WO2002006315A2

WO2002006315A2 - Novel nucleic acid and amino acid sequences

Info

Publication number: WO2002006315A2
Application number: PCT/IL2001/000653
Authority: WO
Inventors: Liat Mintz; Shiri Freilich; Jeanne Bernstein
Original assignee: Compugen Ltd.
Priority date: 2000-07-18
Filing date: 2001-07-17
Publication date: 2002-01-24
Also published as: AU2001278655A1; WO2002006315A3

Abstract

Novel nucleic acid sequences and amino acid sequences are provided. Some of the novel nucleic acid sequences are alternative splice variants of previously known transcripts. Others of these novel nucleic acid sequences are similar to a previously known expressed sequence. The novel nucleic acid and amino acid sequences have a variety of diagnostic and therapeutic applications.

Description

NOVEL NUCLEIC ACID AND AMINO ACID SEQUENCES

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 detection of diseases involved, caused or resulting from said 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 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, 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); CW. 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 estimate to as high as stating 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.

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:

"Novel sequences (NS) - nucleic acid sequence" - the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 128. These sequences are novel, naturally occurring sequences. Some of these sequences are alternative splice variants of known genes. It should be emphasized that these novel variants are naturally occurring, transcribed sequences resulting from alternative splicing of genes and not merely truncated, mutated or fragmented forms of lαiown sequences. Thus the alternative splice variants of the invention have physiological or biological significance regarding the type of tissue, the developmental stage and under which conditions, e.g. a disease state, etc. in which their expression is modulated, i.e., ceased, increased, up-regulated or down-regulated. Other of these NS are homologs of known sequences, i.e. sequences having similarity to known expressed sequences. The similarity does not necessitate identical function as will be explained hereinbelow.

"NS product (NS protein or NS polypeptide) - is an amino acid sequence encoded by the NS nucleic acid sequence. 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 term also includes homologues (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. More specifically, it concerns the amino acid sequence denoted in any one of SEQ ID NO: 129 to SEQ ID NO: 256.

"Nucleic acid sequence" - a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes 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 ofNS nucleic acid sequence" - a 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. Where the NS is a variant sequence obtained by alternative splicing of the original sequence the fragment comprises a sequence which was not included in the original sequence (a sequence that was an intron in the original sequence). An example is when the variant lacks a non-terminal region which was present in the original sequence. Thus the two stretches of nucleotides spanning this region (upstream and downstream) are brought together by splicing, but are spaced from each by that region in the original sequence and are thus not continuous. A continuous stretch of nucleic acids comprising said two stretches of nucleotides, is not present in the original sequence and thus fall under the definition of fragment. Where the NS is a homolog, a fragment of at least 20 b.p. comprises a sequence which includes at least one nucleic acid which does not appear or is different in the original nucleic acid sequence.

"Fragments of NS products" - amino acid sequences coded by the "fragment of the NS nucleic acid sequence " defined above.

"Homologues ofNS" - 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 regions or adjacent to regions where the NS differs from the original sequence, i.e. in regions added, deleted or replaced in the NS as compared to the original sequence.

"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 NS 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 NS 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 NS product denotes a fragment which retains some or all of the immunological properties of the NS product, e.g can bind specific anti-NS product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce said antibodies.

"Optimal alignment" - is defined as an alignment giving the highest percent identity score. Such aligmnent 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 MacVector (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 lαiown 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 X% 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, 70% amino acid sequence identity means that 70% 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.

"Isolated nucleic acid molecule having an variant nucleic acid sequence" - is a nucleic acid molecule that includes the coding NS nucleic acid sequence. Said isolated nucleic acid molecule may include the NS nucleic acid sequence as an independent insert; may include the NS nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the NS coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the NS 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' untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the NS protein coding sequence is a heterologous.

"Expression vector" - refers to a vector that has 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.

"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 NS 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" - where the NS is a variant it is the nucleic acid sequence from which the variant of the invention have been varied as a result of alternative slicing, or, depending on the context, the amino acid sequence encoded thereby. Where the NS is a homolog, it is the transcribed sequence to which the NS shows sequence similarity.

SUMMARY OF THE INVENTION

The present invention is based on the finding of 128 novel transcribed sequences (NS). Some of these sequences are novel splice variants, which are naturally occurring sequences obtained by alternative splicing of lαiown genes. Where the novel sequences of the invention are novel splice variants they are not merely truncated forms, fragments or mutations of known genes, but rather novel sequences which naturally occur within the body of individuals. Each novel splice variant is a result of alternative splicing of an original sequence. Other novel sequences (NS) of the invention are homologs to known sequences showing sequence similarities. The nucleic acid sequences are denoted as SEQ ID NO: 1 to SEQ ID NO: 128.

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. Both where the novel sequences of the invention are variants of alternative splicing or homologs of an original sequence may have the same physiological activity as the original sequence from which they are varied by alternative splicing or to which they show homology (although perhaps at a different level); may have an opposite physiological activity from the activity featured by the original peptide; may have a completely different, unrelated activity to the activity of the original sequence from which they are varied or to which they feature homology; or alternatively may have no activity at all and this may lead to various diseases or pathological conditions. Both in the case where the NS has the same activity as well as an opposite activity as the original sequence, it may differ from the original sequence in its stability, clearance, tissue and cellular localization not necessarily connected to activity.

The novel NS may serve for detection purposes, i.e. their presence, absence or their level may be indicative of a disease, disorder, pathological or normal condition. Where the NS is a variant of alternative splicing 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 NS in various tissues. A certain NS may be expressed mainly in one tissue, while the original sequence from which it has been varied, or to which it shows homology, may be expressed mainly in another tissue. Understanding of the distribution of the NS 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 having high tissue specificity.

The study of the NS 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.

The detection may be the determination of the presence, absence or the level of expression of the NS within a specific cell population, comparing said presence or level between various cell types in a tissue, between different tissues and between individuals. 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: 128, 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), or a molecule comprising a sequence having at least 70%, preferably 80%, most preferably 90% or 95% identity to SEQ ID NO: 1 to SEQ ID NO: 128, provided that the molecule is not completely identical to the original 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 "NS product", 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. More specifically, the amino acid sequences are those denoted as SEQ ID NO: 129 to SEQ ID NO: 256.

The deletions, insertions and modifications should be in regions, or adjacent to regions, wherein the NS differs from the original sequence, for example in regions where the NS has an addition, a deletion or a replacement of at least one nucleic acid as compared to the original sequence.

For example, where the NS 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 NS 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 NS differs from the original sequence, or in regions adjacent to said region. Another example is where the NS is a variant which 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 NS lacks a sequence as compared to the original sequence, or in a region adjacent thereto. Where the NS is a homolog the homologous sequences should be in the regions and sequences where the NS differs from the original sequence.

It should be appreciated that once a person 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 NS as compared to the original sequence, he would have no difficulties in derivating said specific region by addition to it, deleting from it, or substituting some amino acids in it. Thus homologues of NS which are derivated from the NS 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 NS 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). Due to the degenerative nature of the genetic code, a plurality of alternative nucleic acid sequences, beyond those depicted in any one of SEQ ID NO:l to SEQ ID NO: 128, can code for the amino acid sequence of the invention. Those alternative nucleic acid sequences which code for the same amino acid sequences coded by the sequence SEQ ID NO:l to SEQ ID NO:128 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 transfected 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 NS products of the invention and will be explained in a list hereinbelow.

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:l to SEQ ID NO: 128, or complementary to a sequence having at least 70%, preferably 80%, most preferably 90% or even 95% identity to said sequence (with the proviso that sequences identical to the original sequence are excluded) 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.T to SEQ ID NO.T28 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:l to SEQ ID NO: 128 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: 128 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO:l to SEQ ID NO: 128, 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 NS of the invention. The presence of the NS transcript or the level of the NS transcript (identified either by any one of sequences 1 to 128 or by a sequence complementary thereto) may be indicative of a multitude of diseases, disorders and various pathological as well as normal conditions. In addition, the ratio of the level of the transcripts of the NS of the invention may also be compared to that of the transcripts of the original sequences from which they were varied or to which they show homologs, or where the NS is a variant, 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 transfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention. The invention also provides anti-NS product antibodies, namely antibodies directed against the NS product which specifically bind to said NS product. Said antibodies are useful both for diagnostic and therapeutic purposes. For example said antibodies may be as an active ingredient in a pharmaceutical composition as will be explained below. By another alternative, the invention concerns antibodies termed

"distinguishing antibodies " which are directed solely to the amino acid sequences which distinguishes the NS from the original amino acid sequence.

The distinguishing antibodies may be used for detection purposes, i.e. to detect individuals, tissue, conditions (both pathological or physiological) wherein the NS or original sequence are evident or abundant. The antibodies may also be used to distinguish conditions where the level, or ratio of the NS to original sequence is altered.

The distinguishing antibodies may also be used for therapeutical purposes, i.e., to neutralize only the NS product or only the product of the original sequence, as the case may be, without neutralizing the other.

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 present invention also provides pharmaceutical compositions comprising, as an active ingredient, said anti-NS product antibodies. The pharmaceutical compositions comprising said anti-NS 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 NS (either at the transcript or product level) or decreasing the amount of the NS product or blocking its binding to its target, for example, by the neutralizing effect of the antibodies, or by the decrease of the effect of the antisense mRNA in decreasing expression level of the NS product.

Examples of diseases that may be treated or detected according to the first or second aspect of the invention will be given hereinbelow.

According to the third aspect of the invention the present invention provides methods for detecting the level of the transcript (mRNA) of said NS 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 by amplification techniques (such as RT-PCR) utilizing suitable probes; 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 NS 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, as compared to sequences which feature homology or compared to other variant sequences all varied from the same original sequence may be indicative of a plurality of physiological or pathological conditions.

The method, according to this latter aspect, for detection of an NS nucleic acid sequence 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 complex indicates the presence of nucleic acid sequence encoding the NS product in the biological sample.

By another option the present invention provides a method for detection of a nucleic acid sequence in a biological sample the method comprises:

(i) contacting the sample with probes for amplification of any one of

SEQ ID NO: 1 to SEQ ID NO: 128; (ii) proving reagents for amplification;

(iii) detecting the presence of amplified products, said products indicating the presence of NS nucleic acid in the 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, for example, by determining the level of hybridization complexes and then calibrating said levels to determining levels of transcripts of the desired NS in the sample, or by calibrating the amounts of amplified products.

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 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. If desired, the detected level may be compared to that of the original sequence or compared to that of other splice variants, for example, those obtained from the same original sequence by alternative splicing.

Methods for detecting mutations in the region coding for the NS 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 NS 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 NS 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 NS product in said biological sample.

As indicated above, the method can be quantitized to determine the level or the amount of the NS in the sample, alone or in comparison to the level of the original amino acid sequence from which it was varied, or to which it shows homology and qualitative and quantitative results may be used for diagnostic, prognostic and therapy planning purposes.

The pharmaceutical compositions, whether comprising the NS themselves (alone or in an expression vector), comprising sequences complementary thereto (alone or in an expression vector), comprising the amino acid (products) or comprising antibodies to the above, are suitable for the treatment of a plurality of diseases, each one in relation to the activity of the NS itself. A list of possible diseases will be specified hereinbelow.

The detection of diseases utilizing a NS probe (comprising the variant sequence or a sequence complementary thereto) is also in line with the function of the NS. In the following, there shall be a brief summary of those conditions, and diseases in which the pharmaceutical composition of the invention can treat, i.e. cure, ameloriate or prevent. The term "pharmaceutical composition" refers to: sense sequences (SEQ ID NO: 1 to SEQ ID NO: 128, fragments and homologs) and vectors comprising these sequences; anti-sense sequences (complementary to SEQ ID NO: 1 to SEQ ID NO: 128 or complementary to their fragments) and vectors comprising the NS antisense sequences, NS products (proteins or fragments) and NS antibodies.

The following also summarizes those conditions that can be detected by NS probes of the present invention (being SEQ ID NO: 1 to SEQ ID NO: 128 or sequences complementary thereto as well as fragments of any of the above), or by antibodies reactive with the NS product of the invention.

The summary includes the name and accession number of the original sequence of the NS, from which it has been varied by alternative splicing (in that case it is indicated as "new variant") to which it shows homology (in that case it is indicated as "homolog") or being a human homolog to an original sequence not of human origin ( indicated as "ortholog") or which may be any or all the above and in this no indication is given. The summary includes a description of some of the diseases, disorders or pathological conditions believed to be associated with the sequence. In SEQ ID NO: 122 to SEQ ID NO: 128 the Summary also includes a structural analysis of the NS and its comparison to the new sequences. It should be appreciated that the list is merely exemplary and other diseases and disorders may be detected or treated. Unless indicated otherwise, the mention of the disease should be understood that the sequence is appropriate both for treatment of the disease as well as for its detection.

SEQ ID NO. 1: Functional adhesion molecule to gi|3462455 (homolog).

Can be used for the detection and isolation of endothelial cells and for detection of mutation therewith.

SEQ ID NO. 2 and 3: FLDED-1 death effector domain-containing protein homolog to gi|4758144;

FLDED-1 death effector domain-containing protein homolog to gi|475814 the above are homologs of the known gene, and splice variants of one another. Can be used to treat and detect cancer, or degenerative diseases. SEQ ID NO. 4: Progesterone receptor-related protein p23 - gi|5729964 (new variant.)

Can be used to immuno-affinity isolate progesterone receptor complexes.

May also be used in modulation of the progesterone pathway for the treatment of various hormonal conditions wherein a beneficial effect may be obtained by this modulation. May be used for the treatment of breast cancer, as a contraceptive and for the treatment of menopausal disorders; osteoporosis and endometriosis; in treatment of certain carcinomas; and in treatment of women's reproductive diseases.

SEQ ID NO. 5: E1B 19K/Bcl-2-binding protein Nip3 - gi|2511529 (new variant).

Can be used to induce death in cancer cells and also be used for the treatment of degenerative diseases. Probes may be used to detect cancer or degenerative diseases.

SEQ ID NO. 6: eIF4E-like cap-binding protein - gi|4757702 (new variant).

Can be used to regulate (up- or down-regulate) expression of a plurality of genes or to detect abnormalities or pathologies involved or caused by over or under expression of genes.

SEQ ID NO. 7: SECRETORY CARRIER-ASSOCIATED MEMBRANE PROTEIN gi|3914963 (homolog).

Can be used for regulation of the activity of endothelial growth factors receptor (EGFR) and for detection of mutations in said activity which may effect the growth function and differentiation of endothelial cells.

SEQ ID NO. 8: torsin - torsion dystonia gene (DYT1) ATP-binding protein - gi|455754 (homolog). Can be used to detect dystonia, as well as to prevent, cure or alleviate established disorder dystonia condition.

SEQ ID NO. 9: Interferon-inductible PML nuclear bodies-associated protein - gi|206268 (homolog).

Can be used to regulate the binding of nuclear proteins to various other moieties in cells as well as for the detection of various abnormalities caused by non-normal binding of nuclear protein.

SEQ ID NO. 10: ATP binding protein associated with cell differentiation - gi|5031583 (new variant).

Can regulate the differentiation of cells both during developments of tissue and during cancer. Can also be used to detect cancer and various abnormalities in the development of tissues.

SEQ ID NO. 11: Copine VII gi|7656993 (homolog).

Can be used for the treatment of the detection of cancer.

SEQ ID NQ.12: Centaurin-alpha, phosphatidylinositol(3,4,5)trisphosphate binding protein p42IP4 - gi|3281994 (new variant).

May be used for modulation of reentry of cells into cell cycles for various regulation of growth purposes, may be used to regulate vesicle trafficking, to treat and detect diseases associated with vesicle movement of abnormal actin formation.

SEQ ID NO. 13: Melanoma-Associated Antigen MAGE LIKE - gi|3319931 homolog; and

SEQ ID NO. 14: Melanoma-Associated Antigen MAGE LIKE - gi|331991 homolog (genes 13 and 14 are homologs of the original gene above, but splice variants of one another). Can be used in the identification and detection of melanoma, detection of pre-disposition to melanoma and detection of the prognosis of melanoma. In addition, its regulation may be used for the treatment, alleviation or prevention of melanoma.

SEQ ID NO. 15: TIM_HUMAN (P60 TIM) (TRANSFORMING IMMORTALIZED MAMMARY ONCOGENE) - gi|4885633 (homolog).

Can be used to modulate growth of cells both for increasing such growth (in degenerative conditions) as well as for suppressing such growth notably for cancer. In addition, can be used to detect cancer or degenerative conditions.

SEQ ID NO. 16: FGF receptor activating protein 1 - gi| 1518609 (new variant).

Can be used for the treatment of duodenal and skin ulcer; for nerve growth stimulation after damage in strokes, accidents or Alzheimer's disease; to promote angiogenesis, heart muscle regeneration and improved cardiac function in post-infarct hearts; for treatment of severe coronory heart diseases; for the prevention of restenosis following angioplasty and for prevention of atherosclerosis; for treatment of cancer, for prevention and treatment of fibrosis; for use in various tissue repair conditions, for treatment of myocardial fibrosis; atherosclerosis; multiple sclerosis, restenosis, rheumatoid lesions and post-coronary episodes, for treatment of psoriasis, malignant melanoma and ophthalmic disease, including cataracts, macular degeneration and retinal detachment. In addition the probes may be used for the detection of any of the above.

SEQ ID NO. 17: Epithelial protein lost in neoplasm EPLIN - gi|6685009

(homolog).

SEQ ID NO. 18: Epithelial protein lost in neoplasm EPLIN - gi]6685O09 (homolog). SEO ID NO. 19: Epithelial protein lost in neoplasm EPLIN - gi|6685009 (homolog).

SEQ ID NO. 20: Epithelial protein lost in neoplasm EPLIN - gi|6685009 (homolog). SEQ ID NO. 21: Epithelial protein lost in neoplasm EPLIN - gi|6685009 (homolog).

SEQ ID NO. 22: Epithelial protein lost in neoplasm EPLIN - gi|6685009 (homolog).

Can be used to detect cancer (especially oral, prostrate, breast or xenograft tumors) and for the treatment, prevention or alleviation of various types of cancer.

SEQ ID NO. 23: Activator of S phase Kinase - gi|7304903 (homolog).

Can be used for regulation of DNA replication, for treatment of diseases where beneficial effect can be achieved by such regulation.

SEQ ID NO. 24: TRAF interacting protein- TRIP - gi|503219 (new variant).

Through their interaction with the TNF receptor-associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, and cell death.

Can be used for effecting various physiological properties which are typically induced by TNF, especially in connection with inflammation as well as to detect pathological conditions effected by TNF. Can be used in the treatment of cancer, for treatment of inflammatory conditions; can be used as an anti-arthritic; for multiple sclerosis, inflammatory bowl disease and for wasting disorders. SEO ID NO. 25: HUMAN ZINC FINGER PROTEIN 151 (MYC- INTERACTING ZINC FINGER PROTEIN) - gi|4507997 (homolog

Can be used in the treatment of cancer (especially leukemia) and the probe may be used for the detection of cancer.

SEQ ID NO. 26: B-cell CLL/lymphoma 7B B-cell CLL/lymphoma - gi|4502385 (new variant).

Can be used to treat and delete the congenital disorder, Williams Syndrome.

SEQ ID NO. 27: Homolog of Yeast RRP4 (ribosomal RNA processing 4), 3'-5'-exoribonuclease - gi)7657528 (new variant).

SEQ ID NO. 28: Homolog of Yeast RRP4 (ribosomal RNA processing 4), 3'-5'-exoribonuclease - gi|7657528 (new variant). Can be used for the treatment and detection of genetic diseases involving rearrangements of DNA for treatment and detection of cancer, for example in Philadelphia Chromosome-Positive Leukemias.

SEQ ID NO. 29: Thyroid hormone receptor coactivating protein gi|5730053 (variant).

Can be used to treat/detect various abnonnalities relating to thyroid metabolism such as skin disorders, glaucoma and metabolic disorders such as obesity and hypercholesterolaemia.

SEQ ID NO. 30: Fuzzy [Drosophila melanogaster]. gi|2564657 (homolog) SEQ ID NO. 31: Fuzzy [Drosophila melanogaster] . gi|2564657

Can be used to treat and detect problems involving abnormal or pathological development. SEQ ID NO. 32: Pescadillo gi|2194203 (Variant)

Essential for embryonic development, and can be used to regulate and normalize various disruptions in said development. In addition, may be used as a marker for detecting abnormalities in said development of embryongenesis, for example, for non-natal detection purposes prior to abortion decisors.

SEQ ID NO. 33: Similar to Drosophila ring canal protein (kelch) gi|3165570 (homolog)

Can be used to regulate membrane-associated structures as well as to detect pathological conditions involving abnormal structures.

SEQ ID NO. 34: Deltex positive regulator of Notch-signalling pathway - gi|4758202 (homolog).

SEQ ID NO. 35: Deltex positive regulator of Notch-signalling pathway - gi|4758202 (homolog)

Can be used to regulate the ability of undifferentiated precursor cells to respond to signals especially in connection with cancer and for detection of abnormalities of such response.

SEQ ID NO.36: Gene encoding a RING-H2 motif in mouse development - gi|2828790 (homolog).

Can be used for the regulation of development differentiation of mesodermal cells, especially to treat cancers of mesodermal origin in brain, liver, kidney. May also be used for the detection of the above cancers.

SEQ ID NO. 37: Tumor necrosis factor-alpha-induced protein B12 - gi|482321 (homolog).

Can be used for effecting various physiological properties which are typically induced by TNF, especially in connection with inflammation as well as to detect pathological conditions effected by TNF. SEO ID NO. 38: Kelch motif containing protein - gi|7656845 (homolog).

Can be used to regulate communication between cells and flow of cytoplasma.

SEQ ID NO. 39: CDM protein - gi| 5031675 (new variant).

Can be used in connection with detection and treatment of various muscle and movement-related diseases such as muscular dystrophy.

SEQ ID NO. 40: STEROIDOGENIC ACUTE REGULATORY PROTEIN PRECURSOR - gi| 117225 (new variant).

Can be used to regulate steroidogenesis and pathologies relating to production of too much, too little or faulty steroids (both of adrenocortical and of gonadal origin), including such conditions as menapause, sterility, congenital and adrenal hyperplasia.

SEQ ID NO. 41: Fanconi anemia group A (FAA) gene - gi|4503655 (homolog)

Can be used for detection of Fanconi anemia and for its treatment.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC).

SEQ ID NO. 42: Maturation-inducing protein - gi|6723280 (homolog).

Can be used for induction of ovarian maturation, for example in fertility treatments, as well as for detection of pathological conditions (such as infertility) associated with ovarian maturation. By another option may be used as a contraceptive to inhibit ovarian maturation. SEO ID NO. 43: Thymopoietin beta - gi/885684 (variant)

Can be used as immunostimulants for inhibition of growth of cancer cells; for the modulation of interleukins, thymopoietin, production of GM-CSF and other growth factors; for treatment of cytokine-mediated inflammation, and for treatment of Hodgkin's disease, SLE and AIDS. In addition, may be used for the detection of any of the above.

SEQ ID NO. 43: Thymopoietin beta - gi|885684 (new variant). Can be used as immunostimulants for inhibition of growth of cancer cells; for the modulation of interleukins, thymopoietin, GM-CSF and other growth factor production; for treatment of cytokine-mediated inflammation, Hodgkin's disease, SLE, HIV; for treatment of patients suffering from post-traumatic iinmuno-dysfunction syndrome, for modulating the activity of T- and B-cells, and modulation of TNF production. In addition may be used to detect abnormalities in any of the above conditions, or conditions caused or involved with the above.

SEQ ID NO. 44: BONE MORPHOGENETIC PROTEIN 1 - gi|4502421 (variant) Can be used for stimulating bone growth for example for bone replacement purposes; for treatment of fibrotic disease and for remodulation of cartilage and bone tissue (including for implant purposes); for treatment of necrosis; for treatment of osteoporosis. In addition may be used to detect conditions of diseases involving non-normal growth of bone.

SEQ ID NO. 45: Bone-derived growth factor - gi| 1203965 (new variant).

Can be used as bone growth factors; for treatment of osteoporosis and fractures; treatment of metabolic bone disorders and injuries including for implant purposes. In addition may be used to detect conditions of diseases involving non-normal growth of both. SEO ID NO. 46: H beta 58 homolog - gi|729683 (homolog).

Can be used to correlate defects in developmental abnormalities, as well as cancer, and for detection of conditions resulting or caused by developmental abnormalities.

SEQ ID NO. 47: AgX-1 antigen human antigen with sera from infertile patients - gi|688011 (homolog).

Can be used for identification of infertility and for the treatment of infertility both in men and women.

SEQ ID NO. 48 NY-REN- 18 antigen - gi|5360093 (new variant).

SEQ ID NO. 49 NY-REN-18 antigen - gi|5360093 (new variant).

SEQ ID NO. 50 NY-REN- 18 antigen - gi|5360093 (new variant). SEQ ID NO. 51 NY-REN-18 antigen - gi|5360093 (new variant).

SEQ ID NO. 52 NY-REN-18 antigen - gi|5360093 (new variant). Can be used for the detection and treatment of tumors, for detection of pre-disposition to cancer and for the detection and treatment of Peutz-Jeghers Syndrome.

SEQ ID NO. 53: Protein kinase, cAMP-dependent - gi|4506063 (new variant). SEQ ID NO. 54: Similar to cAMP-dependant protein kinase - gi|7160989 (homolog).

SEQ ID NO. 62: cAMP-dependent protein kinase Rl-beta regulatory subunit - gi| 1346362 (new variant).

Can be used in the treatment of head and spinal injury, neurodegenerative disorders, cancer and cardiovascular diseases; for modulation of neurotropin activity, for treatment of tumor (prostate cancer for example) and for the modulation of nerve growth factor; can be used to enhance the treatment of allergies and asthma. In addition may be used to detect any of the above conditions.

SEQ ID NO. 55: PHOSPHOFRUCTOKINASE 1 - gil771743 (Variant) Can be used for modulation of Glycolysis, treatment of Glycogen Storage

Disease VII (Tarui's disease), as well as for the detection of this disease

SEQ ID NO. 56: Ribosomal protein S6 kinase - gi|4506735 (new variant).

Can be used for regulation of cell growth both for decrease of growth such as in cases of cancer and for increase of growth in degenerative diseases.

Modulates cell proliferation, differentiation and apoptosis and may be used for the treatment of cancer based on the above. In addition may be used for detection of the above condition and disorders.

SEQ ID NO. 57: dJ1103G7.3 (novel protein kinase domains containing protein similar to phosphoprotein C8FW) [Homo sapiens] (Neuronal cell death inducible putative kinase) - gi|7263910 (new variant).

SEQ ID NO. 72: Neuronal cell death inducible putative kinase - gi|7263910 (new variant). SEQ ID NO. 58: Mitogen-activated protein kinase 2 - gi|3894094 (homolog).

All the above three may be used in the production of cellular death as well as modulation of cellular response to drugs especially for modulating the cell-surviving effect or the cell destroying (cytotoxic) effect of the drugs. Can be used together with various cytotoxic drugs for example in treatment of cancer, or together with various neuroprotective drugs to modulate their protective activity.

SEQ ID NO. 59: PROTEIN KINASE C, ZETA TYPE - gi|450607 (new variant).

Can be used for the treatment and detection of: neosplastic conditions, hypertension, ischaemia, atherosclerosis, coagulation disorders and inflammatory diseases (including skin inflammatory diseases such as psoriasis), immune diseases, hyper-proliferative diseases and other kinase associated disorders; diabetes and its various side effects, Alzheimer's disease, ischemic brain injury, cancer immune disorders, asthma, lung fibrosis and psoriasis, cardiovascular diseases, artherosclerosis, restinosis, sexual dysfunctions, arthritis, septic shock, inflammatory diseases, reperfusion injury disorders, platelet aggregation (inhibition), neutrophil activation, migraines, can be used for protection against ischemic shock, protection against neuronal death (neuroprotective agents), treatment of trauma, chorea, genescense, epilepsy, neurodegenerative diseases, myocardial infarct angina, AIDS, hypertension, rheumatoid arthritis. In addition may be used for the improvement of memory and hearing, as an antifungal agent, for decreasing organ transplant rejection, and for treatment autoimmune disease including MS, viral infections, thrombosis and diseases of the immune system.

SEQ ID NO. 60: Ser/Thr protein kinase isolog - gi|2262107 (homolog). SEQ ID NO. 61: Ser/Thr protein kinase isolog - gi|2262107 (homolog).

Can be used as anticancer agents, in particular in breast, ovarian and pancreatic cancers, in addition may be used in diabetes and immune disorders. The probes may be used for the detection of any of the above.

SEQ ID NO. 63: Nucleoside diphosphate kinase type 6 (inhibitor of p53-induced apoptosis-alpha) - gi]5031951 (new variant).

Can be used as an antibiotic agent, for various infectious diseases.

SEQ ID NO. 64: Adenylate cyclase - gi|4757724 (new variant).

Can be used for inhibition of platelet aggregation and inhibition of thrombosis, in the treatment of cancer; as broad spectra anti-viral agent for thrombosis, hypertension and as a bronchodilator, in the treatment of congestive heart failure, (to increase the heart's contractile ability), and for the treatment and detection of various CNS, cardiac, inflammatory and reproductive disorders. Can be used as dopamine agonist for the treatment of cholera, as an antiparkinsonian anti-anxiety, as anti-depression and anti-bulimia agent; for the prevention of neuronal death and AIDS. It had additive inhibitory effects against influenza A and B viruses, and can be used in medicaments for treatment of the above.

SEQ ID NO. 65: Protein kinase, AMP-activated, gamma 1 non-catalytic subunit - gi|4506061 (new variant).

Can be used in the diagnosis and treatment of malignancy, hyperlipidaemia, obesity and diabetes mellitus.

SEQ ID NO. 66: Cyclin G associated kinase - gi|4885251 (new variant).

Can be used for the treatment of or detection of cancer.

SEQ ID NO. 67: GLUTAMATE DEHYDROGENASE 1 - gi|4885281 (new variant).

Can be used for modulation of insulin secretion such as in hyperinsulinism cases and hypoglycemia disorders, as well as for the detection of such cases.

SEQ ID NO. 68: BETA-HEXOSAMINIDASE A (HEXA) gi|450437 (Variant)

Can be used for the treatment of diseases relating to excessive storage of products expecially lysomal storage disease such as Tay-Sachs, and for the detection of the above.

SEQ ID NO. 69: Lipase - gi|7473544 (homolog).

Can be used in the treatment of obesity, Type II diabetes, treatment of exocrine pancreatic insufficiency, and for inhibition of fat absorbance from food. In addition may be used for detection or pre-disposition to such diseases. SEQ ID NO. 70: SECRETOGRANIN I - gi|4502807 (new variant).

Can be used for modulation of packaging and secretion of hormones and neuropeptides, for treatment and detection of diseases involving non-normal secretion of the above.

SEQ ID NO. 71: Vesicular membrane protein - gi| 1890141 (Ortholog) (human homolog of mouse vesicular membrane protein - gi| 1890141)

Can be used to identify neuronal cells and for modulation of neuronal organelle transport and may be used for detection or treatment of various neuronal and psychiatric diseases and disorders.

SEQ ID NO. 73: Semaphorin C - gi|2137756 (Ortholog) (human homolog of mouse Semaphorin C - gi|2137756)

Can be used for the modulation of neuronal activity in particular through axon growth and for modulation of immunological activity, and may also be used to detect pathological conditions involving the above.

SEQ ID NO. 74: Neuron-restrictive silencer factor - gi|2135813 (homolog). Can be used for regulation of neurogenesis for treatment of neuronal disorders originating from faulty neurogenesis, and for detection of disorders involving the above.

SEQ ID NO. 75: Slit-3 - gi|4049589 (new variant).

Can be used for the treatment of neuronal diseases involving glia and axon growth and for detection of conditions involving the above.

SEQ ID NO. 76: Tyrosine phosphatase (IA-2/PTP) - gi)4506321 (variant)

Can be used against parasitic infections especially protozan parasite infections, for treatment of diabetes, obesity, AIDS, neurological and inflammatory diseases. Can also be used for detection of any of the above. SEQ ID NO. 77: GLUCOSE-6-PHOSPHATASE (G6PASE) - gi|2352822 (homolog).

Can be used in the treatment of hyperglycaemic conditions for treatment of glycogen storage disease (GSD) conditions and detection of the above, for treatment of diabetes, for treatment of Von Gierk disease. In addition, may be used for the detection of any of the above.

SEQ ID NO. 78: Protein phosphatase dual specificity phosphatase 13 - gi| 1633321 (homolog).

Can be used in regulation of Tyr and Thr/Ser phosphate activities and for the treatment and detection of diseases resulting from abnormalities in those activities.

SEQ ID NO. 79: Metalloproteinase 1, PRSM1 - gi|4506139 (new variant).

Can be used in the treatment of cancer, for regulation of TNF, for treatment of inflammatory conditions; can be used as an antiarthritic; for the treatment of periodontal diseases, for treatment of endotoxaemia, mutliple sclerosis, rheumatoid arthritis, inflammatory bowl disease, treatment of degenerative diseases, for treatment of tumors especially invasive tumors; for treatment of atherosclerosis, vasospastic disorders and diseases associated with enhanced production of endothelin, for treatment of hypertension, corneal ulcertaion, HIV. In addition may be used for the detection of any of the above diseases.

SEQ ID NO. 80: CATHEPSIN B - gi|4503139 (new variant).

Can be used for the treatment of RA, OA, osteoporosis, cancer, malaria, muscular dystrophy, myocardial infarction, Alzheimer's disease and infections, as well as for the detection of any of the above. SEQ ID NO. 82: Hematopoietic-specific IL-2 deubiquitinating enzyme - gi|2739431 (homolog) Gene name: DUB-2

Can be used for regulation of the degradation of the deubiquitinating state of growth factor so as to regulate cytokine specific growth response. This gene belongs to a larger family of cytokine-inducible DUB enzymes DUB-2 encodes a deubiquitinating enzyme, which is induced as an immediate-early gene by interleukin-2. Induced DUB enzyme thereby regulates the degradation or the ubiquitination state of an growth regulatory factor, resulting in a cytokine-specific growth response.

SEQ ID NO. 81: FUR2_DROME Endoprotease FURIN - gi|232114 (homolog).

Can be used as an anti-infective agent in particular as an anti-viral agent.

SEQ ID NO. 82: Hematopoietic-specific IL-2 deubiquitinating enzyme - gi|2739431 (homolog).

SEQ ID NO. 83: Sorting nexin 6 - gi|4689252 (homolog).

SEQ ID NO. 102: SNX9 (Sorting Nexin 9) - gi|5327052 (homolog).

All three can be used for the regulation of degradation of epidermal growth factor receptor and by this regulation of the responses to EGF's platelet-derived growth factors and insulin. May regulate various other types of activities mediated by tyrosine kinase receptors, and may be used to detect diseases involved in abnormalities in these activities.

SEQ ID NO. 84: BETA-SECRETASE PRECURSOR - gi|6912266 (new variant).

Can be used as an anti-ulcer agent for treatment of gastric ulcers; for stimulation of tear production, for increase of fluid and protein secretion by lacrymocytes, as an anti-psychotic agent (especially for schizophrenia and manic disorders), for the treatment of memory loss, as a neuroprotective cerebral ischemia, psychosis and convulsions, for the treatment of breast cancer, anti- inflammatory, depression, anxiety, dystonia, epilepsy, Alzheimer's disease stroke and immune disorders. In addition the probe may be used for detection of any of the above.

SEQ ID NO 85: sigma receptor gi|5032117 (new variant)

Can be used as an anti-ulcer agent for treatment of gastric ulcers; for stimulation of tear production, for increase of fluid and protein secretion by lacrymocytes, as an anti-psychotic agent (especially for schizophrenia and manic disorders), for the treatment of memory loss, as a neuroprotective cerebral ischemia, psychosis and convulsions, for the treatment of breast cancer, anti- inflammatory, depression, anxiety, dystonia, epilepsy, stroke and immune disorders. In addition the probe may be used for detection of any of the above.

SEQ ID NO. 86: Putative G protein-coupled receptor, P40GPRT - gi|5174445 (homolog).

Can be used for regulation of serotonin activity in the CNS; regulation of chemokine activity, for example, during inflammatory, for treatment of renal diseases, hypertension and atherosclerosis, myocardial diseases. In addition may be used to detect any of the above.

SEQ ID NO. 87: Receptor genes for plasminogen related growth factors - gi)3928166 (homolog).

Can be used in modulation of embryogenesis, tissue regeneration and cancer.

SEQ ID NO. 88: CD39-like - gi|4557423 (new variant).

Can be used as an anticoagulant to prevent or treat blood vessel blockage in heart attacks, strokes and thrombosis, for use as an anti-thrombotic. SEQ ID NO. 89: Class I cytokine receptor - gi|4758062 (new variant).

Can be used for the regulation of cytokine activity in immune response, in removal of cells from the CNS and as neuronal protectant.

SEQ ID NO. 90: Interleukin 11 receptor, alpha - gi|4758594 (variant)

Can be used to regulate interleukin activity on lymphohematopoietic cells, and for detection of diseases involved, caused or resulting from abnormal activity.

SEQ ID NO. 91: 5-hydroxytryptamine (serotonin) receptor - gi)6754260 (homolog).

Can be used as antipsychotics (especially schizophrenia); for the treatment of thombosis, diabetic neuropathy and intermittent claudication; as an antidepressant; for the treatment of anxiety and depression; as an antimigraine agent, as an antiemetic; in the reduction of body fat stores in obese individuals; for the treatment of gastro-oesophageal reflux and emesis; for the prevention of cardiovascular diseases, for the treatment of Alzheimer's disease and for the treatment of male premature ejaculation. In addition may be used for the detection of the above conditions.

SEQ ID NO. 92: Vasodilator-stimulated phosphoprotein (VASP) - gi| 1617319 (homolog).

SEQ ID NO. 93: Vasodilator-stimulated phosphoprotein (VASP) -gi| 1617319 (homolog). Can be used for the treatment of infections caused by intracellular pathogens including Listeria monocytogenes for the treatment of atherosclerosis and tumor growth, and for the detection of the above. In addition may be used for limiting the spread of intracellular pathogens. SEQ ID NO. 94: Attractin-2 - gi|4093196 (homolog).

Can be used in the modulation of immune cell interaction, and for detection of diseases involving abnormal interactions.

SEQ ID NO. 95: INHIBIN BETA E CHAIN PRECURSOR (ACTIVIN BETA-E CHAIN) - gi|2072522 (Ortholog).

Can be used as an antisickling agent; as effecting the reproductive system for example as contraceptive both for men and women having reversible effect or for treatment of infertility. Can also be used as a gonadal tumor suppressor; for the treatment of reproduction and haematology disorders. Can be used in in vitro fertilization (IVF), and as a gonadal tumor diagnosis agent for the detection of tumors.

SEQ ID NO. 96: Rho GDP dissociation inhibitor (GDI) alpha - gi|4757768 (new variant).

Can be used in connection with activity of Rho proteins, especially in connection with cancer.

SEQ ID NO. 97: similar to src homology protein 2 Shb2 - gi|4506935 (homolog).

Shb is involved in signal transduction of some ligand activated tyrosine kinase receptors. Protein tyrosine kinases (PTKs) transmit activation signals in almost every cell type, including immune effector cells. Can be used to activate and regulate the activity of a variety of cells including immune effector cells.

SEQ ID NO. 98: Rabphilin 3A-like - gi|5902064 (new variant).

Can be used in regulation of neurotransmitter release, for the treatment of various neuronal and psychiatric diseases as well as for detection of same. SEQ ID NO. 99: Phosphotyrosyl phosphatase activator PTPA - gi| 1082693 (new variant).

Can be used in connection with therapy of diabetes, for autoimmune and transplant rejection disorders, and for the detection of the above.

SEQ ID NO. 100: RAS-LIKE PROTEIN - gi|5454030 (homolog)

Can be used as an adjuvant to immune response's AIDS vaccine; as anticancer vaccine; as solid tumor chemoprotectives, for the treatment of cancer, autoimmune disorders and hypeφroliferative skin disorders; for the treatment of recurrent aphthous stomatitis (RAS) and other oral and non-oral mucosal diseases with an immune aetiology, and for steroid-sparing purposes in mucosal diseases. In addition may be used to detect any of the above.

SEQ ID NO. 101: RHO-GTPASE- ACTIVATING PROTEIN 1 - gi|7706137 (homolog).

May be used in the treatment of delayed vascular spasm, and for the detection of same.

SEQ ID NO. 103: Rac-GTP binding protein-like - gi|7573444 (homolog).

SEQ ID NO. 104: Rac-GTP binding protein-like - gi|7573444 (homolog). Can be used for regulation of cytoskeleton activity, for example, in cancer and such diseases, and for the detection of such diseases.

SEQ ID NO. 105: Sulfate transporter - gi|2626753 (homolog).

Can be used for the treatment and detection of the genetic diseases, diastrophic dysplasia (DTD); for treatment of hearing loss; and treatment of other genetic diseases; in treatment of atelosteogenesis type 2 (AT2) which is a neonatally lethal chondrodysplasia characterised by severe limb shortening and deficient ossification of parts of the skeleton. Other features include facial dysmorphism, cleft palate, talipes, and abducted thumbs and toes. Phenotypic overlap with non-lethal diastrophic dysplasia (DTD) suggested a common aetiology and it has recently been confirmed that both syndromes result from mutations in the DTDST (diastrophic dysplasia sulphate transporter) gene; also used for treatment and detection of pendred syndrome, which is a recessively inherited disorder with the hallmark features of congenital deafness and thyroid goitre; for detection and treatment of bone dysplasias and diastrophic dysplasia (DTD).

SEQ ID NO. 106: SODIUM/BILE ACID COTRANSPORTER - gi|2811069 (homolog). May be used to regulate sodium-dependent reabsorption of bile acids from the lumen of the small intestine and for treatment of diseases involved in primary bile acid malabsorption. (PBAM), an idiopathic intestinal disorder associated with congenital diarrhea, steatorrea, interuption of the enterohepatic circulation of bile acids and reduced plasma cholesterol levels, as well as for the detection of any of the above.

SEQ ID NO. 107: ITM protein multi-membrane spanning polyspecific transporter - gi|3551148 (homolog).

Can be used for a marker of imprinting, i.e. locating whether the allele is inherited from the father or from the mother are expressed.

SEQ ID NO. 108: CTR1_HUMAN HIGH-AFFINITY CATIONIC AMINO ACID TRANSPORTER-1 - gi| 1083250 (homolog).

Function: high-affinity, low capacity permease involved in transport. Can be used for regulation of the transport of the cationic amino acids arginine, lysine and oraithine across the plasma membrane. Can also be used for ecotropic retroviral control and regulation. SEQ ID NO. 109: TXTP_human fricarboxylate transport protein precursor (citrate transport protein) - gi| 1717824 (homolog).

SEQ ID NO. 112: fricarboxylate transport protein precursor (citrate transport protein) (CTP) - gi| 171782 (new variant). SEQ ID NO. 113: fricarboxylate transport protein precursor (citrate transport protein) (CTP) - gi| 1717824 (new variant).

Can be used for the bioenergetics of hepatic cells as it provides a carbon source for fatty acid and sterol biosyntheses, and nad+ for the glycolytic pathway. Can be used to maintain hepatic cells in culture, for example, for implant purposes, and can be used for the treatment and detection of DiGeorge syndrome and velo-cardio-facial syndrome, and improvement of the mental retardation associated with these diseases.

SEQ ID NO. 110: fatty acid transport protein 3; FATP3 - gi|3335567 (homolog).

Can be used for control of fat absorption, for the treatment of obesity. In addition, may be used for the control of hepatic glucose, production and may be used for treatment of diabetes.

SEQ ID NO. Ill: TRP7_HUMAN TRANSIENT RECEPTOR POTENTIAL CHANNEL 7 - gi|4507689 (homolog).

Can be used for the detection and treatment of genetic disorders such as bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holoprosencephaly.

SEQ ID NO. 114: HUMAN SYNTAXIN 6 - gi|5032131 (new variant).

Can be used to regulate neutrophil functions in both inflammation and infection and to treat diseases involved with neutrophil activity. Can modulate the

9+ activity of Ca channels in the CNS thus effecting such properties as learning, memory, neuronal survival, for treatment of neuronal and psychiatric diseases. SEQ ID NO. 118: syntaxin 7 - gi|3123924 (homolog). SEQ ID NO. 119: syntaxin 7 - gi|3123924 (homolog).

Can be used to regulate secretion of various agents by cells and to treat and detect diseases involving abnormal secretion.

SEQ ID NO. 115: fricarboxylate carrier - gi|2144101 (Ortholog) (human homolog of rat fricarboxylate carrier - gi|2144101)

Can be used to maintain hepatic cells in culture, for example, for implant purposes, can be used for the treatment and detection of DiGeorge syndrome and velo-cardio-facial syndrome, and improvement of the mental retardation associated with these diseases.

SEQ ID NO. 116: Contains similarity to equilibratiave nucleoside transporter 1 - gi|3176684 (homolog).

Can be used to regulate nucleoside transport and treat or detect diseases associated with abnormal transport.

SEQ ID NO. 117: Na+/Cl(-)-dependent neurotransmitter transporter gi|400631 (new variant).

Can be used to regulate neurotransitter release for the treatment and detection of various neuronal and psychiatric diseases.

SEQ ID NO. 120: SECRETORY CARRIER-ASSOCIATED MEMBRANE PROTEIN - gi|7021484 (Ortholog) (mouse protein)

Can be used to regulate the activity of endothelial growth factor receptor and by this the activity of EGF itself.

SEQ ID NO. 121: Intestinal membrane A4 protein HOMOLOG Differentiation dependent protein A4-HOMOLOG - gi|4505893 (homolog). Can be used to treat and detect conditions involved with the intestinal epithelium, for example, diseases similar to cystic fibrosis, and diseases involving non normal transport of electrolyes and metabolites through the intestinal epithelium.

SEQ ID NO. 122: MYCN CN-MYC proto-oncogene protein (new variant).

This new variant differs from the original MYCN sequence by replacement of 201 aa at the C-terminus of the original protein in 119 aa, and by deletion of the asp/glu-rich (acidic) domain and the DNA binding domains from the original protein.

Can be used in the treatment of cancer (especially leukemia). The probe may be used for the detection of cancer, or predisposition to cancer.

SEQ ID NO. 123: Calcium/calmodulin-dependent protein kinase type II alpha chain (5915912) (new variant).

This new variant differs from the original calcium/calmodulin-dependent protein kinase type II alpha chain by replacement of 314 aa of the N-terminus of the original protein in 26 aa, and by deletion of the protein kinase domain, ATP binding domain, calmodulin binding domain and the phosphorylation site of the original protein. This kinase may play a role in neurotransmission. This kinase has tissue specificity: alpha camkii is expressed in brain while alpha kap is expressed in skeletal muscle. It belongs to the ser/thr family of protein kinases, subfamily camk. Ser/thr family of protein kinases.

Can be used as anticancer agents, in particular in breast, ovarian and pancreatic cancers, in addition may be used in the treatment of diabetes and immune disorders such as in autoimmune diseases. The probes may be used for the detection of any of the above. SEQ ID NO. 124: Human growth differentiation factor- 12 cDNA (splice variant).

This new splice variant differs from the original human growth differentation factor- 12 cDNA (gi 6680453), which is a liver activin beta e, by replacement of 70 aa of the original protein in 42 aa, thus deleting the potential signal peptide. Activin is a member of the transforming growth factor β (TGF-β) family.

Can be used as an anticancer agent, especially against leukemia (activin induces leukaemic cells change into normal cells), in addition can be used to enhance bone building activity (for example for treatment of osteoporosis) by stimulating osteoblast growth, promotion of hematopoietic activity, for example for the treatment of trombocytopenia, anemia or leukopenia and treatment of nervous system diseases.

SEQ ID NO. 125: Breast cancer associated protein BRAP1 (splice variant).

This new splice variant differs from the original BRAP1 by replacement of 460 aa of the C-terminus of the original protein in 4aa.

This new sequence can be used for the detection of breast and ovary cancers, and for detection and predisposition to these cancers as well as for their use in the treatment of these cancers.

SEQ ID NO. 126: SCG1 (neuron-specific growth-associated protein/stathmin homology) (new variant).

This new variant differs from the original SCG1 sequence by deletion of 12 aa of the N-terminus of the original protein and by deletion of part of the potential membrane attachment domain of the original domain (1-26 aa in the original protein).

Can be used for neuroprotective agent, for the treatment of Alzheimer's disease, multiple sclerosis, peripheral neuropathies (including diabetic neuropathy and motor neuron disorders), traumatic head and spinal injury, cerebral ischemia and stroke. This variant can also be used as anticancer agent, as a memory enhancer, fertility enhancer in fertility treatment, antiparkinsonian agent, antidepressant agent, for the treatment of ADD/ ADHD and for the treatment of male sexual dysfunction.

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SEQ ID NO. 127: SCG1 (neuron-specific growth-associated protein/stathmin homology (new variant).

This new variant differs from the original SCG1 by deletion of 12 aa of the

N-terminus of the original protein and by replacement of 113 aa of the ιo C-terminus of the original protein in 23 aa, thus deleting part of the potential membrane attachment domain of the original domain (1-26 aa in the original protein) and deleting three out of five phosphorylation sites.

Can be used for neuroprotective agent, for the treatment of Alzheimer's disease, multiple sclerosis, peripheral neuropathies (including diabetic ₁₅ neuropathy and motor neuron disorders), traumatic head and spinal injury, cerebral ischemia and stroke. This variant can also be used as anticancer agent, as a memory enhancer, fertility enhancer in fertility treatment, antiparkinsonian agent, antidepressant agent, for the treatment of ADD/ ADHD and for the treatment of male sexual dysfunction.

20

SEQ ID NO. 128: SCG1 (neuron-specific growth-associated protein/stathmin homology (new variant).

This new variant differs from the original SCG1 by deletion of 12 aa of the

N-terminus of the original protein, deletion of aa 67-199 and replacement of ₂₅ 24 aa of the C-terminus of the original protein in 3 aa, thus deleting part of the potential membrane attachment domain of the original domain (1-26 aa in the original protein), and deleting three out of five phosphorylation sites.

Can be used as neuroprotective agent for the treatment of Alzheimer's disease, for multiple sclerosis, peripheral neuropathies (including diabetic ₃₀ neuropathy and motor neuron disorders), traumatic heat and spinal injury, cerebral ischemia and stroke. This new variant can also be used as anticancer agent, memory enhancer, fertility enhancer, antiparkinsonian agent, antidepressant agent, for the treatment of ADD/ ADHD, and for the treatment of male sexual dysfunction.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Example 1: NS nucleic acid sequence

The nucleic acid sequences of the invention include nucleic acid sequences which encode NS 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 70%, preferably 80%, most preferably 90% identity with any one of the sequence identified as SEQ ID NO: 1 to SEQ ID NO: 128 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 5' 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 NS 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 NS 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 31:161 (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 lαiown methods.

As indicated above, the nucleic acid sequence may be substantially a depicted in any one of SEQ ID NO:l to SEQ ID NO: 128 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 coded by the sequence of SEQ ID NO:l to SEQ ID NO: 128, 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 NS 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 lαiown 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 NS nucleic acid sequence for the production of NS products

In accordance with the present invention, nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of NS products.

As will be understood by those of skill in the art, it may be advantageous to produce NS product-encoding nucleotide sequences possessing codons other than those which appear in any one of SEQ ID NO:l to SEQ ID NO: 128 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 NS 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 NS 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 transfected) 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 NS 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 lαiown 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 tetracycline 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 NS product. For example, when large quantities of NS 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(K) (Stratagene), in which the NS 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 WI); 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 NS 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.

NS 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 NS 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 NS 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 NS 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 NS 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 NS 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 an NS product coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where NS 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 NS 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. 11:3561-10, (1980)); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al, J. Mol. Biol, 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. 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, Methods Mol. Biol, 55:121-131, (1995)).

Host cells transformed with a nucleotide sequence encoding NS 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 sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing nucleic acid sequences encoding NS product can be designed with signal sequences which direct secretion of NS product through a prokaryotic or eukaryotic cell membrane. The NS product may also be expressed as a recombinant protein with 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 extension/affinity purification system (Immunex Corp, Seattle, Wash.). The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and NS product is useful to facilitate purification. One such expression vector provides for expression of a fusion protein compromising a NS 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 NS 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 NS 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 NS in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for NS product. Alternatively, the assay may be used to detect soluble NS 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 NS product under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of NS. This assay can be used to distinguish between absence, presence, and excess expression of NS product and to monitor levels of NS expression during therapeutic intervention. In addition, the assay may be used to compare the levels of the NS of the invention to the levels of the original sequence from which it has been varied or to which it shows homology or (where the NS is a splice variant) to levels of other NSs, 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 NS sequences, or diseases in which the ratio of the amount of the original sequence from which the NS was varied or to which it shows homology to the novel NSs of the invention is altered. These sequences can be detected by comparing the sequences of the defective (i.e., mutant) NS coding region with that of a normal coding region. Association of the sequence coding for mutant NS product with abnormal NS product activity may be verified. In addition, sequences encoding mutant NS products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a NS 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 NS 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 lαiown 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 NS 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 NS 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 NS), expression of NS 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 NS 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 NS products. An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the NS 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 NS 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 NS, expression of NS 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 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 transfected 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 lαiown in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 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 NS production or antisense inhibition in response to radiation, eg., radiation therapy for treating tumors. Example II. NS product

The substantially purified NS 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 70%), preferably 80%), most preferably 90%> identity to any one of the sequences coded by the nucleic acid sequence of SEQ ID NO:l to SEQ ID NO: 128 provided that the amino acid sequence is not identical to that of the original sequence from which it has been varied or to which it shows homology. 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 NS 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 70%, preferably 80%, most preferably 90%> sequence identity with any of the products coded by sEQ ID NO: 1 to SEQ ID NO: 128, 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 or to which it features homology. The NS 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 NS 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 NS product. Such fragments, NSs and derivatives are deemed to be within the scope of those skilled in the art from the teachings herein. A. Preparation of NS product

Recombinant methods for producing and isolating the NS product, and fragments of the protein are described above.

In addition to recombinant production, fragments and portions of NS 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 NS product may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

B. Therapeutic uses and compositions utilizing the NS product

The NS product of the invention is generally useful in treating diseases and disorders which are characterized by a lower than normal level of NS expression, and or diseases which can be cured or ameliorated by raising the level of the NS product, even if the level is normal. NS 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.

NS 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. NS 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 lαiown 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.

Example III. Screening methods for medicaments 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 NS product, e.g. activators or deactivators of the NS product of the present invention. Such an assay comprises the steps of providing an NS product encoded by the nucleic acid sequences of the present invention, contacting the NS protein with one or more candidate molecules to determine the candidate molecules modulating effect on the activity of the NS product, and selecting from the molecules a candidate's molecule capable of modulating NS product physiological activity.

The NS 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 NS 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 NS 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 NS product is described in detail by Gey sen 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 NS product or with fragments of NS product and washed. Bound NS product is then detected by methods well known in the art. Substantially purified NS 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 NS 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-NS antibody is affixed to a solid surface such as a microtiter plate and NS product is added. Such an assay can be used to capture compounds which bind to the NS product. Alternatively, such an assay may be used to measure the ability of compounds to influence with the binding of NS product to the NS receptor, and then select those compounds which effect the binding.

Example IV. Anti-NS antibodies/distinguishing antibodies A. Synthesis

In still another aspect of the invention, the purified NS product is used to produce anti-NS antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the NS product. As indicated above, the antibodies may also be directed solely to amino acid sequences present in the NS but not present in the original sequence, or to sequences present only in the original sequence but not in the NS (distinguishing antibodies), or to sequences which differ between the two.

Antibodies to the NS product or to the distinguishing sequence present only in the NS or only in the original sequence (the latter termed "distinguishing antibodies ") 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 NS 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 any sequences coded by the nucleic acid sequence of SEQ ID NO:l to SEQ ID NO: 128 or in distinguishing sequences present only in the NS or only in the original sequence as explained above. 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 NS 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 lαiown in the art can be used for the production of antibodies to NS product.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with NS 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 dinifrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

Monoclonal antibodies to NS protein may be prepared using any technique which provides for the production of antibody molecules by continuous 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)).

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)). 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 NS protein.

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or 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 NS protein may also be generated. For example, such fragments include, but are not 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 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 NS 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 NS 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 NS product or distinguishing antibodies which bind to sequences which distinguish the NS from the original sequence (as explained above) are useful for the diagnosis of conditions or diseases characterized by expression of the NS of the invention (where normally it is not expressed) by over or under expression of NS as well as for detection of diseases in which the proportion between the amount of the NSs of the invention and the original sequence from which it varied or to which it features homology is altered. Alternatively, such antibodies may be used in assays to monitor patients being treated with NS product, or drugs specifically produced utilizing the product. Diagnostic assays for NS protein include methods utilizing the antibody and a label to detect NS 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 lαiown in the art.

A variety of protocols for measuring the NS product, using either polyclonal or monoclonal antibodies specific for the respective protein are lαiown 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 NS 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 NS product expression. Noπnal or standard values for NS product expression are established by combining body fluids or cell extracts taken from normal subjects, preferably human, with antibody to NS 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 NS 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 howNS levels of variable products are responding to drug treatment. 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 NS product in pathological conditions where beneficial effect can be achieved by such a decrease. Again, distinguishing antibodies may be used to neutralize differentially either the NS or the original sequence as the case may be.

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.

Claims

CLAIMS:

1. An isolated nucleic acid sequence, selected from the group consisting of:

(i) the nucleic acid sequence depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 128; 5 (ii) nucleic acid sequence having at least 70% identity with a sequence of

(i) with the proviso that where the nucleic acid sequence is obtained by alternative splicing, it is different from that of an original nucleic acid sequence from which the sequence of (i) has been varied by alternative splicing; and

(iii) fragment 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 sequence of (i) has been varied by alternative splicing or to which it features homology.

2. A nucleic acid sequence according to Claim l(ii) having at least 80% identity. 5

3. A nucleic acid sequence according to Claim 2, having at least 90% identity.

4. An isolated nucleic acid sequence complementary to the nucleic acid sequence of Claim 1.

5. An amino acid sequence selected from the group consisting of:

(i) an amino acid sequence coded by the isolated nucleic acid sequence 0 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, from 5 which the sequence has been varied by alternative splicing, or to which it features homology.

6. An amino acid sequence according to Claim 5, as depicted in any one of SEQ ID NO. 129 to SEQ ID NO: 256.

7. An isolated nucleic acid sequence coding for any one of the amino acid o sequences of Claim 5.

8. A purified antibody which binds specifically to any of the amino acid sequence of Claim 5.

9. An expression vector comprising any one of the nucleic acid sequences of Claim 1 or 7 and control elements for the expression of the nucleic acid sequence in a suitable host.

10. An expression vector comprising any one of the nucleic acid sequences of Claim 4, and control elements for the expression of the nucleic acid sequences in a suitable host.

11. A host cell transfected by the expression vector of Claim 9 or 10.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from:

(i) any one of the nucleic acid sequences of Claim 1 ; (ii) the expression vector of Claim 9; and (iii) any one of the amino acid sequences of Claim 5.

13. A pharmaceutical composition according to Claim 12, for treatment of diseases which can be ameliorated or cured by raising the level of any one of the amino acid sequences of Claim 5.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an agent selected from: (i) any one of the nucleic acid sequences of Claim 4;

(ii) the expression vector of Claim 10 ; and (iii) the purified antibody of Claim 8.

15. A pharmaceutical composition according to Claim 14, for treatment of diseases which can be ameliorated or cured by decreasing the level of any one of the amino acid sequences coded by SEQ ID NO: 1 to SEQ ID NO: 128.

16. A pharmaceutical composition, comprising as an active ingredient a compound selected from:

(i) a nucleic acid sequence according to any one of Claims 1, 2, 3 or 7; (ii) an expression vector comprising the nucleic acid sequence of (i); (iii) a sequence complementary to the nucleic acid sequence of (i); (iv) an expression vector comprising the nucleic acid sequence of (iii); (v) an amino acid sequence encoded by any one of the nucleic acid sequence of (i); and (vi) a purified antibody which binds specifically to any one of the amino 5 acid sequence of (v).

17. A probe for the detection of a nucleic acid sequence comprising a nucleic acid sequence according to any one of Claims 1, 2, 3, 4 or 7.

18. A method for detecting an NS nucleic acid sequence in a biological sample, comprising: 0 (a) hybridizing to nucleic acid material of said biological sample to the probe of Claim 17; and

(b) deteting hybridization complex.

19. A method for determining the level of NS nucleic acid sequences in a biological sample comprising the steps of: ₅ (a) hybridizing to nucleic acid material of said biological sample with any one of the probes of Claim 17; and

(b) determining the amount of hybridization complexes and normalizing said amount to provide the level of the NS nucleic acid sequences in the sample.

20. A method for detection of a nucleic acid sequence in a biological sample the ό method comprises:

(a) contacting the sample with probes for amplification of any one of

SEQ ID NO: 1 to SEQ ID NO: 128;

(b) proving reagents for amplification; and

(c) detecting the presence of amplified products, said products indicating 5 the presence of NS nucleic acid in the sample.

21. A method for determining the ratio between the level of NS of the nucleic acid sequence in a first biological sample and the level of the original sequence, from which the NS has been varied by alternative splicing or to which it features homology, in a second biological sample comprising: (i) determining the level of the NS nucleic acid sequence in the first biological sample according to the method of Claim 19 or 20;

(ii) determining the level of the original sequence in the second biological sample; and (iii) comparing the levels obtained in (i) and (ii) to give said ratio.

22. A method according to Claim 21, wherein said first and said second biological samples are the same sample.

23. A method according to any of Claims 18 to 22, wherein the nucleic acid material of said biological sample are mRNA transcripts.

24. A method according to Claim 22, where the nucleic acid sequence is present in a nucleic acid chip.

25. A method for detecting any one of the amino acid sequences of Claim 5, in a biological sample, comprising:

(a) contacting with said biological sample the antibody of Claim 8, 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.

26. A method for detecting the level of the amino acid sequence of Claim 5, in a biological sample, comprising:

(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 amino acid sequences of Claim 5 present in a first biological sample and the level of the original amino acid sequences, from which they were varied by alternative splicing or to which they feature homology, present in a second biological sample, the method comprising: (a) determining the level of the amino acid sequence of Claim 5 in a first sample by the method of Claim 26;

(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.