser Lys Glu Glu Thr Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser
1970 1975 1980
Tyr Glu Ala Ala Lys Leu Leu Leu Asp His Phe Ala Asn Arg Glu Ile
1985 1990 1995 2000
Thr Asp His Leu Asp Arg Leu Pro Arg Asp Val Ala Gln Glu Arg Leu
2005 2010 2015
His Gln Asp Ile Val Arg Leu Leu Asp Gln Pro Ser Gly Pro Arg Ser
2020 2025 2030
Pro Pro Gly Pro His Gly Leu Gly Pro Leu Leu Cys Pro Pro Gly Ala
2035 2040 2045
Phe Leu Pro Gly Leu Lys Ala Ala Gln Ser Gly Ser Lys Lys Ser Arg
2050 2055 2060
Arg Pro Pro Gly Lys Ala Gly Leu Gly Pro Gln Gly Pro Arg Gly Arg
2065 2070 2075 2080
Gly Lys Lys Leu Thr Leu Ala Cys Pro Gly Pro Leu Ala Asp Ser Ser
2085 2090 2095
Val Thr Leu Ser Pro Val Asp Ser Leu Asp Ser Pro Arg Pro Phe Gly
2100 2105 2110
Gly Pro Pro Ala Ser Pro Gly Gly Phe Pro Leu Glu Gly Pro Tyr Ala
2115 2120 2125
Ala Ala Thr Ala Thr Ala Val Ser Leu Ala Gln Leu Gly Gly Pro Gly
2130 2135 2140
Arg Ala Gly Leu Gly Arg Gln Pro Pro Gly Gly Cys Val Leu Ser Leu
2145 2150 2155 2160
Gly Leu Leu Asn Pro Val Ala Val Pro Leu Asp Trp Ala Arg Leu Pro
2165 2170 2175
Pro Pro Ala Pro Pro Gly Pro Ser Phe Leu Leu Pro Leu Ala Pro Gly
2180 2185 2190
Pro Gln Leu Leu Asn Pro Gly Thr Pro Val Ser Pro Gln Glu Arg Pro
2195 2200 2205
Pro Pro Tyr Leu Ala Val Pro Gly His Gly Glu Glu Tyr Pro Val Ala
2210 2215 2220
Gly Ala His Ser Ser Pro Pro Lys Ala Arg Phe Leu Arg Val Pro Ser
2225 2230 2235 2240
Glu His Pro Tyr Leu Thr Pro Ser Pro Glu Ser Pro Glu His Trp Ala
2245 2250 2255
Ser Pro Ser Pro Pro Ser Leu Ser Asp Trp Ser Glu Ser Thr Pro Ser
2260 2265 2270
Pro Ala Thr Ala Thr Gly Ala Met Ala Thr Thr Thr Gly Ala Leu Pro
2275 2280 2285
Ala Gln Pro Leu Pro Leu Ser Val Pro Ser Ser Leu Ala Gln Ala Gln
2290 2295 2300
Thr Gln Leu Gly Pro Gln Pro Glu Val Thr Pro Lys Arg Gln Val Leu
2305 2310 2315 2320
Ala

3

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

3
ctgcaggtga ggggc 15

4

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

4
cccacacagc ccccc 15

5

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

5
ctgcctgtga gtgcc 15

6

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

6
gcccacaggt gccc 14

7

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

7
tccgaggtga gagg 14

8

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

8
ccctccaggc cctg 14

9

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

9
ttcctggtga gtga 14

10

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

10
cttgttaggg tttg 14

11

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

11
ggacaggtgg gcac 14

12

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

12
tgccacaggc cagt 14

13

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

13
agactggtga gtgg 14

14

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

14
cttcccaggc ctcc 14

15

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

15
ctatcggtga gggg 14

16

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

16
tccggcaggc gcca 14

17

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

17
tggcaggtgg gtgg 14

18

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

18
tgccccaggc ttca 14

19

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

19
cctcgggtga ggac 14

20

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

20
caccccaggc ttca 14

21

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

21
ccgagggtga ggcg 14

22

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

22
ccccacaggc tttg 14

23

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

23
ccacaggtgg gacc 14

24

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

24
gcccctaggt gtga 14

25

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

25
tcacaggtgg gcaa 14

26

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

26
ctccccaggg cccc 14

27

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

27
tggcgggtga gggc 14

28

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

28
cctgccaggt tccg 14

29

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

29
tccagggtgt gtac 14

30

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

30
cccaacagga cgtc 14

31

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

31
ggcaaggtat gccac 15

32

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

32
tacccccagg cccac 15

33

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

33
accccagtga gtgca 15

34

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

34
gtccgcagac ccat 14

35

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

35
ccccaggtgg gcgg 14

36

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

36
cgctccagct cctg 14

37

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

37
tgccaggtgg gtgg 14

38

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

38
ccctccagac gctg 14

39

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

39
agatcggtga gtgg 14

40

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

40
ctttgcaggg gtgc 14

41

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

41
tgtgaggtaa gggg 14

42

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

42
cactgaagtg tctt 14

43

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

43
cgctgggtat gcca 14

44

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

44
tcccccaggg gtgc 14

45

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

45
tctcaggtta acct 14

46

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

46
tcgctcaggt cctc 14

47

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

47
gcccaggtag gtgtg 15

48

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

48
gacccccagc cgttc 15

49

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

49
ttgcaagtga gccc 14

50

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

50
cccaccagcc cggt 14

51

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

51
gatcgggtga gtgac 15

52

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

52
tccctgcagc tcggt 15

53

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

53
tgcggggtgc ggcc 14

54

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

54
tgctcttagg ggagc 15

55

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

55
catgaagtga gaac 14

56

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

56
tccgccagga acat 14

57

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

57
ctaaaggtac tgcc 14

58

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

58
cccctccagg tagag 15

59

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

59
gcccaggtca gtgac 15

60

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

60
ccctgcagat ggct 14

61

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

61
ttccaggtga gata 14

62

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

62
tgtcctagat tctc 14

63

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

63
agcttggtag gttg 14

64

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

64
ccctccaggg aaat 14

65

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

65
agcaaggtga gccc 14

66

14

DNA

Artificial Sequence

Description of Artificial Sequence primer

66
ccccccagga ggag 14

67

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

67
aaggagggag gaggggag 18

68

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

68
tgggggttct tgcactcc 18

69

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

69
ggttcctgcc tcccatga 18

70

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

70
tcctccacct tccttcac 18

71

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

71
acacacaggg cccactggt 19

72

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

72
tgtgctgccc aaccaagcca 20

73

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

73
actgaccaca cccccgacta 20

74

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

74
tagtcggggg tgtggtcagt 20

75

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

75
tcatccacgt cgcttcggca 20

76

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

76
atggacgctt cctctgctc 19

77

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

77
acatagtggc cctgtgtagc 20

78

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

78
atggacgctt cctctgctcc 20

79

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

79
cctctgactc tcctgagtag 20

80

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

80
tgaccatcct tgccccctt 19

81

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

81
ctggcctgtg gcacacagat 20

82

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

82
tggactgctg catctgtgtg 20

83

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

83
acacgcctgt ggcacagtca 20

84

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

84
gagctgcagt cagaatatcg 20

85

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

85
atccatggct ccctgcagag 20

86

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

86
cagagcagga agatctgcct 20

87

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

87
cattcacaga cgacggagct 20

88

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

88
atcgcactcc atccggca 18

89

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

89
acccacctgc catacaga 18

90

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

90
cgttcacacc atagggtagc 20

91

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

91
ccccttccca gacatgtctt 20

92

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

92
cttgtcggac tgtcattgg 19

93

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

93
gtgtactgct ctcaccctt 19

94

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

94
attggtccga ggcctcactt 20

95

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

95
acctggctct cgcagcgtgt 20

96

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

96
ccattcccaa cccctctgtg 20

97

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

97
tgcctgtgct cctggctaca 20

98

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

98
tggccactcc atgccatgtt 20

99

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

99
tctcatggca gccacttgcc 20

100

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

100
atgagtgtgc ttccagccca 20

101

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

101
gcagtgtctg aggctgagaa 20

102

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

102
tccctggcct gactaccttc 20

103

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

103
ctgcagaggg aaggtgaggt 20

104

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

104
aaggctatcc tgcttcc 17

105

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

105
gaggaggagg gaagagaa 18

106

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

106
aggatgtgga cgagtgtgct 20

107

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

107
gcttaatgac tgtgttcc 18

108

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

108
tcagactggg ctaatggggg 20

109

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

109
tcgcagtgga agcctccgta 20

110

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

110
gatgtggatg agtgcctgag 20

111

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

111
gtcctgctct tcaagcaga 19

112

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

112
gatcctccct cccactcctt 20

113

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

113
aggtccccag taactcca 18

114

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

114
actgactcta agtgcttccc 20

115

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

115
agcaggaggt acgtgcatga 20

116

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

116
tgttcctgtg ccactctcct 20

117

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

117
acctcctctt ccctctcct 19

118

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

118
tctgtgtccc actaagctga 20

119

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

119
caagaggaaa tgaagacagc 20

120

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

120
ttcctcttga ccacccctcg 20

121

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

121
tggcaggcac ctgagcgaca 20

122

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

122
caggatacac tggtttgcgc 20

123

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

123
tgccacgtta tggatcagcc 20

124

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

124
gatctacatg ctcccgctcg 20

125

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

125
tactcctcct ccataggccg 20

126

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

126
cgttctgggg tccgcgtt 18

127

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

127
aagcgcagcg gaagaagggc 20

128

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

128
gcccttcttc cgctgcgctt 20

129

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

129
actgcagcgc ctcgcattgc 20

130

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

130
ctgcgaccgc gagtgcaaca 20

131

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

131
atagacagac ggatcgat 18

132

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

132
ctctctgcct caccctt 17

133

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

133
gctggaacgc agtagct 17

134

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

134
tgctcacagt gctgctg 17

135

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

135
cacggctttt ccaggtg 17

136

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

136
tttgagccct ctggtcc 17

137

17

DNA

Artificial Sequence

Description of Artificial Sequence primer

137
aagagcagga agcagag 17

138

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

138
tccctctgct tcctgctctt 20

139

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

139
tcacaaggtc cccgtagtca 20

140

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

140
ctcacatccc ctcttcccat 20

141

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

141
atcacgccca tcatccactg 20

142

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

142
cagcaccaaa gggtg 15

143

15

DNA

Artificial Sequence

Description of Artificial Sequence primer

143
catccctttg ggagg 15

144

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

144
atggcttcac cccgctaatg 20

145

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

145
agccaggtgc aaagcagtct 20

146

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

146
tcagcttggg gcacggactg 20

147

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

147
gcatcggctg tgacagctgt 20

148

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

148
tgttcctgcc atgacccct 19

149

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

149
caggtgacac taacccagtc 20

150

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

150
tcctgacctc tctccccttc 20

151

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

151
aatggcgctg tgccactgct 20

152

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

152
gctactgtta gctggggttt 20

153

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

153
tgatccagca agcgcacgat 20

154

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

154
tcaccgacca cctggaca 18

155

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

155
accaagctgt gccagaga 18

156

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

156
tccaagaaga gcaggagg 18

157

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

157
accaagctgt gccagaga 18

158

20

DNA

Artificial Sequence

Description of Artificial Sequence primer

158
cagtgtctct ggcacagctt 20

159

19

DNA

Artificial Sequence

Description of Artificial Sequence primer

159
tcctgggact gccaggtaa 19

160

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

160
agctgctcaa cccaggga 18

161

18

DNA

Artificial Sequence

Description of Artificial Sequence primer

161
gtggattcgg accagtct 18

162

16

DNA

Artificial Sequence

Description of Artificial Sequence primer

162
gaatcccctg agcact 16

163

16

DNA

Artificial Sequence

Description of Artificial Sequence primer

163
ctaagaactg acgagc 16

Number	Date	Country	Kind
96 09733	Aug 1996	FR
97 04680	Apr 1997	FR

Gene involved in cadasil, method of diagnosis and therapeutic application

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (2)

PCT Information

Foreign Referenced Citations (1)

Non-Patent Literature Citations (12)

Entry
Sambrook et al Molecular Cloning, A Laboratory Manual, 2nd, 16.1-16.81, 1989.*
Rudinger et al., in “Peptide Hormones”, edited by Parsons, J.A., University Park Press, Jun. 1976, p. 6.*
Burgess et al., The Journal of Cell Biology, 111:2129-2138, 1990.*
Lardelli et al., “The Novel Notch Homologue Mouse Notch 3 Lacks specific Epidermal Growth Factor-Repeats and is Expressed in Proliferating Neuroepithelium”, Mech. of Develop., 46:123-136 (1994).
Lindsell et al., “Expression Patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 Genes Identify Ligand-Receptor Pairs that may Function in Neural Development”, Mole. and Cell. Neuroscience, 8:14-27 (1996).
Ducros et al., “Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, Genetic Homogeneity, and Mapping of The Locus Within a 2-cM Interval”, A.m. J. Hum. Genet., 58:171-181 (1996).
Joutel et al., “Notch3 Mutations in Cadasil, A Hereditary Adult-Onset Condition Causing Stroke and Dementia”, Nature, 383:707-710.
Lardelli et al., “Expression of the Notch 3 intracellular Domain in Mouse Central Nervous System Progenitor Cells is Lethal and Leads to Disturbed Neural tube Development”, Mech. of Develop., 59:177-190.
Ophoff et al., “Gene For Familial Hemiplegic Migraine on Chromosome 19p13”, Posters: Molecular Etiology of Disease, vol. 57, No. 4, pp. A222, Abstract 1284 (1995).
Joutel et al., “Identification of Expressed Sequences from the Cadasil Region ON 19p”, Amer. J. of Human Genet., vol. 57, No. 3, pp. A342, Abstract 1985 (1995).
Joutel et al., “Identification of the Cadasil Gene”, Stroke, vol. 28, No. 1, pp. 246, Abstract 65 (1997).
C. Larsson et al., “The Human NOTCH1, 2, and 3 Genes Are Located at Chromosome Positions 9q34, 1p13-p11, and 19p.13.2-p13.1 in Regions of Neoplasia-Associated Translocation,” Genomics, vol. 24, No. 2, pp. 253-258 (1994).