WO2001036632A2 - Variants of alternative splicing - Google Patents

Variants of alternative splicing Download PDF

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Publication number
WO2001036632A2
WO2001036632A2 PCT/IL2000/000766 IL0000766W WO0136632A2 WO 2001036632 A2 WO2001036632 A2 WO 2001036632A2 IL 0000766 W IL0000766 W IL 0000766W WO 0136632 A2 WO0136632 A2 WO 0136632A2
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Prior art keywords
nucleic acid
amino acid
acid sequence
sequence
variant
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PCT/IL2000/000766
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French (fr)
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WO2001036632A3 (en
Inventor
Zurit Levine
Anat David
Idit Azar
Rami Khosravi
Jeanne Bernstein
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Compugen Ltd.
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Priority claimed from IL13297899A external-priority patent/IL132978A0/en
Priority claimed from IL13345599A external-priority patent/IL133455A0/en
Application filed by Compugen Ltd. filed Critical Compugen Ltd.
Priority to AU14108/01A priority Critical patent/AU1410801A/en
Publication of WO2001036632A2 publication Critical patent/WO2001036632A2/en
Publication of WO2001036632A3 publication Critical patent/WO2001036632A3/en
Priority to US11/709,841 priority patent/US20070219125A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/15—Peptidyl-dipeptidases (3.4.15)
    • C12Y304/15001—Peptidyl-dipeptidase A (3.4.15.1)

Definitions

  • the present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above.
  • the present invention further concerns methods for screening for candidate activators or deactivators utilizing said amino acid sequences.
  • AS Alternative splicing
  • Angiotensin I-converting enzyme is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrpphage system.
  • ACE Angiotensin I-converting enzyme
  • ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin.
  • SACE serum ACE activity
  • SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis.
  • nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis
  • extrathoracic granulomatous pathologies such as Gauchers disease and leprosis
  • nongranulomatous disorders such as hyperthyroidism or cholestasis.
  • SACE Strethelial astolic endothelial astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolic astolica, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors.
  • endothelial abnormality such as deep vein thrombosis
  • SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors.
  • angiotensin II increases the production of several autocrine factors, including transforming growth factor betal (TGF-betal), tumor necrosis factor-alpha (TNF-alpha), and platelet-derived growth factor A chain (PDGF).
  • TGF-betal transforming growth factor betal
  • TNF-alpha tumor necrosis factor-alpha
  • PDGF platelet-derived growth factor A chain
  • Angiotensin also increases the release of other growth factors such as endothelin, platelet-activating factor (PAF), and interleukin 6.
  • PAF platelet-activating factor
  • NF-kappaB nuclear factor-kappaB
  • the emerging picture indicates that the actions of angiotensin II may be related to factors that are released or upregulated by angiotensin II, possibly through NF-kappaB.
  • Variant nucleic acid sequence the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 87, native and known genes. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of genes and not merely truncated, mutated or fragmented forms of known sequences which are artificially produced.
  • Angiotensin converting enzyme variant (ACEV) - a sequence shown in SEQ ID NO: 57 or 85 sequences having at least 90% identity (see below) to said sequence and fragments (see below) of the above sequences of least 20 b.p. long.
  • sequences are sequences coding for a novel, naturally occurring, alternative splice variants of the mouse angiotensin converting enzyme which convert angiotensin I to angiotensin II by release of the terminal His-Lew resulting in increase of vasoconstrictor activity of angiotensin.
  • Variant product - also referred at times as the "variant protein” or “variant polypeptide” - is an amino acid sequence encoded by the variant nucleic acid sequence SEQ ID NO: 88 to SEQ ID NO: 174.
  • AACEV product or ACEV protein amino acid coded by the ACEV nucleic acid which is a naturally occurring mRNA sequence obtained as a result of alternative splicing of the ACE gene.
  • the amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein.
  • the variant products are shown in SEQ ID NO: 144 or 172.
  • the term also includes homologies (see below) of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments (see below) of this sequence having at least 10 amino acids. The above two products may be secreted.
  • Nucleic acid sequence a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
  • amino acid sequence - a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
  • the fragment may be a sequence which was previously undescribed in the context of the published RNA and which affects the amino acid sequence encoded by the known gene. For example, where the variant nucleic includes a sequence which was not included in the original sequence (for example a sequence which was an intron in the original sequence) the fragment may contain said additional sequence.
  • the fragment may also be a region which is not an intron, which was not present in the original sequence.
  • the variant lacks a non-terminal region which was present in the original sequence.
  • the two stretches of nucleotides spanning this region are brought together by splicing in the variant, but are spaced from each by the spliced out region in the original sequence and are thus not continuous in the original sequence.
  • a continuous stretch of nucleic acids comprising said two sparing stretches of nucleotides is not present in the original sequence and thus falls under the definition of fragment.
  • Constant substitution refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • Class I Cys
  • Class II Ser, Thr, Pro, Ala, Gly
  • Class III Asn, Asp, Gin, Glu
  • Class IV His, Arg, Lys
  • Class V He, Leu, Val, Met
  • Class VI Phe, Tyr, Trp
  • Non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
  • “Chemically modified” - when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
  • modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
  • Bioly active refers to the variant product having some sort of biological activity, for example, some physiologically measurable effect on target cells, molecules or tissues.
  • immunologically active defines the capability of a natural, recombinant or synthetic variant product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • an immunologically active fragment of variant product denotes a fragment which retains some or all of the immunological properties of the variant product, e.g. can bind specific anti-variant product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce variant.
  • Optimal alignment is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from Mac Vector (TM), operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence). In case of alignments of known gene sequences with that of the new variant, the optimal alignment invariably included aligning the identical parts of both sequences together, then keeping apart and unaligned the sections of the sequences that differ one from the other.
  • Isolated nucleic acid molecule having an variant nucleic acid sequence is a nucleic acid molecule that includes the coding variant nucleic acid sequence.
  • Said isolated nucleic acid molecule may include the variant nucleic acid sequence as an independent insert; may include the variant nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the variant coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the variant nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5' and/or 3 1 untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the variant protein coding sequence is a heterologous.
  • non-coding sequences e.g., introns or control elements, such as promoter and terminator elements or 5' and/or 3 1 untranslated regions
  • Expression vector refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • “Deletion " - is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • “Insertion” or “addition” - is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
  • substitution - replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non- conservative.
  • Antibody - refers to IgG, IgM, IgD, IgA, or IgG antibody. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-variant product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
  • Distinguishing antibody an antibody capable of binding to the variant product and not the original amino acid sequence from which it has been varied, or an antibody capable of binding to the original nucleic acid sequence and not to the variant production.
  • Activator refers to a molecule which mimics the effect of the natural variant product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product.
  • the mechanism may be by any mechanism known to prolonging activities of biological molecules such as binding to receptors; prolonging the lifetime of the molecules; increasing the activity of the molecules on its target; increasing the affinity of molecules to its receptor; inhibiting degradation or proteolysis of the molecules, or mimicking the biological activity of the variants on their targets, etc.
  • Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the variant product.
  • Deactivator or (“Inhibitor”) - refers to a molecule which modulates the activity of the variant product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the variant product. This may be done by any mechanism known to deactivate or inhibit biological molecules such as block of the receptor, block of active site, competition on binding site in target, enhancement of degradation, etc.
  • Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
  • Treating a disease refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
  • Detection refers to a method of detection of a disease, disorder, pathological or normal condition. This term may refer to detection of a predisposition to a disease as well as for establishing the prognosis of the patient by determining the severity of the disease.
  • Probe the variant nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence.
  • the probe may be attached to a solid support or to a detectable label.
  • the present invention is based on the finding of several novel, naturally occurring splice variants, which are naturally occurring sequences obtained by alternative splicing of known genes.
  • novel splice variants of the invention are not merely truncated forms, fragments or mutations of known genes, but rather novel sequences which naturally occur within the body of individuals.
  • the present invention concerns variants of alternative splice variants of angiotensin converting enzyme (ACEV).
  • AAV angiotensin converting enzyme
  • alternative splicing in the context of the present invention and claims refers to: intron inclusion, exon exclusion, addition or deletion of terminal sequences in the variant as compared to the original sequences, as well as to the possibility of "intron retention ".
  • Intron retention is an intermediate stage in the processing of RNA transcripts, where prior to production of fully processed mRNA the intron (naturally spliced in the original sequence) is retained in the variant.
  • These intermediately processed RNAs may have physiological significance and are also within the scope of the invention.
  • novel variant products of the invention including the ACEV-variant (ACEV) may have the same physiological activity as the original peptide from which they have been varied (although perhaps at a different level); may have an opposite physiological activity from the activity featured by the original peptide from which they are varied; may have a completely different, unrelated activity to the activity of the original from which they are varied; or alternatively may have no activity at all and this may lead to various diseases or pathological conditions.
  • the novel variants of the invention may differ from the original sequence, from which they were varied by alternative splicing, by physiological properties not relating directly to their activities such as: tissue localization, temporal pattern of expression, rate of clearance, rate of degradation, manner of up- or down regulation, association with co-factors and cellular elements etc.
  • novel variants may also serve for detection purposes, i.e. their presence or level may be indicative of a disease, disorder, pathological or normal condition or alternatively the ratio between the level variants and the level original peptide from which they were varied, or the ratio to other variants may be indicative to a disease, disorder, pathological or normal condition.
  • a certain variant may be expressed mainly in one tissue, while the original sequence from which it has been varied, or another variant may, be expressed mainly in another tissue. Understanding of the distribution of the variants in various tissues may be helpful in basic research, for understanding the physiological function of the genes as well as may help in targeting pharmaceuticals or developing pharmaceuticals.
  • the study of the variants may also be helpful to distinguish various stages in the life cycles of the same type of cells which may also be helpful for development of pharmaceuticals for various pathological conditions in which cell cycles is non-normal, notably cancer.
  • Detection of various diseases in accordance with the invention is especially useful for detection of diseases which are associated with the function, (over function, under function, or malfunction) of proteins of the original sequence from which each variant of the invention has been obtained by alternative splicing.
  • a list of the original proteins are given in the "Detailed Description" part of the specification.
  • variant of SEQ ID NO: 3 is obtained from an original sequence which is coagulation factor XII
  • this sequence may be used to detect diseases involving excessive or diminished blood coagulation.
  • the detection may by determination of the presence or the level of expression of the variant within a specific cell population, comprising said presence or level between various cell types in a tissue, between different tissues and between individuals.
  • the detection may be used for detection (including disposition) of one of the following diseases.
  • Cardiovascular diseases are cardiovascular diseases.
  • Renal diseases Including hypertension, neurological damage due to cerebral circulatory disorders, peripheral vascular diseases, arteriosclerosis, heart and kidney diseases relating to blood pressure, erection problems and migraine problems relating to circulation functions, heart failures (including recurrent infraction in patients with left ventricular dysfunction), acute phase of myocardial infarction, coronary arterial thrombosis and cardial insufficiency. Renal diseases:
  • Hypertension adrenal injury (particularly in patients with type I or II diabetes), diabetic nepropathy, renal function deterioration in glomerular diseases
  • Muscular diseases Diseases involving growth of smooth muscle cells such as hypertrophy.
  • autoimmune diseases and diseases involving inflammatory mechanisms for example, autoimmune manifestation affects in sarcoidosis, generation of immune complex nephritis, autoimmune encephatomyelitis, marker for chronic fatigue-immune dysfunction syndrome.
  • cancers effected by different growth factors including endothelia, platelet-activating factor (PAF) and interleukin 6.
  • PAF platelet-activating factor
  • interleukin 6 interleukin 6.
  • Nonarcoidotic Pulmonary Granulomatous Diseases are diseases that are associated with the formation of granulomatous lesions that appear especially in the liver, lungs, skin and lymph nodes.
  • Nonarcoidotic Pulmonary Granulomatous Diseases are diseases that are associated with the formation of granulomatous lesions that appear especially in the liver, lungs, skin and lymph nodes.
  • Deep vein thrombosis and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations.
  • the present invention provides by its first aspect, a novel isolated nucleic acid molecule comprising or consisting of any one of the coding sequence SEQ ID NO: 1 to SEQ ID NO: 87, fragments of said coding sequence having at least 20 nucleic acids (provided that said fragments are continuous stretches of nucleotides not present in the original sequence from which the variant was varied), or a molecule comprising a sequence having at least 90%, identity to SEQ ID NO: 1 to SEQ ID NO: 87, provided that the molecule is not completely identical to the original sequence from which the variant was varied.
  • the above variant is that of SEQ ID NO: 57 or SEQ ID NO: 85 being the ACEV nucleic acid sequence.
  • the present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein "variant product" , for example, an amino acid sequence having the sequence as depicted in any one of SEQ ID NO: 88 to SEQ ID NO: 174, fragments of the above amino acid sequence having a length of at least 10 amino acids coded by the above fragments of the nucleic acid sequences, as well as homologues of the above amino acid sequences in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
  • the product is the amino acid sequence of the ACEV as depicted in SEQ ID NO: 144 or 172.
  • deletions, insertions and modifications should be in regions, or adjacent to regions, wherein the variant differs from the original sequence.
  • the invention also concerns homologues of that variant where the additional short stretch is altered for example, it includes only 8 additional amino acids, includes 13 additional amino acids, or it includes 10 additional amino acids, however some of them being conservative or non-conservative substitutes of the original additional 10 amino acids of the novel variants.
  • the changes in the homolog, as compared to the original sequence are in the same regions where the variant differs from the original sequence, or in regions adjacent to said region.
  • the variant lacks a non-terminal region (for example of 20 amino acids) which is present in the original sequence (due for example to exon exclusion).
  • the homologues may lack in the same region only 17 amino acids or 23 amino acids. Again the deletion is in the same region where the variant lacks a sequence as compared to the original sequence, or in a region adjacent thereto.
  • the present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences, (including the fragments and homologues of the amino acid sequences and in particular the ACEV amino acid sequence). Due to the degenerative nature of the genetic code, a plurality of alternative nucleic acid, beyond those depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 87, can code for the amino acid sequence of the invention. Those alternative nucleic acid sequences which code for the same amino acid sequences codes by the sequence SEQ ID NO: 1 to SEQ ID NO: 87 are also an aspect of the of the present invention.
  • the present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells fransfected by said vectors.
  • the present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
  • compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated or cured by raising the level of any one of the variant products of the invention.
  • diseases are diseases which are associated with malfunction or under function of the original sequence (for example, given in the "Detailed Description" part of the specification).
  • SEQ ID NO: 3 and sequences encoded thereby may be used to treat diseases associated with coagulation of blood.
  • the present invention provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of any one of SEQ ID NO: 1 to SEQ ID NO: 87, or complementary to a sequence having at least 90% identity to said sequence (with the proviso added above) or a fragment of said two sequences (according to the above definition of fragment).
  • the complementary sequence may be a DNA sequence which hybridizes with any one of SEQ of ID NO: 1 to SEQ ID NO: 87 or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence.
  • the complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed from any one of SEQ ID NO: 1 to SEQ ID NO: 87 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from any one of SEQ ID NO: 1 to SEQ ID NO: 87 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 87, so as to inhibit its translation.
  • the complementary sequence may also be the mRNA or the fragment of the mRNA itself.
  • the nucleic acids of the second aspect of the invention may be used for therapeutic or diagnostic applications for example as probes used for the detection of the variants of the invention.
  • the presence of the variant transcript or the level of the variant transcript may be indicative of a multitude of diseases, disorders and various pathological as well as normal conditions for example, as indicated above for the variants in general, and for the ACEV in particular.
  • the ratio of the level of the transcripts of the variants of the invention may also be compared to that of the transcripts of the original sequences from which have been varied, or to the level of transcript of other variants, and said ratio may be indicative to a multitude of diseases, disorders and various pathological and normal conditions.
  • the present invention also provides expression vectors comprising any one of the above defined complementary nucleic acid sequences and host cells fransfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
  • the invention also provides anti-variant product antibodies, namely antibodies directed against the variant product which specifically bind to said variant product. Said antibodies are useful both for diagnostic and therapeutic purposes.
  • said antibody may be as an active ingredient in a pharmaceutical composition as will be explained below.
  • the present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences.
  • the pharmaceutical composition thus provides pharmaceutical compositions comprising, as an active ingredient, said anti-variant product antibodies.
  • compositions comprising said anti-variant product antibodies or the nucleic acid molecule comprising said complementary sequence, are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing the variant (either at the transcript or product level) or decreasing the amount of the variant product or blocking its binding to its target, for example, by the neutralizing effect of the antibodies, or by the effect of the antisense mRNA in decreasing the expression level of the variant sequence.
  • ACEV sequence an expression vector comprising said complementary sequence
  • ACEV product or an antibody to the product is any one of the diseases mentioned in connection with the detection aspect above.
  • the present invention provides methods for detecting the level of the transcript (mRNA) of said variant product in a body fluid sample, or in a specific tissue sample, for example by use of probes comprising or consisting of said coding sequences; as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the variant products of the invention.
  • Detection of the level of the expression of the variant of the invention in particular as compared to that of the original sequence from which it was varied or compared to other variant sequences all varied from the same original sequence may be indicative of a plurality of physiological or pathological conditions.
  • a preferred example is the detection of ACEV nucleic acid sequence, ACEV product or anti-ACEV antibody.
  • the method, according to this latter aspect, for detection of a nucleic acid sequence which encodes the variant product in a biological sample comprises the steps of: (a) providing a probe comprising at least one of the nucleic acid sequences defined above;
  • the method as described above is qualitative, i.e. indicates whether the transcript is present in or absent from the sample.
  • the method can also be quantitative, by determining the level of hybridization complexes and then calibrating said levels to determining levels of transcripts of the desired variant in the sample.
  • the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
  • the nucleic acid sequence used in the above method may be a DNA sequence an RNA sequence, etc; it may be a coding or a sequence or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences).
  • RNA transcripts for respective detection of RNA transcripts or coding-DNA sequences.
  • Methods for detecting mutations in the region coding for the variant product are also provided, which may be methods carried-out in a binary fashion, namely merely detecting whether there is any mismatches between the normal variant nucleic acid sequence of the invention and the one present in the sample, or carried-out by specifically detecting the nature and location of the mutation.
  • the present invention also concerns a method for detecting variant product in a biological sample, comprising the steps of:
  • the present invention also concerns a method for detecting anti-variant antibody in a biological sample, comprising: (a) contacting said sample with the variant product of the invention, thereby forming an antibody-antigen complex; and
  • both methods for detection of variant product and for detection of the anti- variant antibody
  • the invention also concerns distinguishing antibodies, i.e. antibodies capable of binding either to the variant product or to the original sequence from which the variant has been varied, while not binding to the original sequence or the variant product respectively. These distinguishing antibodies may be used for detection purposes.
  • the invention also provides a method for identifying candidate compounds capable of binding to the variant product and modulating its activity (being either activators or deactivators).
  • the method includes:
  • the present invention also concerns compounds identified by the above methods described above, which compound may either be an activator of the variant product or a deactivator thereof.
  • Fig. 1 is a comparison between the amino acid sequence of SEQ ID NO: 88 and the original sequence from which it has been varied;
  • Fig. 2 is a comparison between the amino acid sequence of SEQ ID NO: 89 and the original sequence from which it has been varied;
  • Fig. 3 is a comparison between the amino acid sequence of SEQ ID NO: 90 and the original sequence from which it has been varied;
  • Fig. 4 is a comparison between the amino acid sequence of SEQ ID NO: 91 and the original sequence from which it has been varied;
  • Fig. 5 is a comparison between the amino acid sequence of SEQ ID NO: 92 and the original sequence from which it has been varied
  • Fig. 6 is a comparison between the amino acid sequence of SEQ ID NO: 93 and the original sequence from which it has been varied
  • Fig. 7 is a comparison between the amino acid sequence of SEQ ID NO: 94 and the original sequence from which it has been varied;
  • Fig. 8 is a comparison between the amino acid sequence of SEQ ID NO: 95 and the original sequence from which it has been varied;
  • Fig. 9 is a comparison between the amino acid sequence of SEQ ID NO: 96 and the original sequence from which it has been varied;
  • Fig. 10 is a comparison between the amino acid sequence of SEQ ID NO: 97 and the original sequence from which it has been varied;
  • Fig. 11 is a comparison between the amino acid sequence of SEQ ID NO: 97 and the original sequence from which it has been varied;
  • Fig. 12 is a comparison between the amino acid sequence of SEQ ID NO: 99 and the original sequence from which it has been varied;
  • Fig. 13 is a comparison between the amino acid sequence of SEQ ID NO: 100 and the original sequence from which it has been varied
  • Fig. 14 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
  • Fig. 15 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied
  • Fig. 16 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
  • Fig. 17 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
  • Fig. 18 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied;
  • Fig. 19 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
  • Fig. 20 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied;
  • Fig. 21 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
  • Fig. 22 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
  • Fig. 23 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied;
  • Fig. 24 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
  • Fig. 25 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied;
  • Fig. 26 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
  • Fig. 27 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
  • Fig. 28 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied
  • Fig. 29 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
  • Fig. 30 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied;
  • Fig. 31 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
  • Fig. 32 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
  • Fig. 33 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied;
  • Fig. 34 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
  • Fig. 35 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied
  • Fig. 36 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
  • Fig. 37 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
  • Fig. 38 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied;
  • Fig. 39 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
  • Fig. 40 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied
  • Fig. 41 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
  • Fig. 42 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
  • Fig. 43 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied;
  • Fig. 44 is a comparison between the amino acid sequence of SEQ ID NO: 131 and the original sequence from which it has been varied;
  • Fig. 45 is a comparison between the amino acid sequence of SEQ ID NO: 132 and the original sequence from which it has been varied;
  • Fig. 46 is a comparison between the amino acid sequence of SEQ ID NO: 132 and the original sequence from which it has been varied;
  • Fig. 47 is a comparison between the amino acid sequence of SEQ ID NO: 134 and the original sequence from which it has been varied;
  • Fig. 48 is a comparison between the amino acid sequence of SEQ ID NO: 135 and the original sequence from which it has been varied;
  • Fig. 49 is a comparison between the amino acid sequence of SEQ ID NO: 136 and the original sequence from which it has been varied;
  • Fig. 52 is a comparison between the amino acid sequence of SEQ ID NO: 139 and the original sequence from which it has been varied;
  • Fig. 53 is a comparison between the amino acid sequence of SEQ ID NO: 140 and the original sequence from which it has been varied;
  • Fig. 54 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 55 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 56 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 57 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 58 is a comparison between the amino acid sequence of SEQ ID NO: 145 and the original sequence from which it has been varied;
  • Fig. 59 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 60 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 61 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 62 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 63 is a comparison between the amino acid sequence of SEQ ID NO: 150 and the original sequence from which it has been varied;
  • Fig. 64 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 65 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 66 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 67 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 68 is a comparison between the amino acid sequence of SEQ ID NO: 155 and the original sequence from which it has been varied;
  • Fig. 69 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 70 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 71 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 72 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 73 is a comparison between the amino acid sequence of SEQ ID NO: 160 and the original sequence from which it has been varied;
  • Fig. 74 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 75 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 76 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 77 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 78 is a comparison between the amino acid sequence of SEQ ID NO: 165 and the original sequence from which it has been varied;
  • Fig. 79 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 80 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 81 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 82 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 83 is a comparison between the amino acid sequence of SEQ ID NO: 170 and the original sequence from which it has been varied;
  • Fig. 84 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 85 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 86 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 87 is a comparison between the amino acid sequence of SEQ ID NO:
  • Fig. 88 shows immunohistochemical staining with antibodies against a fragment of the ACEV product of SEQ ID NO: 144; expressed in ductal epitilus in salivatory gland (magnification X 100);
  • Fig. 89 shows the same as in Fig. 89 (magnification X 400);
  • Fig. 90 shows immunohistochemical staining with antibodies against a fragment of ACEV product of SEQ ID NO: 144 expressed in salivary glands surrounding the lymph nodes; and
  • Fig. 91 shows RT-PCR results of the ACEV sequence expressed in salivary glands.
  • Gap creation penalty (Gap Weight): 8 Gap extension penalty (GapLength Weight): 2
  • Fig. 1 to 87 show the comparison of each of the variant products depicted in SEQ ID NO: 88 to 174 with the original sequence from which it was varied.
  • SEQ ID 1 Description: Gap between amino acids at the positions 237-247 of the original protein. Missing 6th transmembrane loop of the original Adenosine A2 receptor.
  • Factor XII is a serum glycoprotein that participates in the initiation of blood coagulation, fibrinolysis, and the generation of bradykinin and angiotensin.
  • L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system, the postsynaptic actions of Glu are mediated by a variety of receptors are named according to their selective agonists
  • Glucagon Function Promotes hydrolysis of glycogen and lipids, and raises the blood sugar level.
  • Inhibin is a gonadal glycopeptide that inhibits the secretion of follitropin by the pituitary gland.
  • activin activates the secretion of follitropin. Activin is also important in embryonic axial development.
  • IL6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig-secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation.
  • IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig-secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation.
  • Relaxin is an ovarian hormone that acts with estrogen to
  • TSPIJHUMAN Name Thrombospondin adhesive glycoprotein 0 Function: Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen
  • Truncated exact 1-722 (731aa long compared to 1170aa), last 9 amino acids are different. Missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL, missing CELL ATTACHMENT SITE, missing 1 out of 4 glycosylation sites. Has all other components including signal peptide
  • Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen
  • Truncated exact 1-548 (555aa long compared to 1170) last 7aa different. Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS Ca-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylation sites. Has all other domains (including signal peptide).
  • Truncated exact 1-490 (546aa long compared to 1170) last 56 amino acids are different. Missing 1 out of 3 X TSP TYPE-1 REPEATS (CS-LIKE), Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylations. Has all other domains (including signal peptide).
  • Truncated exact 1-43 laa (459aa long compared to 1170) last 28 amino acids are different. Missing 2 out of 3 X TSP TYPE-1 REPEATS (CS-LIKE), Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylations. Has all other domains (including signal peptide).
  • FACTOR Function May have a role in maintaining the integrity of the vessels. Has growth promoting activity on endothelial cells, angiogenic activity in vivo and chemotactic activity on endothelial cells in vitro.
  • TNF receptor associated factors 1 and 2 (trafl and traf2) to form an heteromeric complex, which is then recruited to the tumor necrosis factor receptor 2 (TNFR2).
  • TNFR2 tumor necrosis factor receptor 2
  • Truncated 305 amino acids compared to 618 aa(protein 2).
  • the new variant contains exact positions 1-299, last 6 amino acids are different.
  • the new variant is missing Zn Finger and half of 3rd BIR repeat.
  • EGF_MOUSE Name PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial tissues
  • EGFJVIOUSE Name PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial
  • EGF_MOUSE Name PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial
  • NMDA receptor subtype of glutamate-gated ion channels possesses high calcium permeability and voltage-dependent sensitivity to magnesium and is mediated by glycine.
  • TRFE_HUMAN Name SEROTRANSFERRIN Function: Iron binding transport proteins SEQ ID : 37
  • SEQ ID : 40 Description: Replacement of 64 C-terminal amino acids of the original protein by alternative 7 amino acids. The resulting new variant is missing the last transmembrane and the cytoplasmic domains. Accession: PACR_HUMAN Name: PITUITARY ADENYLATE CYCLASE ACTIVATING
  • the activity of this receptor is mediated by G proteins which activate adenylyl cyclase. May regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, prolactin, epinephrine.
  • the new variant is missing part of the extracellular domain, the cytoplasmic and the transmembrane domains.
  • NRP_HUMAN Name NEUROPILIN VASCULAR ENDOTHELIAL CELL GROWTH FACTOR 165 RECEPTOR
  • ACH DOMINANT DISEASE
  • ACHONDROPLASIA ACH
  • ACH IS CHARACTERIZED BY A LONG, NARROW TRUNK, SHORT EXTREMITIES, PARTICULARLY IN THE PROXIMAL (RHIZOMELIC) SEGMENTS, A LARGE HEAD WITH FRONTAL BOSSING, HYPOPLASIA OF THE MIDFACE AND A TRIDENT CONFIGURATION OF THE HANDS.
  • CRANIOFACIAL DYSOSTOSIS TYPE I CRANIOFACIAL DYSOSTOSIS TYPE I (CFD1). CHARACTERIZED BY CRANIOSYNOSTOSIS (PREMATURE FUSION OF THE SKULL SUTURES), HYPERTELORISM, EXOPHTHALMOS AND
  • PALPEBRAL FISSURES PTOSIS, HIGHLY ARCHED PALATE, MID-TO-MODERATE SENSORINEURAL HEARING LOSS, NORMAL STATURE,
  • the deleted region includes the C-terminal part of the extracellular domain, the transmembrane domain, the cytoplasmic domain, the protein kinase domain and the two ATP binding domains.
  • the deleted region includes the EGF active chain, 4 out of 9 EGF-like domains within the extracellular region of the protein, the transmembrane and the cytoplasmic regions.
  • NV-20 contains the original signal peptide, part of the extracellular domain, the epidermal growth factor chain (located between the positions 977-1029 of the original protein), and the original transmembrane and the cytoplasmic domains.
  • NV-20 contains the original signal peptide, part of the extracellular domain, the epidermal growth factor chain (located between the positions 977-1029 of the original protein), and the original transmembrane and the cytoplasmic domains.
  • ANGIOTENSIN-CONVERTING ENZYME FUNCTION CONVERTS ANGIOTENSIN I TO ANGIOTENSIN II BY RELEASE OF THE TERMINAL HIS-LEU,THIS RESULTS IN AN INCREASE OF THE VASOCONSTRICTOR ACTIVITY OF ANGIOTENSIN.
  • CATALYTIC ACTIVITY RELEASE OF A C-TERMINAL DIPEPTIDE, OLIGOPEPTIDE-I-XAA-XBB, WHEN XAA IS NOT PRO, AND XBB IS NEITHER ASP NOR GLU. CONVERTS ANGIOTENSIN I TO ANGIOTENSIN II.
  • COF ACTOR BINDS TWO ZINC IONS (BY SIMILARITY).
  • SUBCELLULAR LOCATION TYPE I MEMBRANE PROTEIN.
  • ALTERNATIVE PRODUCTS THE TESTICULAR ANGIOTENSIN- CONVERTING ENZYME IS TRANSCRIBED FROM THE SAME GENE AS THE SOMATIC ISOFORM, PROBABLY FROM AN ALTERNATIVE START SITE.
  • DOMAIN COMPOSED OF THREE DOMAINS: A MODULATING N-TERMINAL DOMAIN, A DNA-BINDING DOMAIN AND A C-TERMINAL STEROID-BINDING DOMAIN. MISCELLANEOUS: IN THE ABSENCE OF LIGAND, STEROID HORMONE RECEPTORS ARE THOUGHT TO BE WEAKLY ASSOCIATED WITH NUCLEAR COMPONENTS; HORMONE BINDING GREATLY INCREASES RECEPTOR AFFINITY. THE HORMONE-RECEPTOR COMPLEX APPEARS TO RECOGNIZE DISCRETE DNA SEQUENCES UPSTREAM OF TRANSCRIPTIONAL START SITES. SIMILARITY: BELONGS TO THE NUCLEAR HORMONE RECEPTORS FAMILY. NR3 SUBFAMILY.
  • FACTOR VII IS CONVERTED TO FACTOR VIIA BY FACTOR XA,
  • SUBUNIT HETERODIMER OF A LIGHT CHAIN AND A HEAVY CHAIN
  • TISSUE SPECIFICITY PLASMA.
  • the deleted region contains 74 amino acids from the C-terminal end of the factor VII light chain, and 26 amino acids from the N-terminal end of the factor VII heavy catalytic chain.
  • the deleted region includes EGF-like 2 domain and the cleavage site (by factor XA, factor XIIA, factor IXA, or thrombin) of the original protein.
  • CDKN2A MUTATIONS ARE INVOLVED IN TUMOR FORMATION IN A WIDE RANGE OF TISSUES.
  • SEQ ID NO : 67 Deletion of 150 amino acids, between the positions 334-485, of the original protein.
  • the deleted region includes the reactive bond (the active site) of the original protein.
  • NV-33 does contain the chemotactic activity domain, the glycosaminoglycan-binding site and the hirudin-like 2 x 11 AA approximate repeats, Asp/Glu rich.
  • FUNCTION SEEMS TO INHIBIT TRYPSIN, FACTOR VII(A)/TISSUE FACTOR, WEAKLY FACTOR XA. HAS NO EFFECT ON THROMBIN. DOMAIN: THIS INHIBITOR CONTAINS THREE INHIBITORY DOMAINS. SIMILARITY: BELONGS TO THE BPTI/KUNITZ FAMILY OF INHIBITORS. HIGHLY SIMILAR TO TPFI.
  • the deleted region includes part of the BPTI/KUNITZ inhibitor domain-3 and the poly-Lysine domain of the original protein.
  • the deleted region includes the active site and part of the BPTI/KUNITZ inhibitor domain-3.
  • the nucleic acid sequences of the invention include nucleic acid sequences which encode variant product and fragments and analogs thereof.
  • the nucleic acid sequences may alternatively be sequences complementary to the above coding sequence, or to a region of said coding sequence. The length of the complementary sequence is sufficient to avoid the expression of the coding sequence.
  • the nucleic acid sequences may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA.
  • the DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.
  • the nucleic acid sequences may also both include dNTPs, rNTPs as well as non naturally occurring sequences.
  • the sequence may also be a part of a hybrid between an amino acid sequence and a nucleic acid sequence.
  • the nucleic acid sequence has at least 90%, identity with any one of the sequence identified as SEQ ID NO: 1 to SEQ ID NO: 174 provided that this sequence is not completely identical with that of the original sequence.
  • the nucleic acid sequences may include the coding sequence by itself.
  • the coding region may be in combination with additional coding sequences, such as those coding for fusion protein or signal peptides, in combination with non-coding sequences, such as introns and control elements, promoter and terminator elements or 5 1 and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or in a vector or host environment in which the variant nucleic acid sequence is introduced as a heterologous sequence.
  • the nucleic acid sequences of the present invention may also have the product coding sequence fused in-frame to a marker sequence which allows for purification of the variant product.
  • the marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767 (1984)).
  • fragments as defined above also referred to herein as oligonucleotides, typically having at least 20 bases, preferably 20-30 bases corresponding to a region of the coding-sequence nucleic acid sequence.
  • the fragments may be used as probes, primers, and when complementary also as antisense agents, and the like, according to known methods.
  • the nucleic acid sequence may be substantially a depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 87 or fragments thereof or sequences having at least 90% identity to the above sequence as explained above.
  • the sequence may be a sequence coding for any one of the amino acid sequence of SEQ ID NO: 88 to SEQ ID NO: 174, or fragments or analogs of said amino acid sequence.
  • the nucleic acid sequences may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify nucleic acid sequences which encode the variant products disclosed above.
  • cDNA libraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
  • the nucleic acid sequences may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al, supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # Kl 802-1).
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T.
  • the primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992; National Biosciences Inc, Madison, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Capture PCR (Lagerstrom, M. et al, PCR Methods Applic. 1:111-19, (1991)) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR. Another method which may be used to retrieve flanking sequences is that of Parker, J.D., et al, Nucleic Acids Res., 19:3055-60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinderTM libraries to "walk in" genomic DNA (PromoterFinderTM; Clontech, Palo Alto, CA).
  • libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs.
  • random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
  • a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • the nucleic acid sequences and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
  • nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of variant products.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of variant product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • nucleic acid sequences of the present invention can be engineered in order to alter a variant product coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the product.
  • alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al, (supra).
  • the present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques.
  • Host cells are genetically engineered (i.e., transduced, transformed or fransfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the variant nucleic acid sequence.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
  • the nucleic acid sequences of the present invention may be included in any one of a variety of expression vectors for expressing a product.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: LTR or SV40 promoter, the E.coli lac or trp promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracy cline or ampicillin resistance in E.coli.
  • the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate expression hosts include: bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. The invention is not limited by the host cells employed.
  • a number of expression vectors may be selected depending upon the use intended for the variant product. For example, when large quantities of variant product are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, multifunctional E.coli cloning and expression vectors such as Bluescript R) (Stratagene), in which the variant polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster J. Biol Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison Wl); and the like.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • the expression of a sequence encoding variant product may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al, Nature 310:511-514. (1984)) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al, EMBO J., 3:17-311, (1987)).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al, EMBO J.
  • Variant product may also be expressed in an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the variant product coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of variant coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
  • the recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which variant protein is expressed (Smith et al, J. Virol 46:584, (1983); Engelhard, E.K. et al, Proc. Nat. Acad. Sci. 91:3224-7, (1994)).
  • a number of viral-based expression systems may be utilized.
  • a variant product coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing variant protein in infected host cells (Logan and Shenk, Proc. Natl Acad. Sci. 81:3655-59, (1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of a variant product coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where variant product coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D. et al, (1994) Results Probl Cell Differ., 20:125-62, (1994); Bittner et al., Methods in Enzymol 153:516-544, (1987)).
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology).
  • Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "pre-pro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express variant product may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M., et al, Cell 11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I., et al, Cell 22:817-23, (1980)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M., et al, Proc. Natl. Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al, J. Mol Biol, 5 150:1-14, (1981)) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman S.C. and R.C. Mulligan, Proc. Natl. Acad. Sci.
  • Host cells transformed with a nucleotide sequence encoding variant product may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the product produced by a recombinant cell may be secreted or contained intracellularly depending on the 0 sequence and/or the vector used.
  • expression vectors containing nucleic acid sequences encoding variant product can be designed with signal sequences which direct secretion of variant product through a prokaryotic or eukaryotic cell membrane.
  • the variant product may also be expressed as a recombinant protein with 5 one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS o extension/affinity purification system (Immunex Corp, Seattle, Wash.).
  • the inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and variant product is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein compromising a variant polypeptide fused to a polyhistidine region separated by an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath, et al., Protein Expression and Purification, 3:263-281, (1992)) while the enterokinase cleavage site provides a means for isolating variant polypeptide from the fusion protein.
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
  • ligand-agarose beads e.g., glutathione-agarose in the case of GST-fusions
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
  • the variant products can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • nucleic acid sequences of the present invention may be used for a variety of diagnostic purposes.
  • the nucleic acid sequences may be used to detect and quantitate expression of the variant in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for variant product.
  • the assay may be used to detect soluble variant in the serum or blood. This assay typically involves obtaining total mRNA from the tissue or serum and contacting the mRNA with a nucleic acid probe.
  • the probe is a nucleic acid molecule of at least 20 nucleotides, preferably 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding variant product under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of variant.
  • This assay can be used to distinguish between absence, presence, and excess expression of variant product and to monitor levels of variant expression during therapeutic intervention.
  • the assay may be used to compare the levels of the variant of the invention to the levels of the original sequence from which it has been varied or to levels of other variants, which comparison may have some physiological meaning.
  • the invention also contemplates the use of the nucleic acid sequences as a diagnostic for diseases resulting from inherited defective variant sequences, or diseases in which the ratio of the amount of the original sequence from which the variant was varied to the novel variants of the invention is altered.
  • These sequences can be detected by comparing the sequences of the defective (i.e., mutant) variant coding region with that of a normal coding region. Association of the sequence coding for mutant variant product with abnormal variant product activity may be verified.
  • sequences encoding mutant variant products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a variant protein deficient strain of HEK293 cells) as yet another means to verify or identify mutations.
  • a functional assay system e.g., colorimetric assay, complementation experiments in a variant protein deficient strain of HEK293 cells
  • nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al, Nature 324:163-166, (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et alProc. Natl. Acad. Sci. USA, 85:4397-4401, (1985)), or by differences in melting temperatures. "Molecular beacons" (Kostrikis L.G.
  • hairpin-shaped, single-stranded synthetic oligo- nucleotides containing probe sequences which are complementary to the nucleic acid of the present invention may also be used to detect point mutations or other sequence changes as well as monitor expression levels of variant product. Such diagnostics would be particularly useful for prenatal testing.
  • Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases.
  • the two probes may be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction.
  • the presence or absence of mutations is then detectable by the presence or absence of ligated probe.
  • oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Patent No. 5,547,839.
  • SBH sequencing by hybridization
  • the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip. The sequence of the target can then be "read" from the pattern of target binding to the array.
  • the nucleic acid sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 20-30 bp) from the variant cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids or using instead radiation hybrids are rapid procedures for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 50 or 60 bases.
  • Verma et al Human Chromosomes: a Manual of Basic Techniques, (1988) Pergamon Press, New York.
  • a sequence Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map data are found, for example, in the OMIM database (Center for Medical Genetics, Johns Hopkins University, Baltimore, MD and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD).
  • the OMIM gene map presents the cytogenetic map location of disease genes and other expressed genes.
  • the OMIM database provides information on diseases associated with the chromosomal location. Such associations include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations in this area with the advent of polygenic diseases, such as cancer, in general and prostate cancer in particular.
  • Therapeutic applications of nucleic acid sequences include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations in this area with the advent of polygenic diseases, such as cancer, in general
  • Nucleic acid sequences of the invention may also be used for therapeutic purposes.
  • expression of variant product may be modulated through antisense technology, which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding variant product.
  • the 5' coding portion of the nucleic acid sequence sequence which codes for the product of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length. Oligonucleotides derived from the transcription start site, e.g. between positions -10 and +10 from the start site, are preferred.
  • An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee et al, Nucl. Acids, Res., 6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan et al, Science 251:1360, (1991)), thereby preventing transcription and the production of the variant products.
  • An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the variant products (Okano J. Neurochem. 56:560, (1991)).
  • the antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo.
  • the antisense may be antisense mRNA or DNA sequence capable of coding such antisense mRNA.
  • the antisense mRNA or the DNA coding thereof can be complementary to the full sequence of nucleic acid sequences coding for the variant protein or to a fragment of such a sequence which is sufficient to inhibit production of a protein product.
  • expression of variant product may be increased by providing coding sequences for coding for said product under the control of suitable control elements ending its expression in the desired host.
  • the nucleic acid sequences of the invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • Cells from a patient may be engineered with a nucleic acid sequence (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA nucleic acid sequence
  • Such methods are well-known in the art.
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be fransfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5-14, (1990)).
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • the genes introduced into cells may be placed under the control of inducible promoters, such as the radiation- inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res., 56(19):4311 (1996)), to stimulate variant production or antisense inhibition in response to radiation, eg., radiation therapy for treating tumors.
  • inducible promoters such as the radiation- inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res., 56(19):4311 (1996)
  • the substantially purified variant product of the invention has been defined above as the product coded from the nucleic acid sequence of the invention.
  • the amino acid sequence is an amino acid sequence having at least 90% identity to any one of the sequences identified as SEQ ID NO: 88 to SEQ ID NO: 174 provided that the amino acid sequence is not identical to that of the original sequence from which it has been varied.
  • the protein or polypeptide may be in mature and/or modified form, also as defined above. Also contemplated are protein fragments having at least 10 contiguous amino acid residues, preferably at least 10-20 residues, derived from the variant product, as well as homologues as explained above.
  • sequence variations are preferably those that are considered conserved substitutions, as defined above.
  • a protein with a sequence having at least 90% sequence identity with any of the products identified as SEQ ID NO: 88 to SEQ ID NO: 174, preferably by utilizing conserved substitutions as defined above is also part of the invention, and provided that it is not identical to the original peptide from which it has been varied.
  • the protein has or contains any one of the sequence identified as SEQ ID NO: 88 to SEQ ID NO: 174.
  • the variant product may be (i) one in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the variant product is fused with another compound, such as a compound to increase the half- life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the protein to its target tissue or target cell population (such as an antibody), or (iv) one in which additional amino acids are fused to the variant product.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • amino acid residues includes a substituent group
  • another compound such as a compound to increase the half- life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the protein to
  • fragments and portions of variant product may be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J., J. Am. Chem. Soc, 85:2149-2154, (1963)).
  • In vitro peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.
  • Fragments of variant product may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • the variant product of the invention is generally useful in treating diseases and disorders which are characterized by a lower than normal level of variant expression, and or diseases which can be cured or ameliorated by raising the level of the variant product, even if the level is normal.
  • Variant products or fragments may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue.
  • the product-containing compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones. .
  • Variant product-containing compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means as well as by nasal application. Variant product-containing compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • the product can be given via intravenous or intraperitoneal injection. Similarly, the product may be injected to other localized regions of the body. The product may also be administered via nasal insufflation. Enteral administration is also possible. For such administration, the product should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
  • Dosage of the product will vary, depending upon the potency and therapeutic index of the particular polypeptide selected.
  • a therapeutic composition for use in the treatment method can include the product in a sterile injectable solution, the polypeptide in an oral delivery vehicle, the product in an aerosol suitable for nasal administration, or the product in a nebulized form, all prepared according to well known methods.
  • Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the product of the invention may also be used to modulate endothelial differentiation and proliferation as well as to modulate apoptosis either ex vivo or in vitro, for example, in cell cultures.
  • Example IV Screening methods for activators and deactivators (inhibitors)
  • the present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, or other molecules, which have a modulating effect on the activity of the variant product, e.g. activators or deactivators of the variant product of the present invention.
  • an assay comprises the steps of providing an variant product encoded by the nucleic acid sequences of the present invention, contacting the variant protein with one or more candidate molecules to determine the candidate molecules modulating effect on the activity of the variant product, and selecting from the molecules a candidate's molecule capable of modulating variant product physiological activity.
  • the variant product, its catalytic or immunogenic fragments or oligopeptides thereof can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell membrane or located intracellularly.
  • the formation of binding complexes, between variant product and the agent being tested, may be measured.
  • the activator or deactivator may work by serving as agonist or antagonist, respectively, of the variant receptor, binding entity or target site, and their effect may be determined in connection with any of the above.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the variant product is described in detail by Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with the full variant product or with fragments of variant product and washed. Bound variant product is then detected by methods well known in the art. Substantially purified variant product can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • Antibodies to the variant product may also be used in screening assays according to methods well known in the art. For example, a "sandwich" assay may be performed, in which an anti-variant antibody is affixed to a solid surface such as a microtiter plate and variant product is added. Such an assay can be used to capture compounds which bind to the variant product. Alternatively, such an assay may be used to measure the ability of compounds to influence with the binding of variant product to the variant receptor, and then select those compounds which effect the binding.
  • Example V Anti-variant antibodies A. Synthesis
  • the purified variant product is used to produce anti-variant antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the variant product.
  • Antibodies to the variant product may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which inhibit dimer formation, are especially preferred for therapeutic use.
  • a fragment of the variant product for antibody induction does not require biological activity but have to feature immunological activity; however, the protein fragment or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids of the sequences specified in any one of SEQ ID NO: 88 to SEQ ID NO: 174. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of variant protein amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to variant product.
  • various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with variant product or any portion, fragment or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to variant protein may be prepared using any technique which provides for the production of antibody molecules by continuous 5 cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell hybridoma technique (Kosbor et al, Immunol. Today 4:72, (1983); Cote et al, Proc. Natl. Acad. Sci. 80:2026-2030, (1983)) and the EBV-hybridoma technique (Cole, et al, Mol Cell Biol. 62:109-120, (1984)).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or
  • Antibody fragments which contain specific binding sites for variant protein may also be generated.
  • such fragments include, but are not
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al, Science
  • Antibodies which specifically bind variant product are useful for the diagnosis of conditions or diseases characterized by expression of the novel variant of the invention (where normally it is not expressed) by over or under expression of variant as well as for detection of diseases in which the proportion between the amount of the variants of the invention and the original sequence from which it varied is altered.
  • such antibodies may be used in assays to monitor patients being treated with variant product, its activators, or its deactivators.
  • Diagnostic assays for variant protein include methods utilizing the antibody and a label to detect variant product in human body fluids or extracts of cells or tissues.
  • the products and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
  • reporter molecules A wide variety of reporter molecules are known in the art.
  • a variety of protocols for measuring the variant product, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on variant product is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of variant product expression.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • Normal or standard values for variant product expression are established by combining body fluids or cell extracts taken from normal subjects, preferably human, with antibody to variant product under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric methods.
  • standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • the antibody assays are useful to determine the level of variant product present in a body fluid sample, in order to determine whether it is being expressed at all, whether it is being overexpressed or underexpressed in the tissue, or as an indication of how variant levels of variable products are responding to drug treatment.
  • the invention concerns methods for determining the presence or level of various anti-variant antibodies in a biological sample obtained from patients, such as blood or serum sample using as an antigen the variant product. Determination of said antibodies may be indicative to a plurality of pathological conditions or diseases.
  • the antibodies may have a therapeutical utility in blocking or decreasing the activity of the variant product in pathological conditions where beneficial effect can be achieved by such a decrease.
  • the antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulin-gene library methods.
  • the antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable.
  • the antibody is administered in an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen.
  • the immunohistochemical staining was performed on mouse salivary gland micron sections.
  • the immunohistochemestry was done using specific polyclonal antibodies designed against the c-terminus of SEQ ID NO: 144 (12 amino-acids), which are unique to said ACEV product and lack in the original ACE protein (Fig 88-a,b,d magnification X 100; Fig 89-b magnification X 400) compared with the pre-immune rabbit's serum (Fig 88-c, Fig. 89-a).
  • ACE was found to express in ductal epifilus (Fig 88-a,b,d, Fig 89-b).
  • RNA Purification and cDNA Synthesis Total RNA was extracted from different mouse tissues using Tri-Reagent System (Molecular Research Center, Inc., Cincinnati, OH). Synthesis of first-strand cDNA was carried out using Oligo(dT)15 (Promega, Medison, Wl), Superscript II (Gibco/BRL, Gaithersburg, MD), Rnasin (Promega, Medison, Wl) and dNTP's (Gibco/BRL, Gaithersburg, MD). + with Superscript II, - without Superscript II.
  • PCR Polymerase Chain Reaction

Abstract

The present invention concerns novel variants, amino acid and nucleic acid sequences obtained by alternative splicing of known sequences, expression vectors and host cells containing the variants' nucleic acid sequence, and antibodies reactive with the variants' products. The invention also concerns pharmaceutical compositions containing any of the above as well as methods of detection. A preferred example is the angiotensin converting enzyme (ACE) variant.

Description

VARIANTS OF ALTERNATIVE SPLICING
FIELD OF THE INVENTION
The present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above. The present invention further concerns methods for screening for candidate activators or deactivators utilizing said amino acid sequences.
BACKGROUND OF THE INVENTION
Alternative splicing (AS) is an important regulatory mechanism in higher eukaryotes (P.A. Sharp, Cell 11, 805-8152 (1994). It is thought to be one of the most important mechanisms for differential expression related to tissue or development stage specificity. It is known to play a major role in numerous biological systems, including human antibody responses, and sex determination in Drosophila, (S. Stamm, M.Q. Zhang, T.G. Marr and D.M. Helfman, Nucleic Acids Research 22, 1515-1526 (1994); B. Chabot, Trends Genet. 12, 472-478 (1996); R.E. Breitbart, A. Andreadis, B. Nadal-Ginard, Annual Rev. Biochem., 56, 467-495 (1987); C.W. Smith, J.G. Patton, B. Nadal-Ginard, Annu. Rev. Genet., 27, 527-577 (1989)).
Until recently it was commonly believed that alternative splicing existed in only a small fraction of genes (about 5%). A recent observation based on literature survey of known genes revises this conservative estimate to as high as an estimate that at least 30% of human genes are alternatively spliced (M.S. Gelfand, I. Dubchak, I. Draluk and M. Zorn, Nucleic Acids Research 27, 301-302 (1999). The importance of the actual frequency of this phenomenon lies not only in the direct impact on the number of proteins created (100,000 human genes, for example, would be translated to a much higher number of proteins), but also in the diversity of functionality derived from the process.
Several mechanisms at different stages may be held responsible for the complexity of higher eukaryote which include: alternative splicing at the transcription level, RNA editing at the post-transcriptional level, and post-translational modifications are the ones characterized to date.
Angiotensin I-converting enzyme (ACE) is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrpphage system. Physiologically, ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin.
Increased serum ACE activity (SACE) has been reported in pathologies involving stimulation of the monocytic cell line, primarily granulomatous diseases. Sarcoidosis is the most frequent and the better studied of these diseases; high SACE is not only a well-established marker for the diagnosis but is also a useful tool for following its course and evaluating the effect of therapy of this disease.
SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis.
Decreased SACE has been reported in vascular pathologies involving an endothelial abnormality, such as deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors.
Various methods have been developed for determining SACE activities. The most widely used is the spectrophotometric assay using hippuryl-histidyl-leucine as substrate. Fluorimetric and radiochemical assays using both classic and novel substrates have been proposed, but they are time consuming, require special apparatus, and are not suited to automation. Kinetic spectrophotometry of furylacryloyl-phenylalanyl-glycyl-glycine hydrolysis is now used extensively because it is easy to automatize. Information obtained in the last decade indicates that angiotensin II increases the production of several autocrine factors, including transforming growth factor betal (TGF-betal), tumor necrosis factor-alpha (TNF-alpha), and platelet-derived growth factor A chain (PDGF). Angiotensin also increases the release of other growth factors such as endothelin, platelet-activating factor (PAF), and interleukin 6. In addition, it increases the "activity" of nuclear factor-kappaB (NF-kappaB) and the synthesis of angiotensinogen. The emerging picture indicates that the actions of angiotensin II may be related to factors that are released or upregulated by angiotensin II, possibly through NF-kappaB.
GLOSSARY
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
"Variant nucleic acid sequence" - the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 87, native and known genes. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of genes and not merely truncated, mutated or fragmented forms of known sequences which are artificially produced. " Angiotensin converting enzyme variant (ACEV)" - a sequence shown in SEQ ID NO: 57 or 85 sequences having at least 90% identity (see below) to said sequence and fragments (see below) of the above sequences of least 20 b.p. long. These sequences are sequences coding for a novel, naturally occurring, alternative splice variants of the mouse angiotensin converting enzyme which convert angiotensin I to angiotensin II by release of the terminal His-Lew resulting in increase of vasoconstrictor activity of angiotensin.
"Variant product - also referred at times as the "variant protein" or "variant polypeptide" - is an amino acid sequence encoded by the variant nucleic acid sequence SEQ ID NO: 88 to SEQ ID NO: 174.
"ACEV product or ACEV protein"- amino acid coded by the ACEV nucleic acid which is a naturally occurring mRNA sequence obtained as a result of alternative splicing of the ACE gene. The amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein. The variant products are shown in SEQ ID NO: 144 or 172. The term also includes homologies (see below) of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments (see below) of this sequence having at least 10 amino acids. The above two products may be secreted.
"Nucleic acid sequence" - a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
"Amino acid sequence" - a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids. "Fragment of variant nucleic acid sequence" and 'fragment of ACEV nucleic acid sequence " - novel short stretch of nucleic acid sequences of at least 20 b.p., which does not appear as a continuous stretch in the original nucleic acid sequence (see below). The fragment may be a sequence which was previously undescribed in the context of the published RNA and which affects the amino acid sequence encoded by the known gene. For example, where the variant nucleic includes a sequence which was not included in the original sequence (for example a sequence which was an intron in the original sequence) the fragment may contain said additional sequence. The fragment may also be a region which is not an intron, which was not present in the original sequence. For example where the variant lacks a non-terminal region which was present in the original sequence. The two stretches of nucleotides spanning this region (upstream and downstream) are brought together by splicing in the variant, but are spaced from each by the spliced out region in the original sequence and are thus not continuous in the original sequence. A continuous stretch of nucleic acids comprising said two sparing stretches of nucleotides is not present in the original sequence and thus falls under the definition of fragment.
"Fragments of variant products" - novel amino acid sequences coded by the "fragment of variant nucleic acid sequence " or "fragment of ACEV nucleic acid sequence " defined above.
"Homologues of variants" - amino acid sequences of variants in which one or more amino acids has been added, deleted or replaced. The addition, deletion or replacement should be in the regions or adjacent to regions where the variant differs from the original sequence (see below).
"Conservative substitution " - refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is a conservative substitution.
"Non-conservative substitution " - refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
"Chemically modified" - when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
"Biologically active" - refers to the variant product having some sort of biological activity, for example, some physiologically measurable effect on target cells, molecules or tissues.
"Immunologically active" defines the capability of a natural, recombinant or synthetic variant product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. Thus, for example, an immunologically active fragment of variant product denotes a fragment which retains some or all of the immunological properties of the variant product, e.g. can bind specific anti-variant product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce variant.
"Optimal alignment" - is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from Mac Vector (TM), operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence). In case of alignments of known gene sequences with that of the new variant, the optimal alignment invariably included aligning the identical parts of both sequences together, then keeping apart and unaligned the sections of the sequences that differ one from the other.
"Having at least 90% identity" - with respect to two amino acid or nucleic acid sequence sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 90% amino acid sequence identity means that 90% of the amino acids in two or more optimally aligned polypeptide sequences are identical, however this definition explicitly excludes sequences which are 100% identical with the original sequence from which the variant of the invention was varied. " Isolated nucleic acid molecule having an variant nucleic acid sequence" - is a nucleic acid molecule that includes the coding variant nucleic acid sequence. Said isolated nucleic acid molecule may include the variant nucleic acid sequence as an independent insert; may include the variant nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the variant coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the variant nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5' and/or 31 untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the variant protein coding sequence is a heterologous.
"Expression vector" - refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
"Deletion " - is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
"Insertion" or "addition" - is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
"Substitution " - replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non- conservative. "Antibody" - refers to IgG, IgM, IgD, IgA, or IgG antibody. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-variant product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
Distinguishing antibody" - an antibody capable of binding to the variant product and not the original amino acid sequence from which it has been varied, or an antibody capable of binding to the original nucleic acid sequence and not to the variant production.
"Activator" - as used herein, refers to a molecule which mimics the effect of the natural variant product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product. The mechanism may be by any mechanism known to prolonging activities of biological molecules such as binding to receptors; prolonging the lifetime of the molecules; increasing the activity of the molecules on its target; increasing the affinity of molecules to its receptor; inhibiting degradation or proteolysis of the molecules, or mimicking the biological activity of the variants on their targets, etc. Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the variant product.
"Deactivator" or ("Inhibitor") - refers to a molecule which modulates the activity of the variant product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the variant product. This may be done by any mechanism known to deactivate or inhibit biological molecules such as block of the receptor, block of active site, competition on binding site in target, enhancement of degradation, etc. Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
"Treating a disease" - refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
"Detection" - refers to a method of detection of a disease, disorder, pathological or normal condition. This term may refer to detection of a predisposition to a disease as well as for establishing the prognosis of the patient by determining the severity of the disease.
"Probe" - the variant nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence. The probe may be attached to a solid support or to a detectable label.
"Original sequence" - the amino acid or nucleic acid sequence from which the variant of the invention have been varied as a result of alternative slicing.
SUMMARY OF THE INVENTION
The present invention is based on the finding of several novel, naturally occurring splice variants, which are naturally occurring sequences obtained by alternative splicing of known genes. The novel splice variants of the invention are not merely truncated forms, fragments or mutations of known genes, but rather novel sequences which naturally occur within the body of individuals.
In particular the present invention concerns variants of alternative splice variants of angiotensin converting enzyme (ACEV). The term "alternative splicing" in the context of the present invention and claims refers to: intron inclusion, exon exclusion, addition or deletion of terminal sequences in the variant as compared to the original sequences, as well as to the possibility of "intron retention ". Intron retention is an intermediate stage in the processing of RNA transcripts, where prior to production of fully processed mRNA the intron (naturally spliced in the original sequence) is retained in the variant. These intermediately processed RNAs may have physiological significance and are also within the scope of the invention.
The novel variant products of the invention, including the ACEV-variant (ACEV), may have the same physiological activity as the original peptide from which they have been varied (although perhaps at a different level); may have an opposite physiological activity from the activity featured by the original peptide from which they are varied; may have a completely different, unrelated activity to the activity of the original from which they are varied; or alternatively may have no activity at all and this may lead to various diseases or pathological conditions. The novel variants of the invention may differ from the original sequence, from which they were varied by alternative splicing, by physiological properties not relating directly to their activities such as: tissue localization, temporal pattern of expression, rate of clearance, rate of degradation, manner of up- or down regulation, association with co-factors and cellular elements etc.
The novel variants may also serve for detection purposes, i.e. their presence or level may be indicative of a disease, disorder, pathological or normal condition or alternatively the ratio between the level variants and the level original peptide from which they were varied, or the ratio to other variants may be indicative to a disease, disorder, pathological or normal condition.
For example, for detectional purposes, it is possible to establish differential expression of various variants in various tissues. A certain variant may be expressed mainly in one tissue, while the original sequence from which it has been varied, or another variant may, be expressed mainly in another tissue. Understanding of the distribution of the variants in various tissues may be helpful in basic research, for understanding the physiological function of the genes as well as may help in targeting pharmaceuticals or developing pharmaceuticals.
The study of the variants may also be helpful to distinguish various stages in the life cycles of the same type of cells which may also be helpful for development of pharmaceuticals for various pathological conditions in which cell cycles is non-normal, notably cancer.
Detection of various diseases in accordance with the invention is especially useful for detection of diseases which are associated with the function, (over function, under function, or malfunction) of proteins of the original sequence from which each variant of the invention has been obtained by alternative splicing. A list of the original proteins are given in the "Detailed Description" part of the specification. Thus, for example, if variant of SEQ ID NO: 3 is obtained from an original sequence which is coagulation factor XII, this sequence may be used to detect diseases involving excessive or diminished blood coagulation. Thus the detection may by determination of the presence or the level of expression of the variant within a specific cell population, comprising said presence or level between various cell types in a tissue, between different tissues and between individuals.
Where the variant in the angiotensin converting enzyme (ACEV) the detection may be used for detection (including disposition) of one of the following diseases.
Cardiovascular diseases:
Including hypertension, neurological damage due to cerebral circulatory disorders, peripheral vascular diseases, arteriosclerosis, heart and kidney diseases relating to blood pressure, erection problems and migraine problems relating to circulation functions, heart failures (including recurrent infraction in patients with left ventricular dysfunction), acute phase of myocardial infarction, coronary arterial thrombosis and cardial insufficiency. Renal diseases:
Hypertension adrenal injury (particularly in patients with type I or II diabetes), diabetic nepropathy, renal function deterioration in glomerular diseases
Muscular diseases: Diseases involving growth of smooth muscle cells such as hypertrophy.
Immune disorders:
Various autoimmune diseases and diseases involving inflammatory mechanisms, for example, autoimmune manifestation affects in sarcoidosis, generation of immune complex nephritis, autoimmune encephatomyelitis, marker for chronic fatigue-immune dysfunction syndrome.
Multiple sclerosis:
Cancer:
Especially those cancers effected by different growth factors including endothelia, platelet-activating factor (PAF) and interleukin 6. Examples of such cancers are tumors of the vascular system, and leukemias.
Diabetes:
Sarcoidosis - a disease of unknown origin characterized by the formation of granulomatous lesions that appear especially in the liver, lungs, skin and lymph nodes. Nonarcoidotic Pulmonary Granulomatous Diseases:
Such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis. (increased sACE)
Vascular Pathologies Involving An Endothelial Abnormality:
Deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations.
Thus the present invention provides by its first aspect, a novel isolated nucleic acid molecule comprising or consisting of any one of the coding sequence SEQ ID NO: 1 to SEQ ID NO: 87, fragments of said coding sequence having at least 20 nucleic acids (provided that said fragments are continuous stretches of nucleotides not present in the original sequence from which the variant was varied), or a molecule comprising a sequence having at least 90%, identity to SEQ ID NO: 1 to SEQ ID NO: 87, provided that the molecule is not completely identical to the original sequence from which the variant was varied. In particular, the above variant is that of SEQ ID NO: 57 or SEQ ID NO: 85 being the ACEV nucleic acid sequence.
The present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein "variant product" , for example, an amino acid sequence having the sequence as depicted in any one of SEQ ID NO: 88 to SEQ ID NO: 174, fragments of the above amino acid sequence having a length of at least 10 amino acids coded by the above fragments of the nucleic acid sequences, as well as homologues of the above amino acid sequences in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified. In particular, the product is the amino acid sequence of the ACEV as depicted in SEQ ID NO: 144 or 172.
The deletions, insertions and modifications should be in regions, or adjacent to regions, wherein the variant differs from the original sequence.
For example, where the variant is different from the original sequence by addition of a short stretch of 10 amino acids, in the terminal or non- terminal portion of the peptide, the invention also concerns homologues of that variant where the additional short stretch is altered for example, it includes only 8 additional amino acids, includes 13 additional amino acids, or it includes 10 additional amino acids, however some of them being conservative or non-conservative substitutes of the original additional 10 amino acids of the novel variants. In all cases the changes in the homolog, as compared to the original sequence, are in the same regions where the variant differs from the original sequence, or in regions adjacent to said region. Another example is where the variant lacks a non-terminal region (for example of 20 amino acids) which is present in the original sequence (due for example to exon exclusion). The homologues may lack in the same region only 17 amino acids or 23 amino acids. Again the deletion is in the same region where the variant lacks a sequence as compared to the original sequence, or in a region adjacent thereto.
It should be appreciated that once a man versed in the art's attention is directed to the importance of a specific region, due to the fact that this region differs in the variant as compared to the original sequence, there is no problem in derivating said specific region by addition to it, deleting from it, or substituting some amino acids in it. Thus homologues of variants which are derivated from the variant by changes (deletion, addition, substitution) only in said region as well as in regions adjacent to it are also a part of the present invention. Generally, if the variant is distinguished from the original sequence by some sort of physiological activity, then the homolog is distinguished from the original sequence in essentially the same manner.
The present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences, (including the fragments and homologues of the amino acid sequences and in particular the ACEV amino acid sequence). Due to the degenerative nature of the genetic code, a plurality of alternative nucleic acid, beyond those depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 87, can code for the amino acid sequence of the invention. Those alternative nucleic acid sequences which code for the same amino acid sequences codes by the sequence SEQ ID NO: 1 to SEQ ID NO: 87 are also an aspect of the of the present invention.
The present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells fransfected by said vectors. The present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
These pharmaceutical compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated or cured by raising the level of any one of the variant products of the invention. In particular, those diseases are diseases which are associated with malfunction or under function of the original sequence (for example, given in the "Detailed Description" part of the specification). Thus for example, SEQ ID NO: 3 and sequences encoded thereby may be used to treat diseases associated with coagulation of blood.
By a second aspect, the present invention provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of any one of SEQ ID NO: 1 to SEQ ID NO: 87, or complementary to a sequence having at least 90% identity to said sequence (with the proviso added above) or a fragment of said two sequences (according to the above definition of fragment). The complementary sequence may be a DNA sequence which hybridizes with any one of SEQ of ID NO: 1 to SEQ ID NO: 87 or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence. The complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed from any one of SEQ ID NO: 1 to SEQ ID NO: 87 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from any one of SEQ ID NO: 1 to SEQ ID NO: 87 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 87, so as to inhibit its translation. The complementary sequence may also be the mRNA or the fragment of the mRNA itself.
The nucleic acids of the second aspect of the invention may be used for therapeutic or diagnostic applications for example as probes used for the detection of the variants of the invention. The presence of the variant transcript or the level of the variant transcript may be indicative of a multitude of diseases, disorders and various pathological as well as normal conditions for example, as indicated above for the variants in general, and for the ACEV in particular. In addition or alternatively, the ratio of the level of the transcripts of the variants of the invention may also be compared to that of the transcripts of the original sequences from which have been varied, or to the level of transcript of other variants, and said ratio may be indicative to a multitude of diseases, disorders and various pathological and normal conditions.
The present invention also provides expression vectors comprising any one of the above defined complementary nucleic acid sequences and host cells fransfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
The invention also provides anti-variant product antibodies, namely antibodies directed against the variant product which specifically bind to said variant product. Said antibodies are useful both for diagnostic and therapeutic purposes. For example said antibody may be as an active ingredient in a pharmaceutical composition as will be explained below.
The present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences. The pharmaceutical composition thus provides pharmaceutical compositions comprising, as an active ingredient, said anti-variant product antibodies.
The pharmaceutical compositions comprising said anti-variant product antibodies or the nucleic acid molecule comprising said complementary sequence, are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing the variant (either at the transcript or product level) or decreasing the amount of the variant product or blocking its binding to its target, for example, by the neutralizing effect of the antibodies, or by the effect of the antisense mRNA in decreasing the expression level of the variant sequence. Examples of diseases which can be treated either with ACEV sequence, an expression vector comprising that sequence, a sequence complementary to the
ACEV sequence, an expression vector comprising said complementary sequence,
ACEV product or an antibody to the product is any one of the diseases mentioned in connection with the detection aspect above.
According to the third aspect of the invention the present invention provides methods for detecting the level of the transcript (mRNA) of said variant product in a body fluid sample, or in a specific tissue sample, for example by use of probes comprising or consisting of said coding sequences; as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the variant products of the invention. Detection of the level of the expression of the variant of the invention in particular as compared to that of the original sequence from which it was varied or compared to other variant sequences all varied from the same original sequence may be indicative of a plurality of physiological or pathological conditions. A preferred example is the detection of ACEV nucleic acid sequence, ACEV product or anti-ACEV antibody.
The method, according to this latter aspect, for detection of a nucleic acid sequence which encodes the variant product in a biological sample, comprises the steps of: (a) providing a probe comprising at least one of the nucleic acid sequences defined above;
(b) contacting the biological sample with said probe under conditions allowing hybridization of nucleic acid sequences thereby enabling formation of hybridization complexes; (c) detecting hybridization complexes, wherein the presence of the complexes indicates the presence of nucleic acid sequence encoding the variant product in the biological sample.
The method as described above is qualitative, i.e. indicates whether the transcript is present in or absent from the sample. The method can also be quantitative, by determining the level of hybridization complexes and then calibrating said levels to determining levels of transcripts of the desired variant in the sample.
Both qualitative and quantitative determination methods can be used for diagnostic, prognostic and therapy planning purposes. By a preferred embodiment the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
The nucleic acid sequence used in the above method may be a DNA sequence an RNA sequence, etc; it may be a coding or a sequence or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences). By quantization of the level of hybridization complexes and calibrating the quantified results it is possible also to detect the level of the transcript in the sample.
Methods for detecting mutations in the region coding for the variant product are also provided, which may be methods carried-out in a binary fashion, namely merely detecting whether there is any mismatches between the normal variant nucleic acid sequence of the invention and the one present in the sample, or carried-out by specifically detecting the nature and location of the mutation.
The present invention also concerns a method for detecting variant product in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of the invention, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said antibody-antigen complex correlates with the presence of variant product in said biological sample.
Many diseases are diagnosed by detecting the presence of antibodies against a protein characterizing the disease in the blood, serum or any other body fluid of the patient. The present invention also concerns a method for detecting anti-variant antibody in a biological sample, comprising: (a) contacting said sample with the variant product of the invention, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said antibody-antigen complex correlates with the presence of anti-variant antibody in the sample.
As indicated above, both methods (for detection of variant product and for detection of the anti- variant antibody) can be quantitized to determine the level or the amount of the variant or antibody in the sample, alone or in comparison to the level of the original amino acid sequence from which it was varied or compared to the level of antibodies against the original amino acid sequence, and qualitative and quantitative results may be used for diagnostic, prognostic and therapy planning purposes.
The invention also concerns distinguishing antibodies, i.e. antibodies capable of binding either to the variant product or to the original sequence from which the variant has been varied, while not binding to the original sequence or the variant product respectively. These distinguishing antibodies may be used for detection purposes.
By yet another aspect the invention also provides a method for identifying candidate compounds capable of binding to the variant product and modulating its activity (being either activators or deactivators). The method includes:
(i) providing a protein or polypeptide comprising an amino acid sequence substantially as depicted in any one of SEQ ID NO: 88 to 174, or a fragment of such a sequence;
(ii) contacting a candidate compound with said amino acid sequence; (iii) measuring the physiological effect of said candidate compound on the activity of the amino acid sequences and selecting those compounds which show a significant effect on said physiological activity.
The present invention also concerns compounds identified by the above methods described above, which compound may either be an activator of the variant product or a deactivator thereof. BRIEF DESCRIPTION OF THE DRAWINGS
In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Fig. 1 is a comparison between the amino acid sequence of SEQ ID NO: 88 and the original sequence from which it has been varied;
Fig. 2 is a comparison between the amino acid sequence of SEQ ID NO: 89 and the original sequence from which it has been varied;
Fig. 3 is a comparison between the amino acid sequence of SEQ ID NO: 90 and the original sequence from which it has been varied;
Fig. 4 is a comparison between the amino acid sequence of SEQ ID NO: 91 and the original sequence from which it has been varied;
Fig. 5 is a comparison between the amino acid sequence of SEQ ID NO: 92 and the original sequence from which it has been varied; Fig. 6 is a comparison between the amino acid sequence of SEQ ID NO: 93 and the original sequence from which it has been varied;
Fig. 7 is a comparison between the amino acid sequence of SEQ ID NO: 94 and the original sequence from which it has been varied;
Fig. 8 is a comparison between the amino acid sequence of SEQ ID NO: 95 and the original sequence from which it has been varied;
Fig. 9 is a comparison between the amino acid sequence of SEQ ID NO: 96 and the original sequence from which it has been varied;
Fig. 10 is a comparison between the amino acid sequence of SEQ ID NO: 97 and the original sequence from which it has been varied; Fig. 11 is a comparison between the amino acid sequence of SEQ ID
NO: 98 and the original sequence from which it has been varied;
Fig. 12 is a comparison between the amino acid sequence of SEQ ID NO: 99 and the original sequence from which it has been varied;
Fig. 13 is a comparison between the amino acid sequence of SEQ ID NO: 100 and the original sequence from which it has been varied; Fig. 14 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
Fig. 15 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied; Fig. 16 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
Fig. 17 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
Fig. 18 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied;
Fig. 19 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
Fig. 20 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied; Fig. 21 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
Fig. 22 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
Fig. 23 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied;
Fig. 24 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
Fig. 25 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied; Fig. 26 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
Fig. 27 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
Fig. 28 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied; Fig. 29 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
Fig. 30 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied; Fig. 31 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
Fig. 32 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
Fig. 33 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied;
Fig. 34 is a comparison between the amino acid sequence of SEQ ID 1 and the original sequence from which it has been varied;
Fig. 35 is a comparison between the amino acid sequence of SEQ ID 2 and the original sequence from which it has been varied; Fig. 36 is a comparison between the amino acid sequence of SEQ ID 3 and the original sequence from which it has been varied;
Fig. 37 is a comparison between the amino acid sequence of SEQ ID 4 and the original sequence from which it has been varied;
Fig. 38 is a comparison between the amino acid sequence of SEQ ID 5 and the original sequence from which it has been varied;
Fig. 39 is a comparison between the amino acid sequence of SEQ ID 6 and the original sequence from which it has been varied;
Fig. 40 is a comparison between the amino acid sequence of SEQ ID 7 and the original sequence from which it has been varied; Fig. 41 is a comparison between the amino acid sequence of SEQ ID 8 and the original sequence from which it has been varied;
Fig. 42 is a comparison between the amino acid sequence of SEQ ID 9 and the original sequence from which it has been varied;
Fig. 43 is a comparison between the amino acid sequence of SEQ ID 0 and the original sequence from which it has been varied; Fig. 44 is a comparison between the amino acid sequence of SEQ ID NO: 131 and the original sequence from which it has been varied;
Fig. 45 is a comparison between the amino acid sequence of SEQ ID NO: 132 and the original sequence from which it has been varied; Fig. 46 is a comparison between the amino acid sequence of SEQ ID
NO: 133 and the original sequence from which it has been varied;
Fig. 47 is a comparison between the amino acid sequence of SEQ ID NO: 134 and the original sequence from which it has been varied;
Fig. 48 is a comparison between the amino acid sequence of SEQ ID NO: 135 and the original sequence from which it has been varied;
Fig. 49 is a comparison between the amino acid sequence of SEQ ID NO: 136 and the original sequence from which it has been varied;
Fig. 50 is a comparison between the amino acid sequence of SEQ ID NO: 137 and the original sequence from which it has been varied; Fig. 51 is a comparison between the amino acid sequence of SEQ ID
NO: 138 and the original sequence from which it has been varied;
Fig. 52 is a comparison between the amino acid sequence of SEQ ID NO: 139 and the original sequence from which it has been varied;
Fig. 53 is a comparison between the amino acid sequence of SEQ ID NO: 140 and the original sequence from which it has been varied;
Fig. 54 is a comparison between the amino acid sequence of SEQ ID NO:
141 and the original sequence from which it has been varied;
Fig. 55 is a comparison between the amino acid sequence of SEQ ID NO:
142 and the original sequence from which it has been varied; Fig. 56 is a comparison between the amino acid sequence of SEQ ID NO:
143 and the original sequence from which it has been varied;
Fig. 57 is a comparison between the amino acid sequence of SEQ ID NO:
144 and the original sequence from which it has been varied;
Fig. 58 is a comparison between the amino acid sequence of SEQ ID NO: 145 and the original sequence from which it has been varied; Fig. 59 is a comparison between the amino acid sequence of SEQ ID NO:
146 and the original sequence from which it has been varied;
Fig. 60 is a comparison between the amino acid sequence of SEQ ID NO:
147 and the original sequence from which it has been varied; Fig. 61 is a comparison between the amino acid sequence of SEQ ID NO:
148 and the original sequence from which it has been varied;
Fig. 62 is a comparison between the amino acid sequence of SEQ ID NO:
149 and the original sequence from which it has been varied;
Fig. 63 is a comparison between the amino acid sequence of SEQ ID NO: 150 and the original sequence from which it has been varied;
Fig. 64 is a comparison between the amino acid sequence of SEQ ID NO:
151 and the original sequence from which it has been varied;
Fig. 65 is a comparison between the amino acid sequence of SEQ ID NO:
152 and the original sequence from which it has been varied; Fig. 66 is a comparison between the amino acid sequence of SEQ ID NO:
153 and the original sequence from which it has been varied;
Fig. 67 is a comparison between the amino acid sequence of SEQ ID NO:
154 and the original sequence from which it has been varied;
Fig. 68 is a comparison between the amino acid sequence of SEQ ID NO: 155 and the original sequence from which it has been varied;
Fig. 69 is a comparison between the amino acid sequence of SEQ ID NO:
156 and the original sequence from which it has been varied;
Fig. 70 is a comparison between the amino acid sequence of SEQ ID NO:
157 and the original sequence from which it has been varied; Fig. 71 is a comparison between the amino acid sequence of SEQ ID NO:
158 and the original sequence from which it has been varied;
Fig. 72 is a comparison between the amino acid sequence of SEQ ID NO:
159 and the original sequence from which it has been varied;
Fig. 73 is a comparison between the amino acid sequence of SEQ ID NO: 160 and the original sequence from which it has been varied; Fig. 74 is a comparison between the amino acid sequence of SEQ ID NO:
161 and the original sequence from which it has been varied;
Fig. 75 is a comparison between the amino acid sequence of SEQ ID NO:
162 and the original sequence from which it has been varied; Fig. 76 is a comparison between the amino acid sequence of SEQ ID NO:
163 and the original sequence from which it has been varied;
Fig. 77 is a comparison between the amino acid sequence of SEQ ID NO:
164 and the original sequence from which it has been varied;
Fig. 78 is a comparison between the amino acid sequence of SEQ ID NO: 165 and the original sequence from which it has been varied;
Fig. 79 is a comparison between the amino acid sequence of SEQ ID NO:
166 and the original sequence from which it has been varied;
Fig. 80 is a comparison between the amino acid sequence of SEQ ID NO:
167 and the original sequence from which it has been varied; Fig. 81 is a comparison between the amino acid sequence of SEQ ID NO:
168 and the original sequence from which it has been varied;
Fig. 82 is a comparison between the amino acid sequence of SEQ ID NO:
169 and the original sequence from which it has been varied;
Fig. 83 is a comparison between the amino acid sequence of SEQ ID NO: 170 and the original sequence from which it has been varied;
Fig. 84 is a comparison between the amino acid sequence of SEQ ID NO:
171 and the original sequence from which it has been varied;
Fig. 85 is a comparison between the amino acid sequence of SEQ ID NO:
172 and the original sequence from which it has been varied; Fig. 86 is a comparison between the amino acid sequence of SEQ ID NO:
173 and the original sequence from which it has been varied;
Fig. 87 is a comparison between the amino acid sequence of SEQ ID NO:
174 and the original sequence from which it has been varied; Fig. 88 shows immunohistochemical staining with antibodies against a fragment of the ACEV product of SEQ ID NO: 144; expressed in ductal epitilus in salivatory gland (magnification X 100);
Fig. 89 shows the same as in Fig. 89 (magnification X 400); Fig. 90 shows immunohistochemical staining with antibodies against a fragment of ACEV product of SEQ ID NO: 144 expressed in salivary glands surrounding the lymph nodes; and
Fig. 91 shows RT-PCR results of the ACEV sequence expressed in salivary glands.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example I: Comparison of variants with original sequences
Original sequences were obtained from GenBank Version 110. Comparison between the original sequences and the novel variant sequences was made using the Pileup application from the GCG suite version 10.0 (January 1999), with the default values:
Gap creation penalty (Gap Weight): 8 Gap extension penalty (GapLength Weight): 2
The comparison is shown in Fig. 1 to 87 which show the comparison of each of the variant products depicted in SEQ ID NO: 88 to 174 with the original sequence from which it was varied.
The following is a list which gives the name and the description of each original sequence from which the alternative splice variant has been varied by alternative splicing. The description is followed by the internal reference to the novel variant (NV-NV... or NV-... etc.) and a short comparison between the variant and the original sequence. It should be noticed that several splice variants may have been originated from the same parent sequence by several different alternative splicings. The following table summarizes the accession number of the original sequence, the terminology of the new variant (RN-NV... or NV-...) and the description of the difference between the new variant and the original sequence. Table
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Identification of the original sequence from which the novel
Variant was variant
The following is the explanation of the definition to be used in the following:
Accession: Accession number of the original sequence in the GeneBank database
Name: Name of the original sequence in the database
Function: Physiological activity.
SEQ ID NO : Sequence number of variant
Description: the difference between the variant and the original sequence.
Accession: AA2A_HUMAN Name: Adenosine A2 receptor Function: Receptor for adenosine.
SEQ ID 1 Description: Gap between amino acids at the positions 237-247 of the original protein. Missing 6th transmembrane loop of the original Adenosine A2 receptor.
Accession: ASMJHUMAN Name: SPHINGOMYELIN PHOSPHODIESTERASE Function: Converts sphingomyelin to ceramide.
SEQ ID : 2
Description: Insertion of 2 amino acids after amino acid at the position 34 and insertion of 54 amino acids after amino acid at the position 492 of the original SPHINGOMYELIN PHOSPHODIESTERASE protein.
Accession: FA12_HUMAN
Name: COAGULATION FACTOR XII
Function: Factor XII is a serum glycoprotein that participates in the initiation of blood coagulation, fibrinolysis, and the generation of bradykinin and angiotensin.
SEQ ID : 3
Description: Alternative 10 C-terminal amino acids. Has part of catalytic domain missing 1 active site.
Accession: GCSR_HUMAN Name: GRANULOCYTE COLONY STIMULATING FACTOR receptor
Function: Receptor for granulocyte colony-stimulating factor (g- csf).
In addition it may function in some adhesion or recognition events at the cell surface.
SEQ ID : 4
Description: Deletion of 62amino acids between the positions 320-382 of the original GRANULOCYTE COLONY STIMULATING FACTOR receptor. The deletion is in the EXTRACELLULAR domain in one of the FIBRONECTIN TYPE-III domains Rl.
Accession: GCSR HUMAN Name: GRANULOCYTE COLONY STIMULATING FACTOR receptor
Function: Receptor for granulocyte colony-stimulating factor (g- csf).
In addition it may function in some adhesion or recognition events at the cell surface.
SEQ ID : 5
Description: Insertion of 37 amino acids in the extracellular domain after the position 574 of the original GRANULOCYTE COLONY STIMULATING FACTOR receptor.
Accession: GLR2 HUMAN
Name: Glutamate receptor 2
Function: L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system, the postsynaptic actions of Glu are mediated by a variety of receptors are named according to their selective agonists
SEQ ID : 6
Description: Replacement of 88 C-terminal amino acids of the original glutamate receptor 2 by alternative 42 amino acids. Has most of domains, might be missing 4th transmembrane domain.
Accession: GLUC_HUMAN Name: Glucagon Function: Promotes hydrolysis of glycogen and lipids, and raises the blood sugar level.
SEQ ID : 7 Description Gap; 156aa compared to 180aa; exact 1-108; gap 108-132; exact 132-180. Missing almost whole GLUCAGON-LIKE PEPTIDE 1
Accession: IHBA_HUMAN Name: Inhibin; erythroid differentiation factor Function: Inhibin is a gonadal glycopeptide that inhibits the secretion of follitropin by the pituitary gland. On the other hand activin activates the secretion of follitropin. Activin is also important in embryonic axial development.
SEQ ID : 8
Description: Replacement of 128 N-terminal amino acids of the original inhibin protein by alternative 5 amino acids. The deleted part contains propep and glycosylation site of the original protein. The resulting new variant sustains the inhibin beta chain.
Accession: IL6_HUMAN Name: Interleukin 6 Function: IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig-secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation.
SEQ ID : 9
Description: Deletion of 17 amino acids between the positions 79-96 of the original interleukin 6 protein. Has all necessary domains.
Accession: IL6_HUMAN Name: Interleukin 6 Function: IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig-secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation. SEQ ID : 10
Description: Deletion of 55 amino acids between the positions 6-61 of the original protein. Has only the beginning of signal peptide; has disulfide bonds 5 and carbohydrate region.
Accession: REL1_HUMAN
Name: Relaxin
Function: Relaxin is an ovarian hormone that acts with estrogen to
10 produce dilatation of the birth canal in many mammals.
SEQ ID : 11
Description: Insertion of 35 amino acids after the amino acid at the position 15 70 of the original relaxin protein. The insertion is in the connecting peptide.
Accession: SY04_HUMAN Name: SMALL INDUCIBLE CYTOKINE A4, MACROPHAGE INFLAMMATORY PROTEIN 1-BETA 0 Function: Monokine with inflammatory and chemokinetic properties
SEQ ID : 12
Description: Deletion of 5 amϊno acids between the positions 65-69 of the 5 original protein. Replacement of the amino acid at the position 70 of the original protein by an alternative amino acid. Missing part of strand.
Accession: TSPIJHUMAN Name: Thrombospondin adhesive glycoprotein 0 Function: Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen
SEQ ID : 13 5
Description: Truncated exact 1-722 (731aa long compared to 1170aa), last 9 amino acids are different. Missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL, missing CELL ATTACHMENT SITE, missing 1 out of 4 glycosylation sites. Has all other components including signal peptide
Accession: TSP1_HUMAN Name: Thrombospondin adhesive glycoprotein
Function: Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen
SEQ ID : 14
Description: Truncated exact 1-548 (555aa long compared to 1170) last 7aa different. Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS Ca-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylation sites. Has all other domains (including signal peptide).
Accession: TSP1_HUMAN Name: Thrombospondin adhesive glycoprotein Function: Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen
SEQ ID : 15
Description: Truncated exact 1-490 (546aa long compared to 1170) last 56 amino acids are different. Missing 1 out of 3 X TSP TYPE-1 REPEATS (CS-LIKE), Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylations. Has all other domains (including signal peptide).
Accession: TSP1_HUMAN Name: Thrombospondin adhesive glycoprotein Function Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type v collagen SEQ ID : 16
Description: Truncated: exact 1-43 laa (459aa long compared to 1170) last 28 amino acids are different. Missing 2 out of 3 X TSP TYPE-1 REPEATS (CS-LIKE), Missing 3 X EGF-TYPE REPEATS, missing 7 X TSP TYPE-3 REPEATS CA-BINDING domain C-TERMINAL missing CELL ATTACHMENT SITE, missing all diSulfide bonds, missing 2 out of 4 glycosylations. Has all other domains (including signal peptide).
Accession: TYPH HUMAN Name: PLATELET-DERIVED ENDOTHELIAL CELL GROWTH FACTOR
Function: May have a role in maintaining the integrity of the blood vessels. Has growth promoting activity on endothelial cells, angiogenic activity in vivo and chemotactic activity on endothelial cells in vitro.
CATALYSES THE REVERSIBLE PHOSPHOROLYSIS OF THYMIDINE. THE PRODUCED MOLECULES ARE THEN UTILIZED AS CARBON AND ENERGY SOURCES OR IN THE RESCUE OF PYRIMIDINE BASES FOR NUCLEOTIDE SYNTHESIS. SIMILARITY: - BELONGS TO THYMIDINE/ PYRIMIDINE-NUCLEOSIDE PHOSPHORYLASES
FAMILY.
SEQ ID : 17
Description: Deletion of 119 amino acids between the positions 333-452 of the original protein. The resulting new variant is missing the 3rd repeat of the original protein.
Accession: TYPH_HUMAN Name: PLATELET-DERIVED ENDOTHELIAL CELL GROWTH
FACTOR Function: May have a role in maintaining the integrity of the vessels. Has growth promoting activity on endothelial cells, angiogenic activity in vivo and chemotactic activity on endothelial cells in vitro.
CATALYSES THE REVERSIBLE PHOSPHOROLYSIS OF THYMIDINE. THE PRODUCED MOLECULES ARE THEN UTILIZED AS CARBON AND ENERGY SOURCES OR IN THE RESCUE OF PYRIMIDINE BASES FOR NUCLEOTIDE SYNTHESIS. SIMILARITY: BELONGS TO THYMIDINE/ PYRIMIDINE-NUCLEOSIDE PHOSPHORYLASES
FAMILY.
SEQ ID : 18
Description: Replacement of 48 amino acids between the positions 216-264 of the original protein by alternative 9 amino acids.
Accession: TYPH HUMAN Name: PLATELET-DERIVED ENDOTHELIAL CELL GROWTH
FACTOR
Function: May have a role in maintaining the integrity of the vessels.
Has growth promoting activity on endothelial cells, angiogenic activity in vivo and chemotactic activity on endothelial cells in vitro.
CATALYSES THE REVERSIBLE PHOSPHOROLYSIS
OF THYMIDINE. THE PRODUCED MOLECULES ARE
THEN UTILIZED AS CARBON AND ENERGY
SOURCES OR IN THE RESCUE OF PYRIMIDINE
BASES FOR NUCLEOTIDE SYNTHESIS.
SIMILARITY: BELONGS TO THYMIDINE/
PYRIMIDINE-NUCLEOSIDE PHOSPHORYLASES
FAMILY. SEQ ID : 19
Description: Deletion of 119 amino acids between the positions 333-452, missing 3rd repeat of the original protein. Replacement of 48 amino acids between the positions 216-264 of the original protein by alternative 9 amino acids.
Accession: IC1_HUMAN Name: PLASMA PROTEASE Cl INHIBITOR Function: Activation of the cl complex is under control of the cl-.
Inhibitor. IT FORMS A PROTEOLYTICALLY INACTIVE
STOICHIOMETRIC COMPLEX WITH THE C1R OR CIS
PROTEASES. MAY PLAY A POTENTIALLY CRUCIAL
ROLE IN REGULATING IMPORTANT
PHYSIOLOGICAL PATHWAYS INCLUDING
COMPLEMENT ACTIVATION, BLOOD
COAGULATION, FIBRINOLYSIS AND THE
GENERATION OF KININS.
PTM: HIGHLY GLYCOSYLATED (49%).
SIMILARITY: BELONGS TO THE SERPIN FAMILY.
SEQ ID NO : 20
Description: Deletion of 19 amino acids between the positions 29-48 of the original protein. Missing 1 glycosylation out of 14.
Accession: PTI6 TUMAN
Name: PLACENTAL THROMBIN INHIBITOR
Function: Cytoplasmic antiproteinase.
SIMILARITY: BELONGS TO THE SERPIN FAMILY.
OV-SERPIN SUBFAMILY.
SEQ ID : 21
Description: Deletion of 261 N-terminal amino acids of the original protein (the first possible Met is at the position 261). The new variant has 116 amino acids compared to 376 in the original protein (exact 261-376), including the active site. Accession: PTI6_HUMAN Name: PLACENTAL THROMBIN INHIBITOR Function: Cytoplasmic antiproteinase
SIMILARITY: BELONGS TO THE SERPIN FAMILY.
OV-SERPIN SUBFAMILY.
SEQ ID : 22
Description: Deletion of 57 amino acids between the positions 267-325 of the original protein. The resulting new variant contains the active site.
Accession: PTI6_HUMAN
Name: PLACENTAL THROMBIN INHIBITOR
Function: Cytoplasmic antiproteinase
SEQ ID : 23
Description: Deletion of 189 amino acids between the positions 89-278 of the original protein. The resulting new variant contains the active site.
Accession: PTI6_HUMAN
Name: PLACENTAL THROMBIN INHIBITOR
Function: Cytoplasmic antiproteinase
SEQ ID : 24
Description: Replacement of 376 C-terminal amino acids of the original protein by alternative 5 amino acids. The resulting new variant doesn't contain the active site.
Accession: IAP2_HUMAN
Name: INHIBITOR OF APOPTOSIS PROTEIN 2
Function: Apoptotic suppressor. The BIR motifs region interacts with
TNF receptor associated factors 1 and 2 (trafl and traf2) to form an heteromeric complex, which is then recruited to the tumor necrosis factor receptor 2 (TNFR2). SEQ ID : 25
Description: Truncated: 305 amino acids compared to 618 aa(protein 2). The new variant contains exact positions 1-299, last 6 amino acids are different. Two SNIPs in positions 235 and 241 of the original protein. The new variant is missing Zn Finger and half of 3rd BIR repeat.
Accession: SET JUMAN Name: PHOSPHATASE 2A INHIBITOR I2PP2A Function: May be involved in the generation of intracellular signaling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules. Potent inhibitor of protein phosphatase 2a.
SEQ ID : 26
Description: Extra 83 amino acids in the N-terminus of the protein. The added sequence has predicted potential transmembrane domain (probable signal peptide?)
Accession: SET_HUMAN
Name: PHOSPHATASE 2A INHIBITOR I2PP2A
Function: May be involved in the generation of intracellular signaling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules. Potent inhibitor of protein phosphatase 2a.
SEQ ID : 27
Description: Replacement of 24 C-terminal amino acids of the original protein by alternative 8 amino acids. Missing part of ASP/GLU-RICH and BREAKPOINT FOR TRANSLOCATION TO FORM SET-CAN
ONCOGENE.
Accession: CDNC_HUMAN Name: CYCLIN-DEPENDENT KINASE INfflBITOR 1C Function: POTENT TIGHT-BINDING INHIBITOR OF SEVERAL GI CYCLIN/CDK COMPLEXES (CYCLIN E-CDK2, CYCLIN D2-CDK4, AND , CYCLIN A-CDK2) AND, TO LESSER EXTENT, OF THE MITOTIC CYCLIN B-CDC2. NEGATIVE REGULATOR OF CELL PROLIFERATION. MAY PLAY A ROLE IN MAINTENANCE OF THE NONPROLIFERATIVE STATE THROUGHOUT LIFE. SUBCELLULAR LOCATION: NUCLEAR (BY SIMILARITY).
DISEASE: CDKN1C MUTATIONS ARE INVOLVED IN TUMOR FORMATION.
SEQ ID : 28
Description: Deletion of 178 amino acids at the positions 97-275 of the original protein. Insertion of 121 amino acids at the N-terminus. The resulting new variant is missing PAPA repeats.
Accession: F13B_MOUSE Name: COAGULATION FACTOR XIII B CHAIN Function: The B chain of factor XIII is not catalytically active, but is thought to stabilize the a subunits and regulate the rate of transglutaminase formation by thrombin
SEQ ID : 29
Description
Deletion of 87 C-terminal amino acids of the original protein. SNIP at position 236 (L->V). The resulting new variant is missing the last shushi repeat.
Accession: EGF_MOUSE
Name: PRO-EPIDERMAL GROWTH FACTOR
Function: Stimulates the growth of various epidermal and epithelial tissues.
SEQ ID : 30 Description: Deletion of 641 amino acids between the positions 67-708 of the original protein. Missing 4 EGF-like domains, 2 glycosylations, 9 diSulfide bonds.
Accession: EGF MOUSE
Name: PRO-EPIDERMAL GROWTH FACTOR
Function: Stimulates the growth of various epidermal and epithelial
Tissues
SEQ ID : 31
Description: Deletion of 641 amino acids between the positions 67-708, and deletion of 45 amino acids between the positions 1020-1065 of the original protein. Missing 4 EGF-like domains, 2 glycosylations, 9diSulfide bonds. Missing transmembrane domain.
Accession: EGF_MOUSE Name: PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial tissues
SEQ ID : 32
Description:
Deletion of 641 amino acids between the positions 67-708 of the original protein. Missing 4 EGF-like domains, 2 glycosylations, 9 diSulfide bonds. Replacement of 419 C-terminal amino acids by 5 amino acids.
Accession: EGFJVIOUSE Name: PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial
Tissues
SEQ ID : 33
Description: Deletion of 841 amino acids between the positions 18-859 of the original protein. Missing 5 EGF-like domains and 2 glycosylation sites.
Accession: EGF_MOUSE Name: PRO-EPIDERMAL GROWTH FACTOR Function: Stimulates the growth of various epidermal and epithelial
Tissues SEQ ID : 34
Description: Deletion of 774 amino acids between the positions 5-779 of the original protein. Missing signal peptide, 5 EGF-like domains, and 2 glycosylation sites.
Accession: P53_MOUSE
Name: CELLULAR TUMOR ANTIGEN P53
Function: Acts as a tumor suppressor in many tumor types. Induces growth arrest or apoptosis depending on the physiological circumstances or cell type, but both activities are involved in tumor suppression.
SEQ ID : 35
Description: Deletion of 336 N-terminal amino acids of the original protein. Missing ASP/GLU-RICH (ACIDIC), missing hydrophobic domain, missing NUCLEAR LOCALIZATION SIGNAL , missing 1 out of 2 PHOSPHORYLATION sites.
Accession: NME3_HUMAN Name: GLUTAMATE [NMDA] RECEPTOR SUBUNIT
EPSILON 3
Function: NMDA receptor subtype of glutamate-gated ion channels possesses high calcium permeability and voltage-dependent sensitivity to magnesium and is mediated by glycine.
SEQ ID : 36
Description: Deletion of 381 N-terminal amino acids of the original protein. Missing 2 out of 4 glycosylation sites.
Accession: TRFE_HUMAN Name: SEROTRANSFERRIN Function: Iron binding transport proteins SEQ ID : 37
Description: Deletion of 34 amino acids between the positions 654-689 of the original protein. Loss of disulfide bond.
Accession: TRFE TUMAN
Name: SEROTRANSFERRIN
Function: Iron binding transport proteins
SEQ ID : 38
Description: Deletion of 52 amino acids between the positions 447-499 of the original protein. Loss of disulfide bond.
Accession: BAA23795 Name: Brain ryanodine receptor
SEQ ID : 39
Description: Replacement of 83 C-terminal amino acids from probable cytoplasmic domain of the original protein by alternative 4 amino acids. Resulting in truncated new variant: 4787 compared to 4866, exact 1-4783 with last 4 amino acids different.
Accession: VIPS IUMAN Name: VASOACTIVE INTESTINAL POLYPEPTIDE
RECEPTOR 2
Function: This is a receptor for VIP as well as PACAP-38 and -27, the activity of this receptor is mediated by G proteins which activate adenylyl cyclase. Can be coupled to phospholipase C.
SEQ ID : 40 Description: Replacement of 64 C-terminal amino acids of the original protein by alternative 7 amino acids. The resulting new variant is missing the last transmembrane and the cytoplasmic domains. Accession: PACR_HUMAN Name: PITUITARY ADENYLATE CYCLASE ACTIVATING
POLYPEPTIDE TYPE RECEPTOR
Function: This is a receptor for PACAP-27 and PACAP-38. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. May regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, prolactin, epinephrine.
SEQ ID : 41
Description: Deletion of 22 amino acids between the positions 88-110 of the original protein. The deletion is in extracellular loop.
Accession: NRPJHUMAN Name: NEUROPILIN VASCULAR ENDOTHELIAL CELL GROWTH FACTOR 165 RECEPTOR
Function: Calcium-independent cell adhesion molecule that function during the formation of certain neuronal circuits. Binds to semaphorin III and to the VEGF 165 isoform of VEGF
SEQ ID : 42
Description: Deletion of 540 C-terminal amino acids of the original protein, resulting in truncated new variant (383 compared to 923 amino acids).
The new variant is missing part of the extracellular domain, the cytoplasmic and the transmembrane domains.
Accession: NRP_HUMAN Name: NEUROPILIN VASCULAR ENDOTHELIAL CELL GROWTH FACTOR 165 RECEPTOR
Function: Calcium-independent cell adhesion molecule that during the formation of certain neuronal circuits. Binds to semaphorin III and to the VEGF 165 isoform of VEGF R2 NV43
Description: Replacement of 595 C-terminal amino acids of the original protein by alternative 11 amino acids. The resulting new variant is truncated (339 compared to 923 amino acids, exact 1-328 with last 11 amino acids different), and is missing part of the extracellular domain, the cytoplasmic and the transmembrane domains.
Accession: gi| 1899200 Name: N-METHYL D-ASPARTATE RECEPTOR SUBTYPE 2A Function: NMDA RECEPTOR SUBTYPE OF GLUTAMATE-
GATED ION CHANNELS POSSESSES HIGH CALCIUM
PERMEABILITY AND VOLTAGE-DEPENDENT
SENSITIVITY TO MAGNESIUM AND IS MEDIATED
BY GLYCINE.
SUBUNIT: HETERODIMER OF AN EPSILON SUBUNIT
AND A ZETA SUBUNIT.
SUBCELLULAR LOCATION: INTEGRAL MEMBRANE
PROTEIN.
SIMILARITY: BELONGS TO THE LIGAND-GATED
IONIC CHANNELS FAMILY.
SEQ ID : 44:
Description: Deletion of 114 amino acids between the positions
1257-1372 of the original protein.
Accession: VIPS_HUMAN Name: VASOACTIVE INTESTINAL POLYPEPTIDE
RECEPTOR 2
Function: THIS IS A RECEPTOR FOR VIP AS WELL AS
PACAP-38 AND -27, THE ACTIVITY OF THIS
RECEPTOR IS MEDIATED BY G PROTEINS WHICH
ACTIVATE ADENYLYL CYCLASE. CAN BE
COUPLED TO PHOSPHOLIPASE C.
SUBCELLULAR LOCATION: INTEGRAL
MEMBRANE PROTEIN. SIMILARITY: BELONGS TO FAMILY 2 OF G-PROTEIN COUPLED RECEPTORS.
SEQ ID : 45:
Description: Replacement of 56 C-terminal amino acids from the cytoplasmic domain of the original protein by alternative 73 amino acids.
SEQ ID : 46
Description: Replacement of 56 C-terminal amino acids from the cytoplasmic domain of the original protein by alternative 70 amino acids.
Accession: IG1R ΪUMAN Name: INSULIN-LIKE GROWTH FACTOR I RECEPTOR
PRECURSOR
Function: THIS RECEPTOR BINDS INSULIN-LIKE GROWTH
FACTOR I (IGF I) WITH A HIGH AFFINITY AND IGF II
WITH A LOWER AFFINITY. IT HAS A
TYROSINE-PROTEIN KINASE ACTIVITY.
CATALYTIC ACTIVITY: ATP + A PROTEIN TYROSINE
= ADP + PROTEIN TYROSINE PHOSPHATE.
SUBUNIT: TETRAMER OF 2 ALPHA AND 2 BETA
CHAINS LINKED BY DISULFIDE BONDS. THE ALPHA
CHAINS
CONTRIBUTE TO THE FORMATION OF THE LIGAND-
BINDING DOMAIN, WHILE THE BETA CHAIN
CARRIES THE KINASE DOMAIN.
SUBCELLULAR LOCATION: TYPE I MEMBRANE
PROTEIN.
SIMILARITY: BELONGS TO THE INSULIN RECEPTOR
FAMILY OF TYROSINE- PROTEIN KINASES.
SIMILARITY: CONTAINS 2 FIBRONECTIN TYPE
HI-LIKE DOMAINS. SEQ ID : 47
Description: Deletion of 22 amino acids between the positions 1268-1291 of the original protein. The deleted fragment is part of the cytoplasmic domain of INSULIN-LIKE GROWTH FACTOR I RECEPTOR, BETA-CHAIN.
Accession: NRP_HUMAN Name: NEUROPILIN Function: CALCIUM-INDEPENDENT CELL ADHESION
MOLECULE THAT FUNCTION DURING THE
FORMATION OF CERTAIN NEURONAL CIRCUITS.
BINDS TO SEMAPHORIN III AND TO THE VEGF 165
ISOFORM OF VEGF.
SUBCELLULAR LOCATION: TYPE I MEMBRANE
PROTEIN.
SIMILARITY: CONTAINS 2 CUB DOMAINS.
SIMILARITY: CONTAINS 2 F5/8 TYPE C DOMAINS.
SIMILARITY: CONTAINS 1 MAM DOMAIN.
SEQ ID : 48
Description: Replacement of 282 C-terminal amino acids of the original protein, including the transmembrane domain and the MAM domain, by alternative 3 amino acids.
SEQ ID : 49
Description: Deletion of 83 amino acids between the positions 538-622 of the original protein. The deleted region includes part of the F5/8 TYPE C 2 domain and part of the MAM domain.
SEQ ID : 50
Description: Deletion of 385 C-terminal amino acids of the original protein, including the transmembrane domain and the MAM domain. Accession: FGR3_HUMAN
Name: FIBROBLAST GROWTH FACTOR RECEPTOR 3
Function: SUBCELLULAR LOCATION: TYPE I MEMBRANE PROTEIN.
DISEASE: DEFECTS IN FGFR3 ARE THE CAUSE OF THE AUTOMOSOMAL DOMINANT DISEASE ACHONDROPLASIA (ACH); THE MOST FREQUENT FORM OF SHORT-LIMB DWARFISM. ACH IS CHARACTERIZED BY A LONG, NARROW TRUNK, SHORT EXTREMITIES, PARTICULARLY IN THE PROXIMAL (RHIZOMELIC) SEGMENTS, A LARGE HEAD WITH FRONTAL BOSSING, HYPOPLASIA OF THE MIDFACE AND A TRIDENT CONFIGURATION OF THE HANDS.
DISEASE: DEFECTS IN FGFR3 ARE A CAUSE OF CROUZON SYNDROME, ALSO CALLED
CRANIOFACIAL DYSOSTOSIS TYPE I (CFD1). CHARACTERIZED BY CRANIOSYNOSTOSIS (PREMATURE FUSION OF THE SKULL SUTURES), HYPERTELORISM, EXOPHTHALMOS AND
EXTERNAL STRABISMUS, PARROT-BEAKED NOSE, SHORT UPPER LIP, HYPOPLASTIC MAXILLA, AND A RELATIVE- MANDIBULAR PROGNATHISM. DISEASE: DEFECTS IN FGFR3 ARE A CAUSE OF THANATOPHORIC DYSPLASIA (TD) (ALSO KNOWN AS THANATOPHORIC DWARFISM), THE MOST COMMON NEONATAL LETHAL SKELETAL DYSPLASIA, AFFECTED INDIVIDUALS DISPLAY FEATURES SIMILAR TO THOSE SEEN IN HOMOZYGOUS ACHONDROPLASIA. IT CAUSES SEVERE SHORTENING OF THE LIMBS WITH MACROCEPHALY, NARROW THORAX AND SHORT RIBS. IN THE MOST COMMON SUBTYPE (TD1), FEMUR ARE CURVED, WHILE IN TD2, STRAIGHT FEMURS ARE ASSOCIATED WITH CLOVERLEAF SKULL. DISEASE: DEFECTS IN FGFR3 ARE A CAUSE OF CRANIOSYNOSTOSIS ADELAIDE TYPE (CRS3), A FORM OF CORONAL SYNOSTOSIS (CS) CHARACTERIZED BY CRANIOSYNOSTOSIS, MIDFACE HYPOPLASIA, DOWNSLANDING
PALPEBRAL FISSURES, PTOSIS, HIGHLY ARCHED PALATE, MID-TO-MODERATE SENSORINEURAL HEARING LOSS, NORMAL STATURE,
BRADYDACTYLY, BROAD BIG TOES. RADIOLOGICAL Y HANDS AND FEET SHOW
THIMBLE-LIKE MIDDLE PHALANGES, CONED EPIPHYSES, AND CARPAL AND TARSAL FUSIONS. DISEASE: DEFECTS IN FGFR3 ARE A CAUSE OF THE AUTOSOMAL DOMINANT DISEASE HYPO- CHONDROPLASIA CHARACTERIZED BY
DISPROPORTIONATE SHORT STATURE. IT RESEMBLE ACHONDROPLASIA, BUT WITH A LESS SEVERE PHENOTYPE. SIMILARITY: BELONGS TO THE FIBROBLAST GROWTH FACTOR RECEPTOR FAMILY.
SIMILARITY: CONTAINS 3 IMMUNOGLOBULIN- LIKE DOMAINS.
SEQ ID : 51
Description: Replacement of 496 C-terminal amino acids of the original protein by alternative 79 amino acids.. The deleted region includes the C-terminal part of the extracellular domain, the transmembrane domain, the cytoplasmic domain, the protein kinase domain and the two ATP binding domains.
EGF MOUSE
EPIDERMAL GROWTH FACTOR
FUNCTION: THE GROWTH FACTOR STIMULATES THE GROWTH OF VARIOUS EPIDERMAL AND EPITHELIAL TISSUES IN VIVO AND IN VITRO AND OF SOME FIBROBLASTS IN CELL CULTURE. SUBCELLULAR LOCATION: TYPE I MEMBRANE PROTEIN. SIMILARITY: CONTAINS 8 COMPLETE AND ONE INCOMPLETE EGF-LIKE DOMAINS.
SEQ ID NO : 52
Deletion of 144 amino acids between the positions 1020-1165, including the transmembrane domain and part of the cytoplasmic domain of the original protein.
SEQ ID NO : 53
Replacement of 418 C-terminal amino acids of the original protein by alternative 5 amino acids. The deleted region includes the EGF active chain, 4 out of 9 EGF-like domains within the extracellular region of the protein, the transmembrane and the cytoplasmic regions.
SEQ ID NO : 54
Deletion of 641 amino acids between the positions 66-707 of the original protein. The deleted region is in the extracellular part of the protein and it includes 4 out of 9 EGF-like domains. NV-20 contains the original signal peptide, part of the extracellular domain, the epidermal growth factor chain (located between the positions 977-1029 of the original protein), and the original transmembrane and the cytoplasmic domains.
SEQ ID NO : 55
Deletion of 842 amino acids between the positions 17-859 of the original protein (including replacement of the amino acid in the position 859 by an alternative one). The deleted region is in the extracellular part of the protein and it includes 5 out of 9 EGF-like domains. NV-20 contains the original signal peptide, part of the extracellular domain, the epidermal growth factor chain (located between the positions 977-1029 of the original protein), and the original transmembrane and the cytoplasmic domains.
ACE_MOUSE
ANGIOTENSIN-CONVERTING ENZYME FUNCTION: CONVERTS ANGIOTENSIN I TO ANGIOTENSIN II BY RELEASE OF THE TERMINAL HIS-LEU,THIS RESULTS IN AN INCREASE OF THE VASOCONSTRICTOR ACTIVITY OF ANGIOTENSIN. CATALYTIC ACTIVITY: RELEASE OF A C-TERMINAL DIPEPTIDE, OLIGOPEPTIDE-I-XAA-XBB, WHEN XAA IS NOT PRO, AND XBB IS NEITHER ASP NOR GLU. CONVERTS ANGIOTENSIN I TO ANGIOTENSIN II. COF ACTOR: BINDS TWO ZINC IONS (BY SIMILARITY). SUBCELLULAR LOCATION: TYPE I MEMBRANE PROTEIN. ALTERNATIVE PRODUCTS: THE TESTICULAR ANGIOTENSIN- CONVERTING ENZYME IS TRANSCRIBED FROM THE SAME GENE AS THE SOMATIC ISOFORM, PROBABLY FROM AN ALTERNATIVE START SITE.
SIMILARITY: BELONGS TO PEPTIDASE FAMILY M2 (ZINC METALLOPROTEASE).
SEQ ID NO : 56
Replacement of 77 C-terminal amino acids of the original protein, including the entire transmembrane and cytoplasmic domains, by alternative 14 amino acids.
ESRl_MOUSE
ESTROGEN RECEPTOR
FUNCTION: THE STEROID HORMONES AND THEIR RECEPTORS ARE INVOLVED IN THE REGULATION OF EUKARYOTIC GENE EXPRESSION AND AFFECT CELLULAR PROLIFERATION AND DIFFERENTIATION IN TARGET TISSUES. SUBUNIT: HOMODIMER. SUBCELLULAR LOCATION: NUCLEAR.
DOMAIN: COMPOSED OF THREE DOMAINS: A MODULATING N-TERMINAL DOMAIN, A DNA-BINDING DOMAIN AND A C-TERMINAL STEROID-BINDING DOMAIN. MISCELLANEOUS: IN THE ABSENCE OF LIGAND, STEROID HORMONE RECEPTORS ARE THOUGHT TO BE WEAKLY ASSOCIATED WITH NUCLEAR COMPONENTS; HORMONE BINDING GREATLY INCREASES RECEPTOR AFFINITY. THE HORMONE-RECEPTOR COMPLEX APPEARS TO RECOGNIZE DISCRETE DNA SEQUENCES UPSTREAM OF TRANSCRIPTIONAL START SITES. SIMILARITY: BELONGS TO THE NUCLEAR HORMONE RECEPTORS FAMILY. NR3 SUBFAMILY.
SEQ ID : 57
Replacement of 229 C-terminal amino acids of the original protein, including part of the steroid-binding domain, by an alternative 12 amino acids.
FA7_MOUSE
COAGULATION FACTOR VII PRECURSOR
FUNCTION: CIRCULATES IN THE BLOOD IN A ZYMOGEN FORM.
FACTOR VII IS CONVERTED TO FACTOR VIIA BY FACTOR XA,
FACTOR XIIA, FACTOR IXA, OR THROMBIN BY MINOR PROTEOLYSIS.
IN THE PRESENCE OF TISSUE FACTOR AND CALCIUM IONS, FACTOR
VIIA THEN CONVERTS FACTOR X TO FACTOR XA BY LIMITED PROTEOLYSIS. FACTOR VIIA WILL ALSO CONVERT FACTOR IX TO
FACTOR IXA IN THE PRESENCE OF TISSUE FACTOR AND CALCIUM
(BY SIMILARITY).
CATALYTIC ACTIVITY: HYDROLYSES ONE ARG-|-ILE BOND IN
FACTOR X TO FORM FACTOR XA. SUBUNIT: HETERODIMER OF A LIGHT CHAIN AND A HEAVY CHAIN
LINKED BY A DISULFIDE BOND (BY SIMILARITY).
TISSUE SPECIFICITY: PLASMA.
PTM: THE VITAMIN K-DEPENDENT, ENZYMATIC CARBOXYLATION
OF SOME GLUTAMIC ACID RESIDUES ALLOWS THE MODIFIED PROTEIN TO BIND CALCIUM (BY SIMILARITY).
SIMILARITY: CONTAINS 2 EGF-LIKE DOMAINS.
SIMILARITY: BELONGS TO PEPTIDASE FAMILY SI; ALSO KNOWN AS
THE TRYPSIN FAMILY.
SEQ ID : 58
Deletion of 101 amino acids, between the positions 119-220 of the original protein. The deleted region contains 74 amino acids from the C-terminal end of the factor VII light chain, and 26 amino acids from the N-terminal end of the factor VII heavy catalytic chain. The deleted region includes EGF-like 2 domain and the cleavage site (by factor XA, factor XIIA, factor IXA, or thrombin) of the original protein.
CAL0_MOUSE
CALCITONIN PRECURSOR
FUNCTION: CAUSES A RAPID BUT SHORT-LIVED DROP IN THE LEVEL OF CALCIUM AND PHOSPHATE IN BLOOD BY PROMOTING THE INCORPORATION OF THOSE IONS IN THE BONES.
ALTERNATIVE PRODUCTS: THE CALCITONIN PRECURSOR AND THE CALCITONIN RELATED PEPTIDE PRECURSOR ARE OBTAINED BY TISSUE-SPECIFIC SPLICING OF THE SAME GENE. SIMILARITY: BELONGS TO THE CALCITONIN FAMILY.
SEQ ID NO : 59
Deletion of 33 amino acids, spanning the positions 18-50, between the signal and the calcitonin peptide in the original precursor protein.
gi 2826776
VESICULAR INHIBITORY AMINO ACID TRANSPORTER
function=:"uptake of GABA and glycine into synaptic vesicles"
SEQ ID NO : 60
Replacement of the last 7 C-terminal amino acids of the original protein by alternative 1 1 amino acids.
PTI6JHUMAN
PLACENTAL THROMBIN INHIBITOR
CYTOPLASMIC ANTIPROTEINASE, PROTEASE INHIBITOR 6.
SIMILARITY: BELONGS TO THE SERPIN FAMILY. OV-SERPIN SUBFAMILY. SEQ ID NO : 61
Replacement of the last 4 C-terminal amino acids of the original protein by alternative 28 amino acids.
SEQ ID NO : 62
Replacement of the last 16 C-terminal amino acids of the original protein by alternative 12 amino acids.
RIN1_HUMAN
RAS INTERACTION INTERFERENCE PROTEIN 1
(RAS INHIBITOR JC99 (FRAGMENT)
SEQ ID NO : 63
Replacement of 158 last C-terminal amino acids of the original protein by alternative 71 amino acids with probable transmembrane region.
CDNC_HUMAN
CYCLIN-DEPENDENT KINASE INHIBITOR 1C P57
FUNCTION: POTENT TIGHT-BINDING INHIBITOR OF SEVERAL GI CYCLIN/CDK COMPLEXES (CYCLIN E-CDK2, CYCLIN D2-CDK4, AND CYCLIN A-CDK2) AND, TO LESSER EXTENT, OF THE MITOTIC CYCLIN B-CDC2. NEGATIVE REGULATOR OF CELL PROLIFERATION. MAY PLAY A ROLE IN MAINTENANCE OF THE NONPROLIFERATIVE STATE THROUGHOUT LIFE.
SUBCELLULAR LOCATION: NUCLEAR (BY SIMILARITY). DISEASE: CDKN1C MUTATIONS ARE INVOLVED IN TUMOR
FORMATION.
SEQ ID NO : 64
Addition of 121 amino acids at the N-terminus of the protein. CDN2_HUMAN
CYCLIN-DEPENDENT KINASE 4 INHIBITOR A (CDK4D (MULTIPLE TUMOR SUPPRESSOR 1) (MTS1
FUNCTION: INTERACTS STRONGLY WITH CDK4 AND CDK6. INHIBITS ITS ABILITY TO INTERACT WITH CYCLINS D. COULD ACT AS A
NEGATIVE REGULATOR OF THE PROLIFERATION OF NORMAL
CELLS.
SUBUNIT: HETERODIMER WITH CDK4 OR CDK6.
DISEASE: CDKN2A MUTATIONS ARE INVOLVED IN TUMOR FORMATION IN A WIDE RANGE OF TISSUES.
SIMILARITY: BELONGS TO THE CDKN2 FAMILY OF
CYCLIN-DEPENDENT KINASE INHIBITORS.
SIMILARITY: CONTAINS 4 ANK REPEATS.
SEQ ID NO : 65
Replacement of 5 amino acids at the positions 18, 24, 27, 30, 37 of the original protein by alternative amino acids. Replacement of last 4 C-terminal amino acids of the original protein by alternative 20 amino acids.
CDN5_HUMAN
CYCLIN-DEPENDENT KINASE 4 INHIBITOR B
(MULTIPLE TUMOR SUPPRESSOR 2)
FUNCTION: INTERACTS STRONGLY WITH CDK4 AND CDK6. POTENT
INHIBITOR. POTENTIAL EFFECTOR OF TGF-BETA INDUCED CELL CYCLE ARREST.
SUBUNIT: HETERODIMER OF P14 WITH CDK4.
DISEASE: CDKN2B MUTATIONS ARE INVOLVED IN TUMOR
FORMATION.
SIMILARITY: BELONGS TO THE CDKN2 FAMILY OF CYCLIN-DEPENDENT KINASE INHIBITORS.
SIMILARITY: CONTAINS 2 ANK REPEATS. SEQ ID NO : 66
Replacement of the last 6 C-terminal amino acids of the original protein by alternative 52 amino acids.
HEP2_HUMAN
HEPARIN COFACTOR II PRECURSOR PROTEASE INHIBITOR LEUSERPIN 2
FUNCTION: THROMBIN INHIBITOR ACTIVATED BY THE GLYCOSAMINOGLYCANS, HEPARIN OR DERMATAN SULFATE. IN THE PRESENCE OF THE LATTER, HC-II BECOMES THE PREDOMINANT THROMBIN INHIBITOR IN PLACE OF ANTITHROMBIN III (AT). ALSO INHIBITS CHYMOTRYPSIN, BUT IN A GLYCOSAMINOGLYCAN- ΓNDEPENDENT MANNER.
FUNCTION: PEPTIDES AT THE N-TERMINAL OF HC-II HAVE CHEMOTACTIC ACTIVITY FOR BOTH MONOCYTES AND NEUTROPHILS. TISSUE SPECIFICITY: EXPRESSED PREDOMINANTLY IN LIVER, DOMAIN: THE N-TERMINAL ACIDIC REPEAT REGION MEDIATES, IN
PART, THE
GLYCOSAMINOGLYCAN-ACCELERATED THROMBIN INHIBITION. DISEASE: DEFECTS IN HCF2 ARE ASSOCIATED WITH THROMBOSIS (THROMBOPHILIA).
SIMILARITY: BELONGS TO THE SERPIN FAMILY.
SEQ ID NO : 67 Deletion of 150 amino acids, between the positions 334-485, of the original protein. The deleted region includes the reactive bond (the active site) of the original protein. NV-33 does contain the chemotactic activity domain, the glycosaminoglycan-binding site and the hirudin-like 2 x 11 AA approximate repeats, Asp/Glu rich.
TFP2JHUMAN
TISSUE FACTOR PATHWAY INHIBITOR 2 PRECURSOR
FUNCTION: SEEMS TO INHIBIT TRYPSIN, FACTOR VII(A)/TISSUE FACTOR, WEAKLY FACTOR XA. HAS NO EFFECT ON THROMBIN. DOMAIN: THIS INHIBITOR CONTAINS THREE INHIBITORY DOMAINS. SIMILARITY: BELONGS TO THE BPTI/KUNITZ FAMILY OF INHIBITORS. HIGHLY SIMILAR TO TPFI.
SEQ ID NO : 68
Replacement of 36 C-terminal amino acids of the original protein by alternative 12 amino acids. The deleted region includes part of the BPTI/KUNITZ inhibitor domain-3 and the poly-Lysine domain of the original protein.
SEQ ID NO : 69
Deletion of 25 amino acids, between the positions 153-178 of the original protein, and replacement of the amino acid at the position 179 by alternative one. The deleted region includes the active site and part of the BPTI/KUNITZ inhibitor domain-3.
TFPIJHUMAN
TISSUE FACTOR PATHWAY INHIBITOR PRECURSOR (TFPD
SEQ H) NO : 70
Replacement of 95 C-terminal amino acids of the original protein, containing the entire BPTI/KUNITZ inhibitor-3 domain, by aternative 16 amino acids.
Example II: Variant nucleic acid sequence
The nucleic acid sequences of the invention include nucleic acid sequences which encode variant product and fragments and analogs thereof. The nucleic acid sequences may alternatively be sequences complementary to the above coding sequence, or to a region of said coding sequence. The length of the complementary sequence is sufficient to avoid the expression of the coding sequence. The nucleic acid sequences may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand. The nucleic acid sequences may also both include dNTPs, rNTPs as well as non naturally occurring sequences. The sequence may also be a part of a hybrid between an amino acid sequence and a nucleic acid sequence.
In a general embodiment, the nucleic acid sequence has at least 90%, identity with any one of the sequence identified as SEQ ID NO: 1 to SEQ ID NO: 174 provided that this sequence is not completely identical with that of the original sequence. The nucleic acid sequences may include the coding sequence by itself. By another alternative the coding region may be in combination with additional coding sequences, such as those coding for fusion protein or signal peptides, in combination with non-coding sequences, such as introns and control elements, promoter and terminator elements or 51 and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or in a vector or host environment in which the variant nucleic acid sequence is introduced as a heterologous sequence.
The nucleic acid sequences of the present invention may also have the product coding sequence fused in-frame to a marker sequence which allows for purification of the variant product. The marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767 (1984)).
Also included in the scope of the invention are fragments as defined above also referred to herein as oligonucleotides, typically having at least 20 bases, preferably 20-30 bases corresponding to a region of the coding-sequence nucleic acid sequence. The fragments may be used as probes, primers, and when complementary also as antisense agents, and the like, according to known methods.
As indicated above, the nucleic acid sequence may be substantially a depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 87 or fragments thereof or sequences having at least 90% identity to the above sequence as explained above. Alternatively, due to the degenerative nature of the genetic code, the sequence may be a sequence coding for any one of the amino acid sequence of SEQ ID NO: 88 to SEQ ID NO: 174, or fragments or analogs of said amino acid sequence.
A. Preparation of nucleic acid sequences
The nucleic acid sequences may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify nucleic acid sequences which encode the variant products disclosed above. cDNA libraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
The nucleic acid sequences may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al, supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # Kl 802-1).
Alternatively, the technique of "restriction-site" PCR (Gobinda et al. PCR Methods Applic. 2:318-22, (1993)), which uses universal primers to retrieve flanking sequence adjacent a known locus, may be employed. First, genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase. Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al., Nucleic Acids Res. 16:8186, (1988)). The primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992; National Biosciences Inc, Plymouth, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
Capture PCR (Lagerstrom, M. et al, PCR Methods Applic. 1:111-19, (1991)) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR. Another method which may be used to retrieve flanking sequences is that of Parker, J.D., et al, Nucleic Acids Res., 19:3055-60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinder™ libraries to "walk in" genomic DNA (PromoterFinder™; Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA. Genomic libraries are useful for extension into the 5' nontranslated regulatory region. The nucleic acid sequences and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
B. Use of variant nucleic acid sequence for the production of variant products
In accordance with the present invention, nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of variant products.
As will be understood by those of skill in the art, it may be advantageous to produce variant product-encoding nucleotide sequences possessing codons other than those which appear in any one of SEQ ID NO: 1 to SEQ ID NO: 87 which are those which naturally occur in the human genome. Codons preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al. Nuc Acids Res., 17:477-508, (1989)) can be selected, for example, to increase the rate of variant product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
The nucleic acid sequences of the present invention can be engineered in order to alter a variant product coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the product. For example, alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.
The present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al, (supra).
The present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transformed or fransfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the variant nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
The nucleic acid sequences of the present invention may be included in any one of a variety of expression vectors for expressing a product. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: LTR or SV40 promoter, the E.coli lac or trp promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracy cline or ampicillin resistance in E.coli.
The vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. Examples of appropriate expression hosts include: bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. The invention is not limited by the host cells employed.
In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the variant product. For example, when large quantities of variant product are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E.coli cloning and expression vectors such as Bluescript R) (Stratagene), in which the variant polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster J. Biol Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison Wl); and the like.
In the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. For reviews, see Ausubel et al (supra) and Grant et al, (Methods in Enzymology 153:516-544, (1987)).
In cases where plant expression vectors are used, the expression of a sequence encoding variant product may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al, Nature 310:511-514. (1984)) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al, EMBO J., 6:307-311, (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al, EMBO J. 3:1671-1680, (1984); Broglie et al, Science 224:838-843, (1984)); or heat shock promoters (Winter J and Sinibaldi R.M., Results Probl. Cell Differ., 17:85-105, (1991)) may be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For reviews of such techniques, see Hobbs S. or Murry L.E. (1992) in McGraw Hill Yearbook of Science and Technology, McGraw Hill, New York, N.Y., pp 191-196; or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York, N.Y., pp 421-463.
Variant product may also be expressed in an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The variant product coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of variant coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which variant protein is expressed (Smith et al, J. Virol 46:584, (1983); Engelhard, E.K. et al, Proc. Nat. Acad. Sci. 91:3224-7, (1994)).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a variant product coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing variant protein in infected host cells (Logan and Shenk, Proc. Natl Acad. Sci. 81:3655-59, (1984). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Specific initiation signals may also be required for efficient translation of a variant product coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where variant product coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D. et al, (1994) Results Probl Cell Differ., 20:125-62, (1994); Bittner et al., Methods in Enzymol 153:516-544, (1987)).
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology). Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
A host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "pre-pro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express variant product may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M., et al, Cell 11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I., et al, Cell 22:817-23, (1980)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M., et al, Proc. Natl. Acad. Sci. 77:3567-70, (1980)); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al, J. Mol Biol, 5 150:1-14, (1981)) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman S.C. and R.C. Mulligan, Proc. Natl. Acad. Sci.
10 85:8047-51, (1988)). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrates, luciferin and ATP, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. et. al,
15 Methods Mol Biol, 55:121-131, (1995)).
Host cells transformed with a nucleotide sequence encoding variant product may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The product produced by a recombinant cell may be secreted or contained intracellularly depending on the 0 sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing nucleic acid sequences encoding variant product can be designed with signal sequences which direct secretion of variant product through a prokaryotic or eukaryotic cell membrane.
The variant product may also be expressed as a recombinant protein with 5 one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS o extension/affinity purification system (Immunex Corp, Seattle, Wash.). The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and variant product is useful to facilitate purification. One such expression vector provides for expression of a fusion protein compromising a variant polypeptide fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath, et al., Protein Expression and Purification, 3:263-281, (1992)) while the enterokinase cleavage site provides a means for isolating variant polypeptide from the fusion protein. pGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
The variant products can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
C. Diagnostic applications utilizing nucleic acid sequences The nucleic acid sequences of the present invention may be used for a variety of diagnostic purposes. The nucleic acid sequences may be used to detect and quantitate expression of the variant in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for variant product. Alternatively, the assay may be used to detect soluble variant in the serum or blood. This assay typically involves obtaining total mRNA from the tissue or serum and contacting the mRNA with a nucleic acid probe. The probe is a nucleic acid molecule of at least 20 nucleotides, preferably 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding variant product under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of variant. This assay can be used to distinguish between absence, presence, and excess expression of variant product and to monitor levels of variant expression during therapeutic intervention. In addition, the assay may be used to compare the levels of the variant of the invention to the levels of the original sequence from which it has been varied or to levels of other variants, which comparison may have some physiological meaning.
The invention also contemplates the use of the nucleic acid sequences as a diagnostic for diseases resulting from inherited defective variant sequences, or diseases in which the ratio of the amount of the original sequence from which the variant was varied to the novel variants of the invention is altered. These sequences can be detected by comparing the sequences of the defective (i.e., mutant) variant coding region with that of a normal coding region. Association of the sequence coding for mutant variant product with abnormal variant product activity may be verified. In addition, sequences encoding mutant variant products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a variant protein deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once mutant genes have been identified, one can then screen populations of interest for carriers of the mutant gene. Individuals carrying mutations in the nucleic acid sequence of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al, Nature 324:163-166, (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et alProc. Natl. Acad. Sci. USA, 85:4397-4401, (1985)), or by differences in melting temperatures. "Molecular beacons" (Kostrikis L.G. et al, Science 279:1228-1229, (1998)), hairpin-shaped, single-stranded synthetic oligo- nucleotides containing probe sequences which are complementary to the nucleic acid of the present invention, may also be used to detect point mutations or other sequence changes as well as monitor expression levels of variant product. Such diagnostics would be particularly useful for prenatal testing.
Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases. The two probes may be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction. The presence or absence of mutations is then detectable by the presence or absence of ligated probe. Also suitable for detecting mutations in the variant product coding sequence are oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Patent No. 5,547,839. In a typical method, the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip. The sequence of the target can then be "read" from the pattern of target binding to the array.
D. Gene mapping utilizing nucleic acid sequences
The nucleic acid sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 20-30 bp) from the variant cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids or using instead radiation hybrids are rapid procedures for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al, Human Chromosomes: a Manual of Basic Techniques, (1988) Pergamon Press, New York.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in the OMIM database (Center for Medical Genetics, Johns Hopkins University, Baltimore, MD and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD). The OMIM gene map presents the cytogenetic map location of disease genes and other expressed genes. The OMIM database provides information on diseases associated with the chromosomal location. Such associations include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations in this area with the advent of polygenic diseases, such as cancer, in general and prostate cancer in particular. E. Therapeutic applications of nucleic acid sequences
Nucleic acid sequences of the invention may also be used for therapeutic purposes. Turning first to the second aspect of the invention (i.e. inhibition of expression of variant), expression of variant product may be modulated through antisense technology, which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding variant product. For example, the 5' coding portion of the nucleic acid sequence sequence which codes for the product of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length. Oligonucleotides derived from the transcription start site, e.g. between positions -10 and +10 from the start site, are preferred. An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee et al, Nucl. Acids, Res., 6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan et al, Science 251:1360, (1991)), thereby preventing transcription and the production of the variant products. An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the variant products (Okano J. Neurochem. 56:560, (1991)). The antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo. The antisense may be antisense mRNA or DNA sequence capable of coding such antisense mRNA. The antisense mRNA or the DNA coding thereof can be complementary to the full sequence of nucleic acid sequences coding for the variant protein or to a fragment of such a sequence which is sufficient to inhibit production of a protein product.
Turning now to the first aspect of the invention, i.e. expression of variant, expression of variant product may be increased by providing coding sequences for coding for said product under the control of suitable control elements ending its expression in the desired host. The nucleic acid sequences of the invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The products of the invention as well as any activators and deactivators compounds (see below) which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy. " Cells from a patient may be engineered with a nucleic acid sequence (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a product of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be fransfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5-14, (1990)). The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
The genes introduced into cells may be placed under the control of inducible promoters, such as the radiation- inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res., 56(19):4311 (1996)), to stimulate variant production or antisense inhibition in response to radiation, eg., radiation therapy for treating tumors.
Example III. Variant product
The substantially purified variant product of the invention has been defined above as the product coded from the nucleic acid sequence of the invention. Preferably the amino acid sequence is an amino acid sequence having at least 90% identity to any one of the sequences identified as SEQ ID NO: 88 to SEQ ID NO: 174 provided that the amino acid sequence is not identical to that of the original sequence from which it has been varied. The protein or polypeptide may be in mature and/or modified form, also as defined above. Also contemplated are protein fragments having at least 10 contiguous amino acid residues, preferably at least 10-20 residues, derived from the variant product, as well as homologues as explained above.
The sequence variations are preferably those that are considered conserved substitutions, as defined above. Thus, for example, a protein with a sequence having at least 90% sequence identity with any of the products identified as SEQ ID NO: 88 to SEQ ID NO: 174, preferably by utilizing conserved substitutions as defined above is also part of the invention, and provided that it is not identical to the original peptide from which it has been varied. In a more specific embodiment, the protein has or contains any one of the sequence identified as SEQ ID NO: 88 to SEQ ID NO: 174. The variant product may be (i) one in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the variant product is fused with another compound, such as a compound to increase the half- life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the protein to its target tissue or target cell population (such as an antibody), or (iv) one in which additional amino acids are fused to the variant product. Such fragments, variants and derivatives are deemed to be within the scope of those skilled in the art from the teachings herein.
A. Preparation of variant product
Recombinant methods for producing and isolating the variant product, and fragments of the protein are described above.
In addition to recombinant production, fragments and portions of variant product may be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J., J. Am. Chem. Soc, 85:2149-2154, (1963)). In vitro peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Fragments of variant product may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
B. Therapeutic uses and compositions utilizing the variant product
The variant product of the invention is generally useful in treating diseases and disorders which are characterized by a lower than normal level of variant expression, and or diseases which can be cured or ameliorated by raising the level of the variant product, even if the level is normal. Variant products or fragments may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue. The product-containing compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones. .
Variant product-containing compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means as well as by nasal application. Variant product-containing compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
The product can be given via intravenous or intraperitoneal injection. Similarly, the product may be injected to other localized regions of the body. The product may also be administered via nasal insufflation. Enteral administration is also possible. For such administration, the product should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
The foregoing exemplary administration modes will likely require that the product be formulated into an appropriate carrier, including ointments, gels, suppositories. Appropriate formulations are well known to persons skilled in the art.
Dosage of the product will vary, depending upon the potency and therapeutic index of the particular polypeptide selected.
A therapeutic composition for use in the treatment method can include the product in a sterile injectable solution, the polypeptide in an oral delivery vehicle, the product in an aerosol suitable for nasal administration, or the product in a nebulized form, all prepared according to well known methods. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The product of the invention may also be used to modulate endothelial differentiation and proliferation as well as to modulate apoptosis either ex vivo or in vitro, for example, in cell cultures.
Example IV. Screening methods for activators and deactivators (inhibitors)
The present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, or other molecules, which have a modulating effect on the activity of the variant product, e.g. activators or deactivators of the variant product of the present invention. Such an assay comprises the steps of providing an variant product encoded by the nucleic acid sequences of the present invention, contacting the variant protein with one or more candidate molecules to determine the candidate molecules modulating effect on the activity of the variant product, and selecting from the molecules a candidate's molecule capable of modulating variant product physiological activity. The variant product, its catalytic or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques. The fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell membrane or located intracellularly. The formation of binding complexes, between variant product and the agent being tested, may be measured. Alternatively, the activator or deactivator may work by serving as agonist or antagonist, respectively, of the variant receptor, binding entity or target site, and their effect may be determined in connection with any of the above. Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the variant product is described in detail by Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the full variant product or with fragments of variant product and washed. Bound variant product is then detected by methods well known in the art. Substantially purified variant product can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
Antibodies to the variant product, as described in Example VI below, may also be used in screening assays according to methods well known in the art. For example, a "sandwich" assay may be performed, in which an anti-variant antibody is affixed to a solid surface such as a microtiter plate and variant product is added. Such an assay can be used to capture compounds which bind to the variant product. Alternatively, such an assay may be used to measure the ability of compounds to influence with the binding of variant product to the variant receptor, and then select those compounds which effect the binding. Example V. Anti-variant antibodies A. Synthesis
In still another aspect of the invention, the purified variant product is used to produce anti-variant antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the variant product.
Antibodies to the variant product may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which inhibit dimer formation, are especially preferred for therapeutic use.
A fragment of the variant product for antibody induction does not require biological activity but have to feature immunological activity; however, the protein fragment or oligopeptide must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids of the sequences specified in any one of SEQ ID NO: 88 to SEQ ID NO: 174. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of variant protein amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to variant product.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with variant product or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.
Monoclonal antibodies to variant protein may be prepared using any technique which provides for the production of antibody molecules by continuous 5 cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell hybridoma technique (Kosbor et al, Immunol. Today 4:72, (1983); Cote et al, Proc. Natl. Acad. Sci. 80:2026-2030, (1983)) and the EBV-hybridoma technique (Cole, et al, Mol Cell Biol. 62:109-120, (1984)).
10 Techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can also be used (Morrison et al, Proc. Natl. Acad. Sci. 81:6851-6855, (1984); Neuberger et al, Nature 312:604-608, (1984); Takeda et al, Nature 314:452-454, (1985)).
15 Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies specific for the variant protein.
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or
20 panels of highly specific binding reagents as disclosed in Orlandi et al. (Proc. Natl. Acad. Sci. 86:3833-3837, 1989)), and Winter G and Milstein C, (Nature 349:293-299, (1991)).
Antibody fragments which contain specific binding sites for variant protein may also be generated. For example, such fragments include, but are not
25 limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab*)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al, Science
30 256:1275-1281, (1989)). B. Diagnostic applications of antibodies
A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between the variant product and its specific antibody and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific variant product is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D.E., et al, (J. Exp. Med. 158:1211, (1983)).
Antibodies which specifically bind variant product are useful for the diagnosis of conditions or diseases characterized by expression of the novel variant of the invention (where normally it is not expressed) by over or under expression of variant as well as for detection of diseases in which the proportion between the amount of the variants of the invention and the original sequence from which it varied is altered. Alternatively, such antibodies may be used in assays to monitor patients being treated with variant product, its activators, or its deactivators. Diagnostic assays for variant protein include methods utilizing the antibody and a label to detect variant product in human body fluids or extracts of cells or tissues. The products and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known in the art.
A variety of protocols for measuring the variant product, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on variant product is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of variant product expression. Normal or standard values for variant product expression are established by combining body fluids or cell extracts taken from normal subjects, preferably human, with antibody to variant product under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation may be quantified by various methods, preferably by photometric methods. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
The antibody assays are useful to determine the level of variant product present in a body fluid sample, in order to determine whether it is being expressed at all, whether it is being overexpressed or underexpressed in the tissue, or as an indication of how variant levels of variable products are responding to drug treatment.
By another aspect the invention concerns methods for determining the presence or level of various anti-variant antibodies in a biological sample obtained from patients, such as blood or serum sample using as an antigen the variant product. Determination of said antibodies may be indicative to a plurality of pathological conditions or diseases.
C. Therapeutic uses of antibodies
In addition to their diagnostic use the antibodies may have a therapeutical utility in blocking or decreasing the activity of the variant product in pathological conditions where beneficial effect can be achieved by such a decrease.
The antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulin-gene library methods. The antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable. Typically, the antibody is administered in an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen.
Although the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.
Example VI. Expression of ACEV
(a) Immunohistochemical staining:
The immunohistochemical staining was performed using Histostain plus Kit (Zymed Laboratories Inc.). Mouse salivary gland micron sections were prepared using a
R. Gung microtome and fixed on superfrost plus slides with 2% Tespa.
Deparaffinization was performed in xylene for 10 min. Dehydration was performed three times in absolute ethanol and once 95% ethanol. The slides were washed in DDW and then incubated with 3% H2O for 5 min. Subsequently, the slide were washed in DDW and twice in 0.05M TrisHCl pH 7.6 (Optimax wash Buffer, BioGenex). The rest of the procedure was performed following the manufacturer's instructions. The results are shown in Figs. 88 and 89.
The immunohistochemical staining was performed on mouse salivary gland micron sections. The immunohistochemestry was done using specific polyclonal antibodies designed against the c-terminus of SEQ ID NO: 144 (12 amino-acids), which are unique to said ACEV product and lack in the original ACE protein (Fig 88-a,b,d magnification X 100; Fig 89-b magnification X 400) compared with the pre-immune rabbit's serum (Fig 88-c, Fig. 89-a). ACE was found to express in ductal epifilus (Fig 88-a,b,d, Fig 89-b).
The same procedure was repeated for mouse lymph node sectors stained with pre-immune serum (Fig. 90a, 90c - magnifications X 100, X 200, respectively) and immune serum (Fig. 90b and 90c magnifications X 100, X 200 respectively). The results show positive staining in salivary glands. (b) RT - PCR
RNA Purification and cDNA Synthesis Total RNA was extracted from different mouse tissues using Tri-Reagent System (Molecular Research Center, Inc., Cincinnati, OH). Synthesis of first-strand cDNA was carried out using Oligo(dT)15 (Promega, Medison, Wl), Superscript II (Gibco/BRL, Gaithersburg, MD), Rnasin (Promega, Medison, Wl) and dNTP's (Gibco/BRL, Gaithersburg, MD). + with Superscript II, - without Superscript II.
Polymerase Chain Reaction (PCR): PCR was performed using Expand Long Template PCR system (Roche). As a template cDNA from different tissues was used. The PCR reaction on PTC-225 (MJ Research, Inc.). PCR products were analyzed on an automated DNA sequencer ABI Prizem 310 Genetic Analyzer (Perkin Elmer).
The results are shown in Fig. 91. As can be seen the ACEV of the invention was expressed in skin, lung, heart, thymus, spleen, bone marrow and brain tissue

Claims

CLAIMS:
1. An isolated nucleic acid sequence, of an alternative splicing variant, selected from the group consisting of:
(i) the nucleic acid sequence depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 87;
(ii) nucleic acid sequences having at least 90% identity with the sequence of (i) with the proviso that each sequence is different than the original nucleic acid sequence from which the sequences of (i) have been varied by alternative splicing; and (Hi) fragments of (i) or (ii) of at least 20 b.p., provided that said fragment contains a sequence which is not present, as a continuous stretch of nucleotides, in the original nucleic acid sequence from which the sequences of (i) have been varied by alternative splicing.
2. An isolated nucleic acid sequence complementary to the nucleic acid sequence of Claim 1.
3. An amino acid sequence selected from the group consisting of:
(i) an amino acid sequence coded by the isolated nucleic acid sequence of alternative splice variants of Claim 1;
(ii) homologues of the amino acid sequences of (i) in which one or more amino acids has been added, deleted, replaced or chemically modified in the region or adjacent to the region where the amino acid sequences differs from the original amino acid sequence, coded by the original nucleic acid sequence from which the variant has been varied.
4. An amino acid sequence according to Claim 3, as depicted in any one of SEQ ID NO: 88 to SEQ ID NO: 174.
5. An isolated nucleic acid sequence coding for any one of the amino acid sequences of Claim 3 or 4.
6. A purified antibody which binds specifically to any of the amino acid sequence of Claim 3 or 4.
7. An expression vector comprising any one of the nucleic acid sequences of Claim 1 or 5 and control elements for the expression of the nucleic acid sequence in a suitable host.
8. An expression vector comprising any one of the nucleic acid sequences of Claim 2, and control elements for the expression of the nucleic acid sequences in a suitable host.
9. A host cell fransfected by the expression vector of Claim 7 or 8.
10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from the group consisting of: (i) the expression vector of Claim 7; and
(ii) any one of the amino acid sequences of Claim 3 or 4.
11. A pharmaceutical composition according to Claim 10, for treatment of diseases which can be ameliorated or cured by raising the level of any one of the amino acid sequences depicted in SEQ ID NO: 88 to SEQ ID NO: 174.
12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from the group consisting of: (i) any one of the nucleic acid sequences of Claim 2; (ii) the expression vector of Claim 8; and (iii) the purified antibody of Claim 6.
13. A pharmaceutical composition according to Claim 12, for treatment of diseases which can be ameliorated or cured by decreasing the level of any one of the amino acid sequences depicted in SEQ ID NO: 88 to SEQ ID NO: 174.
14. A method for detecting an variant nucleic acid sequence in a biological sample, comprising the steps of: (a) hybridizing to nucleic acid material of said biological sample any one of the nucleic acid sequences of Claim 1 or 2; and (b) detecting said hybridization complex; wherein the presence of said hybridization complex correlates with the presence of an variant nucleic acid sequence in the said biological sample.
15. A method for determining the level of variant nucleic acid sequences in a biological sample comprising the steps of: (a) hybridizing to nucleic acid material of said biological sample any one of the nucleic acid sequences of Claim 1 or 2; and
(b) determining the amount of hybridization complexes and normalizing said amount to provide the level of the variant nucleic acid sequences in the
5 sample.
16. A method for determining the ratio between the level of variant of the nucleic acid sequence in a first biological sample and the level of the original sequence from which the variant has been varied by alternative splicing in a second biological sample comprising: ιo (a) determining the level of the variant nucleic acid sequence in the first biological sample according to the method of Claim 15;
(b) determining the level of the original sequence in the second biological sample; and
(c) comprising the levels obtained in (a) and (b) to give said ratio.
15 17. A method according to Claim 16, wherein said first and said second biological samples are the same sample.
18. A method according to any of Claims 14 to 17, wherein the nucleic acid material of said biological sample are mRNA transcripts.
19. A method according to Claim 18, where the nucleic acid sequence is present 0 in a nucleic acid chip.
20. A method for identifying candidate compounds capable of binding to the variant product and modulating its activity the method comprising:
(i) providing any one of the amino acid sequences as defined in Claim 3 or 4; 5 (ϋ) contacting a candidate compound with said amino acid sequence;
(iii) determining the effect of said candidate compound on the biological activity of said protein or polypeptide and selecting those compounds which show a significant effect on said biological activity.
21. A method according to Claim 20, wherein the compound is an activator and 0 the measured effect is increase in the biological activity.
22. A method according to Claim 20, wherein the compound is an deactivator and the effect is decrease in the biological activity.
23. An activator of any one of the amino acid sequences of Claim 3 or 4.
24. An deactivator of any one of the amino acid sequences of Claims 3 or 4.
5 25. A method for detecting any one of the amino acid sequences of Claim 3 or 4 in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of Claim 8, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex
10 wherein the presence of said antibody-antigen complex correlates with the presence of the desired amino acid in said biological sample.
26. A method for detecting the level of the amino acid sequence of any one of Claim 3 or 4 in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of Claim 8, 15 thereby forming an antibody-antigen complex; and
(b) detecting the amount of said antibody-antigen complex and normalizing said amount to provide the level of said amino acid sequence in the sample.
27. A method for determining the ratio between the level of any one of the 20 amino acid sequences of Claims 3 or. 4 present in a first biological sample and the level of the original amino acid sequences from which they were varied by alternative splicing, present in a second biological sample, the method comprising:
(a) determining the level of the amino acid sequences of Claims 3 or 4 into a first sample by the method of Claim 26; 25 (b) determining the level of the original amino acid sequence in the second sample; and
(c) comparing the level obtained in (a) and (b) to give said ratio.
28. A method according to Claim 27, wherein said first and said second biological samples are the same sample.
30
29. A method for detecting any one of the antibodies of Claim 6 in a biological sample comprising the steps of: (a) contacting said biological sample with any one of the amino acid sequences of Claim 3 or 4 thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said antibody-antigen complex correlates with the presence of the antibody in said biological sample.
30. A method for detecting the level of any one of the antibodies of Claim 6 in a biological sample comprising the steps of:
(a) contacting said biological sample with any one of the amino acid sequences of Claim 3; (b) detecting the amount of said antibody-antigen complex and normalizing said amount to provide the levels of said antibody in the sample.
31. An isolated nucleic acid sequence according to Claim 1 of an alternative splicing variant of an angiotensin converting enzyme (ACEV) selected from the group consisting of: (i) the nucleic acid sequence depicted in SEQ ID NO: 57 or SEQ ID
NO: 85;
(ii) nucleic acid sequences having at least 90% identity with the sequence of (i) with the proviso that each sequence is different than the original nucleic acid sequence from which the sequences of (i) have been varied by alternative splicing; and
(iii) fragments of (i) or (ii) of at least 20 b.p., provided that said fragment contains a sequence which is not present, as a continuous stretch of nucleotides, in the original nucleic acid sequence from which the sequences of (i) have been varied by alternative splicing.
32. An isolated nucleic acid sequence complementary to the nucleic acid sequence of Claim 31.
33. An amino acid sequence selected from the group consisting of:
(i) an amino acid sequence coded by the isolated nucleic acid sequence of alternative splice variants of Claim 31; (ϋ) homologues of the amino acid sequences of (i) in which one or more amino acids has been added, deleted, replaced or chemically modified in the region or adjacent to the region where the amino acid sequences differs from the original amino acid sequence, coded by the original nucleic acid sequence from which the variant has been varied.
34. An amino acid sequence according to Claim 33, as depicted in SEQ ID NO: 144 or SEQ ID NO: 172.
35. An isolated nucleic acid sequence coding for any one of the amino acid sequences of Claim 33 or 34.
36. A purified antibody which binds specifically to any of the amino acid sequence of Claim 33 or 34.
37. An expression vector comprising any one of the nucleic acid sequences of Claim 31 or 35 and control elements for the expression of the nucleic acid sequence in a suitable host.
38. An expression vector comprising any one of the nucleic acid sequences of Claim 32, and control elements for the expression of the nucleic acid sequences in a suitable host.
39. A host cell fransfected by the expression vector of Claim 37 or 38.
40. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from the group consisting of:
(i) the expression vector of Claim 37; and (ϋ) any one of the amino acid sequences of Claim 33 or 34.
41. A pharmaceutical composition according to Claim 40, for treatment of diseases which can be ameliorated or cured by raising the level of any one of the amino acid sequences depicted in SEQ ID NO: 144 or SEQ ID NO: 172.
42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from the group consisting of:
(i) any one of the nucleic acid sequences of Claim 32; (ii) the expression vector of Claim 38 ; and (iii) the purified antibody of Claim 36.
43. A pharmaceutical composition according to Claim 42, for treatment of diseases which can be ameliorated or cured by decreasing the level of the amino acid sequences depicted in SEQ ID NO: 144 or SEQ ID NO: 172.
44. A pharmaceutical composition according to Claim 40 or 42 for the treatment of a disease selected from: cardiovascular disorders, congestive heart failure, hypertension, renal hypertension, diabetes, multiple sclerosis, sarcoidosis, nonsarcoidotic pulmonary granulomatous diseases, vascular pathologies involving an endothelial abnormality and cancer.
45. A method for detecting an variant nucleic acid sequence in a biological sample, comprising the steps of:
(a) hybridizing to nucleic acid material of said biological sample any one of the nucleic acid sequences of Claim 31 or 32; and (b) detecting said hybridization complex; wherein the presence of said hybridization complex correlates with the presence of an variant nucleic acid sequence in the said biological sample.
46. A method for determining the level of variant nucleic acid sequences in a biological sample comprising the steps of: (a) hybridizing to nucleic acid material of said biological sample any one of the nucleic acid sequences of Claim 31 or 32; and
(b) determining the amount of hybridization complexes and normalizing said amount to provide the level of the variant nucleic acid sequences in the sample.
47. A method for determining the ratio between the level of variant of the nucleic acid sequence in a first biological sample and the level of the original sequence from which the variant has been varied by alternative splicing in a second biological sample comprising:
(a) determining the level of the variant nucleic acid sequence in the first biological sample according to the method of Claim 46;
(b) determining the level of the original sequence in the second biological sample; and
(c) comprising the levels obtained in (a) and (b) to give said ratio.
48. A method according to Claim 47, wherein said first and said second biological samples are the same sample.
49. A method according to any of Claims 45 to 48, wherein the nucleic acid material of said biological sample are mRNA transcripts.
50. A method according to Claim 49, where the nucleic acid sequence is present in a nucleic acid chip.
51. A method for identifying candidate compounds capable of binding to the variant product and modulating its activity the method comprising:
(i) providing any one of the amino acid sequences as defined in Claim 33 or 34;
(ii) contacting a candidate compound with said amino acid sequence; (iii) determining the effect of said candidate compound on the biological activity of said protein or polypeptide and selecting those compounds which show a significant effect on said biological activity.
52. A method according to Claim 51, wherein the compound is an activator and the measured effect is increase in the biological activity.
53. A method according to Claim 51, wherein the compound is an deactivator and the effect is decrease in the biological activity.
54. An activator of any one of the amino acid sequences of Claim 33 or 34.
55. An deactivator of any one of the amino acid sequences of Claims 33 or 34.
56. A method for detecting any one of the amino acid sequences of Claim 33 or 34 in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of Claim 38, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said antibody-antigen complex correlates with the presence of the desired amino acid in said biological sample.
57. A method for detecting the level of the amino acid sequence of any one of Claim 33 or 34 in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of Claim 38, thereby forming an antibody-antigen complex; and (b) detecting the amount of said antibody-antigen complex and normalizing said amount to provide the level of said amino acid sequence in the sample.
58. A method for determining the ratio between the level of any one of the amino acid sequences of Claims 33 or 34 present in a first biological sample and the level of the original amino acid sequences from which they were varied by alternative splicing, present in a second biological sample, the method comprising: (a) determining the level of the amino acid sequences of Claims 33 or 34 into a first sample by the method of Claim 57; (b) determining the level of the original amino acid sequence in the second sample; and
(c) comparing the level obtained in (a) and (b) to give said ratio.
59. A method according to Claim 58, wherein said first and said second biological samples are the same sample.
60. A method for detecting any one of the antibodies of Claim 36 in a biological sample comprising the steps of:
(a) contacting said biological sample with any one of the amino acid sequences of Claim 3 or 4 thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said .antibody-antigen complex correlates with the presence of the antibody in said biological sample.
61. A method for detecting the level of any one of the antibodies of Claim 36 in a biological sample comprising the steps of:
(a) contacting said biological sample with any one of the amino acid sequences of Claim 33 ;
(b) detecting the amount of said antibody-antigen complex and normalizing said amount to provide the levels of said antibody in the sample.
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