WO2002059259A2 - Methods for discovering secreted and transmembrane proteins - Google Patents

Abstract

This invention provides a differential display method for identifying at least one secreted or transmembrane protein comprising the following steps: obtaining mRNA from at least two samples; synthesizing cDNA from said mRNA of each sample; contacting said cDNA from each sample with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second primer which contains at least ATG sequence, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain an amplified products; detecting said amplified products; and comparing said amplified products from each sample thereby identifying a distinctive amplified products coding for at least one secreted or transmembrane protein.

Description

METHODS FOR DISCOVERING SECRETED AND TRANSMEMBRANE

PROTEINS

FIELD OF THE INVENTION The present invention relates to a differential display method for discovering novel secreted and/or transmembrane proteins.

BACKGROUND OF THE INVENTION Secreted and transmembrane located proteins are particularly important for cell processes and play an important role in determining its phenotype. Such proteins are singularly critical molecules and participate in a multitude of biological processes including cell growth, cell proliferation, cell differentiation and cell death. These proteins act as mediators for the transfer of signals from the external environment into the cell itself, thereby modulating the genes expression. For example, the secreted protein insulin can bind to the insulin receptor located on the cell membrane and thus alter gene expression within the insulin-stimulated cell. Similarly, the secreted growth hormone protein binds to the growth hormone receptor, thereby initiating a signaling cascade, which ultimately results in alterations in gene expression profiles and changes in the cell phenotype. The detection of new, biologically relevant, transmembrane and secreted protein molecules is, thus, one of the leading and important goals in biotechnological research today. The quintessential importance of this class of proteins makes them attractive targets for therapeutic interventions in pathological states. It is thus, abundantly clear that a major goal in the design and in the development of new therapies is the identification and characterization of secreted and transmembrane proteins and the genes that encode these proteins.

An example for a membrane-bound therapeutic protein is erbB₂ protein. The erbB₂ cell membrane-located receptor is overexpressed in a subset of human breast cancers, which make it an attractive target for cytotoxic antibodies, that bind to erbB₂ expressing cells and destroying them. The secreted cytokines for example are an important class of secreted proteins that can act on specific cells to elicit a particular physiological response, such as for example, without being limited, secretion of proteins, which are necessary for differentiation, cell proliferation, cell death or inflammation processes. Cytokines, namely, lymphokines, hematopoietins, interleukins, Colony Stimulating Factor (CSF) are stimulating factors and they are potential therapeutic agents. It should be noted in this respect, that recently, erythropoietin (hematopoietic cytokine) was found to be efficient in treating anemia and G-CSF and GM-CSF (cytokines) are important agents for treating pathological states, characterized by a rapid decline in the amount of white blood cells.

Thus, considerable effort is invested in discovering novel secreted and membrane-bound proteins and in identifying the role they play under pathological conditions. In the past, novel secreted cytokines and membrane-bound proteins were identified by measuring a particular cell for a measurable biological response. Thus, the method of discovering new proteins has been limited by the availability of the assays, and if a novel cytokine or a membrane protein has an activity that is immeasurable by a known assay, these proteins remain undetectable.

Using a different approach, secreted and membrane bound proteins have also been cloned using subtractive hybridization to construct and screen cDNA libraries, or have been cloned using PCR (Polymerase Chain Reaction) and gel electrophoresis that detect differentially expressed genes. The major drawback of these procedures is that mRNA of the secreted and membrane-bound proteins represent only a minor fraction of all mRNAs that are differentially expressed. Furthermore, these procedures do not focus, a priori, on secreted or on membrane bound proteins. Thus, the identification of the relevant DNA clones according the above procedure is not time and cost effective as well as being complicated. U.S. Pat. No. 5,525,486 and U.S. Pat. No. 5,536,637 teach methods that enable the identification of cDNAs encoding for a signal sequence, which is capable of directing the secretion of a particular protein from certain cell types. These methods enable the identification of secreted proteins. However, they failed to discover the biological functions of these proteins and to identify the pathological states in which they may play important roles.

Another recent procedure (Kojima T. and Kitamura T., 1999, Nature, 17, 487-490) is based on the identification of hydrophobic domains present in secreted and transmembrane proteins. In the secreted proteins, a hydrophobic signal peptide usually appears at the amino terminal of the protein and in cell-membrane anchored proteins an additional hydrophobic domain is located in the transmembrane domains. This identification method is very time consuming and does not provide information about the biological functionality of the identified proteins.

Screening hundreds of thousands of Expressed Sequence Tags (ESTs) generated by large-scale sequencing require extremely fast and powerful computational tools. But even with such tools the accurate identification of secreted and transmembrane proteins is severely hampered by the fact that the order of leucine, valine, isoleucine and other hydrophobic residues within the hydrophobic domains of such proteins seems to be irrelevant to function. As a result of this variation, hydrophobic domains cannot be identified readily by the usual sequence analysis methods like FASTA or BLAST that are based on the conservation of sequences. Furthermore as ESTs are severely biased for 3' (carboxyl) terminal sequences, such computational screening procedures are unlikely to reveal potential signal peptides present at the amino terminal of secreted proteins.

There is thus a recognized need for an uncomplicated method that will identify transmembrane and secreted proteins which can play important roles in a number of different biological processes such as, without being limited, malignant transformation of cells, hypertension, erythropoiesis, control of food intake and the like.

SUMMARY OF THE INVENTION

This invention provides in one embodiment a differential display method for identifying at least one secreted or transmembrane protein which overcomes the limitations and disadvantages of prior methods known in the art and will be cost effective and time effective.

This invention provides in one embodiment a differential display method for identifying at least one secreted or transmembrane protein comprising the following steps: obtaining mRNA from at least two samples; synthesizing cDNA from said mRNA of each sample; contacting said cDNA from each sample with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second primer, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain an amplified products; detecting said amplified products; and comparing said amplified products from each sample thereby identifying a distinctive amplified products coding for at least one secreted or transmembrane protein.

This invention provides in another embodiment, a method of screening samples for the presence of at least one secreted or transmembrane protein comprising the following steps: contacting said cDNA from each sample with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second oligonucleotide primer, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain an amplified products; detecting said amplified products; and comparing said amplified products from said sample to amplified products derived from known samples thereby identifying distinctive amplified products coding for a at least one secreted or transmembrane protein. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended drawings in which:

Figure 1 is a presentation of the insert of a differentially displayed RT-PCR product.

Figure 2 is a single peptide sequence and nucleotide sequence of selected secreted proteins.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

This invention provides a differential display method and a method of screening samples which selectively focus on mRNA molecules which code for transmembrane and/or secreted protein molecules that are differentially expressed in diverse conditions. The following described method is a unique, simple and time-effective method that enables to simultaneously determine a number of secreted and transmembrane proteins, which play key roles in defined biological processes and pathologies.

As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across the cell membrane into the extracellular environment, including transport as a result of signal sequences in its amino acid terminus. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially from the cell in which they are expressed. "Secreted" proteins also include without limitation, proteins which are transported across any membrane inside the cell.

It should be noted that the terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to the translation product of a oligonucleic (e.g. a gene product). As use hereinabove and in the claims section, the term "transmembrane protein" is a protein that has hydrophobic domains which enable the anchoring of the protein to the membrane.

The term "downstream antisense primer" is refers hereinabove and in the claims section to a primer which is in the 3' direction with regard to reference sequence.

The term "upstream sense primer" is refers hereinabove and in the claims section to a primer which is in the 5' direction with regard to reference sequence. The term "sense" relates to the DNA strand that is identical to the RNA fragment from which the protein is translated.

As used herein, the term "oligonucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

The invention is based on the occurrence of particular amino acids within the hydrophobic sequences located in transmembrane and signal peptide domains of transmembrane and secreted proteins. More particularly, these hydrophobic sequences contain leucine-rich motif, i.e a string of either two, three, four or more leucines (see for example, Figure 1 and Figure 2). Although there are six different codons for leucine, a search of leucine codon usage within the hydrophobic domain of the signal peptide of known secreted proteins, unambiguously demonstrates marked preferred leucine codon usage within the signal peptide, which is restricted in most cases to the codons CTC, CTG, or more repeated units (see Figure 2). Moreover, in many cases, the codons for other amino acids located in this area consist of similar oligonucleic sequence. This fact enables anyone who is skilled in the art to use a downstream antisense primer or upstream sense primer which are complementary to an oligonucleic sequence coding for a string of leucines and have a good probability of hybridizing to a stretch of nucleotides within an mRNA species coding for a transmembrane or secreted protein containing a string of leucine residues and other hydrophobic amino acid residues.

Partial cDNA molecules complementary to mRNA species coding for secreted and/or transmembrane proteins can thus be amplified by PCR using downstream antisense primer which includes at least CAG or GAG unit, where

C and G nucleotides are randomly exchangeable (SEQ ID: No 1), or using upstream sense primer which includes at least CTG or CTC unit.

The upstream sense oligonucleotide sequence (upstream sense primer) in one embodiment is planned to correspond at its 3' terminus to the initiating methionine residue which is located upstream (usually within a distance of 5-30 amino acids) to the hydrophobic sequence of the signal peptide. As there is only a single codon (ATG), which codes for methionine, the upstream sense primer will include at its 3' end the sequence ATG. To allow for hybridization of the upstream sense primer a minimum length of ten nucleotides is required. As it is known that the five or so nucleotide residues present at the 3' terminus of the oligonucleic acid are critical for determining the PCR product specificity, the upstream primer is synthesized with two invariant nucleic acids at the -1 and -2 positions, relative to the A in the 3' terminal ATG. In order to cover all possible combinations (CC, GC, AC, etc.) a total of 16 different upstream sense primers are synthesized, with each position at the remaining five 5' terminal nucleic acid residues (positions -3 to -7) consisting of a mixture of all four nucleic acids (see SEQ.ID No: 2-17).

In another embodiment, the upstream sense or the downstream antisense oligonucleotide primers contains a nucleotide sequence coding for a consensus motif found in transmembrane proteins. In one embodiment, said consensus motif is Trp-Ser-X-Trp-Ser in which X may be any amino acid residue. The downstream antisense primer coding for Trp-Ser-X-Trp-Ser is the oligonucleotide sequence of SEQ ID No.18. The upstream sense primer comprising the oligonucleotide sequence of SEQ ID No.19.

In one embodiment, the downstream antisence primer comprises at least one CAG or GAG nucleotide unit and the upstream sense primer comprises the oligonucleotide sequence of SEQ ID No. 19.

In another embodiment, the downstream antisence primer comprises the oligonucleotide sequence of SEQ ID No. 18 and the upstream sense primer comprises at least one CTG or CTC nucleotide unit.

Yet, in another embodiment, the downstream antisence primer comprises at least one CAG or GAG nucleotide unit and the upstream sense primer comprises at least ATG nucleotide sequence.

The method of screening samples for the presence of at least one secreted or transmembrane protein comprises the following steps: obtaining mRNA from at least two samples; synthesizing cDNA from said mRNA of each sample; contacting said cDNA from each sample with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second oligonucleotide primer, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain an amplified products; separating the said amplified products, detecting said amplified products; and comparing said amplified product from each sample thereby identifying a distinctive amplified products coding for at least one secreted or transmembrane protein. As refer hereinabove and in the claims section the term "obtaining" mRNA refers to either receive mRNA or extract mRNA from any sample by methods which are detailed below or other methods known in the art.

Further, in accordance with an embodiment of the present invention the sample is derived from a plant or any other organism including human or a mammal and can be without being limited a tissue biopsy, a cell or blood.

Further, in accordance with an embodiment of the present invention, the cell is a cancer cell.

Further, in accordance with an embodiment of the present invention the cancer cell is derived from lung cancer, breast cancer, prostate cancer, ovarian cancer or a colon cancer.

As refer hereinabove and in the claims section "cDNA" is a complementary DNA that is made by using the enzyme reverse transcriptase and an RNA template. The step of "synthesizing" refer to step of building cDNA complementary to the mRNA template.

As refer hereinabove and in the claims section the step of "contacting" refers to adding the cDNA from each sample to the primers in appropriate conditions, so as to allow adjoining of the primers with the cDNA.

As refer hereinabove and in the claims section, the step of "amplifying" refer to the selective replication of a cDNA in greater number than usual.

As refer herein above and in the claims section, the step of "separating" refer to the step of separation of the products using for example, gel electrophoresis. As refer hereinabove and in the claims section, the step of "detecting" refer to the step of noticing, which is done for example by visualization of the amplified product's bands.

As refer hereinabove and in the claims section, the step of "comparing" refers to the step of searching for differences between the amplified products derived from the at least two samples.

The sample can be a tissue biopsy sample or a sample of blood, plasma, serum or the like. The sample should be treated to extract the mRNA molecules contained therein.

The term "RNA" refers to an oligonucleic acid in which the sugar is ribose, as opposed to deoxyribose in DNA. RNA is intended to include any nucleic acid, which can be entrapped by ribosomes and translated into protein.

The term "mRNA" refers to messenger RNA molecules.

RNA can be extracted from cells or tissues according to methods known in the art. In a preferred embodiment, RNA can be extracted from monolayers of mammalian cells grown in tissue culture, cells in suspension or from mammalian tissue. RNA can be extracted from such sources by, e.g., treating the cells with proteinase K in the presence of SDS. In another embodiment, RNA is extracted by organic solvents. In yet another embodiment, RNA is extracted by differential precipitation to separate high molecular weight RNA from other nucleic acids.

RNA can also be extracted from a specific cellular compartment, e.g., nucleus or the cytoplasm. In such methods, the nucleus is either isolated for purification of

RNA therefrom, or the nucleus is discarded for purification of cytoplasmic RNA.

Further details regarding these and other RNA extraction protocols are set forth, e.g., in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,

Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989). For instance, RNA can be extracted by a method using guanidium thiocyanate and purification of the RNA on a cesium chloride gradient. Accordingly, tissue or cells are lysed in the presence of guanidium thiocyanate and the cell lysate is loaded on a cushion of cesium chloride (CsCI) and centrifuged at high speed, such that the RNA is recovered in the pellet and the DNA is left in the supernatant after the centrifugation. The RNA can then be recovered by ethanol precipitation. This method is set forth in details, e.g., in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989).

In order to prevent RNA from being degraded by nucleases, e.g., by RNAases, that may be present, the extraction of RNA, and reactions involving RNA are performed in "RNAase free conditions". Various methods known in the art can be used to maintain RNAase free conditions. For example, during RNA extraction, potent denaturing agents, such as guanidium hydrochloride and guanidium thiocyanate can be used to denature and thereby inactivate nucleases. Reducing agents, e.g., β-mercaptoethanol, can also be used to inactivate ribonucleases. This combination of agents is particularly useful when isolating RNA from tissues rich in ribonucleases, e.g., pancreas

Other reagents that can be added to a solution containing RNA to prevent degradation of the RNA include RNAase inhibitors, also referred to herein as "protein inhibitor of RNAases", e.g., Rnasin RTM which can be obtained, from Promega Corp. (Madison, Wis.) (e.g., Cat #N2514). Protein inhibitors of RNAases are preferably not included during extraction of RNA using potent denaturing agents (since these will also denature the protein inhibitor of RNAases). However, it is preferable to include such protein inhibitors of RNAases during RNA extraction using more gentle methods of cell lysis and RNAse inhibitors are preferably present at all stages during the subsequent purification of RNA. Yet another reagent that can be added to a solution containing RNA to prevent degradation of the RNA include vanadyl-ribonucleoside complexes. The complexes formed between the oxovanadium IV ion and any of the four ribonucleosides are transition-state analogs that bind to many RNAases and inhibit their activity almost completely. The four vanadyl-ribonucleoside complexes are preferably added to intact cells and preferably used at a concentration of 10 mM during all stages or RNA extraction and purification. Yet in another embodiment, macaloid is used to absorb RNAases.

In one embodiment, cDNA is synthesize from the mRNA. This step is performed, without being limited, by Reverse Transcriptase polymerization chain reaction (RT/PCR), which produce single stranded DNA molecule using RNA as a template. The technique of reverse transcription can be used to amplify cDNA transcribed from mRNA encoding for secreted and transmembrane proteins. The method of RT/PCR is well known in the art (for example, see Watson and Fleming,) and can be performed as follows: Total cellular RNA is isolated by, for example, the standard guanidium isothiocyanate method and the total RNA is reverse transcribed. The reverse transcription method involves synthesis of DNA on a template of RNA using a reverse transcriptase enzyme and a 3' end primer. Typically, the primer contains an oligo(dT) sequence.

The cDNA is than contacting with at least one downstream antisense primer and at least one upstream sense primer that were described above to form a cDNA hybrid.

The term "cDNA-hybrid" is refer hereinabove in the specification and in the claims section to cDNA which is hybridizes to the first and the second primers that were defined above. The cDNA-hybrid is then amplified using the PCR method and the above described first and second specific primers. (Belyavsky et al, Nucl Acid Res 17:2919-2932, 1989; Krug and Berger, Methods in Enzymology, Academic Press, N.Y., Vol.152, pp. 316-325, 1987 which are incorporated by reference). An oligonucleic acid molecule is "hybridizable" to another oligonucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the oligonucleic acid molecule can anneal to the other oligonucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

In one embodiment for preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°C, can be used, (e.g., 5 times SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5 times SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5 times or 6 times SCC. High stringency hybridization conditions correspond to the highest Tm e.g., 50% formamide, 5 times or 6 times SCC.

Hybridization requires that the two oligonucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing oligonucleic acids depends on the length of the oligonucleic acids and the degree of complementation, variables well known in the art. For hybridization with shorter oligonucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., 11.7-11.8). In one embodiment the length for a hybridizable oligonucleic is at least about 10 nucleotides.

In a specific embodiment, the hybridization conditions are as described in the methods section: step 1- 94°C - 4 minutes, step 2- 40 cycles of 94°C for 30 seconds, 42°C for 60 seconds, 72°C for 20 seconds and step 3- 72°C for 5 minutes. The Polymerase Chain Reaction method is performed using the first (the downstream antisense primer) and the second (the upstream sense primer) that are complementary to the two termini regions of the DNA segment to be amplified. The upstream and downstream primers are typically more than 10 base pairs in length and hybridize to the termini regions for replication of the oligonucleotide sequence. Therefore, the primers need not reflect the exact sequence of the template, but must be sufficiently complementary to selectively hybridize with the strand being amplified.

The polymerization is catalyzed by a DNA-Taq-Polymerase in the presence of four deoxynucleotide triphosphates, one of which is radioactive or labeled with fluorescent markers, or nucleotide analogs to produce double-stranded DNA molecules. The double strands are then separated by any denaturing method including physical, chemical or enzymatic. Commonly, the method of physical denaturation is used involving heating the oligonucleic, typically to temperatures from about 80°C to 105°C for times ranging from less than 1 to 10 minutes. The process is repeated for the desired number of cycles (as is exemplified in the example section).

The resulting amplified product is subjected to gel electrophoresis or other size separation techniques and may be detected by ethidum bromide staining (Sambrook, et al., 1989).

Detection of the resulting bands is usually accomplished by exposure of the gel to X-ray film (autoradiography). The amplified products that are obtained from the two samples are compared. The distinctive amplified products, which refer hereinabove in the specifications and in the claims are amplified products which appear in one sample and not in the other or alternatively are over expressed in one sample but not in the other. The step of comparing the separated PCR products from the at least two samples is conducted as follows: Most of the bands are excepted to appear in the at least two samples and with equal intensity. However some of the bands may appear in one sample and not in the other or may appear in both but with different expression level. This different bands can be isolated from the gel, subcloned and sequenced.

The sequence oligonucleotides are submitted to an homology search in order to find their identity to other known polypeptide sequences. "Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity. Such a search may reveal that some of the sequences are identical to known sequences for secreted or transmembrane protein. However, new sequences, can be discovered, i.e. sequences that are not identical or have low degree of relatedness to known polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences.

This will reveal whether the distinctive amplified products are known mRNA coding for secreted and transmembrane proteins or whether they are novel transmembrane or secreted proteins. Alternatively, it is possible to transcribe the proteins from the mRNA and to assess their biological activity in methods that are well known in the art. The location of these new proteins within the cell, can be assessed by for example, the synthesis of labeled antibodies for these protein and by the use of microscopy techniques.

Any method known in the art for detecting proteins can be used. Such methods include, but are not limited to immunodiffusion, immunoelectrophoresis, immunochemical methods, binder-ligand assays, immunohistochemical techniques, agglutination and complement assays (for example see Basic and Clinical Immunology, Sites and Terr, eds., Appleton & Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference).

The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activity. Alternatively, they can have demonstrate a new biological activity. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

There is thus provided, in accordance with another embodiment of the present invention, a method for identifying a new secreted and/or transmembrane protein operative in a altered biological system. As biological systems are always in flux and, at the same time, strive to retain homeostasis, the term "an altered biological system" refers to a system that has been modified from its present status such that the component cells of the system will engage strategies to bring the system into its original state alternatively, the "altered cells" are forced to produce proteins that are quantitatively or qualitatively differ from those produce by "normal cells". Such strategies invariably involve secreted and/or membrane proteins.

The primers list in accordance with the embodiments of this Invention is as follows:

Downstream antisense primer: Primer 1 (19 bases) GGAATTCCAGCAGCAGCAG G G G G

Upstream sense primers:

Pr mer 2 (10 bases) XXXXTGATG Pr mer 3 (10 bases) XXXXGGATG Pr mer 4 (10 bases) XXXXACATG Pri imer 5 (10 bases) XXXXCCATG Pr mer 6 (10 bases) XXXXTCATG Pr mer 7 (10 bases) XXXXGCATG Pr mer 8 (10 bases) XXXXAAATG Pri imer 9 (10 bases) XXXXCAATG Pr mer 10 (10 bases) XXXXTAATG Pr mer 11 (10 bases) XXXXGAATG Pr mer 12 (10 bases) XXXXATATG Pr mer 13 (10 bases) XXXXCTATG Pr mer 14 (10 bases) XXXXTTATG Pr mer 15 (10 bases) XXXXGTATG Pr mer 16 (10 bases) XXXXAGATG Pr mer 17 (10 bases) XXXXCGATG Primer 18 Downstream antisense primer: (12 bases) CCA XXX XC/GT/A CCA Primer 19 upstream sense primer: (12 bases) TGG T/AC/GX XXX TGG

All primers are written from the 5' terminus to the 3' terminus, left to right. X stands for a mixture of 4 nucleic acids- A, G, T and C.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 1.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 2.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 3.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 4. Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 5.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 6.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 7.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 8.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 9. Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 10.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 11.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 12.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 13. Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 14.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 15. Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 16.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 17.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 18.

Further, in accordance with an embodiment of the present invention the oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 19.

It will be appreciated that the present invention is not limited by what has been described hereinabove and that numerous modifications, all of which fall within the scope of the present invention, exist.

It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above.

EXAMPLES

EXAMPLE 1 In the following described experiment total RNA was isolated from two different cell lines: (a) from human T cells and (b) from human mammary epithelial cells in order to identify new and/or differentially expressed secreted and transmembrane proteins.

MATERIALS AND METHODS RNA extraction from cells: The Gibco/BRL TRIZOL RNA kit was used to isolate RNA from the cell.

Reverse transcriptase reaction: mRNA - 50μg/ml, final concentration. ddH2O - to complete to a final volume of 20μl.

Oligo dT primer (15mer) - 10μM, final concentration.

The above components were incubated at 70°C for 10 minutes. The following was added and Incubated at 45°C for 60 minutes:

DTT - 0.01 M, final concentration.

Rnasin -1 unit (1μl)/per reaction dNTPs - 0.2mM, final concentration.

5 x buffer - diluted 1 :4. (Promega 5x buffer) Reverse transcriptase- - 5U/20μl reaction mixture (Promega). Inactivation of the enzyme- 95°C for 4 minutes.

The mixture was kept at 4°C.

The PCR mixture:

Downstream antisense primer - 2.5 μM, final concentration (62.5 pmoles). Upstream sense primer - 10 μM, final concentration. dNTPs - 2 μM, final concentration

³³P-dATP(2000Ci/mmol,Amersham)- 5μCi/25 μl reaction mixture

10 x PCR buffer -2.5μl

RT reaction - 2μl/25μl PCR reaction mixture Taq Gold DNA Polymerase - 2U/25μl reaction mixture/

Total volume - 25μl

PCR conditions:

Step 1- 94°C - 4 minutes

Step 2- 40 cycles of- 94°C -30 seconds 42°C -60 seconds

72°C -20 seconds

Step 3- 72°C - 5 minutes.

Stopping PCR reaction:

Twelve μl of stop buffer was added to each 25 μl of PCR reaction. Heating of reaction to 70°C for 5 minutes.

Acrylamide gel (sequencing gel):

Urea - 6M final concentration Acrylamid: Bis Acrylamid (25:1) - 10% final concentration

5 x TBE - final concentration 1xTBE

10% APS, TEMED added to catalyze acrylamide polymerization.

Loading sample: 1 volume sample + 4 volumes loading buffer (90% formamide in TBE).

Heat to 70°C for 5 minutes.

Gel exposure:

The gel was marked with radioactive marker ink for later alignment.

The gel was exposed to film at -70°C for about 3 days. The desired band was cut from the gel and placed in an Eppendorf tube.

DNA elution from the gel and its precipitation:

The DNA band was eluted by adding 100 μl of double distilled water and boiling for 5 minutes.

Precipitation of the eluted DNA is performed by adding 20μg glycogen followed by the addition of ethanol and sodium acetate pH 5 to final concentrations of

75% and 0.1M respectively.

Over night precipitation at -20°C, ethanol washes and resuspension of precipitated DNA with 10μl of ddH₂O.

Reamplification of DNA fragments by PCR: Downstream antisense primer - 62.5pmoles

Appropriate sense upstream primer - 62.5pmoles dNTPs - 100uM, final concentration.

10 x PCR buffer - diluted 10 times. ddH₂O - added to a final volume of 25μl. DNA - 3.5ul/ 25μl reaction mixture.

Taq DNA polymerase Gold - 2U/ 25 μl reaction mixture.

Total volume - 25ul.

Separation and purification of the PCR products by acrylamide gel electrophoresis: The re-amplified PCR products were run in a 10% acrylamide gel prepared with

TAE (Tris-acetate-EDTA) buffer. The DNA products were visualized by soaking the gel in TAE buffer containing 0.2μg/ml ethidium bromide. A small well was then cut in the acrylamide gel in front of the required DNA band and the DNA electroeluted into the well. The eluted DNA was then ethanol precipitated as described above.

EXPERIMENTAL PROCEDURE

RNA from each cell type was transcribed separately, by reverse transcriptase, into cDNA using, as a universal primer Oligo-dT composed of 15 residues of thymidylic acid. Aliquots of the cDNA were then distributed to sixteen separate tubes. Each tube contained, in addition to the four oligonucleics (wherein one was radioactively labeled), an identical downstream antisense primer and one of sixteen possible upstream sense primers (see specifications for a detailed explanation).

The downstream antisense primer sequence (SEQ. ID No:1) contained four tandem antisense codons devised to be complementary to four tandem leucine codons of the sequence CTG/C where the third oligonucleic of the codon is either G or C. In addition, this downstream antisense codon contained a 3' terminal EcoR1 site (GAATTC) in order to facilitate subsequent cloning of the selected PCR product into a convenient vector.

The upstream sense primers (sixteen in total) were terminated at their 3' terminus with the sequence ATG, whereas the two nucleic acids immediately upstream to the terminal ATG consisted of all 16 possible permutations (primers SEQ. ID Nos. 2-17). To allow for a reasonable length capable of forming a relatively stable hybrid, the upstream oligonucleic was synthesized as a decamer in which the first 5 positions (starting at the 5' terminus) consisted of a mixture of all four nucleic acid s at each position.

The cDNA was aliquoted into each of sixteen tubes and then subjected to 40 PCR cycles (see Methods). The resulting PCR products were subsequently resolved by acrylamide gel electrophoresis under denaturing conditions and the radioactive bands were visualized by exposing the gel to X-ray film. The autoradiograph was then scrutinized in order to identify bands appearing in the one sample but not in the other. These bands should derive from mRNAs coding for transmembrane/secreted proteins expressed in the one test sample and not in the other.

EXPERIMENTAL RESULTS

As expected, most of the bands appeared with equal intensity in both samples meaning that the mRNAs relate for these RT-PCR products are expressed at equal levels in both samples. However, some bands were observed that were relatively specific for one or the other sample and these represent the differentially expressed mRNAs coding for putative transmembrane/secreted proteins expressed in the one test sample and not in the other. In order to identify the differentially appearing bands, five of these PCR products were chosen and they were isolated from the gel, subcloned and sequenced. Demonstration that these PCR products derive from mRNAs coding for either transmembrane or secreted proteins would in fact provide "proof of the principle". The nucleic acids sequences obtained were then submitted to a BLAST homology search. Of the five differentially expressed sequences thus analyzed two were found to be part of known mRNAs coding for transmembrane proteins- one is the nip3 protein and the other is a ribosome binding protein (see Figure 1 for nucleic acids sequences and the inferred translated amino sequence of the inserts). Notably, both of these proteins are in fact transmembrane proteins- the nip3 protein is bound to the mitochondrial membrane and functions as a proapoptotic protein. The ribosome binding protein is a non-glycosylated membrane protein characteristic of rough microsomes and is believed to play a role in the ribosome-membrane association. The protein data bank did not provide perfect matches for the additional three differentially expressed PCR, which may be related to yet unidentified transmembrane/secreted proteins.

Thus, the above described experiment proves that differentially expressed RT-PCR products code for transmembrane proteins, and the validity of present invention method for identifying transmembrane and secreted proteins has been approved.

Claims

WHAT IS CLAIMED IS

1. A differential display method for identifying at least one secreted or transmembrane protein comprising the following steps:

obtaining mRNA from at least two samples; synthesizing cDNA from said mRNA of each sample; contacting said cDNA from each sample with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second oligonucleotide primer, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain amplified products; detecting said amplified products; and comparing said amplified products from each sample thereby identifying distinctive amplified products coding for at least one secreted or transmembrane protein.

2. The method of claim 1 , wherein the step of comparing the amplified products further comprising the step of characterizing said distinctive amplified products coding for at least one secreted or transmembrane protein.

3. The method of claim 2 further comprising submitting the distinctive amplified products to a sequence homology search.

4. The method of claim 1 , wherein the step of detecting said amplified products further comprising the step of separating said amplified products.

5. The method of claim 4 wherein said step of separating said amplified products further comprising the step of visualizing said amplified products.

6. The method of claim 1 , wherein said first primer is a downstream antisense primer.

7. The method of claim 6, wherein said downstream antisense primer comprises at least one CAG or GAG nucleotide unit.

8. The method of claim 1 , wherein said first primer is an upstream sense primer.

9. The method of claim 8, wherein said upstream sense primer comprises at least one CTG or CTC nucleotide unit.

10. The method of claim 1 , wherein said leucine-rich motif comprises at least two leucine residues.

1 1. The method of claim 1 , wherein said second primer contains a nucleotide sequence coding for a consensus motif found in transmembrane proteins.

12. The method of claim 11 , wherein said consensus motif is Trp-Ser-X-Trp-Ser in which X may be any amino acid residue.

13. The method of claim 11 , wherein said second primer is a downstream antisense primer.

14. The method of claim 13, wherein said downstream antisense primer comprising the oligonucleotide sequence of SEQ ID No.18.

15. The method of claim 11 , wherein said second primer is a upstream sense primer.

16. The method of claim 15, wherein said upstream sense primer comprising the oligonucleotide sequence of SEQ ID No.19.

17. The method of claim 1 , wherein said second primer is an upstream sense primer comprising at least ATG nucleotide sequence.

18. The method of claim 17, wherein said second primer further comprises at least two additional nucleotides.

19. The method of claim 18, wherein said additional nucleotides are at -1 and -2 positions relative to the ATG sequence.

20. The method of claim 19, wherein said additional nucleotides are selected from the group consisting of A, T, C and G.

21 . The method of claim 1 , wherein said two samples are a pair of a sample under normal conditions and a sample under pathogenic conditions, or a sample under normal conditions and a sample under stress conditions, or a sample derived from a healthy individual and a sample derived from a non healthy individual.

22. A method of screening samples for the presence of at least one secreted or transmembrane protein comprising the following steps: obtaining mRNA from a sample; synthesizing cDNA from said mRNA; contacting said cDNA with at least one first primer, said first primer hybridizes to a oligonucleic sequence coding for a leucine-rich motif, and at least one second oligonucleotide primer, so as to form a cDNA- hybrid molecules; amplifying said cDNA-hybrid molecules, so as to obtain amplified products; detecting said amplified products; and comparing said amplified products from said sample to the amplified products derived from known samples, thereby identifying distinctive amplified products coding for a at least one secreted or transmembrane protein.

23. The method of claim 22, wherein the step of comparing the amplified products further comprising the step of characterizing said distinctive amplified products coding for at least one secreted or transmembrane protein.

D 24. The method of claim 23 further comprising submitting the distinctive amplified products to a sequence homology search.

25. The method of claim 22, wherein the step of detecting said amplified products further comprising the step of separating said amplified products. 0

26. The method of claim 25 wherein said step of separating said amplified products further comprising the step of visualizing said amplified products.

27. The method of claim 22, wherein said two samples are a pair of a sample s under normal conditions and a sample under pathogenic conditions, or a sample under normal conditions and a sample under stress conditions, or a sample derived from a healthy individual and a sample derived from a non healthy individual.

0 28. The method of claim 22, wherein said second primer contains a nucleotide sequence coding for a consensus motif found in transmembrane proteins.

29. The method of claim 28, wherein said consensus motif is 5 Trp-Ser-X-Trp-Ser in which X may be any amino acid residue.

30. The method of claim 29, wherein said second primer is a downstream antisense primer.

0 31. The method of claim 30, wherein said downstream antisense primer comprising the oligonucleotide sequence of SEQ ID No.18.

32. The method of claim 29, wherein said second primer is a upstream sense primer.

33. The method of claim 32, wherein said upstream sense primer comprising the oligonucleotide sequence of SEQ ID No.19.

34. The method of claim 22, wherein said second primer is an upstream sense primer comprising at least ATG nucleotide sequence.

35. The method of claim 34, wherein said second primer further comprises at least two additional nucleotides.

36. The method of claim 35, wherein said additional nucleotides are at -1 and -2 positions relative to the ATG sequence.

37. The method of claim 36, wherein said additional nucleotides are selected from the group consisting of A, T, C and G.

38. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 1.

39. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 2.

40. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 3.

41. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 4.

42. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 5.

43. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 6.

44. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 7.

45. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 8.

46. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 9.

47. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 10.

48. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No.

11.

49. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 12.

50. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 13.

51. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 14.

52. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 15.

53. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 16.

54. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 17.

55. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 18.

56. An oligonuclotide comprising the oligonucleic sequence of SEQ ID No. 19.