Patent application title: METHOD OF DETECTING A CANCER CELL BY ABERRANT EXPRESSION OF A HUMAN K+ ION CHANNEL
Inventors:
Luis Angel Pardo-Fernandez (Gottingen, DE)
Walter Stuhmer (Gottingen, DE)
Synnove Beckh (Gottingen, DE)
Andrea Bruggemann (Frankfurt, DE)
Donato Del Camino Fernandez-Miranda (Boston, MA, US)
Araceli Sanchez Perez (Gottingen, DE)
Rudiger Weseloh (Gottingen, DE)
Assignees:
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds antigen or epitope whose amino acid sequence is disclosed in whole or in part (e.g., binds specifically-identified amino acid sequence, etc.)
Publication date: 2010-01-28
Patent application number: 20100021467
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Patent application title: METHOD OF DETECTING A CANCER CELL BY ABERRANT EXPRESSION OF A HUMAN K+ ION CHANNEL
Inventors:
Walter Stuhmer
Luis Angel Pardo-Fernandez
Synnove Beckh
Andrea Bruggemann
Donato Del Camino Fernandez-Miranda
Araceli Sanchez Perez
Rudiger Weseloh
Agents:
ROPES & GRAY LLP
Assignees:
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
Origin: NEW YORK, NY US
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Patent application number: 20100021467
Abstract:
The present invention relates to a novel human K+ ion channel, to
nucleic acid molecules encoding the same and to vectors comprising said
nucleic acid molecules. The invention additionally relates to antibodies
specifically directed to the novel K+ ion channel and to
pharmaceutical compositions and diagnostic kits containing at least one
of the above-mentioned components. Furthermore, the present invention
relates to methods of treating a disease caused by malfunction of the
polypeptide of the present invention or by the (over)expression of the
nucleic acid molecule of the invention comprising administering an
inhibitor of said (over)expression or of ion channel function or an
inhibitor abolishing said malfunction to a patient in need thereof.
Methods of devising drugs for treating or preventing the above-mentioned
disease, methods of inhibiting cell proliferation and methods of
prognosing cancer are additional embodiments comprised by the present
invention. The invention also envisages specific antisense or gene
therapies on the basis of the nucleic acid molecule of the invention for
inhibiting undesired cellular proliferation, for example, in connection
with cancer or in neurodegenerative diseases.Claims:
1-32. (canceled)
33. A method for detecting the presence of a cancer cell in a human subject comprising:(a) obtaining a tissue sample that is not derived from brain or placenta tissue from said subject and preparing an analyte from said tissue;(b) incubating an antibody that binds specifically to a polypeptide comprising the amino acid sequence of SEQ ID NO:3 with said analyte; and(c) detecting the presence of a cancer cell by the binding of said antibody to a polypeptide in said analyte.
Description:
[0001]This application is a division of co-pending U.S. patent application
Ser. No. 11/633,085, filed Nov. 30, 2006, which is continuation of U.S.
patent application Ser. No. 10/188,287, filed Jul. 1, 2002, now
abandoned; which is a divisional of U.S. patent application Ser. No.
09/694,777, filed Oct. 23, 2000, now U.S. Pat. No. 6,638,736; which is a
continuation of International Application PCT/EP99/02695, filed Apr. 21,
1999, which application designated the United States, the specifications
of all of which are hereby incorporated by reference. Applicants have
incorporated by reference herein "substitutesequence.txt", which was
created on Sep. 28, 2009 and has a size of 75,888 bytes.
BACKGROUND OF THE INVENTION
[0002]The present invention relates to a novel human K+ ion channel, to nucleic acid molecules encoding the same and to vectors comprising said nucleic acid molecules. The invention additionally relates to antibodies specifically directed to the novel K+ ion channel and to pharmaceutical compositions and diagnostic kits containing at least one of the above-mentioned components. Furthermore, the present invention relates to methods of treating a disease caused by malfunction of the polypeptide of the present invention or by the (over)expression of the nucleic acid molecule of the invention comprising administering an inhibitor of said (over)expression or of ion channel function or an inhibitor abolishing said malfunction to a patient in need thereof. Methods of devising drugs for treating or preventing the above-mentioned disease, methods of inhibiting cell proliferation and methods of prognosing cancer are additional embodiments comprised by the present invention. The invention also envisages specific antisense or gene therapies on the basis of the nucleic acid molecule of the invention for inhibiting undesired cellular proliferation, for example, in connection with cancer or in neurodegenerative diseases.
[0003]Potassium channels are a relevant factor in the regulation of the resting potential of cells, and this has been regarded as their major role in excitable and non-excitable tissues. On the other hand, the explanation for their ubiquitous presence and the impressive variability in their properties remains elusive. A reasonable hypothesis is that potassium channels are present in all cell types because they have in addition some "housekeeping" role, for example in cell proliferation1. Their implication in the regulation of the cell division cycle has been tested repeatedly, and some experimental evidence has been presented2,3. However, especially since both depolarization and hyperpolarization of the membrane potential during cell cycle have been reported as depending on cell type1,4, there is no general model to explain the function of potassium channels in cell cycle. Two mechanisms have been proposed to explain the role of K+ channels: they either influence the intracellular Ca2+ concentration, or control cell volume (17, 18). Both mechanisms would indirectly influence cell proliferation. A member of the eag family has also been proposed to be preferentially expressed in cancer cells (19) Several potassium channel blockers have been tested for their capability to block cancer cell proliferation, and some of them have even been used as coadjuvants for tumor chemotherapy, specially in multidrug-resistant tumors. Nevertheless, the lack of identification of a particular potassium channel directly involved in the control of cell proliferation has, up to date, precluded the description of more specific and effective treatment protocols.
[0004]Thus, the technical problem underlying the present invention was to identify a biological component within the conglomerate of potassium channels with their various effects on cell cycle division that allows an unambiguous assignment to cellular proliferation, with a specific view to human cellular proliferation. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
[0005]Accordingly, the present invention relates to a nucleic acid molecule comprising a nucleic acid molecule encoding a (poly)peptide having a function of the human K+ ion eag channel which is
[0006](a) a nucleic acid molecule comprising a nucleic acid molecule encoding the polypeptide having the amino acid sequence of SEQ ID: No 3 or 4;
[0007](b) a nucleic acid molecule comprising the nucleic acid molecule having the DNA sequence of SEQ ID: No 13 or 14;
[0008](c) a nucleic acid molecule hybridizing to the complementary strand of a nucleic acid molecule of (a) or (b); or
[0009](d) a nucleic acid molecule being degenerate to the sequence of the nucleic acid molecule of (c).
[0010]The nucleic acid molecule of the invention encodes a (poly)peptide which is or comprises the human homologues of the rat eag channel. In this regard the term "a nucleic acid molecule comprising a nucleic acid molecule encoding a (poly)peptide having a function of the human K+ ion eag channel" may mean that said first mentioned nucleic acid molecule solely encodes said (poly)peptide. Thus, it may be identical to said second mentioned nucleic acid molecule. Alternatively, it may comprise regulatory regions or other untranslated regions. In a further embodiment, said first mentioned nucleic acid may comprise heterologous nucleic acid which may encode heterologous proteinaceous material thus giving rise, e.g., to fusion proteins. It is further to be noted that the DNA sequences of SEQ ID NO: 13 and 14 are splice variants of the nucleic acid sequence encoding the (poly)peptide of the invention. The corresponding amino acid sequences are depicted in SEQ ID NO: 3 and 4.
[0011]The term "having a function of a human K+ ion eag channel", as used in connection with the present invention, has the following meaning: The channel has a single channel conductance in asymmetrical potassium, at 0 mV of about 6 pS. This value clearly distinguishes the human channel from the rat channel for which a value of about 7 pS was measured. In addition or in the alternative, the above term may have the following meaning: The channel has a IC50 of about 1 mM to quinidine when expressed in Xenopus laevis oocytes, as compared to 400 μM for reag. Further, when measuring voltage-dependence of activation in high extracellular potassium using a two-electrode voltage-clamp it was found that in a conductance-voltage plot, the voltage for half-activation is shifted by about 40 mV or more to the right in the heag channel with respect to the reag channel (see FIG. 13). On the basis of the above features, either alone or in combination, a differentiation based on function between the human ion channel of the invention and the prior art channels, in particular of the rat ion channel, is possible for the person skilled in the art without further ado. Preferably, the channel has all recited functions. The above values refer to values that are obtainable with the experimental set-up described in this specification. Alterations of experimental parameters such as the employment of a different expression system may, as is well known to the person skilled in the art, also change the above values. Yet, these embodiments are also comprized by the scope of the present invention.
[0012]The term "hybridizing" as used in accordance with the present invention relates to stringent or non-stringent hybridization conditions. Preferably, it relates to stringent conditions. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory (1989) N.Y., Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (eds) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington D.C., (1985). Hybridizing molecules or molecules falling under alternative (d), supra, also comprise fragments of the molecules identified in (a) or (b) wherein the nucleotide sequence need not be identical to its counterpart in SEQ ID 13 or 14, said fragments having a function as indicated above.
[0013]An example of one such stringent hybridization condition is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4×SSC at 42° C. Examples of such non-stringent hybridization conditions are 4×SSC at 50° C. or hybridization with 30-40% formamide at 42° C. Complementary strands of hybridizing molecules comprise those which encode fragments, analogues or derivatives of the polypeptide of the invention and differ, for example, by way of amino acid and/or nucleotide deletion(s), insertion(s), substitution(s), addition(s) and/or recombination(s) or any other modification(s) known in the art either alone or in combination from the above-described amino acid sequences or their underlying nucleotide sequence(s). Using the PESTFIND program (Rogers, Science 234 (1986), 364-368), PEST sequences (rich in proline, glutamic acid, serine, and threonine) can be identified, which are characteristically present in unstable proteins. Such sequences may be removed from the polypeptide of the invention in order to increase the stability and optionally the activity of the proteins. Methods for introducing such modifications in the nucleic acid molecules according to the invention are well-known to the person skilled in the art. The invention also relates to nucleic acid molecules the sequence of which differs from the nucleotide sequence of any of the above-described nucleic acid molecules due to the degeneracy of the genetic code. All such fragments, analogues and derivatives encoding the protein of the invention are included within the scope of the present invention, as long as the essential characteristic immunological and/or biological properties as defined above remain unaffected in kind, that is the novel nucleic acid molecules of the invention include all nucleotide sequences encoding proteins or peptides which have at least a part of the primary structural conformation for one or more epitopes capable of reacting with antibodies to said polypeptide which are encoded by a nucleic acid molecule as set forth above and which have comparable or identical characteristics in terms of biological activity. Part of the invention is therefore also concerned with nucleic acid molecules encoding a polypeptide comprising at least a functional part of the above identified polypeptide encoded by a nucleic acid sequence comprised in a nucleic acid molecule according to the invention.
[0014]The present inventors have recently described a potassium channel (reag) which is strongly downregulated immediately after the activation of cyclin dependent kinases (key molecules in the cell cycle regulation), in both G1-S and G2-M transitions5. The K+ current is inhibited following activation of cyclin-dependent kinases due to a voltage-dependent sodium block, which is not apparent in all phases of the cell cycle. The experiments presented here are aimed to determine whether eag, in addition to being regulated by the cell cycle, is also able to directly influence cell proliferation and growth (20). In accordance with the present invention and with a view to the development of a suitable system for assessing (disease-related) proliferation in human cells, it was further attempted to study whether the implication of the channel in the cell cycle goes in both directions, such that it is not only regulated by but also regulator of the progression of the cell cycle.
[0015]The results obtained in this rat derived ion channel system show that in three different cell lines obtained from different species (Chinese hamster --CHO--, human --HEK293-, and mouse --NIH3T3-), the rate of proliferation is faster when the channel is overexpressed after transfection of the cells with a plasmid containing the channel DNA under the control of the cytomegalovirus promoter. FIG. 1 and FIG. 18a show the increase in metabolic activity in cultures of CHO cells in the presence of normal concentrations of fetal calf serum (10% FCS). Under these normal conditions, reag transfected cells grow several folds faster than untransfected cells (WT).
[0016]FIG. 2 shows a comparable experiment at very low concentrations of fetal calf serum (0.5% FCS). These low serum concentrations do not allow wild-type cells to grow; after a few hours, the cells start to die. However, reag transfected cells are able to proliferate under the same conditions. The ability to overcome the growth arrest induced by the absence of growth factors is one of the typical properties of malignant transformation (cf FIG. 18).
[0017]Not only the metabolic activity can be used to trace the proliferation in culture. The measurement of DNA synthesis is a more direct estimation of the rate of cell growth, since only cells entering S phase (committed to divide) synthesize DNA. Also DNA synthesis becomes serum-independent in reag transfected cells, i.e., the growth is maintained in the absence of growth factors (while it induces the programmed death of non-transfected cells). This is depicted in FIG. 3, were the incorporation of 5-Bromo-2'-deoxyuridine.7-10 (BrdU) was used to monitor DNA synthesis in the presence of 10 or 0.5% FCS in CHO cells. As opposed to wild-type or cells transfected with an inactivating voltage-dependent potassium channel from rat brain (Kv1.4), there are no significant differences in the amount of DNA synthesized in the presence of normal or low FCS concentrations in reag-expressing cells. Similar experiments were done using epidermal growth factor (EGF) in HEK-293 cells or platelet-derived growth factor (PDGF) in CHO cells, with essentially the same result. The pure growth factors were used to avoid the complexity introduced by the use of whole serum.
[0018]To test the effects of eag on cell proliferation more directly, DNA synthesis was measured through incorporation of 5-Bromo-deoxyuridine (BrdU) in cells synchronized in the S-phase of the cell cycle by means of thymidine arrest (23). Consistent with the above mentioned findings, when the S-phase of the cell cycle was allowed to proceed, reag expressing CHO cells (CHOrEAG) showed higher metabolic activity (FIG. 18B) and increased BrdU incorporation (FIG. 18C). These results suggest that more eag-transfected cells entered the S-phase during the arrested period and/or DNA synthesis was elevated, in any case indicating a faster proliferation rate in CHOrEAG cells. In the presence of low serum, BrdU incorporation was significantly higher in CHOrEAG than in wild type cells (FIG. 18C).
[0019]Yet another cell line, NIH3T3, has been frequently used for tumor transformation assays, since these cells are very strongly contact-inhibited, (i.e., their growth is stopped when the culture reaches confluency). This results in a homogeneous monolayer in wild-type cells. The malignant transformation of the line (through oncogene expression) usually induces the loss of this property, and NIH3T3 cells start forming colonies composed of several layers of cells. This can be seen after the transfection with reag DNA, which induced the formation of such foci in several independent clones (FIGS. 4A and B). Another standard test for transforming activity is the ability of NIH3T3 cells to grow in colonies when no substrate for attachment is available. To test this, cells are plated in an agar-containing medium, where the agar will prevent contacts between the cells and the surface of the plate. Under these conditions, wild-type NIH3T3 cells were unable to grow, while cells expressing reag formed large colonies even detectable by simple visual inspection of the plate. Table I shows that reag- (but not rKv1.4-) transfected cells formed colonies in a semisolid medium containing 0.3% agar (24,25), regardless of the vector used for transfection (FIG. 14). All of the above results indicate a transforming potential of eag.
[0020]Altogether, the results obtained from transfected cells indicate that reag can, at least under certain conditions, display oncogenic properties.
[0021]Once the transforming ability of reag was determined in accordance with the invention, the expression of the respective channel in human cancer cells was investigated. For this investigation, the cell line MCF-7 was used, which was initially obtained from a pleural effusion of a breast adenocarcinoma. The line is estrogen receptor positive as well as estrogen-sensitive and relatively well differentiated. The strategy followed was first to test electrophysiologically and pharmacologically for the presence of a functional current similar to eag, and then to try an identification of the corresponding channel at the molecular level. However, conventional approaches for such an identification failed.
[0022]Namely, in most cells, the current density was too low to allow reliable measurements of the whole cell current. Low current density precluded an accurate measurement of channel properties using a whole cell configuration for the patch champ. Therefore, due to said low current densities encountered, another approach was resorted to. Due to such a low number of channels per cell, it is only possible to characterize the functional properties of a channel by a special patch champ method, excising patches of membranes containing one (or a few) channels and allowing characterization on a single molecule level. This approach relied on single-channel measurements in order to also compare properties at the single-molecule level such as single channel conductance, pharmacological properties, voltage dependence, and mean open times. Indeed, a channel with several properties compatible with reag in terms of kinetics, voltage-dependence, and pharmacology in most membrane patches could thus be identified. FIG. 5 shows whole-cell currents obtained from a MCF7 cell under nystatin patch conditions and single channel currents, together with their current-voltage relationship. Despite differences in kinetics at very depolarized voltages, the voltage dependence of the channel in human cells is highly reminiscent to the voltage-dependence of the reag channel. Moreover, the single channel properties of the putative human-eag are also very similar to those of reag.
[0023]Furthermore, standard approaches to isolate the said channel on a molecular level also were not successful. Several other groups have attempted and/or are still attempting to isolate the gene coding for a human eag without success and this in spite of the fact that the rat eag channel has already been published in 1994. For example, Warmke and Ganetzky (Proc. Natl. Acad. Sci. USA 91 (1994), 3428-3442) specifically set out to clone the human eag gene using conventional technology. They were, however, unsuccessful and cloned a novel, eag related gene which they termed h-erg (also referred to as HERG). Further, Wymore et al., Circulation Res. 80 (1997), 261-268, reported that no eag specific clones could be detected in a cDNA library from human heart in spite of the fact that primers for amplification were used that were conserved across the entire eag/erg superfamily. Thus, the standard approach with degenerated oligonucleotides based on the sequence of members of the family revealed itself unsuccessful, although HERG was systematically detected by other researchers in the field. Significantly, most of these approaches to clone the human eag gene were made with brain libraries. The conclusion from these combined prior art data was that the human eag gene could not be cloned by conventional technology using the most obvious source, namely brain tissue. The repeated isolation of HERG clones instead is most probably due to the relative abundance of HERG transcripts in brain libraries, and also to the high homology between the two channels. Consequently, a different strategy had to be devised to direct the screening more specifically to eag channels. First, as described herein above, a cell line expressing a channel functionally similar to reag was identified. Then degenerated oligonucleotides based on conserved sequences between rat, bovine and mouse eag, but divergent from HERG were designed. Using these primers, the cDNA obtained from MCF7 cells by PCR was amplified, and a band of the expected size was cloned in a suitable vector and sequenced. The amplified fragment corresponded to approximately 400 bp within the core region of the channel protein, and shared 90% identity to the reag sequence at the DNA level, and 99% at the amino acid level. However, at this stage it was still quite unclear what the thus identified clone corresponded to. For example, it was quite possible that a further member of the eag family had been identified. This is in particular true in view of the fact that despite of a number of attempts with brain libraries, nobody had been able to clone the human eag gene and that the MCF7 line is a breast cancer derived line.
[0024]Since MCF7 cells are immortal cells, it is assumed that a number of genes is mutated. Ab initio, it could have been expected that the human eag channel, if at all expressed in this cell line, was mutated. Under this assumption, it was quite uncertain whether this cell line could at all be used for the isolation of the desired gene.
[0025]Due to the prior art failures to clone human eag gene from brain libraries and the above recited uncertainties with immortalized cell lines, another source for a library was in need. The 400 bp fragment was therefore used to screen a normal human breast cDNA library. Due to the presence of eag is breast cancer cells, such a library was expected to comprise heag clones. Surprisingly, however, after screening 2×106 phages, no human-eag clones could be identified in said library. This rises the possibility that the channel is expressed only in tumor cells, and not in normal tissue. Specific oligonucleotides, namely 5'-CCAAACACACACACCAGC (SEQ ID NO: 5) and 5'-CGTGGATGTTATCTTTTTGG (SEQ ID NO: 6), were designed to check for heag fragments by PCR amplification directly from the above library, but no evidence for the presence of any eag clones in this library was found. In view of the above discussed prior art results, it came as a further surprise that the same primers detected heag in a normal human brain cDNA library, that was therefore screened. First, the probe obtained from MCF7 cells was used to check 106 phages. This procedure allowed to isolate a 1.6 kbp fragment from human eag. This fragment was then used as a probe for the screening of 2×106 phages from the same library. Several independent clones were isolated, but none of them was a full-length clone. Furthermore, only one clone contained the 5' end of the sequence, while two of them contained the 3' end and part of the 3' non-coding region. It is likely that the abundance of restriction sites in the nucleic acid sequence encoding the channel has induced this extensive fragmentation of the cDNA. For example, when EcoRI was used to extract the inserts of the library that was cloned in λ-gt10 phage at the EcoRI site, this conventional approach systematically failed to find the 5' end of the molecule (there is an EcoRI site at position 400 of the clone). The pooled positive clones were therefore screened again by PCR, trying to amplify the start codon, and only by this means was it possible to isolate one phage that contained this ATG. Two splice variants of heag were cloned, both expressed in brain tissue. The sequences obtained for heag 1 and heag 2 and their deduced amino acid sequences are shown in FIGS. 10 and 11, and compared to other members of the family.
[0026]The deduced amino acid sequence is identical to the sequence published after the priority date of the present invention by Occhidoro (27) and is 97.7% identical to reag. As mentioned, a second (81 bp longer) splice variant (heag 2) was also isolated analogous to that reported for bovine and mouse eag channels (28), the splice insertion being identical in all three species. The chromosomal localization of heag was determined by FISH detection (29) to map to chromosome 1q32.1-32.3 (see also ref. 26).
[0027]To further check the possibility that heag is not expressed in normal mammary gland, as opposed to MCF-7 cancer cells, we performed single-tube RT-PCR experiments using total RNA from human brain, human mammary gland, and MCF-7 cells (FIG. 12), using as primers two oligonucleotides designed to discriminate between the two splice variants of heag. In human brain, two splice variants were detected, while only the short one was expressed in MCF-7 cells (this, together with the lack of amplification in the absence of reverse transcriptase, rules out a possible contamination by genomic DNA of the RNA preparation). No heag signal was detected in normal mammary gland RNA with this highly sensitive technique. This result was totally unexpected, because preliminary results had suggested that expression was present in tumor cells from the same organ. Further, after Southern blot analysis of the RT-PCR products a faint band hybridizing with a heag probe in mammary gland was identified. Accordingly, it is quite difficult to make a strong statement on the total absence of heag message in breast in view of these contradictory experimental data.
[0028]Furthermore, electrophyiological properties (21, 30) of heag were tested in Xenopus oocytes. As described above, they did not differ significantly from those or reag with the above mentioned exceptions, e.g. a shift in activation of 40 mV to more depolarized potentials when both channels were measured under identical conditions. The electrophysiological observations of heag channels expressed in Xenopus oocytes correlate well to hose reported by Bijlenga et al. (31).
[0029]The present invention also relates to a nucleic acid molecule specifically hybridizing to the nucleic acid molecule of the invention which comprises the sequence 5'-GGGAGGATGACCATGGCT (SEQ ID NO: 7).
[0030]This embodiment of the present invention is particularly useful for specific antisense therapies for inhibiting cell proliferation as will be discussed in more detail herein below (e.g. in Example 5). In addition, this embodiment of the nucleic acid molecule of the invention can, naturally, also be used as a probe for specifically detecting heag mRNA in tissues, for example, by employing the Northern Blot technology. The analysis of heag mRNA expression in various tissues by Northern blot revealed a strong hybridization signal of approximately 9.2 kb in brain and a weak signal of similar size in placenta. Heart, lung, liver, skeletal muscle, kidney and pancreas were negative even following long exposures. In addition, total RNA from human brain, heart, trachea, adrenal gland, liver, kidney, skeletal muscle and mammary gland, and spinal cord poly(A)+ RNA, as well as total RNA from the adenovirus-transformed line 293 (a human non-tumoral cell line) were assayed by single-tube RT-PCR and Southern blot. Under these experimental conditions, heag was detected in brain only, where both splice variants were identified (FIG. 15; Example 3).
[0031]The preferential expression of heag in brain was intriguing since the first cDNA had been isolated from an epithelial tumor cell line (MCF-7) and not from brain tissue (see above). To elucidate the presence of heag in other tumoral cell lines, total RNA was prepared from HeLa (cervix carcinoma), SHSY-5Y (neuroblastoma), and lines from mammary gland tumors: COLO-824 (carcinoma), EFM-19 (carcinoma), and BT-474 (ductal carcinoma). Total RNA from brain, MCF-7 cells, 293 cells and RNA from cultures of mammary gland epithelial cells (included to circumvent the mixed cell populations in whole mammary gland) served as controls. All cell lines were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and maintained following the DSMZ catalog guidelines. Normal human mammary epithelial cells were purchased from BioWhittaker. The primers were designed to amplify different bands for heag 1 and heag 2, thus allowing us to rule out false positives due to genomic DNA contamination (controls in the absence of reverse transcriptase were also performed). HeLa, SHSY-5Y, EFM-19 and MCF-7 RNA exhibited an heag band, whereas COLO-824 and BT-474 signals were indistinguishable from background (FIG. 15B). Cultured epithelial cells and 293 cells (FIG. 15A) were negative. As discussed above, it could be shown in accordance with the present invention that reag transfected cells can display oncogenic properties. Thus, to determine whether the expression of heag is advantageous for tumor cells in vivo, subcutaneous implants of CHO cells expressing the channel (CHOhEAG cells) into the flank of female scid (severe combined immunodeficiency, 32) mice were performed and it could be shown that expression of heag represents an advantage for the proliferation of tumor cells in vivo, since CHOhEAG tumors grow faster and are more aggressive than CHOKv tumors.
[0032]Thus, the embodiment of the nucleic acid molecule of the present invention may be employed in the quantitative and qualitative analysis of the expression level of human eag in various disease states detectable in a tissue that may be indicative of, for example, cancer (in particular mamma carcinoma, neuroblastoma), psoriasis, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, lateral amyotrophic sclerosis or multiple sclerosis.
[0033]In a preferred embodiment of the nucleic acid molecule of the invention, said nucleic acid molecule is DNA, such as genomic DNA. Whereas the present invention also comprises synthetic or semi-synthetic DNA molecules or derivatives thereof, such as peptide nucleic acid, the most preferred DNA molecule of the invention is cDNA.
[0034]In a further preferred embodiment of the present invention, said nucleic acid molecule is RNA, preferably mRNA.
[0035]Another preferred embodiment of the nucleic acid molecule of the invention encodes a fusion protein. For example, the nucleic acid molecule of the invention can be fused in frame to a detectable marker such as FLAG or GFP.
[0036]The invention further relates to a vector, particularly plasmid, cosmids, viruses and bacteriophages comprising the nucleic acid molecule of the invention. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Thus the polynucleotide of the invention can be operatively linked in said vector to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL).
[0037]Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors and gene targeting or transfer vectors are well-known in the art and can be adapted for specific purposes of the invention by the person skilled in the art. Thus, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vectors of the invention into targeted cell populations. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
[0038]The invention furthermore relates to a host transformed with the vector of the invention. Said host may be a prokaryotic or eukaryotic cell; see supra. The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. In this respect, it is also to be understood that the recombinant DNA molecule of the invention can be used for "gene targeting" and/or "gene replacement", for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press. Preferably, the host is a mammalian cell, a fungal cell, a plant cell, an insect cell or a bacterial cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of the protein of the present invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The genetic constructs and methods described therein can be utilized for expression the protein of the present invention in prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The polypeptides of the invention can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies. As regards mammalian cells, HEK 293, CHO, HeLa and NIH 3T3 are preferred. As regards insect cells, it is most preferred to use Spodoptera frugiperda cells, whereas the most preferred bacterial cells are E. coli cells.
[0039]The invention also relates to a method of producing the (poly)peptide encoded by the nucleic acid molecule of the invention comprising culturing the host of the invention and isolating the produced (poly)peptide.
[0040]Depending on the vector constructing employed, the (poly)peptide of the invention may be exported to the culture medium or maintained within the host. Suitable protocols for obtaining the (poly)peptide produced are well-known in the art for both ways of (poly)peptide production.
[0041]The present invention furthermore relates to a (poly)peptide encoded by the nucleic acid molecule of the invention or produced by the method of the invention. The new channel is envisaged to show a structure having a short amino-terminal region, probably intracellular, five membrane-spanning segments, a hydrophobic hairpin entering the membrane, a sixth transmembrane segment, and a long C-terminal cytoplasmic part comprising a cyclic-nucleotide binding consensus sequence, a nuclear localization consensus sequence, and a hydrophobic domain probably forming a coiled-coil structure. The polypeptide of the invention may also be a functional fragment of the human K+ ion channel. By "functional fragment" polypeptides are meant that exhibit any of the activity of heag as described above. Using recombinant DNA technology, fragments of the (poly)peptide of the invention can be produced. These fragments can be tested for the desired function, for example, as indicated above, using a variety of assay systems such as those described in the present invention. Preferably, said fragments comprise the C-terminal portion of the novel ion channel.
[0042]The present invention also relates to an antibody specifically directed to the (poly)peptide of the invention. The antibody of the invention specifically discriminates between the human eag channel and the prior art channels such as mouse and rat eag and preferably binds to epitopes in the C-terminal part of the ion channel. The term "antibody", as used in accordance with the invention, also relates to antibody fragments or derivatives such as F(ab)2, Fab', Fv or scFv fragments; see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, N.Y. Preferably, the antibody of the invention is a monoclonal antibody.
[0043]The invention also relates to a pharmaceutical composition comprising the nucleic acid molecule of the invention, the vector of the invention, the polypeptide of the invention and/or the antibody of the invention and a pharmaceutically acceptable carrier and/or diluent and/or excipient.
[0044]Examples of suitable pharmaceutical carriers and diluents as well as of excipients are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the patient in need thereof at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 1012 copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
[0045]It is envisaged by the present invention that the various polynucleotides and vectors of the invention are administered either alone or in any combination using standard vectors and/or gene delivery systems, and optionally together with a pharmaceutically acceptable carrier or excipient. Subsequent to administration, said polynucleotides or vectors may be stably integrated into the genome of the subject. On the other hand, viral vectors may be used which are specific for certain cells or tissues and persist in said cells or tissues. Suitable pharmaceutical carriers and excipients are, as has been stated above, well known in the art. The pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of diseases, which are related to the undesired (over)expression of the above identified nucleic acid molecule of the invention. In a preferred embodiment the pharmaceutical composition comprises antisense oligodesoxynucleotides, as for example described in example 5, capable of regulating, preferably decreasing heavy expression.
[0046]Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises the polynucleotide or vector of the invention in gene therapy. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Gene therapy, which is based on introducing therapeutic genes, for example for vaccination into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivery systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 81996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729).
[0047]Standard methods for transfecting cells with recombinant DNA are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469. Gene therapy may be carried out by directly administering the recombinant DNA molecule or vector of the invention to a patient or by transfecting cells with the polynucleotide or vector of the invention ex vivo and infusing the transfected cells into the patient. Furthermore, research pertaining to gene transfer into cells of the germ line is one of the fastest growing fields in reproductive biology. Gene therapy, which is based on introducing therapeutic genes into cells by ex vivo or in vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in vitro or in vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., WO94/29469, WO 97/00957 or Schaper (Current Opinion in Biotechnology 7 (1996), 635-640) and references cited above. The polynucleotides and vectors comprised in the pharmaceutical composition of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) containing said recombinant DNA molecule into the cell. Preferably, said cell is a germ line cell, embryonic cell, stem cell or egg cell or derived therefrom. An embryonic cell can be for example an embryonic stem cell as described in, e.g., Nagy, Proc. Natl. Acad. Sci. USA 90 (1993) 8424-8428.
[0048]It is to be understood that the introduced polynucleotides and vectors of the invention express the (poly)peptide of the invention after introduction into said cell and preferably remain in this status during the lifetime of said cell. For example, cell lines which stably express the polynucleotide under the control of appropriate regulatory sequences may be engineered according to methods well known to those skilled in the art. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the polynucleotide or vector of the invention and a selectable marker, either on the same or separate vectors. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. Such engineered cell lines are particularly useful in screening methods or methods for identifying an inhibitor of the polypeptide of the present invention as described below.
[0049]A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl. Acad. Sci. USA 77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA 78 (1981), 1527), gpt, which confers resistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981), 2072), neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, J. Mol. Biol. 150 (1981), 1), hygro, which confers resistance to hygromycin (Santerre, Gene 30 (1984), 147), Shble, which confers resistance to Zeocin® (Mulsant, Somat. Cell. Mol. Genet. 14 (1988), 243-252 or puromycin (pat, puromycin N-acetyl transferase). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.). Cells to be used for ex vivo gene therapy are well known to those skilled in the art. For example, such cells include for example cancer cells present in blood or in a tissue or preferably the corresponding stem cells.
[0050]Furthermore, the invention relates to a diagnostic composition comprising the nucleic acid molecule of the invention, the vector of the invention, the polypeptide of the invention and/or the antibody of the invention.
[0051]The diagnostic composition of the invention is useful in detecting the onset or progress of diseases related to the undesired expression or overexpression of the nucleic acid molecule of the invention. As has been pointed out herein above, such diseases are interrelated or caused by an increased or ongoing cellular proliferation. Accordingly, the diagnostic composition of the invention may be used for assessing the onset or the disease status of cancer. Having thus an early criterium for tumor activity, suitable counter-measures can immediately be applied. Such an immediate action will, of course, significantly improve the prognosis of the patient. These considerations equally apply to the diagnosis of metastases and recurrent tumors.
[0052]On the other hand, not all types of tumors may be characterized by an undesired expression or overexpression of the nucleic acid molecule of the invention. Alternatively, said (over)expression may occur only in certain stages, such as early stages, of tumor development. Therefore, the diagnostic composition of the invention may also or alternatively be employed as a means for the classification of tumors or of the developmental status of a tumor. Naturally, the or most of the applications of the composition of the invention described here for tumors also apply to other diseases interrelated with or caused by the undesired (over)expression of the nucleic acid molecule of the invention.
[0053]Furthermore, a disease as recited throughout this specification also could be caused by a malfunction of the polypeptide of the present invention. Said disease could be interrelated or caused by, for example, an increased or reduced gene dosis of the polypeptide of the present invention, an increased or reduced activity of said polypeptide e.g. due to a modification in the primary amino acid sequence as compared to the corresponding wild-type polypeptide in a cell or tissue or a loss of the regulation of the activity of said polypeptide. Said disease might further be caused by an incorrect expression of the polypeptide during cell cycle progression or cell development. For example, mutated binding sites to intracellular or extracellular compounds, e.g. ions or second messengers or regulatory proteins, might result in a malfunction of the polypeptide of the present invention as it changes the binding characteristics for said compounds regulating the activity of said polypeptide. Malfunction could also be caused by defective modifications sites, for example, phosphorylation or glycosylation sites. It also might be caused by incorrect splicing events and therefore by expression of a truncated or extended polypeptides, for example, if heag 1 is expressed instead of heagh 2 or vice versa.
[0054]Thus, in a further embodiment the diagnostic composition described above could also be used to detect a malfunction of the polypeptide of the present invention.
[0055]In a further embodiment, the invention relates to a method for preventing or treating a disease which is caused by the malfunction of the polypeptide of the invention, comprising introducing an inhibitor of the expression of the nucleic acid molecule of the present invention or an inhibitor or a modifying agent of the malfunction of the (poly)peptide of the present invention or a nucleic acid molecule coding heag or a polypeptide having heag activity into a mammal affected by said disease or being suspected of being susceptible to said disease. Methods for introduction of a nucleic acid molecule of the present invention encoding heag into a cell or subject, i.e. gene therapy, are described within this specification as well as methods for the identification of inhibitors of the expression of a nucleic acid molecule of the present invention. Furthermore, inhibitors or modifying agents of the malfunction of the polypeptide of the present invention can be identified according to methods for the identification of inhibitors inhibitors of the polypeptide of the present invention known to a person skilled in the art (see below). For example, some genetic changes causing a malfunction of the polypeptide of the present invention lead to altered protein conformational states. Mutant proteins could possess a tertiary structure that renders them far less capable of fascilitating ion transport. Restoring the normal or regulated conformation of mutated proteins is the most elegant and specific means to correct these molecular defects. Pharmacological manipulations thus may aim at restoration of wild-type conformation of the protein. Thus, the polynucleotides and encoded proteins of the present invention may also be used to design and/or identify molecules which are capable of activating the wild-type function of a derivative of the polypeptide of the present invention displaying said malfunction.
[0056]The doses and routes for the administration for the treatment of a patient in need thereof have already been discussed herein above in connection with the pharmaceutical composition of the invention. Diseases that may be treated using the method of the present invention comprise any diseases that are correlated with cellular proliferation. Preferred diseases that fall into this category are tumor diseases such as cancer (breast cancer, neuroblastoma etc.), psoriasis, and degenerative diseases, especially those of the nervous system such as Alzheimer's disease, multiple sclerosis, lateral amyotrophic sclerosis, and Parkinson's disease.
[0057]Preferably, said inhibitor of the expression or overexpression of said nucleic acid molecule is the nucleic acid molecule of the invention that hybridizes to the nucleic acid molecule encoding the ion channel of the invention or fragment thereof. For example, this nucleic acid molecule can be an antisense oligodesoxynucleotide (ODN). The inventors could show that antisense ODNs treatment significantly reduces DNA synthesis of several tumor cells, e.g. EFM cells, SHSY-5Y cells and HeLa cells (Example 5). Thus, in a preferred embodiment the nucleic acid molecule comprises antisense ODNs.
[0058]In a further preferred embodiment, said inhibitor of polypeptide function is the antibody of the invention or a drug. Said drug can be histamine receptor H1 inhibitor. Preferably, said drug inhibits active heag, for example, acts as use-dependent, probably open-channel blocker, preferably said drug is astemizole or terfenadine. Further suitable drugs can be identified or designed by the person skilled in the art on the basis of the teachings of the present invention. Preferably, the drug will have an affinity to heag channel in the mM range, more preferable in the nM range or lower. Preferably, the drug has no effect on other channels, for example on cardiac channels.
[0059]In a further preferred embodiment of the invention, said method further comprises prior to the introduction step,
[0060](a) obtaining cells from the mammal infected by said disease and, after said introduction step, wherein said introduction is effected into said cells,
[0061](b) reintroducing said cells into said mammal or into a mammal of the same species.
[0062]This embodiment of the present invention is particularly useful for gene therapy purposes which will reduce the treatment duration largely and increase the effectivity and reduce (even eliminate) side effects. In addition, this embodiment of the method of the invention can also be employed in the context or in combination with conventional medical therapy. The removal from and the reintroduction into said mammal may be carried out according to standard procedures.
[0063]Preferably, the above referenced cell is a germ cell, an embryonic cell or an egg cell or a cell derived from any of these cells.
[0064]The invention further relates to a method of designing a drug for the treatment of a disease which is caused by the undesired expression or overexpression of the nucleic acid molecule of the invention comprising:
[0065](a) identification of a specific and potent drug;
[0066](b) identification of the binding site of said drug by site-directed mutagenesis and chimeric protein studies;
[0067](c) molecular modeling of both the binding site in the (poly)peptide and the structure of said drug; and
[0068](d) modifications of the drug to improve its binding specificity for the (poly)peptide.
[0069]The term "specific and potent drug" as used herein refers to a drug that potently and specifically blocks heag function.
[0070]All techniques employed in the various steps of the method of the invention are conventional or can be derived by the person skilled in the art from conventional techniques without further ado. Thus, biological assays based on the herein identified features of the ion channel of the invention may be employed to assess the specificity or potency of the drugs wherein the decrease of one or more activities of the ion channel may be used to monitor said specificity or potency. Steps (b) and (d) can be carried out according to conventional protocols described, for example, in K. L. Choi, C. Mossman, J. Aube & G. Yellen. The International Quaternary Ammonium Receptor Site of Shaker Potassium Channels. Neuron 10, 533-541 (1993), C.-C. Shieh & G. E. Kirsch: Mutational Analysis of Ion Conduction and Drug Binding Sites in the Inner Mouth of Voltage-Gated K+-Channels. Biophys. J. 67, 2316-2325 (1994), or C. Miller: The Charybdotoxin Family of K+-Channel-Blocking Peptide. Neuron 15, 5-10 (1995).
[0071]For example, identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the (poly)peptide primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.
[0072]As regards step (c), the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (b)) and computer simulations of the structure of the binding site (since a potassium channel has recently been crystallized in the art, this can now be done by the person skilled in the art without further ado) provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other in the heag molecule.
[0073]Finally, in step (d) the drug can be modified to improve its binding affinity or its potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of heag and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction; additionally, if those interactions occur with a region of heag that is not conserved with other channel proteins, it is conceivable that an improvement of that interaction while other binding factors are weakened will improve the specificity of the drug.
[0074]Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptide of the invention by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of inhibitors of the polypeptide of the invention can be used for the design of peptidomimetic inhibitors, e.g., in combination with the (poly)peptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
[0075]An exemplary strategy for identifying a specific inhibitor that may be used in accordance with the present invention is provided in the appended examples.
[0076]The invention also relates to a method of identifying an inhibitor of the expression of the nucleic acid of the invention or of a function of the (poly)peptide of the invention comprising:
[0077](a) testing a compound for the inhibition or reduction of translation wherein said compound is selected from antisense oligonucleotides and ribozymes; or
[0078](b) testing a compound for the inhibition of transcription wherein said compound binds to the promoter region of the gene encoding the (poly)peptide of the invention and preferably with transcription factor responsive elements thereof; or
[0079](c) testing peptides or antibodies suspected to block the proliferative activity of the (poly)peptide of the invention for said blocking activity.
[0080]As regards alternative (b) referred to above, it may be advantageous to first characterize the promoter region and locate transcription factor responsive sequences in it. Then it would be possible to genetically manipulate the promoter to render it more sensitive to repressors or less sensitive to enhancers. Turning now to alternative (c), it may be advantageous to first locate the part or parts of the ion channel of the invention implicated in the generation of proliferation disorders. Compounds that have been positive in one of the test systems are, prima facie, useful as inhibitors.
[0081]Peptidomimetics, phage display and combinatorial library techniques are well-known in the art and can be applied by the person skilled in the art without further ado to the improvement of the drug or inhibitor that is identified by the basic method referred to herein above.
[0082]In a further embodiment, the present invention relates to a method of inhibiting cell proliferation comprising applying an inhibitor to expression of the nucleic acid of the invention or the (poly)peptide of the invention. The method of the invention may be carried out in vitro, ex vivo or when application is to a subject, in vivo.
[0083]The present invention also relates to a method of prognosing cancer and/or neurodegenerative diseases and/or psoriasis comprising assessing the expression of the nucleic acid molecule of the invention or assessing the quantitative presence of the (poly)peptide of the invention. In a preferred embodiment said cancer is a mamma carcinoma or neuroblastoma, in a more preferred embodiment said cancer is breast adenocarcinoma, breast carcinoma ductal type, or cervix carcinoma. In a further embodiment said neurodegenerative diseases is Alzheimer's disease, Parkinson's disease, lateral amytrophic sclerosis or multiple sclerosis.
[0084]The method of the invention may be carried out in vitro, in vivo, or ex vivo. Suitable protocols for carrying out the method of the invention are well-known in the art and include, as regards in vitro techniques, Northern blotting for the assessment of the level of mRNA or the analysis of tissue by microscopic techniques using, for example, antibodies that specifically recognize the (poly)peptide of the invention. One or more these techniques may be combined with PCR based techniques which may also or in combination with further (conventional) techniques be used for the above recited assessment.
[0085]In a preferred embodiment of the above-mentioned methods of the invention, said mammal is a human, rat or mouse.
[0086]The present invention further relates to the use of the nucleic acid molecules of the invention in gene therapy. As has been pointed out here above, gene therapy may be designed to inhibit cell proliferation and thus treat any disease affected thereby such as cancer or psoriasis in a specific way. The invention particularly envisages two independent lines carrying out such gene therapy protocols:
[0087](a) Mutagenesis of the channel together with chemical engineering of H1 antagonists (preferably of astemizole) in order to obtain a drug specific for human eag;
[0088](b) Quantitative and qualitative analysis of the expression levels of eag in cancer tissue, in order to design a diagnostic and/or prognostic method. This would also allow the design of genetic therapies against specific tumors.
[0089]For example, the nucleic acid molecule may be introduced in vivo into cells using a retroviral vector (Naldini et al., Science 272 (1996), 263-267; Mulligan, Science 260 (1993), 926-932) or another appropriate vector. Likewise, in accordance with the present invention cells from a patient can be isolated, modified in vitro using standard tissue culture techniques and reintroduced into the patient. Such methods comprise gene therapy or gene transfer methods which have been referred to herein above.
[0090]Finally, the present invention relates to a kit comprising the nucleic acid molecule specifically hybridizing to the nucleic acid molecule encoding the (poly)peptide of the invention, the vector of the invention, the polypeptide of the invention and/or the antibody of the invention.
[0091]The kit of the invention can, inter alia, be employed in a number of diagnostic methods referred to above. The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transformed host cells and transgenic plant cells, plant tissue or plants. Furthermore, the kit may include buffers and substrates for reporter genes that may be present in the recombinant gene or vector of the invention. The kit of the invention may advantageously be used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications referred to herein, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
[0092]Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
The figures show:
[0093]FIG. 1. Proliferation of wild-type (circles) and reag-expressing CHO cells as a function of time. Cells were plated in 96-well dishes and at the indicated times the tetrazolium salt MTT6 (50 μg/ml) was added to the plates. After four hours incubation in humidified atmosphere (37° C., 5% CO2), the reaction was stopped by addition of 2 volumes of 10% SDS in 1M HCl. The blue formazan crystals produced in living cells were solubilized overnight, and the resulting color was measured as optical density at the indicated wavelength. Possible non-specific effects of the transfection on the cell proliferation can be neglected, since a) the results were comparable in three independent cell lines from different species (rat, hamster and human); b) transfection with different independent clones gave the same results, and c) transfection with a different potassium channel (Kv1.4) in the same vector (thus with a tendency to recombine at the same site) gave results comparable to WT and did not reproduce the effects of the reag transfection.
[0094]FIG. 2. Proliferation of wild type (circles) and reag expressing (triangles) CHO cells, in the presence of 0.5% FCS. This serum concentration is not able to sustain growth of normal cells, but transfected cells complete almost three cycles. Methods as for FIG. 1.
[0095]FIG. 3. DNA synthesis in CHO cells expressing different potassium channels, in the presence of normal (10%) or low (0.5%) concentrations of FCS. In control cells, WT or cells transfected with Kv1.4, the levels of DNA synthesis drop significantly in the presence of low serum concentration, whereas reag expressing cells maintain the same replication levels as in high serum concentrations.
[0096]FIG. 4. (A) Photographs of plates with wild type, Kv1.4 transfected or reag transfected NIH3T3 cells. The cells were seeded at low density, and allowed to grow under standard conditions until wild-type cells reached confluence. The cells were then fixed with methanol and stained with Giemsa blue. Under those conditions, both wild type and Kv1.4-expressing cells grow in a monolayer, whereas reag expressing cells form foci. (B) Foci formation of reag-transfected N1H-3T3 cells compared to cells transfected with rKv1.4 and to wild type cells. The vector control (pcDNA3 transfected cells) yielded a similar phenotype as wild type cells (not shown). Transient transfection was carried out using calcium phosphate (33). Cells were maintained in rich medium until control cells reached confluence, then fixed with methanol and stained with Giemsa blue.
[0097]FIG. 5. Currents elicited by depolarizations in MCF7 cells under voltage clamp conditions. Left traces are whole cell currents, right traces have been obtained in an excised outside-out patch. Both the macroscopic currents and the I-V relationships (C and D) are reminiscent of reag currents.
[0098]FIG. 6. Single channel activity in an outside-out membrane patch voltage-clamped at 0 mV, in the presence or the absence of 5 μM astemizole. The pipette solution contained 140 mM KCl, 10 mM BAPTA, 10 mM HEPES pH 7.2; the bath solution contained 140 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 2.5 mM KCl, 10 HEPES pH 7.2.
[0099]FIG. 7. A. DNA synthesis in MCF7 cells under different eag blockers. B. HEK293 DNA synthesis levels in the presence of astemizole, glibenclamide and terfenadine.
[0100]FIG. 8. Dose-response curve for the effects of two H1 antagonists on DNA synthesis in MCF7 cells (IC50 7 and 10 mM for LY 91241 and astemizole respectively).
[0101]FIG. 9. Fluorescence images of control (untreated, A) and astemizole-treated (B) MCF7 cells, stained with Hoechst 33342. Notice in B the smaller surface of the nuclei, and a much lower cell density (due to cell death).
[0102]FIG. 10. Nucleotide sequence of human-eag cDNA (heag; SEQ ID NO: 1) from human brain compared to the rat sequence (reag; SEQ ID NO: 20) and bovine sequence (beag; SEQ ID NO: 19). Those positions showing a different nucleotide in any of the sequences are shaded.
[0103]FIG. 11. Amino acid sequences of both splice variants (heag1 (SEQ ID NO: 3) and heag2 (SEQ ID NO: 4)) obtained from human eag cDNA translation, compared to the corresponding bovine (beag1; SEQ ID NO: 21; beag2; SEQ ID NO: 22), mouse (meag; SEQ ID NO: 23) and rat (reag; SEQ ID NO: 24) sequences. The black boxes indicate a different residue in any of the sequences.
[0104]FIG. 12. RT-PCR from human brain, human mammary gland and MCF-7 cells total RNA. The amplification produced two specific fragments corresponding to the expected sizes for heag 1 and 2 in brain, and the band corresponding to heag 1 in MCF-7 cells, while no amplification was detected in normal breast RNA.
[0105]FIG. 13. Voltage-dependence of activation in high extracellular potassium, two-electrode voltage-clamp: In the conductance-voltage plot, the voltage for half-activation is shifted by 40 mV to the right in the heag channel with respect to the reag channel.
[0106]FIG. 14. Colony formation in semisolid medium of NIH-3T3 cells transfected with the indicated DNAs. Cells were plated in regular medium containing 0.3% agar onto a layer of 0.55% agar medium. Colonies larger than 0.1 mm in diameter were scored 14 days after transfection. The average number of colonies in at least ten counted microscope fields is expressed per μg DNA used in the transfection (except for the lanes "Transfection buffer" and "No treatment", where the numbers are absolute values). reag and Kv1.4 were transfected using either pcDNA3 or pTracer CMV vectors.
[0107]FIG. 15. (A) Southern blot of RT-PCR products of RNAs from different human tissues and 293 cells. Transferrin receptor (TFR) signals are shown at the bottom. (B) Southern blot analysis of RT-PCR products of total RNAs from different human cell lines and mammary epithelial cells in primary culture (Epith. cells). TRF signals are shown at the bottom.
[0108]FIG. 16. (A) Treatment of heag expressing tumor cell lines with antisense ODNs. (B) heag current in SHSY-5Y neuroblastoma cells (C) Current density in SHSY-5Y cells treated with antisense ODNs (D) Inhibition of DNA synthesis in human cancer cells (EFM-19, HeLa and SHSY-5Y) by antisense ODNs directed against heag.
[0109]FIG. 17. (A) Subcutaneous implantation of CHOhEAG cells induced aggressive tumors that grew rapidly and soon broke the skin of the carrier mice. The photograph was taken in the third week post-implantation of 2×106 cells. (B,C) The average mass of CHOhEAG tumors was significantly greater than that of the CHOKv tumors both two weeks (B; mean±S.E.M.; p=0.002) or three weeks post-implantation (C; mean±S.E.M.; p=0.03) (D) CHOhEAG and (E) CHOKv tumors photographed in situ. The main macroscopic differences are the darker color and the fixation to the skin of the CHOhEAG tumor. (F, G) CHOhEAG (F) and CHOKv (G) tumors were cut open to show the great extent of necrosis (arrowheads) in the former. (H, I) The greater degree of necrosis and the fixation to the skin are also evident microscopically after paraffin embedding and hematoxylin-eosin staining. The histology is comparable in both micrographs, but in (H) a much bigger necrotic area is observed (arrowheads), and there is no border between the subcutaneous fat and the tumor. (Scale bars, 100 μm) (J) As a quantitative measurement of these images, the average width of the vital area in CHOKv tumors was significantly larger than that of CHOhEAG tumors (mean±S.E.M.; p<0.0005).
[0110]FIG. 18: Proliferation assays of rEAG-transfected CHO cells (A-C). Growth curves of CHO cells transfected with rEAG (circles) as compared to naive cells (triangles) in 10% (filled symbols) or 0.5% (open symbols) fetal calf serum. The values are referred to the ones measured after 12 h in culture (time 0 in the plot), and represent mean±S.E.M. of eight wells in the same plate. Cell lines were established by selection through the G-418 resistance encoded in the pcDNA3 vector. MTT hydrolysis (22) was used to measure metabolic activity of viable cells. Serum was carefully diluted 12 hours after plating. (B) Increase in metabolic activity during the first 12 hours after removal of S-phase block. For cell synchronization, 2 mM thymidine was added to the culture medium for 12 h. Thymidine was removed from the medium for additional 12 h, and then a second arresting pulse was applied for 12 h. Cells were then trypsinized and plated for metabolic activity and DNA synthesis determination. (C) BrdU incorporationi during the first 12 hours after removal of S-phase block for 12 h incubation in 10% FCS, or in the presence of 0.5% FCS (24 h incubation). BrdU incorporation was measured using the Boehringer-Mannheim "BrdU labeling and detection kit", following the indications of the manufacturer. The bars represent mean±S.D. for wild-type CHO cells (open bars), Kv1.4-transfected (shaded bars) and eag-transfected (solid bars). The incorporation of BrdU is quantified as optical density at 405 nm (reference 490 nm) produced on ABTS® substrate by peroxidase coupled to the anti BrdU antibody.
[0111]The examples illustrate the invention.
EXAMPLE 1
Cloning of the K+ Ion Channel
[0112]mRNA was purified from total RNA obtained from MCF-7 cells following standard procedures. Then, cDNA was prepared by reverse transcription with Superscript II reverse transcriptase; this cDNA was used as a template for PCR amplification using degenerate oligonucleotides designed to match highly conserved eag sequences. After amplification, a SacII/SacII fragment from rat eag was used as a probe for Southern blot analysis of the results. Those bands showing positive hybridization were subsequently cloned in pGEM-T vector (Promega) and sequenced. All of them gave sequences corresponding to HERG.
[0113]Specific oligonucleotides engineered to avoid HERG cDNA amplification were then designed, taking into account rat, mouse and bovine eag. We looked for sequences having high homology between the various eag clones but with maximal divergence to the HERG sequence.
[0114]The sequences of the oligonucleotides were the following:
TABLE-US-00001 (SEQ ID NO: 8) 5'-CAGAA(T, C)AA(T, C)GTGGC(A, C, T, G,)TGGCT (SEQ ID NO: 9) 5'-TCACT(G, A)AAGATCTATA(A, G)TC
[0115]After PCR amplification, the band of the expected size was cloned into pGEMT and sequenced. The sequence obtained showed high homology to rat eag (nucleotides 942-1108).
[0116]This band was labeled and used as a probe to screen a mammary gland cDNA library. After screening of 2×106 phages, no positive clones were found.
[0117]We then used specific oligonucleotides to analyze cDNA using PCR from human heart and human brain (obtained from total RNA purchased from Clontech). Two PCR products from brain were sequenced, and the sequence corresponded to two alternatively spliced variants of eag. To further test the possibility of cloning the full length molecule from the human brain, we performed PCR analysis of a human cDNA library, and compared this result to the same experiment in the human mammary gland library (both from Clontech). Only the brain library gave positive results.
[0118]Subsequently, the amplified fragment was employed to screen the human brain library (2 rounds, 106 phages) and several clones that were cloned into the pBSK-vector were found and sequenced. All of them corresponded to the central part of the molecule, but were missing the 5' and 3' ends. The longest of these positive clones was used to prepare a probe and re-screen the library (again two rounds, 2×106 clones).
[0119]The sequences obtained in this case corresponded to part of the coding sequence (approximately 400 bp 5' were missing until the initiation codon) and a long 3' untranslated sequence. Since the fragment close to the 5' end of the molecule started in all cases with an EcoRI site, it was suspected that the site was actually present in the heag sequence, and that is was lost in the subcloning of the fragments into vectors for sequencing.
[0120]To obtain the full length sequence, we pooled those phages that carried fragments close to the 5' end and analyzed them by PCR amplification, using the sequence 3' to the mentioned EcoRI site and a sequence from lambda gt10 as primers for the PCR. After successive fractionation of the pools, two phages that carried the 5' end of the coding sequence were obtained, and one of them contained part of the 5' untranslated region.
[0121]Once we knew the complete sequence, we assembled the whole clone starting from two phages, one of them containing the 3' UTR and most of the coding sequence, and the other containing the 5' end. The first fragment was extracted from the phage by SphI/HindIII digestion, and subcloned into pBKS-- to produce pBKSheag 1. In this was, a 1.2 kbp SphI-SphI fragment was also removed from the clone, and it was necessary to reintroduce it afterwards. The fragment containing the 5' end was extracted by HindIII/MunI digestion. This fragment was ligated with a HindIII/MunI digest of pBKSheag 1. Only using this procedure were we able to obtain the full length clone in a single plasmid. We then needed to reintroduce the SphI-SphI fragment since we had deleted one of the SphI sites. Subsequently, an EagI/NotI fragment was subcloned into the NotI site of pcDNA3 vector, to eliminate the contaminating phage sequences and to obtain a vector suitable for functional expression of the channel. Finally obtained sequences are depicted in sequence listing as SEQ ID No. 1 and SEQ ID No. 2.
EXAMPLE 2
Identification of Inhibitors that Specifically Bloc the Action of Human eag
[0122]Another member of the eag family, HERG 1-6, has been related to a familiar form of long QT syndrome (LQT). This has allowed to identify several blockers of HERG based on their ability to induce LQT-type arrythmias. Thus, certain histamine H1 receptor blockers, such as astemizole and terfenadine, as well as class III antiarrythmic drugs (dofetilide, E-4031) are potent and specific blockers of HERG15,17. However, for eag channels, specific blockers have not yet been described. Due to the sequence similarity between HERG and eag channels, both groups of drugs on reag were tested in accordance with the present invention. The H1 blockers also affect reag, whereas the channel is rather insensitive to class III antiarrythmics (dofetilide). This provides a useful tool to selectively block eag-type channels and to discard possible effects of HERG channels (which are also present in MCF7 cells). The effect of one of these drugs (astemizole 5 μM) is shown on single putative human eag channels in FIG. 6.
[0123]It was further tested whether several reag and other potassium channel blockers are able to inhibit growth of MCF7 cells. As a "positive" control glibenclamide, a blocker of the ATP-sensitive potassium channel was also included, since it has been described to inhibit the proliferation of this cell line2. To determine the rate of DNA synthesis, cells were plated on 96-well microtiter plates at a density of ≈105 cells/ml and in the absence of growth factors. After 24 hours starvation, cells were stimulated by addition of 10% FCS in the presence of BrdU. The amount of BrdU incorporated into the newly synthesized DNA was determined using a commercial antibody (Boehringer Mannheim). The drugs tested were added either at the same time or 12 hours prior to the stimulation. In a different human cell line, HEK293, the addition of 10 μM astemizole or 100 μM glibenclamide did not reduce significantly the DNA synthesis, while terfenadine (10 μM) produced a strong inhibition. For this reason, only effects of astemizole (and its closely related analog LY91241) were considered, and those produced by terfenadine (although MCF7 cells are significantly more sensitive to growth inhibition by terfenadine than the control cells) discarded. In MCF7 cells, 5 μM astemizole reduced the DNA synthesis by 40%, while the same concentration of the HERG-specific blocker dofetilide produced no significant effects. Ten times higher concentrations (50 μM) of other potassium channel blockers (quinidine or glibenclamide) where required to induce a similar effect. A dose-response curve for astemizole effects on DNA synthesis in MCF7 cells is depicted in FIG. 8. The half-maximal effect was obtained for 10 μM astemizole.
[0124]In an attempt to clarify the mechanism underlying the proliferation inhibition in MCF7 cells, the nuclear morphology of cells treated with 5 μM astemizole were checked, using the supravital nuclear stain Hoechst 33342. After 24 hours of treatment, most cells showed nuclear condensation and fragmentation, typical features of apoptotic cell death (FIG. 9).
[0125]In conclusion, a human counterpart of the reag channels are present in human cancer cells, and they have the ability to induce malignant transformation in several different cell types.
EXAMPLE 3
Expression of heag in Different Human Tissues
[0126]500 ng total RNA from different tissues (or 5 ng polyA+ RNA, for spinal cord) were reverse transcribed and amplified using a pair of oligonucleotides of the sequences 5'-CGCATGAACTACCTGAAGACG (SEQ ID NO: 10) (forward) and 5'-TCTGTGGATGGGGCGATGTTC (SEQ ID NO: 11) (reverse). The amplified DNA was analyzed by Southern blot using a specific human eag probes (a 1.5 kb EcoRI fragment from the core of the channel). Among the RNAs tested, only brain total RNA gave positive signals. RNAs from spinal cord, adrenal gland, skeletal muscle, heart trachea, liver, kidney and mammary gland were negative. The integrity of the RNA was checked using transferrin amplification. Using the same approach, the expression of heag in several tumoral human cell lines was checked, in: MCF-7 (breast adenocarinoma), BT-474 (breast ductal carcinoma, from a solid tumor, EFM-19 (breast carcinoma, ductal type, from pleural fluid), COLO-824 (breast carcinoma, ductal type, from pleural fluid), SHSY5Y (neuroblastoma).
[0127]In contrast to normal tissues, all the cancer cell lines tested were found positive for heag expression.
[0128]Further, Southern blot of RT-PCR products of RNAs from different human tissues and 293 cells show that only in RNA from brain the two bands corresponding to heag A and B could be amplified and identified. Transferrin receptor (TFR) signals are shown at the bottom (FIG. 15A). Furthermore, a Southern blot analysis of RT-PCR products of total RNAs from different human cell lines an mammary epithelial cells in primary culture (Epith. cells). TRF signals are shown at the bottom. RNAs from different cell lines (34) and commercial RNAs from human tissues (Clontech) were subjected to single-tube RT-PCR (35). Total RNA was used with the exception of spinal cord, where poly(A)+ RNA was used (primer sequences were: forward: 5'-CGCATGAACTACCTGAAGACG (SEQ ID NO: 10) and reverse: 5'-TCTGTGGATGGGGCGATGTTC (SEQ ID NO: 11). 5'-TCAGCCCAGCAGAAGCATTAT (SEQ ID NO: 17) and reverse: 5'-CTGGCAGCGTGTGAGAGC (SEQ ID NO: 18) were used to control RNA and PCR performance.). Specific primers for TFR were used to control RNA and PCR performance. These ODNs were designed according to the published TFR sequence (36), starting at exon 11 and spanning to exon 19 (37). This, together with the amplification of two heag splice fragments and controls in the absence of reverse transcriptase, excludes a false positive due to genomic DNA contamination. 50 μl (heag) or 15 μl (TFR) of PCR reactions were analyzed in 2% agarose gels. DNA was transferred to membranes and consecutively hybridized at high stringency with [32P]-dCTP labeled random primed probes consisting of a 980 bp heag fragment and the TFR fragment amplified from brain RNA.
EXAMPLE 4
Expression of Heag In Vivo
[0129]To determine whether the expression of heag is advantageous for tumor cells in vivo, the inventors preformed subcutaneous implants of CHO cells expressing the channel (CHOhEAG cells) into the flank of female scid (severe combined immunodeficiency, 33) mice. CHOKv cells were used as a control. Therefore, 2×106 CHOhEAG or CHO-Kv1.4 cells suspended in 100 μl PBS were implanted subcutaneously on the flank of 6-8 week old female Fox Chase scid mice (C.B-17/Icr sicd/scid) obtained from Bomholtgard, Ry, Denmark. The presence of tumors was checked every second day by tactile inspection of every mouse. After two or three weeks, the animals were sacrificed by cervical dislocation and the tumors dissected and fixed in paraformaldehyde for subsequent paraffin inclusion and staining. The identity of the CHOhEAG cells was established by UV illumination of the tumors to evoke fluorescence from the green fluorescence protein encoded in the pTracer vector (Invitrogen). One week after the implantation, all CHOhEAG-injected mice carried tumors detectable by palpation, while no mass greater than 1 mm was observed in the controls. During the second week post-implantation, the heag-expressing tumors reached in excess of 5 mm in diameter and visibly emerged through the skin in most cases (FIG. 17A); the mice were sacrificed after two (N=6) or three weeks (N=7). Only one of the 11 control animals used was free of visible tumors; all 13 CHOhEAG-injected animals showed tumors. The average mass (FIG. 17B, C) of the heag-expressing tumors was significantly larger than that of controls, especially two weeks following implantation (FIG. 17B). From macroscopic observation, the tumors appeared friable and hemorragic; the CHOhEAG tumors were darker than the controls and were adhered to the skin (FIG. 17D, E) in all CHOhEAG-injected mice at two weeks. Six of seven mice exhibited similar characteristics at three weeks. In contrast, the tumor could be easily dissected from the skin in all of the control mice after two weeks, and in five out of six mice at three weeks. The tissue below the tumor appeared unaffected in all cases. The dark color was due to great extent of intratumoral necrosis (FIG. 17 F, G, arrows), confirmed by histology (FIG. 17 H, I, arrowheads), indicating a faster growth of CHOhEAG tumors. The thickness of the vital area in the EAG-expressing tumors was significantly smaller than in the controls (FIG. 17J). The rapid growth of the tumor can account for the massive intratumoral necrosis in the CHOhEAG group. This could also explain the enhanced difference found in the mass of the tumors two weeks after implantation, since CHOhEAG tumors would cease growth due to massive necrosis. These data strongly suggest that expression of heag tumors grow faster and are more aggressive than CHOKv tumors.
EXAMPLE 5
Inhibition of heag
[0130]It is assumed that expression of heag in some tumor cells is not the consequence of their abnormal growth, but that this K+ channel is necessary for their proliferation. Therefore, inhibition of heag expression with antisense oligodeoxynucleotides (ODNs) should decrease the proliferation rate in these tumor cells. Therefore, a 19-mer antisense phosphorothioate ODN (5'-CAGCCATGGTCATCCTCCC) (SEQ ID NO: 15) spanning the putative initiation codon of heag was used to test inhibition of proliferation. The sense ODN and a scrambled sequence (gtcggtaccagtaggaggg) (SEQ ID NO: 16) were used as controls. Data shown in FIG. 16A confirms the efficiency of the antisense ODN treatment in reducing the heag mRNA content in EFM cells. A reduction in heag mediated K+ currents in SHSY-5Y cells by treatment with antisense ODN is shown in FIGS. 16B and C.
[0131]Treatment of heag expressing tumor cell lines with antisense ODNs significantly reduced the yield of amplified PCR products. EFM-19 cells were treated with 10 μg/ml DAC30 (lanes "C") or 10 μg/ml DAC30 (Eurogentec) plus 1 μM antisense ODN (lanes "AS") overnight, total RNA was extracted and assayed under the same conditions as described in Example 3, with ODNs designed to either amplify heag or the transferrin receptor. The arrows in FIG. 16A mark the expected sizes of the amplified fragments. Further, to dissect the heag current in SHSY-5Y neuroblastoma cells, the inventors utilized the voltage-dependence of the activation of eag (30) in the presence of extracellular Mg2+. The current was measured after a depolarization to +60 mV from -120 mV (FIG. 16B, gray lines). The first part of the subtracted trace (FIG. 16B, black line) corresponds to eag current that has not yet activated when the holding potential is very negative (-120 mV), but becomes evident if the holding potential is -60 mV. The average current between 19 and 21 ms was chosen to determine the current density. The current density in SHSY-5Y cells treated with antisense ODNs was significantly reduced as compared to control cells (The electrophysiological determinations were performed using standard protocols in the whole cell configuration of the patch-clamp technique (Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. Pflugers Arch Eur. J. Physiol 391, 85 (1981)), with an extracellular solution containing (mM) 140 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 Hepes/NaOH pH 7.2, 10 glucose. The pipette solution was (mM) 140 KCl, 10 BAPTA, 10 Hepes/KOH pH 7.2.). The cells were treated overnight with antisense ODN 1 μM containing fluorescein-labeled ODN. The currents were determined 1 to 3 days later in cells showing fluorescence in their nuclei. The bars in FIG. 16C represent mean±S.E.M. for 9 cells (control) or 25 cells (antisense). Only the outward currents were evaluated in the analysis. Furthermore, the inhibition of DNA synthesis in human cancer cells (EFM-19, HeLa and SHSY-5Y) by antisense ODNs directed against heag was investigated. DNA synthesis is expressed relative to BrdU incorporation in the absence of ODNs. The uptake conditions into cells using fluorescein labeled antisense ODN was optimized. Cells were seeded in 96-well plates at a density of 105 cells/ml. One day after plating, the cells were washed with culture medium and the ODN was added (final concentration 10 μM). The ODN had previously been mixed with 20 μg/ml of the transfection ragenent DAC-30 (Eurogentec) in serum-free medium and allowed to incubate at room temperature for 20-30 min. The mixture was then added as a 1:1 dilution in culture medium and maintained in contact with cells overnight. After this incubation, the cells were washed and labeled with BrdU (100 μM) for 2 h. Incorporation was detected using the kit from Boehringer Mannheim and measured as OD units at 405 nm (reference 490 nm) after subtraction of the non-specific background incorporation. (FIG. 16D). The bars indicate mean+S.D. for eight wells per condition in a representative experiment.
GLOSSARY AND LIST OF ABBREVIATIONS
TABLE-US-00002 [0132]Cell lines: CHO CHO-K1 (ATCC CCL 61) Chinese hamster Cricetulus griseus ovary HEK293 293 (ATCC CRL 1573) Transformed primary human embryonal kidney NIH3T3 (ATCC CRL 1658) Embryo Swiss mouse fibroblasts MCF7 (ATCC HTB 22) Human breast adenocarcinoma WT Wild-type cells Genes and gene products eag ether-a-go-gopotassium channel HERG Human-Eag-Related Gene. Codes for an inwardly rectifying potassium channel mainly expressed in heart. Kv1.4 Inactivating voltage-dependent potassium channel. Initially cloned from rat brain, it is present in many other tissues. Others EGF Epidermal growth factor PDGF Platelet-derived growth factor FCS Fetal calf serum I-V relation Current-Voltage relation LQT Long Q-T (interval between Q and T waves in the electrocardiogram). Induces severe arrythmias due to repolarization defects. BrdU 5-Bromo-2'-deoxyuridine. Structure analog of thymidine. IC50 Concentration that produces 50% inhibition RT-PCR. Polymerase Chain Reaction of cDNA produced by reverse transcription in the same tube.
REFERENCES
[0133]1. Moody, W. J. (1995). Critical periods of early development created by the coordinate modulation of ion channel properties. Perspect Dev Neurobiol 2, 309-315. [0134]2. Woodfork, K. A., Wonderlin, W. F., Peterson, V. A., and Strobl, J. S. (1995). Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture. J Cell Physiol 162, 163-71. [0135]3. Lepple Wienhues, A., Berweck, S., Bohmig, M., Leo, C. P., Meyling, B., Garbe, C., and Wiederholt, M. (1996). K+ channels and the intracellular calcium signal in human melanoma cell proliferation. J Membr Biol 151, 149-57. [0136]4. Wonderlin, W. F., Woodfork, K. A., and Strobl, J. S. (1995). Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle. J Cell Physiol 165, 177-85. [0137]5. Bruggemann, A., Stuhmer, W., and Pardo, L. A. (1997). Mitosis-promoting factor-mediated suppression of a cloned delayed rectifier potassium channel expressed in Xenopus oocytes. Proc Nat Acad Sci USA 94, 537-542. [0138]6. Mosmann, T. (1983). Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63. [0139]7. Muir, D., Varon, S., and Manthorpe, M. (1990). An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Anal. Biochem. 185, 377-82. [0140]8. Magaud, J. P., Sargent, I., and Mason, D. Y. (1988). Detection of human white cell proliferative responses by immunoenzymatic measurement of bromodeoxyuridine uptake. J. Immunol. Meth. 106, 95-100. [0141]9. Huong, P. L., Kolk, A. H., Eggelte, T. A., Verstijnen, C. P., Gilis, H., and Hendriks, J. T. (1991). Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation. J. Immunol. Meth. 140, 243-8. [0142]10. Ellwart, J., and Dormer, P. (1985). Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect. Cytometry 6, 513-20. [0143]11. Benson, D. W., MacRae, C. A., Vesely, M. R., Walsh, E. P., Seidman, J. G., Seidman, C. E., and Satler, C. A. (1996). Missense mutation in the pore region of HERG causes familial long QT syndrome. Circulation 93, 1791-5. [0144]12. Curran, M. E., Splawski, I., Timothy, K. W., Vincent, G. M., Green, E. D., and Keating, M. T. (1995). A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795-803. [0145]13. Sanguinetti, M. C., Jiang, C., Curran, M. E., and Keating, M. T. (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81, 299-307. [0146]14. Spector, P. S., Curran, M. E., Keating, M. T., and Sanguinetti, M. C. (1996). Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides. Circ Res 78, 499-503. [0147]15. Trudeau, M. C., Warmke, J. W., Ganetzky, B., and Robertson, G. A. (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269, 92-5. [0148]16. Suessbrich, H., Waldegger, S., Lang, F., and Busch, A. E. (1996). Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole. FEBS Lett 385, 77-80. [0149]17. For example, see: DeCoursey, T. E., Chandy, K. G., Gupta, S., Cahalan, M. D. Nature 307, 465 (1984); Mauro, T., Dixon, D. B., Komuves, L., Hanley, K., Pappone, P. A. J. Invest. Dermat. 108, 864 (1997). [0150]18. Rouzaire-Dubois, B. and Dubois, J. M. J. Physiol. 510, 93 (1998). [0151]19. Arcangeli, A., L. Bianchi, et al. J. Physiol. 489, 455-471 (1995). Bianchi, L. et al., Cancer Res. 58, 815-822 (1998). [0152]20. Bruggemann, A., Stuhmer, W., Pardo, L. A. Proc. Natl. Acad. Sci. USA. 94, 537 (1997). Pardo, L. A., Bruggemann, A., Camacho, J., Stuhmer, W. J. Cell. Biol. 143, 767 (1998). [0153]21. Ludwig, J., et al., EMBO J. 13, 4451 (1994). [0154]22. Mosmann, T. J. Immunol. Meth. 65, 55 (1983). Cells were incubated with MTT (Thiazolyl blue) 50 μg/ml for 4 h, and the formazan crystals were solubilized in 10% SDS, 10 mM HCl for 12-14 h. The optical density at 570 nm was measured using an ELISA reader, with a reference wavelength of 650 nm. [0155]23. Stein, G. S. et al., in Cell Growth and Apoptosis. A Practical Approach, G. P. Studzinski, Ed. (IRL Press, Oxford, 1995) pp. 193-203. [0156]24. Jainchill, J. L., Aaronson, S. A., Todaro, G. J. J. Virol. 4, 549 (1969). [0157]25. Hermouet, S., Merendino, J. J., Jr., Gutkind, S., Spiegel, A. M. Proc. Natl. Acad. Sci. USA 88, 10455 (1991). [0158]26. GenBank Accession Numbers for the sequences reported in this paper are AF078741 (heag A) and AF078742 (heag B). [0159]27. Occhiodoro, T. et al., FEBS Lett. 434, 177 (1998). [0160]28. Warmke, J. W. and Ganetzki, B. Proc. Natl. Acad. Sci. USA. 91, 3438 (1994). Frings, S., et al., J. Gen. Physiol. 111, 583 (1998). [0161]29. Heng, H. H. Q., Squire, J., Tsui, L.-C. Proc. Natl. Acad. Sci. USA, 89, 9509 (1992); Heng, H. H. Q., and Tsui, L.-C. Chromosoma, 102, 325 (1993). We acknowledge Dr. Henry Heng (See DNA Biotech, Downsview, Ontario, Canada) for FISH analysis. [0162]30. Terlau, H., et al., Pflugers Arch.--Eur. J. Physiol., 432, 301 (1996). Terlau, H., Heinemann, S. H., Stuhmer, W., Pongs, O., Ludwig, J. J. Physiol., 502, 537-543 (1997). Meyer, R. and Heinemann, S. H. J. Physiol. 508, 49 (1998). [0163]31. Bijlenga, P., et al. J. Physiol. 512, 317 (1998). [0164]32. Bosma, G. C., et al. Nature 301, 527-530 (1987) [0165]33. Chen, C. and Okayama, H. Mol. Cell. Biol. 7, 2745 (1987). [0166]34. Chomczynski, P. and Sacchi, N. Anal. Biochem. 162, 156 (1987). [0167]35. Soto, F., et al., Proc. Natl. Acad. Sci. USA. 93, 3684 (1996). [0168]36. McClelland, A., Kuhn, L. C., Ruddle, F. H. Cell 39, 267 (1984). Schneider, C., Owen, M. J., Banville, D., Williams, J. G. Nature 311, 675 (1984). [0169]37. Evans, P. and Kemp, J. Gene 199, 123 (1997).
Sequence CWU
1
2413002DNAHomo sapiens 1aattccgggc ccgccggacc ccgagctgct gggaggatga
ccatggctgg gggcaggagg 60ggactagtgg cccctcaaaa cacgtttctg gagaatattg
ttcggcggtc caatgatact 120aattttgtgt tggggaatgc tcagatagtg gactggccta
ttgtgtacag caatgatgga 180ttttgcaagc tgtctggcta tcacagggca gaagtgatgc
aaaaaagcag cacctgcagt 240tttatgtatg gggagctgac tgataaagac acgattgaaa
aagtgcggca aacatttgag 300aactatgaga tgaattcctt tgaaattctg atgtacaaga
agaacaggac acctgtgtgg 360ttctttgtga aaattgctcc aattcgaaac gaacaggata
aagtggtttt atttctttgc 420actttcagtg acataacagc tttcaaacag ccaattgagg
atgattcatg taaaggctgg 480gggaagtttg ctcggctgac aagagcactg acaagcagca
ggggtgtcct gcagcagctg 540gctccaagcg tgcaaaaagg cgagaatgtc cacaagcact
cccgcctggc agaggtccta 600cagctgggct cagacatcct tccccagtac aagcaagagg
caccaaagac tccccctcac 660atcatcttac attattgtgt ttttaagacc acgtgggatt
ggatcatctt gatcttgacc 720ttctatacag ccatcttggt cccttataat gtctccttca
aaaccaggca gaataatgtg 780gcctggctgg ttgttgatag catcgtggat gttatctttt
tggtggacat tgtgctcaat 840tttcatacca cctttgttgg accagcaggg gaggtgattt
ctgaccccaa acttatccgc 900atgaactacc tgaagacgtg gtttgtgatt gaccttctgt
cctgtttgcc atatgatgtc 960atcaacgctt ttgagaacgt ggatgagggc atcagcagcc
tgttcagctc tctaaaagtt 1020gtccggctgc tccgtcttgg gcgagtggcc cgtaagctgg
accactacat tgaatatgga 1080gctgctgtgc tggtcctgct ggtgtgtgtg tttgggctgg
ctgcacactg gatggcctgc 1140atctggtaca gcattgggga ctatgagatc tttgacgagg
acaccaagac aatccgcaac 1200aacagctggc tgtaccaact agcgatggac attggcaccc
cttaccagtt taatgggtct 1260ggctcaggga agtgggaagg tggtcccagc aagaattctg
tctacatctc ctcgttgtat 1320ttcacaatga ccagcctcac cagtgtgggc tttgggaaca
tcgccccatc cacagacatt 1380gagaagatct ttgcagtggc catcatgatg attggctcac
ttctctatgc caccatcttc 1440gggaatgtga cgactatttt ccaacagatg tatgccaaca
ccaacagata ccatgagatg 1500ctcaacagtg ttcgggactt cctgaagctc taccaggtgc
caaaaggatt gagtgagcga 1560gtaatggatt atattgtgtc cacttggtcc atgtccagag
gcattgacac agagaaggtc 1620ctgcagatct gccccaagga catgagagcc gacatctgcg
tgcacctgaa ccgcaaggtg 1680ttcaaggagc acccggcctt ccggctggcc agtgatggct
gcctccgggc actggccatg 1740gagttccaga cggtgcactg tgccccaggg gacctcatct
accatgcagg agagagcgtt 1800gacagcctct gctttgtggt ttctggctcc ctggaggtga
tccaagatga tgaggtggtg 1860gccattctag gaaaaggaga cgtgtttgga gatgtgttct
ggaaggaagc cacccttgcc 1920cagtcctgtg ccaatgttag ggccttgacc tactgtgatc
tgcatgtgat caagcgggat 1980gccctgcaga aagtgctgga attctacacg gccttctccc
attccttctc ccggaacctg 2040attctgacgt acaacttgag gaagaggatt gtgttccgga
agatcagcga tgtgaaacgt 2100gaagaggaag aacgcatgaa acgaaagaat gaggcccccc
tgatcttgcc cccggaccac 2160cctgtccggc gcctcttcca gagattccga cagcagaaag
aggccaggct ggcagctgag 2220agagggggcc gggacctgga tgacctagat gtggagaagg
gcaatgtcct tacagagcat 2280gcctccgcca accacagcct cgtgaaggcc agcgtggtca
ccgtgcgtga gagtcctgcc 2340acgcccgtat ccttccaggc agcctccacc tccggggtgc
cagaccacgc aaagctacag 2400gcgccagggt ccgagtgcct gggccccaag gggggcgggg
gcgattgtgc caagcgcaaa 2460agctgggccc gcttcaaaga tgcttgcggg aagagtgagg
actggaacaa ggtgtccaag 2520gctgagtcga tggagacact tcccgagagg acaaaagcgt
caggcgaggc cacactgaag 2580aagacagact cgtgtgacag tggcatcacc aagagcgact
tgcgcctgga caacgtgggt 2640gaggccagga gtccccagga tcggagtccc atcctggcag
aggtcaagca ttcgttctac 2700cccatccctg agcagacgct gcaggccaca gtcctggagg
tgaggcacga gctgaaggag 2760gacatcaagg ccttaaacgc caaaatgacc aatattgaga
aacagctctc tgagatactc 2820aggatattaa cttccagaag atcctctcag tctcctcagg
agttgtttga aatatcgagg 2880ccacagtccc cagaatcaga gagagacatt tttggagcca
gctgagaggt ctatttaaaa 2940aaaaagtcag agacagatac ctccaaccct gccgtcacca
ccacccctac cacccggaat 3000tc
300223083DNAHomo sapiens 2aattccgggc ccgccggacc
ccgagctgct gggaggatga ccatggctgg gggcaggagg 60ggactagtgg cccctcaaaa
cacgtttctg gagaatattg ttcggcggtc caatgatact 120aattttgtgt tggggaatgc
tcagatagtg gactggccta ttgtgtacag caatgatgga 180ttttgcaagc tgtctggcta
tcacagggca gaagtgatgc aaaaaagcag cacctgcagt 240tttatgtatg gggagctgac
tgataaagac acgattgaaa aagtgcggca aacatttgag 300aactatgaga tgaattcctt
tgaaattctg atgtacaaga agaacaggac acctgtgtgg 360ttctttgtga aaattgctcc
aattcgaaac gaacaggata aagtggtttt atttctttgc 420actttcagtg acataacagc
tttcaaacag ccaattgagg atgattcatg taaaggctgg 480gggaagtttg ctcggctgac
aagagcactg acaagcagca ggggtgtcct gcagcagctg 540gctccaagcg tgcaaaaagg
cgagaatgtc cacaagcact cccgcctggc agaggtccta 600cagctgggct cagacatcct
tccccagtac aagcaagagg caccaaagac tccccctcac 660atcatcttac attattgtgt
ttttaagacc acgtgggatt ggatcatctt gatcttgacc 720ttctatacag ccatcttggt
cccttataat gtctccttca aaaccaggca gaataatgtg 780gcctggctgg ttgttgatag
catcgtggat gttatctttt tggtggacat tgtgctcaat 840tttcatacca cctttgttgg
accagcaggg gaggtgattt ctgaccccaa acttatccgc 900atgaactacc tgaagacgtg
gtttgtgatt gaccttctgt cctgtttgcc atatgatgtc 960atcaacgctt ttgagaacgt
ggatgaggtt agtgccttta tgggtgatcc agggaagatt 1020ggttttgctg atcagattcc
accaccactg gaggggagag agagtcaggg catcagcagc 1080ctgttcagct ctctaaaagt
tgtccggctg ctccgtcttg ggcgagtggc ccgtaagctg 1140gaccactaca ttgaatatgg
agctgctgtg ctggtcctgc tggtgtgtgt gtttgggctg 1200gctgcacact ggatggcctg
catctggtac agcattgggg actatgagat ctttgacgag 1260gacaccaaga caatccgcaa
caacagctgg ctgtaccaac tagcgatgga cattggcacc 1320ccttaccagt ttaatgggtc
tggctcaggg aagtgggaag gtggtcccag caagaattct 1380gtctacatct cctcgttgta
tttcacaatg accagcctca ccagtgtggg ctttgggaac 1440atcgccccat ccacagacat
tgagaagatc tttgcagtgg ccatcatgat gattggctca 1500cttctctatg ccaccatctt
cgggaatgtg acgactattt tccaacagat gtatgccaac 1560accaacagat accatgagat
gctcaacagt gttcgggact tcctgaagct ctaccaggtg 1620ccaaaaggat tgagtgagcg
agtaatggat tatattgtgt ccacttggtc catgtccaga 1680ggcattgaca cagagaaggt
cctgcagatc tgccccaagg acatgagagc cgacatctgc 1740gtgcacctga accgcaaggt
gttcaaggag cacccggcct tccggctggc cagtgatggc 1800tgcctccggg cactggccat
ggagttccag acggtgcact gtgccccagg ggacctcatc 1860taccatgcag gagagagcgt
tgacagcctc tgctttgtgg tttctggctc cctggaggtg 1920atccaagatg atgaggtggt
ggccattcta ggaaaaggag acgtgtttgg agatgtgttc 1980tggaaggaag ccacccttgc
ccagtcctgt gccaatgtta gggccttgac ctactgtgat 2040ctgcatgtga tcaagcggga
tgccctgcag aaagtgctgg aattctacac ggccttctcc 2100cattccttct cccggaacct
gattctgacg tacaacttga ggaagaggat tgtgttccgg 2160aagatcagcg atgtgaaacg
tgaagaggaa gaacgcatga aacgaaagaa tgaggccccc 2220ctgatcttgc ccccggacca
ccctgtccgg cgcctcttcc agagattccg acagcagaaa 2280gaggccaggc tggcagctga
gagagggggc cgggacctgg atgacctaga tgtggagaag 2340ggcaatgtcc ttacagagca
tgcctccgcc aaccacagcc tcgtgaaggc cagcgtggtc 2400accgtgcgtg agagtcctgc
cacgcccgta tccttccagg cagcctccac ctccggggtg 2460ccagaccacg caaagctaca
ggcgccaggg tccgagtgcc tgggccccaa ggggggcggg 2520ggcgattgtg ccaagcgcaa
aagctgggcc cgcttcaaag atgcttgcgg gaagagtgag 2580gactggaaca aggtgtccaa
ggctgagtcg atggagacac ttcccgagag gacaaaagcg 2640tcaggcgagg ccacactgaa
gaagacagac tcgtgtgaca gtggcatcac caagagcgac 2700ttgcgcctgg acaacgtggg
tgaggccagg agtccccagg atcggagtcc catcctggca 2760gaggtcaagc attcgttcta
ccccatccct gagcagacgc tgcaggccac agtcctggag 2820gtgaggcacg agctgaagga
ggacatcaag gccttaaacg ccaaaatgac caatattgag 2880aaacagctct ctgagatact
caggatatta acttccagaa gatcctctca gtctcctcag 2940gagttgtttg aaatatcgag
gccacagtcc ccagaatcag agagagacat ttttggagcc 3000agctgagagg tctatttaaa
aaaaaagtca gagacagata cctccaaccc tgccgtcacc 3060accaccccta ccacccggaa
ttc 30833962PRTHomo sapiens
3Met Thr Met Ala Gly Gly Arg Arg Gly Leu Val Ala Pro Gln Asn Thr1
5 10 15Phe Leu Glu Asn Ile Val
Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25
30Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser
Asn Asp Gly 35 40 45Phe Cys Lys
Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50
55 60Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp
Lys Asp Thr Ile65 70 75
80Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu
85 90 95Ile Leu Met Tyr Lys Lys
Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100
105 110Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val
Leu Phe Leu Cys 115 120 125Thr Phe
Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser 130
135 140Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr
Arg Ala Leu Thr Ser145 150 155
160Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln Lys Gly Glu
165 170 175Asn Val His Lys
His Ser Arg Leu Ala Glu Val Leu Gln Leu Gly Ser 180
185 190Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro
Lys Thr Pro Pro His 195 200 205Ile
Ile Leu His Tyr Cys Val Phe Lys Thr Thr Trp Asp Trp Ile Ile 210
215 220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu
Val Pro Tyr Asn Val Ser225 230 235
240Phe Lys Thr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser
Ile 245 250 255Val Asp Val
Ile Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260
265 270Phe Val Gly Pro Ala Gly Glu Val Ile Ser
Asp Pro Lys Leu Ile Arg 275 280
285Met Asn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290
295 300Pro Tyr Asp Val Ile Asn Ala Phe
Glu Asn Val Asp Glu Gly Ile Ser305 310
315 320Ser Leu Phe Ser Ser Leu Lys Val Val Arg Leu Leu
Arg Leu Gly Arg 325 330
335Val Ala Arg Lys Leu Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu
340 345 350Val Leu Leu Val Cys Val
Phe Gly Leu Ala Ala His Trp Met Ala Cys 355 360
365Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile Phe Asp Glu Asp
Thr Lys 370 375 380Thr Ile Arg Asn Asn
Ser Trp Leu Tyr Gln Leu Ala Met Asp Ile Gly385 390
395 400Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser
Gly Lys Trp Glu Gly Gly 405 410
415Pro Ser Lys Asn Ser Val Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr
420 425 430Ser Leu Thr Ser Val
Gly Phe Gly Asn Ile Ala Pro Ser Thr Asp Ile 435
440 445Glu Lys Ile Phe Ala Val Ala Ile Met Met Ile Gly
Ser Leu Leu Tyr 450 455 460Ala Thr Ile
Phe Gly Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala465
470 475 480Asn Thr Asn Arg Tyr His Glu
Met Leu Asn Ser Val Arg Asp Phe Leu 485
490 495Lys Leu Tyr Gln Val Pro Lys Gly Leu Ser Glu Arg
Val Met Asp Tyr 500 505 510Ile
Val Ser Thr Trp Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val 515
520 525Leu Gln Ile Cys Pro Lys Asp Met Arg
Ala Asp Ile Cys Val His Leu 530 535
540Asn Arg Lys Val Phe Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp545
550 555 560Gly Cys Leu Arg
Ala Leu Ala Met Glu Phe Gln Thr Val His Cys Ala 565
570 575Pro Gly Asp Leu Ile Tyr His Ala Gly Glu
Ser Val Asp Ser Leu Cys 580 585
590Phe Val Val Ser Gly Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val
595 600 605Ala Ile Leu Gly Lys Gly Asp
Val Phe Gly Asp Val Phe Trp Lys Glu 610 615
620Ala Thr Leu Ala Gln Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr
Cys625 630 635 640Asp Leu
His Val Ile Lys Arg Asp Ala Leu Gln Lys Val Leu Glu Phe
645 650 655Tyr Thr Ala Phe Ser His Ser
Phe Ser Arg Asn Leu Ile Leu Thr Tyr 660 665
670Asn Leu Arg Lys Arg Ile Val Phe Arg Lys Ile Ser Asp Val
Lys Arg 675 680 685Glu Glu Glu Glu
Arg Met Lys Arg Lys Asn Glu Ala Pro Leu Ile Leu 690
695 700Pro Pro Asp His Pro Val Arg Arg Leu Phe Gln Arg
Phe Arg Gln Gln705 710 715
720Lys Glu Ala Arg Leu Ala Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp
725 730 735Leu Asp Val Glu Lys
Gly Asn Val Leu Thr Glu His Ala Ser Ala Asn 740
745 750His Ser Leu Val Lys Ala Ser Val Val Thr Val Arg
Glu Ser Pro Ala 755 760 765Thr Pro
Val Ser Phe Gln Ala Ala Ser Thr Ser Gly Val Pro Asp His 770
775 780Ala Lys Leu Gln Ala Pro Gly Ser Glu Cys Leu
Gly Pro Lys Gly Gly785 790 795
800Gly Gly Asp Cys Ala Lys Arg Lys Ser Trp Ala Arg Phe Lys Asp Ala
805 810 815Cys Gly Lys Ser
Glu Asp Trp Asn Lys Val Ser Lys Ala Glu Ser Met 820
825 830Glu Thr Leu Pro Glu Arg Thr Lys Ala Ser Gly
Glu Ala Thr Leu Lys 835 840 845Lys
Thr Asp Ser Cys Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu 850
855 860Asp Asn Val Gly Glu Ala Arg Ser Pro Gln
Asp Arg Ser Pro Ile Leu865 870 875
880Ala Glu Val Lys His Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu
Gln 885 890 895Ala Thr Val
Leu Glu Val Arg His Glu Leu Lys Glu Asp Ile Lys Ala 900
905 910Leu Asn Ala Lys Met Thr Asn Ile Glu Lys
Gln Leu Ser Glu Ile Leu 915 920
925Arg Ile Leu Thr Ser Arg Arg Ser Ser Gln Ser Pro Gln Glu Leu Phe 930
935 940Glu Ile Ser Arg Pro Gln Ser Pro
Glu Ser Glu Arg Asp Ile Phe Gly945 950
955 960Ala Ser4989PRTHomo sapiens 4Met Thr Met Ala Gly
Gly Arg Arg Gly Leu Val Ala Pro Gln Asn Thr1 5
10 15Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp
Thr Asn Phe Val Leu 20 25
30Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly
35 40 45Phe Cys Lys Leu Ser Gly Tyr His
Arg Ala Glu Val Met Gln Lys Ser 50 55
60Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile65
70 75 80Glu Lys Val Arg Gln
Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85
90 95Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val
Trp Phe Phe Val Lys 100 105
110Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys
115 120 125Thr Phe Ser Asp Ile Thr Ala
Phe Lys Gln Pro Ile Glu Asp Asp Ser 130 135
140Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr
Ser145 150 155 160Ser Arg
Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln Lys Gly Glu
165 170 175Asn Val His Lys His Ser Arg
Leu Ala Glu Val Leu Gln Leu Gly Ser 180 185
190Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro Lys Thr Pro
Pro His 195 200 205Ile Ile Leu His
Tyr Cys Val Phe Lys Thr Thr Trp Asp Trp Ile Ile 210
215 220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu Val Pro
Tyr Asn Val Ser225 230 235
240Phe Lys Thr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser Ile
245 250 255Val Asp Val Ile Phe
Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260
265 270Phe Val Gly Pro Ala Gly Glu Val Ile Ser Asp Pro
Lys Leu Ile Arg 275 280 285Met Asn
Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290
295 300Pro Tyr Asp Val Ile Asn Ala Phe Glu Asn Val
Asp Glu Val Ser Ala305 310 315
320Phe Met Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp Gln Ile Pro Pro
325 330 335Pro Leu Glu Gly
Arg Glu Ser Gln Gly Ile Ser Ser Leu Phe Ser Ser 340
345 350Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg
Val Ala Arg Lys Leu 355 360 365Asp
His Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val Leu Leu Val Cys 370
375 380Val Phe Gly Leu Ala Ala His Trp Met Ala
Cys Ile Trp Tyr Ser Ile385 390 395
400Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys Thr Ile Arg Asn
Asn 405 410 415Ser Trp Leu
Tyr Gln Leu Ala Met Asp Ile Gly Thr Pro Tyr Gln Phe 420
425 430Asn Gly Ser Gly Ser Gly Lys Trp Glu Gly
Gly Pro Ser Lys Asn Ser 435 440
445Val Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr Ser Leu Thr Ser Val 450
455 460Gly Phe Gly Asn Ile Ala Pro Ser
Thr Asp Ile Glu Lys Ile Phe Ala465 470
475 480Val Ala Ile Met Met Ile Gly Ser Leu Leu Tyr Ala
Thr Ile Phe Gly 485 490
495Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala Asn Thr Asn Arg Tyr
500 505 510His Glu Met Leu Asn Ser
Val Arg Asp Phe Leu Lys Leu Tyr Gln Val 515 520
525Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr Ile Val Ser
Thr Trp 530 535 540Ser Met Ser Arg Gly
Ile Asp Thr Glu Lys Val Leu Gln Ile Cys Pro545 550
555 560Lys Asp Met Arg Ala Asp Ile Cys Val His
Leu Asn Arg Lys Val Phe 565 570
575Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp Gly Cys Leu Arg Ala
580 585 590Leu Ala Met Glu Phe
Gln Thr Val His Cys Ala Pro Gly Asp Leu Ile 595
600 605Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys Phe
Val Val Ser Gly 610 615 620Ser Leu Glu
Val Ile Gln Asp Asp Glu Val Val Ala Ile Leu Gly Lys625
630 635 640Gly Asp Val Phe Gly Asp Val
Phe Trp Lys Glu Ala Thr Leu Ala Gln 645
650 655Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys Asp
Leu His Val Ile 660 665 670Lys
Arg Asp Ala Leu Gln Lys Val Leu Glu Phe Tyr Thr Ala Phe Ser 675
680 685His Ser Phe Ser Arg Asn Leu Ile Leu
Thr Tyr Asn Leu Arg Lys Arg 690 695
700Ile Val Phe Arg Lys Ile Ser Asp Val Lys Arg Glu Glu Glu Glu Arg705
710 715 720Met Lys Arg Lys
Asn Glu Ala Pro Leu Ile Leu Pro Pro Asp His Pro 725
730 735Val Arg Arg Leu Phe Gln Arg Phe Arg Gln
Gln Lys Glu Ala Arg Leu 740 745
750Ala Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp Leu Asp Val Glu Lys
755 760 765Gly Asn Val Leu Thr Glu His
Ala Ser Ala Asn His Ser Leu Val Lys 770 775
780Ala Ser Val Val Thr Val Arg Glu Ser Pro Ala Thr Pro Val Ser
Phe785 790 795 800Gln Ala
Ala Ser Thr Ser Gly Val Pro Asp His Ala Lys Leu Gln Ala
805 810 815Pro Gly Ser Glu Cys Leu Gly
Pro Lys Gly Gly Gly Gly Asp Cys Ala 820 825
830Lys Arg Lys Ser Trp Ala Arg Phe Lys Asp Ala Cys Gly Lys
Ser Glu 835 840 845Asp Trp Asn Lys
Val Ser Lys Ala Glu Ser Met Glu Thr Leu Pro Glu 850
855 860Arg Thr Lys Ala Ser Gly Glu Ala Thr Leu Lys Lys
Thr Asp Ser Cys865 870 875
880Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu Asp Asn Val Gly Glu
885 890 895Ala Arg Ser Pro Gln
Asp Arg Ser Pro Ile Leu Ala Glu Val Lys His 900
905 910Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu Gln Ala
Thr Val Leu Glu 915 920 925Val Arg
His Glu Leu Lys Glu Asp Ile Lys Ala Leu Asn Ala Lys Met 930
935 940Thr Asn Ile Glu Lys Gln Leu Ser Glu Ile Leu
Arg Ile Leu Thr Ser945 950 955
960Arg Arg Ser Ser Gln Ser Pro Gln Glu Leu Phe Glu Ile Ser Arg Pro
965 970 975Gln Ser Pro Glu
Ser Glu Arg Asp Ile Phe Gly Ala Ser 980
985518DNAArtificial SequenceDescription of Artificial Sequence Synthetic
DNA 5ccaaacacac acaccagc
18620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 6cgtggatgtt atctttttgg
20718DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 7gggaggatga ccatggct
18820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic DNA 8cagaayaayg tggcntggct
20919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic DNA
9tcactraaga tctatartc
191021DNAArtificial SequenceDescription of Artificial Sequence Synthetic
DNA 10cgcatgaact acctgaagac g
211121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 11tctgtggatg gggcgatgtt c
211218DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 12gggaggatga ccatggct
18132886DNAHomo sapiens 13atgaccatgg
ctgggggcag gaggggacta gtggcccctc aaaacacgtt tctggagaat 60attgttcggc
ggtccaatga tactaatttt gtgttgggga atgctcagat agtggactgg 120cctattgtgt
acagcaatga tggattttgc aagctgtctg gctatcacag ggcagaagtg 180atgcaaaaaa
gcagcacctg cagttttatg tatggggagc tgactgataa agacacgatt 240gaaaaagtgc
ggcaaacatt tgagaactat gagatgaatt cctttgaaat tctgatgtac 300aagaagaaca
ggacacctgt gtggttcttt gtgaaaattg ctccaattcg aaacgaacag 360gataaagtgg
ttttatttct ttgcactttc agtgacataa cagctttcaa acagccaatt 420gaggatgatt
catgtaaagg ctgggggaag tttgctcggc tgacaagagc actgacaagc 480agcaggggtg
tcctgcagca gctggctcca agcgtgcaaa aaggcgagaa tgtccacaag 540cactcccgcc
tggcagaggt cctacagctg ggctcagaca tccttcccca gtacaagcaa 600gaggcaccaa
agactccccc tcacatcatc ttacattatt gtgtttttaa gaccacgtgg 660gattggatca
tcttgatctt gaccttctat acagccatct tggtccctta taatgtctcc 720ttcaaaacca
ggcagaataa tgtggcctgg ctggttgttg atagcatcgt ggatgttatc 780tttttggtgg
acattgtgct caattttcat accacctttg ttggaccagc aggggaggtg 840atttctgacc
ccaaacttat ccgcatgaac tacctgaaga cgtggtttgt gattgacctt 900ctgtcctgtt
tgccatatga tgtcatcaac gcttttgaga acgtggatga gggcatcagc 960agcctgttca
gctctctaaa agttgtccgg ctgctccgtc ttgggcgagt ggcccgtaag 1020ctggaccact
acattgaata tggagctgct gtgctggtcc tgctggtgtg tgtgtttggg 1080ctggctgcac
actggatggc ctgcatctgg tacagcattg gggactatga gatctttgac 1140gaggacacca
agacaatccg caacaacagc tggctgtacc aactagcgat ggacattggc 1200accccttacc
agtttaatgg gtctggctca gggaagtggg aaggtggtcc cagcaagaat 1260tctgtctaca
tctcctcgtt gtatttcaca atgaccagcc tcaccagtgt gggctttggg 1320aacatcgccc
catccacaga cattgagaag atctttgcag tggccatcat gatgattggc 1380tcacttctct
atgccaccat cttcgggaat gtgacgacta ttttccaaca gatgtatgcc 1440aacaccaaca
gataccatga gatgctcaac agtgttcggg acttcctgaa gctctaccag 1500gtgccaaaag
gattgagtga gcgagtaatg gattatattg tgtccacttg gtccatgtcc 1560agaggcattg
acacagagaa ggtcctgcag atctgcccca aggacatgag agccgacatc 1620tgcgtgcacc
tgaaccgcaa ggtgttcaag gagcacccgg ccttccggct ggccagtgat 1680ggctgcctcc
gggcactggc catggagttc cagacggtgc actgtgcccc aggggacctc 1740atctaccatg
caggagagag cgttgacagc ctctgctttg tggtttctgg ctccctggag 1800gtgatccaag
atgatgaggt ggtggccatt ctaggaaaag gagacgtgtt tggagatgtg 1860ttctggaagg
aagccaccct tgcccagtcc tgtgccaatg ttagggcctt gacctactgt 1920gatctgcatg
tgatcaagcg ggatgccctg cagaaagtgc tggaattcta cacggccttc 1980tcccattcct
tctcccggaa cctgattctg acgtacaact tgaggaagag gattgtgttc 2040cggaagatca
gcgatgtgaa acgtgaagag gaagaacgca tgaaacgaaa gaatgaggcc 2100cccctgatct
tgcccccgga ccaccctgtc cggcgcctct tccagagatt ccgacagcag 2160aaagaggcca
ggctggcagc tgagagaggg ggccgggacc tggatgacct agatgtggag 2220aagggcaatg
tccttacaga gcatgcctcc gccaaccaca gcctcgtgaa ggccagcgtg 2280gtcaccgtgc
gtgagagtcc tgccacgccc gtatccttcc aggcagcctc cacctccggg 2340gtgccagacc
acgcaaagct acaggcgcca gggtccgagt gcctgggccc caaggggggc 2400gggggcgatt
gtgccaagcg caaaagctgg gcccgcttca aagatgcttg cgggaagagt 2460gaggactgga
acaaggtgtc caaggctgag tcgatggaga cacttcccga gaggacaaaa 2520gcgtcaggcg
aggccacact gaagaagaca gactcgtgtg acagtggcat caccaagagc 2580gacttgcgcc
tggacaacgt gggtgaggcc aggagtcccc aggatcggag tcccatcctg 2640gcagaggtca
agcattcgtt ctaccccatc cctgagcaga cgctgcaggc cacagtcctg 2700gaggtgaggc
acgagctgaa ggaggacatc aaggccttaa acgccaaaat gaccaatatt 2760gagaaacagc
tctctgagat actcaggata ttaacttcca gaagatcctc tcagtctcct 2820caggagttgt
ttgaaatatc gaggccacag tccccagaat cagagagaga catttttgga 2880gccagc
2886142967DNAHomo
sapiens 14atgaccatgg ctgggggcag gaggggacta gtggcccctc aaaacacgtt
tctggagaat 60attgttcggc ggtccaatga tactaatttt gtgttgggga atgctcagat
agtggactgg 120cctattgtgt acagcaatga tggattttgc aagctgtctg gctatcacag
ggcagaagtg 180atgcaaaaaa gcagcacctg cagttttatg tatggggagc tgactgataa
agacacgatt 240gaaaaagtgc ggcaaacatt tgagaactat gagatgaatt cctttgaaat
tctgatgtac 300aagaagaaca ggacacctgt gtggttcttt gtgaaaattg ctccaattcg
aaacgaacag 360gataaagtgg ttttatttct ttgcactttc agtgacataa cagctttcaa
acagccaatt 420gaggatgatt catgtaaagg ctgggggaag tttgctcggc tgacaagagc
actgacaagc 480agcaggggtg tcctgcagca gctggctcca agcgtgcaaa aaggcgagaa
tgtccacaag 540cactcccgcc tggcagaggt cctacagctg ggctcagaca tccttcccca
gtacaagcaa 600gaggcaccaa agactccccc tcacatcatc ttacattatt gtgtttttaa
gaccacgtgg 660gattggatca tcttgatctt gaccttctat acagccatct tggtccctta
taatgtctcc 720ttcaaaacca ggcagaataa tgtggcctgg ctggttgttg atagcatcgt
ggatgttatc 780tttttggtgg acattgtgct caattttcat accacctttg ttggaccagc
aggggaggtg 840atttctgacc ccaaacttat ccgcatgaac tacctgaaga cgtggtttgt
gattgacctt 900ctgtcctgtt tgccatatga tgtcatcaac gcttttgaga acgtggatga
ggttagtgcc 960tttatgggtg atccagggaa gattggtttt gctgatcaga ttccaccacc
actggagggg 1020agagagagtc agggcatcag cagcctgttc agctctctaa aagttgtccg
gctgctccgt 1080cttgggcgag tggcccgtaa gctggaccac tacattgaat atggagctgc
tgtgctggtc 1140ctgctggtgt gtgtgtttgg gctggctgca cactggatgg cctgcatctg
gtacagcatt 1200ggggactatg agatctttga cgaggacacc aagacaatcc gcaacaacag
ctggctgtac 1260caactagcga tggacattgg caccccttac cagtttaatg ggtctggctc
agggaagtgg 1320gaaggtggtc ccagcaagaa ttctgtctac atctcctcgt tgtatttcac
aatgaccagc 1380ctcaccagtg tgggctttgg gaacatcgcc ccatccacag acattgagaa
gatctttgca 1440gtggccatca tgatgattgg ctcacttctc tatgccacca tcttcgggaa
tgtgacgact 1500attttccaac agatgtatgc caacaccaac agataccatg agatgctcaa
cagtgttcgg 1560gacttcctga agctctacca ggtgccaaaa ggattgagtg agcgagtaat
ggattatatt 1620gtgtccactt ggtccatgtc cagaggcatt gacacagaga aggtcctgca
gatctgcccc 1680aaggacatga gagccgacat ctgcgtgcac ctgaaccgca aggtgttcaa
ggagcacccg 1740gccttccggc tggccagtga tggctgcctc cgggcactgg ccatggagtt
ccagacggtg 1800cactgtgccc caggggacct catctaccat gcaggagaga gcgttgacag
cctctgcttt 1860gtggtttctg gctccctgga ggtgatccaa gatgatgagg tggtggccat
tctaggaaaa 1920ggagacgtgt ttggagatgt gttctggaag gaagccaccc ttgcccagtc
ctgtgccaat 1980gttagggcct tgacctactg tgatctgcat gtgatcaagc gggatgccct
gcagaaagtg 2040ctggaattct acacggcctt ctcccattcc ttctcccgga acctgattct
gacgtacaac 2100ttgaggaaga ggattgtgtt ccggaagatc agcgatgtga aacgtgaaga
ggaagaacgc 2160atgaaacgaa agaatgaggc ccccctgatc ttgcccccgg accaccctgt
ccggcgcctc 2220ttccagagat tccgacagca gaaagaggcc aggctggcag ctgagagagg
gggccgggac 2280ctggatgacc tagatgtgga gaagggcaat gtccttacag agcatgcctc
cgccaaccac 2340agcctcgtga aggccagcgt ggtcaccgtg cgtgagagtc ctgccacgcc
cgtatccttc 2400caggcagcct ccacctccgg ggtgccagac cacgcaaagc tacaggcgcc
agggtccgag 2460tgcctgggcc ccaagggggg cgggggcgat tgtgccaagc gcaaaagctg
ggcccgcttc 2520aaagatgctt gcgggaagag tgaggactgg aacaaggtgt ccaaggctga
gtcgatggag 2580acacttcccg agaggacaaa agcgtcaggc gaggccacac tgaagaagac
agactcgtgt 2640gacagtggca tcaccaagag cgacttgcgc ctggacaacg tgggtgaggc
caggagtccc 2700caggatcgga gtcccatcct ggcagaggtc aagcattcgt tctaccccat
ccctgagcag 2760acgctgcagg ccacagtcct ggaggtgagg cacgagctga aggaggacat
caaggcctta 2820aacgccaaaa tgaccaatat tgagaaacag ctctctgaga tactcaggat
attaacttcc 2880agaagatcct ctcagtctcc tcaggagttg tttgaaatat cgaggccaca
gtccccagaa 2940tcagagagag acatttttgg agccagc
29671519DNAArtificial SequenceDescription of Artificial
Sequence Antisense phosphorothioate ODN 15cagccatggt catcctccc
191619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic scrambled
sequence 16gtcggtacca gtaggaggg
191721DNAArtificial SequenceDescription of Artificial Sequence
Primer 17tcagcccagc agaagcatta t
211818DNAArtificial SequenceDescription of Artificial Sequence
Primer 18ctggcagcgt gtgagagc
18193041DNABovine sp. 19gtgccgggac gccccccaga ccccgagctg ccgggaggat
gaccatggct gggggcagga 60agggactggt ggccccgcaa aacacgtttc tggagaatat
tgtccggcgg tccaatgata 120ctaactttgt tttggggaat gcccagatag tggactggcc
tatcgtgtac agcaatgatg 180gattttgcaa gctgtctggc tatcacaggg cggaagtgat
gcaaaaaagc agtacatgca 240gttttatgta tggggagctg accgataaag ataccattga
aaaagtgcgg caaacctttg 300agaactatga gatgaattcc tttgaaattc tgatgtacaa
gaagaacagg acacctgtgt 360ggttctttgt gaaaattgct ccaattcgaa acgaacagga
taaagtggtt ttatttcttt 420gcactttcag tgacataacc gctttcaaac agccgattga
agatgattca tgtaaaggct 480gggggaagtt cgctcggctg accagagcac tgacgagcag
ccggggtgtc ctgcagcagc 540tggctcccag cgtgcagaaa ggcgagaacg tccacaagca
ctcccgtctg gccgaggttc 600tgcagctggg ctcagacatc cttccccagt acaagcaaga
ggcaccaaag actcccccgc 660acatcatctt acactactgc gtttttaaga ccacgtggga
ctggatcatc ctgatcctaa 720ccttctacac agccatcctg gttccttaca acgtctcctt
taaaaccagg cagaacaacg 780tggcctggct ggttgtggac agcatcgtgg atgtcatttt
tttggtggac attgtgctga 840attttcacac cacttttgtt ggacccgctg gggaggtgat
ttctgacccc aaactcattc 900gcatgaacta cctgaagacg tggtttgtga ttgaccttct
gtcctgtttg ccctatgacg 960tcatcaacgc ttttgagaac gtggatgagg gcatcagcag
cctgttcagc tctctgaaag 1020ttgtccggct gctccgcctg ggacgcgtgg cccggaagct
ggaccactac atcgagtatg 1080gagctgccgt gctggtcctg ctggtgtgtg tgttcgggct
ggccgctcac tggatggcct 1140gcatttggta cagcatcggg gactatgaga tcttcgacga
ggacaccaag accatccgca 1200acaacagctg gctctaccag ctggccatgg acattggcac
cccttaccag tttaacgggt 1260ctggctcagg gaagtgggaa gggggtccca gcaagaattc
cgtctacatc tcctcgttgt 1320atttcaccat gaccagcctc accagcgtgg gctttgggaa
catcgccccg tccacagaca 1380ttgagaagat ctttgccgtg gccatcatga tgattggctc
cctcctctat gccaccatct 1440ttgggaatgt gacgaccatt ttccaacaga tgtacgccaa
caccaacagg taccatgaga 1500tgctcaacag tgtccgggac ttcttgaagc tctaccaggt
gcccaagggg ctgagcgagc 1560gagtcatgga ttacatcgtg tccacctggt ccatgtccag
aggcattgac acagagaagg 1620tcctgcagat ctgccccaag gacatgagag cggacatctg
cgtgcaccta aaccgcaagg 1680tcttcaagga gcacccagcc tttcggctgg ccagcgacgg
ctgcctgcgg gcactggcca 1740tggagttcca gacggtgcac tgcgcccctg gggacctcat
ctaccacgca ggggagagcg 1800tcgacagcct gtgcttcgtg gtctccggct ccctggaggt
gatccaggat gacgaggtgg 1860tggccattct agggaaagga gacgtgtttg gagacgtgtt
ctggaaggaa gccacccttg 1920cccagtcctg tgccaatgtg agggccttga cctactgtga
cctccatgtg atcaagcggg 1980acgccctgca gaaagtgctg gaattctaca cagccttctc
ccactccttc tcccggaacc 2040tcattctcac ctacaacttg aggaagcgga tcgtgttccg
gaagatcagt gacgtgaaac 2100gggaggagga ggagcgcatg aagcggaaga atgaggcccc
cctgatcctg ccgcccgacc 2160accccgtccg gcggctcttc cagaggttcc gccagcagaa
ggaagccagg ctggccgcgg 2220agaggggcgg gcgggacttg gacgacctgg acgtggagaa
gggcagcgtc ctcaccgagc 2280acagccacca cggcctggcg aaggccagcg tcgtcaccgt
ccgagagagc cctgccacgc 2340ccgtggcctt cccggcggcc gctgccccgg cggggctgga
tcacgcccgg ctgcaggcgc 2400ctggggccga gggcctgggc cccaaggccg gcggggccga
ctgcgccaag cgcaagggct 2460gggcccgctt caaggatgcc tgcgggcagg ctgaggactg
gagcaaggtg tccaaggccg 2520agtccatgga aacgctcccc gagaggacga aggccgccgg
cgaggccaca ctcaagaaga 2580cggactcgtg cgacagcggc atcaccaaga gcgacctgcg
tctggacaac gtgggcgagg 2640ccagaagccc ccaggaccgg agccccatct tggcggaggt
caagcactcc ttctacccca 2700tccccgagca gacgctgcag gccgccgtcc tggaggtgaa
gcacgagctc aaggaggaca 2760tcaaggcctt gagcaccaag atgacgagca ttgagaaaca
gctctctgag atactcagga 2820tattaacctc cagaagatcc tctcagtcgc ctcaggagct
atttgaaata tcgaggcccc 2880agtccccaga gtcagagaga gacatttttg gcgcaagctg
agaggtctgt tgtaaaaaaa 2940aagaaaaaaa atccaagatg acaaaaacct accgtcctgc
cctagacacc accacacaca 3000cacctacatg accaacaacc ttcaaagtag gcttttccca a
3041203041DNARattus sp. 20tgcggtgaga cacggcgccg
gacgccccca gagccccagc agtagggagg atgaccatgg 60ctggcggccg gcggggacta
gtggccccgc agaacacatt tctggagaac atcgtgcggc 120ggtccaacga cactaatttt
gtgttgggga atgcccagat cgtggactgg cccatcgtgt 180acagcaatga tggattctgc
aagctgtctg gctaccaccg agcggaagtg atgcaaaaga 240gtagcgcctg cagttttatg
tatggagagc tgaccgacaa ggacacggtt gaaaaggttc 300gccagacctt tgagaactac
gagatgaact ccttcgaaat tctgatgtac aagaagaaca 360ggacacctgt gtggtttttt
gtgaagatcg ctccaatcag gaacgaacag gataaagtgg 420ttctgttcct ttgcactttc
agtgacataa cggcattcaa gcagcccatt gaggacgact 480cctgcaaagg ttgggggaag
tttgctcgac tgacgagagc tctgacaagc agcaggggag 540tcctgcagca gctggccccc
agtgtgcaga agggtgagaa tgttcacaag cactcgcgcc 600tggcagaggt cctgcagctg
ggttcagaca tcctccccca gtacaagcaa gaggcgccaa 660agacaccccc tcacatcatc
ctacactact gtgtctttaa gaccacatgg gattggatca 720tcttgatcct gaccttctac
acagccatcc tggtccctta caacgtctcc tttaaaacca 780ggcagaataa cgtggcctgg
ctggtggtgg acagcatcgt ggatgtcatc tttttggtgg 840acattgtctt gaattttcac
accacctttg tcgggccagc gggggaagtg atctctgacc 900ccaaacttat ccgcatgaac
tacctgaaga cgtggtttgt gatcgacctt ctctcctgtt 960tgccatatga cgtcatcaac
gcttttgaga acgtggatga gggcatcagc agcctgttca 1020gttctctgaa agtcgtgcgg
ctgctccgtc tcggacgagt ggcccgcaag ctggaccatt 1080atatcgagta cggagcggcg
gtactggtcc tgctggtgtg cgtgttcggg ctggctgccc 1140actggatggc ctgcatctgg
tacagcattg gggattatga gatctttgat gaagacacca 1200agaccatccg taacaacagc
tggctctacc aactggcatt ggacattggc actccatacc 1260agtttaatgg gtctggttcg
gggaagtggg aaggcgggcc aagcaagaac tccgtataca 1320tttcctcgct gtacttcacc
atgacaagtc tcaccagtgt gggctttggt aacatcgccc 1380catccacaga catcgagaag
atcttcgccg tagccatcat gatgattggc tcccttctgt 1440atgccaccat ctttgggaat
gtgacgacca ttttccagca gatgtatgcc aacaccaaca 1500ggtatcatga gatgctcaac
agcgtccggg atttcctgaa gctctaccag gtgcccaagg 1560ggctgagcga gcgggtcatg
gactacattg tgtctacctg gtccatgtcc cgcggcatcg 1620acacggagaa ggtcctgcaa
atctgcccca aggacatgcg agctgacatt tgcgtacacc 1680tgaaccgaaa agtgttcaaa
gaacaccccg ccttccggct ggccagcgat ggttgcctga 1740gggccttggc catggagttc
cagacagtac actgcgcccc aggggacctc atctatcacg 1800ccggggagag tgtggacagc
ctctgcttcg tggtctcggg ctccctggag gtgatccagg 1860atgatgaggt ggtggccatc
ctagggaaag gagatgtgtt tggggatgtt ttctggaagg 1920aggctaccct tgcacagtcc
tgcgctaatg tccgggcctt gacctactgt gacctgcacg 1980tgatcaagag ggatgccctg
cagaaagtgc tagaattcta cacagccttc tcccactcct 2040tctcccggaa cctgattctc
acctacaatc tgaggaagag gattgtgttc cggaagatca 2100gcgacgtgaa acgagaagaa
gaggagagga tgaaacggaa gaacgaggcc ccccttatcc 2160tgcctcctga ccaccctgtc
aggaggctct tccaaaggtt ccgccagcag aaagaagcca 2220ggctggcagc cgagagaggt
ggccgggacc tggatgacct ggatgtagag aagggcaatg 2280ccctcacgga ccatacctca
gccaaccaca gcctggtgaa ggccagtgtg gtcacggtgc 2340gtgagagtcc cgccacgcct
gtgtccttcc aggcagcctc cacctccaca gtgtcagacc 2400acgccaagct gcatgcaccg
ggatctgagt gcctaggtcc caaggcaggc ggtggcgacc 2460ctgccaagcg caaaggctgg
gcccggttca aagatgcctg tgggaagggt gaggattgga 2520acaaggtgtc caaggcagag
tccatggaga cgcttcccga gaggacaaag gcatcgggcg 2580aggccacgct gaagaagaca
gactcctgtg acagtggaat caccaagagt gacctgcgct 2640tggacaatgt gggtgaggcc
aggagtcccc aggaccggag ccccatcttg gccgaggtca 2700agcattcttt ctaccccatc
cccgagcaga cactgcaggc cacagtgctg gaggtgaagc 2760atgagctgaa ggaagacatc
aaggccttga atgccaaaat gacctccatt gagaagcagc 2820tgtctgagat cctcaggata
ctcatgtcca gagggtcctc ccagtctccg caggacacgt 2880gtgaggtctc caggccccag
tccccagagt cagacagaga catttttggg gcaagctgag 2940aggatcattt caaaacaaac
aaacaaaaaa atcaaagaca aaagcctgcc ccctgcccct 3000gacacttcct accgcaccaa
acacatgacc aacaactttc a 304121960PRTBovine sp.
21Met Thr Met Ala Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr1
5 10 15Phe Leu Glu Asn Ile Val
Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25
30Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser
Asn Asp Gly 35 40 45Phe Cys Lys
Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50
55 60Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp
Lys Asp Thr Ile65 70 75
80Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu
85 90 95Ile Leu Met Tyr Lys Lys
Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100
105 110Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val
Leu Phe Leu Cys 115 120 125Thr Phe
Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser 130
135 140Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr
Arg Ala Leu Thr Ser145 150 155
160Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln Lys Gly Glu
165 170 175Asn Val His Lys
His Ser Arg Leu Ala Glu Val Leu Gln Leu Gly Ser 180
185 190Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro
Lys Thr Pro Pro His 195 200 205Ile
Ile Leu His Tyr Cys Val Phe Lys Thr Thr Trp Asp Trp Ile Ile 210
215 220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu
Val Pro Tyr Asn Val Ser225 230 235
240Phe Lys Thr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser
Ile 245 250 255Val Asp Val
Ile Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260
265 270Phe Val Gly Pro Ala Gly Glu Val Ile Ser
Asp Pro Lys Leu Ile Arg 275 280
285Met Asn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290
295 300Pro Tyr Asp Val Ile Asn Ala Phe
Glu Asn Val Asp Glu Gly Ile Ser305 310
315 320Ser Leu Phe Ser Ser Leu Lys Val Val Arg Leu Leu
Arg Leu Gly Arg 325 330
335Val Ala Arg Lys Leu Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu
340 345 350Val Leu Leu Val Cys Val
Phe Gly Leu Ala Ala His Trp Met Ala Cys 355 360
365Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile Phe Asp Glu Asp
Thr Lys 370 375 380Thr Ile Arg Asn Asn
Ser Trp Leu Tyr Gln Leu Ala Met Asp Ile Gly385 390
395 400Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser
Gly Lys Trp Glu Gly Gly 405 410
415Pro Ser Lys Asn Ser Val Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr
420 425 430Ser Leu Thr Ser Val
Gly Phe Gly Asn Ile Ala Pro Ser Thr Asp Ile 435
440 445Glu Lys Ile Phe Ala Val Ala Ile Met Met Ile Gly
Ser Leu Leu Tyr 450 455 460Ala Thr Ile
Phe Gly Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala465
470 475 480Asn Thr Asn Arg Tyr His Glu
Met Leu Asn Ser Val Arg Asp Phe Leu 485
490 495Lys Leu Tyr Gln Val Pro Lys Gly Leu Ser Glu Arg
Val Met Asp Tyr 500 505 510Ile
Val Ser Thr Trp Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val 515
520 525Leu Gln Ile Cys Pro Lys Asp Met Arg
Ala Asp Ile Cys Val His Leu 530 535
540Asn Arg Lys Val Phe Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp545
550 555 560Gly Cys Leu Arg
Ala Leu Ala Met Glu Phe Gln Thr Val His Cys Ala 565
570 575Pro Gly Asp Leu Ile Tyr His Ala Gly Glu
Ser Val Asp Ser Leu Cys 580 585
590Phe Val Val Ser Gly Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val
595 600 605Ala Ile Leu Gly Lys Gly Asp
Val Phe Gly Asp Val Phe Trp Lys Glu 610 615
620Ala Thr Leu Ala Gln Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr
Cys625 630 635 640Asp Leu
His Val Ile Lys Arg Asp Ala Leu Gln Lys Val Leu Glu Phe
645 650 655Tyr Thr Ala Phe Ser His Ser
Phe Ser Arg Asn Leu Ile Leu Thr Tyr 660 665
670Asn Leu Arg Lys Arg Ile Val Phe Arg Lys Ile Ser Asp Val
Lys Arg 675 680 685Glu Glu Glu Glu
Arg Met Lys Arg Lys Asn Glu Ala Pro Leu Ile Leu 690
695 700Pro Pro Asp His Pro Val Arg Arg Leu Phe Gln Arg
Phe Arg Gln Gln705 710 715
720Lys Glu Ala Arg Leu Ala Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp
725 730 735Leu Asp Val Glu Lys
Gly Ser Val Leu Thr Glu His Ser His His Gly 740
745 750Leu Ala Lys Ala Ser Val Val Thr Val Arg Glu Ser
Pro Ala Thr Pro 755 760 765Val Ala
Phe Pro Ala Ala Ala Ala Pro Ala Gly Leu Asp His Ala Arg 770
775 780Leu Gln Ala Pro Gly Ala Glu Gly Leu Gly Pro
Lys Ala Gly Gly Ala785 790 795
800Asp Cys Ala Lys Arg Lys Gly Trp Ala Arg Phe Lys Asp Ala Cys Gly
805 810 815Gln Ala Glu Asp
Trp Ser Lys Val Ser Lys Ala Glu Ser Met Glu Thr 820
825 830Leu Pro Glu Arg Thr Lys Ala Ala Gly Glu Ala
Thr Leu Lys Lys Thr 835 840 845Asp
Ser Cys Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu Asp Asn 850
855 860Val Gly Glu Ala Arg Ser Pro Gln Asp Arg
Ser Pro Ile Leu Ala Glu865 870 875
880Val Lys His Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu Gln Ala
Ala 885 890 895Val Leu Glu
Val Lys His Glu Leu Lys Glu Asp Ile Lys Ala Leu Ser 900
905 910Thr Lys Met Thr Ser Ile Glu Lys Gln Leu
Ser Glu Ile Leu Arg Ile 915 920
925Leu Thr Ser Arg Arg Ser Ser Gln Ser Pro Gln Glu Leu Phe Glu Ile 930
935 940Ser Arg Pro Gln Ser Pro Glu Ser
Glu Arg Asp Ile Phe Gly Ala Ser945 950
955 96022987PRTBovine sp. 22Met Thr Met Ala Gly Gly Arg
Lys Gly Leu Val Ala Pro Gln Asn Thr1 5 10
15Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn
Phe Val Leu 20 25 30Gly Asn
Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35
40 45Phe Cys Lys Leu Ser Gly Tyr His Arg Ala
Glu Val Met Gln Lys Ser 50 55 60Ser
Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile65
70 75 80Glu Lys Val Arg Gln Thr
Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85
90 95Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp
Phe Phe Val Lys 100 105 110Ile
Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys 115
120 125Thr Phe Ser Asp Ile Thr Ala Phe Lys
Gln Pro Ile Glu Asp Asp Ser 130 135
140Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr Ser145
150 155 160Ser Arg Gly Val
Leu Gln Gln Leu Ala Pro Ser Val Gln Lys Gly Glu 165
170 175Asn Val His Lys His Ser Arg Leu Ala Glu
Val Leu Gln Leu Gly Ser 180 185
190Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro Lys Thr Pro Pro His
195 200 205Ile Ile Leu His Tyr Cys Val
Phe Lys Thr Thr Trp Asp Trp Ile Ile 210 215
220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu Val Pro Tyr Asn Val
Ser225 230 235 240Phe Lys
Thr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser Ile
245 250 255Val Asp Val Ile Phe Leu Val
Asp Ile Val Leu Asn Phe His Thr Thr 260 265
270Phe Val Gly Pro Ala Gly Glu Val Ile Ser Asp Pro Lys Leu
Ile Arg 275 280 285Met Asn Tyr Leu
Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290
295 300Pro Tyr Asp Val Ile Asn Ala Phe Glu Asn Val Asp
Glu Val Ser Ala305 310 315
320Phe Met Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp Gln Ile Pro Pro
325 330 335Pro Leu Glu Gly Arg
Glu Ser Gln Gly Ile Ser Ser Leu Phe Ser Ser 340
345 350Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg Val
Ala Arg Lys Leu 355 360 365Asp His
Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val Leu Leu Val Cys 370
375 380Val Phe Gly Leu Ala Ala His Trp Met Ala Cys
Ile Trp Tyr Ser Ile385 390 395
400Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys Thr Ile Arg Asn Asn
405 410 415Ser Trp Leu Tyr
Gln Leu Ala Met Asp Ile Gly Thr Pro Tyr Gln Phe 420
425 430Asn Gly Ser Gly Ser Gly Lys Trp Glu Gly Gly
Pro Ser Lys Asn Ser 435 440 445Val
Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr Ser Leu Thr Ser Val 450
455 460Gly Phe Gly Asn Ile Ala Pro Ser Thr Asp
Ile Glu Lys Ile Phe Ala465 470 475
480Val Ala Ile Met Met Ile Gly Ser Leu Leu Tyr Ala Thr Ile Phe
Gly 485 490 495Asn Val Thr
Thr Ile Phe Gln Gln Met Tyr Ala Asn Thr Asn Arg Tyr 500
505 510His Glu Met Leu Asn Ser Val Arg Asp Phe
Leu Lys Leu Tyr Gln Val 515 520
525Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr Ile Val Ser Thr Trp 530
535 540Ser Met Ser Arg Gly Ile Asp Thr
Glu Lys Val Leu Gln Ile Cys Pro545 550
555 560Lys Asp Met Arg Ala Asp Ile Cys Val His Leu Asn
Arg Lys Val Phe 565 570
575Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp Gly Cys Leu Arg Ala
580 585 590Leu Ala Met Glu Phe Gln
Thr Val His Cys Ala Pro Gly Asp Leu Ile 595 600
605Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys Phe Val Val
Ser Gly 610 615 620Ser Leu Glu Val Ile
Gln Asp Asp Glu Val Val Ala Ile Leu Gly Lys625 630
635 640Gly Asp Val Phe Gly Asp Val Phe Trp Lys
Glu Ala Thr Leu Ala Gln 645 650
655Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys Asp Leu His Val Ile
660 665 670Lys Arg Asp Ala Leu
Gln Lys Val Leu Glu Phe Tyr Thr Ala Phe Ser 675
680 685His Ser Phe Ser Arg Asn Leu Ile Leu Thr Tyr Asn
Leu Arg Lys Arg 690 695 700Ile Val Phe
Arg Lys Ile Ser Asp Val Lys Arg Glu Glu Glu Glu Arg705
710 715 720Met Lys Arg Lys Asn Glu Ala
Pro Leu Ile Leu Pro Pro Asp His Pro 725
730 735Val Arg Arg Leu Phe Gln Arg Phe Arg Gln Gln Lys
Glu Ala Arg Leu 740 745 750Ala
Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp Leu Asp Val Glu Lys 755
760 765Gly Ser Val Leu Thr Glu His Ser His
His Gly Leu Ala Lys Ala Ser 770 775
780Val Val Thr Val Arg Glu Ser Pro Ala Thr Pro Val Ala Phe Pro Ala785
790 795 800Ala Ala Ala Pro
Ala Gly Leu Asp His Ala Arg Leu Gln Ala Pro Gly 805
810 815Ala Glu Gly Leu Gly Pro Lys Ala Gly Gly
Ala Asp Cys Ala Lys Arg 820 825
830Lys Gly Trp Ala Arg Phe Lys Asp Ala Cys Gly Gln Ala Glu Asp Trp
835 840 845Ser Lys Val Ser Lys Ala Glu
Ser Met Glu Thr Leu Pro Glu Arg Thr 850 855
860Lys Ala Ala Gly Glu Ala Thr Leu Lys Lys Thr Asp Ser Cys Asp
Ser865 870 875 880Gly Ile
Thr Lys Ser Asp Leu Arg Leu Asp Asn Val Gly Glu Ala Arg
885 890 895Ser Pro Gln Asp Arg Ser Pro
Ile Leu Ala Glu Val Lys His Ser Phe 900 905
910Tyr Pro Ile Pro Glu Gln Thr Leu Gln Ala Ala Val Leu Glu
Val Lys 915 920 925His Glu Leu Lys
Glu Asp Ile Lys Ala Leu Ser Thr Lys Met Thr Ser 930
935 940Ile Glu Lys Gln Leu Ser Glu Ile Leu Arg Ile Leu
Thr Ser Arg Arg945 950 955
960Ser Ser Gln Ser Pro Gln Glu Leu Phe Glu Ile Ser Arg Pro Gln Ser
965 970 975Pro Glu Ser Glu Arg
Asp Ile Phe Gly Ala Ser 980 98523989PRTMus sp.
23Met Thr Met Ala Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr1
5 10 15Phe Leu Glu Asn Ile Val
Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25
30Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser
Asn Asp Gly 35 40 45Phe Cys Lys
Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50
55 60Ser Ala Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp
Lys Asp Thr Val65 70 75
80Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu
85 90 95Ile Leu Met Tyr Lys Lys
Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100
105 110Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val
Leu Phe Leu Cys 115 120 125Thr Phe
Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser 130
135 140Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr
Arg Ala Leu Thr Ser145 150 155
160Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln Lys Gly Glu
165 170 175Asn Val His Lys
His Ser Arg Leu Ala Glu Val Leu Gln Leu Gly Ser 180
185 190Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro
Lys Thr Pro Pro His 195 200 205Ile
Ile Leu His Tyr Cys Val Phe Lys Thr Thr Trp Asp Trp Ile Ile 210
215 220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu
Val Pro Tyr Asn Val Ser225 230 235
240Phe Lys Thr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser
Ile 245 250 255Val Asp Val
Ile Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260
265 270Phe Val Gly Pro Ala Gly Glu Val Ile Ser
Asp Pro Lys Leu Ile Arg 275 280
285Met Asn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290
295 300Pro Tyr Asp Val Ile Asn Ala Phe
Glu Asn Val Asp Glu Val Ser Ala305 310
315 320Phe Met Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp
Gln Ile Pro Pro 325 330
335Pro Leu Glu Gly Arg Glu Ser Gln Gly Ile Ser Ser Leu Phe Ser Ser
340 345 350Leu Lys Val Val Arg Leu
Leu Arg Leu Gly Arg Val Ala Arg Lys Leu 355 360
365Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val Leu Leu
Val Cys 370 375 380Val Phe Gly Leu Ala
Ala His Trp Met Ala Cys Ile Trp Tyr Ser Ile385 390
395 400Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr
Lys Thr Ile Arg Asn Asn 405 410
415Ser Trp Leu Tyr Gln Leu Ala Leu Asp Ile Gly Thr Pro Tyr Gln Phe
420 425 430Asn Gly Ser Gly Ser
Gly Lys Trp Glu Gly Gly Pro Ser Lys Asn Ser 435
440 445Val Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr Ser
Leu Thr Ser Val 450 455 460Gly Phe Gly
Asn Ile Ala Pro Ser Thr Asp Ile Glu Lys Ile Phe Ala465
470 475 480Val Ala Ile Met Met Ile Gly
Ser Leu Leu Tyr Ala Thr Ile Phe Gly 485
490 495Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala Asn
Thr Asn Arg Tyr 500 505 510His
Glu Met Leu Asn Ser Val Arg Asp Phe Leu Lys Leu Tyr Gln Val 515
520 525Pro Lys Gly Leu Ser Glu Arg Val Met
Asp Tyr Ile Val Ser Thr Trp 530 535
540Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val Leu Gln Ile Cys Pro545
550 555 560Lys Asp Met Arg
Ala Asp Ile Cys Val His Leu Asn Arg Lys Val Phe 565
570 575Lys Glu His Pro Ala Phe Arg Leu Ala Ser
Asp Gly Cys Leu Arg Ala 580 585
590Leu Ala Met Glu Phe Gln Thr Val His Cys Ala Pro Gly Asp Leu Ile
595 600 605Tyr His Ala Gly Glu Ser Val
Asp Ser Leu Cys Phe Val Val Ser Gly 610 615
620Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val Ala Ile Leu Gly
Lys625 630 635 640Gly Asp
Val Phe Gly Asp Val Phe Trp Lys Glu Ala Thr Leu Ala Gln
645 650 655Ser Cys Ala Asn Val Arg Ala
Leu Thr Tyr Cys Asp Leu His Val Ile 660 665
670Lys Arg Asp Ala Leu Gln Lys Val Leu Glu Phe Tyr Thr Ala
Phe Ser 675 680 685His Ser Phe Ser
Arg Asn Leu Ile Leu Thr Tyr Asn Leu Arg Lys Arg 690
695 700Ile Val Phe Arg Lys Ile Ser Asp Val Lys Arg Glu
Glu Glu Glu Arg705 710 715
720Met Lys Arg Lys Asn Glu Ala Pro Leu Ile Leu Pro Pro Asp His Pro
725 730 735Val Arg Arg Leu Phe
Gln Arg Phe Arg Gln Gln Lys Glu Ala Arg Leu 740
745 750Ala Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp Leu
Asp Val Glu Lys 755 760 765Gly Asn
Ala Leu Thr Asp His Thr Ser Ala Asn His Gly Leu Ala Lys 770
775 780Ala Ser Val Val Thr Val Arg Glu Ser Pro Ala
Thr Pro Val Ala Phe785 790 795
800Gln Ala Ala Thr Thr Ser Thr Met Ser Asp His Ala Lys Leu His Ala
805 810 815Pro Gly Ser Glu
Cys Leu Gly Pro Lys Ala Val Ser Cys Asp Pro Ala 820
825 830Lys Arg Lys Gly Trp Ala Arg Phe Lys Asp Ala
Cys Gly Gln Ala Glu 835 840 845Asp
Trp Ser Lys Val Ser Lys Ala Glu Ser Met Glu Thr Leu Pro Glu 850
855 860Arg Thr Lys Ala Pro Gly Glu Ala Thr Leu
Lys Lys Thr Asp Ser Cys865 870 875
880Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu Asp Asn Val Gly
Glu 885 890 895Thr Arg Ser
Pro Gln Asp Arg Ser Pro Ile Leu Ala Glu Val Lys His 900
905 910Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu
Gln Ala Ala Val Leu Glu 915 920
925Val Lys Tyr Glu Leu Lys Glu Asp Ile Lys Ala Leu Asn Ala Lys Met 930
935 940Thr Ser Ile Glu Lys Gln Leu Ser
Glu Ile Leu Arg Ile Leu Met Ser945 950
955 960Arg Gly Ser Ala Gln Ser Pro Gln Glu Thr Gly Glu
Ile Ser Arg Pro 965 970
975Gln Ser Pro Glu Ser Asp Arg Asp Ile Phe Gly Ala Ser 980
98524962PRTRattus sp. 24Met Thr Met Ala Gly Gly Arg Lys Gly
Leu Val Ala Pro Gln Asn Thr1 5 10
15Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val
Leu 20 25 30Gly Asn Ala Gln
Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35
40 45Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Glu Val
Met Gln Lys Ser 50 55 60Ser Ala Cys
Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Val65 70
75 80Glu Lys Val Arg Gln Thr Phe Glu
Asn Tyr Glu Met Asn Ser Phe Glu 85 90
95Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe
Val Lys 100 105 110Ile Ala Pro
Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys 115
120 125Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro
Ile Glu Asp Asp Ser 130 135 140Cys Lys
Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr Ser145
150 155 160Ser Arg Gly Val Leu Gln Gln
Leu Ala Pro Ser Val Gln Lys Gly Glu 165
170 175Asn Val His Lys His Ser Arg Leu Ala Glu Val Leu
Gln Leu Gly Ser 180 185 190Asp
Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro Lys Thr Pro Pro His 195
200 205Ile Ile Leu His Tyr Cys Val Phe Lys
Thr Thr Trp Asp Trp Ile Ile 210 215
220Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu Val Pro Tyr Asn Val Ser225
230 235 240Phe Lys Thr Arg
Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser Ile 245
250 255Val Asp Val Ile Phe Leu Val Asp Ile Val
Leu Asn Phe His Thr Thr 260 265
270Phe Val Gly Pro Ala Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg
275 280 285Met Asn Tyr Leu Lys Thr Trp
Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295
300Pro Tyr Asp Val Ile Asn Ala Phe Glu Asn Val Asp Glu Gly Ile
Ser305 310 315 320Ser Leu
Phe Ser Ser Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg
325 330 335Val Ala Arg Lys Leu Asp His
Tyr Ile Glu Tyr Gly Ala Ala Val Leu 340 345
350Val Leu Leu Val Cys Val Phe Gly Leu Ala Ala His Trp Met
Ala Cys 355 360 365Ile Trp Tyr Ser
Ile Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys 370
375 380Thr Ile Arg Asn Asn Ser Trp Leu Tyr Gln Leu Ala
Leu Asp Ile Gly385 390 395
400Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser Gly Lys Trp Glu Gly Gly
405 410 415Pro Ser Lys Asn Ser
Val Tyr Ile Ser Ser Leu Tyr Phe Thr Met Thr 420
425 430Ser Leu Thr Ser Val Gly Phe Gly Asn Ile Ala Pro
Ser Thr Asp Ile 435 440 445Glu Lys
Ile Phe Ala Val Ala Ile Met Met Ile Gly Ser Leu Leu Tyr 450
455 460Ala Thr Ile Phe Gly Asn Val Thr Thr Ile Phe
Gln Gln Met Tyr Ala465 470 475
480Asn Thr Asn Arg Tyr His Glu Met Leu Asn Ser Val Arg Asp Phe Leu
485 490 495Lys Leu Tyr Gln
Val Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr 500
505 510Ile Val Ser Thr Trp Ser Met Ser Arg Gly Ile
Asp Thr Glu Lys Val 515 520 525Leu
Gln Ile Cys Pro Lys Asp Met Arg Ala Asp Ile Cys Val His Leu 530
535 540Asn Arg Lys Val Phe Lys Glu His Pro Ala
Phe Arg Leu Ala Ser Asp545 550 555
560Gly Cys Leu Arg Ala Leu Ala Met Glu Phe Gln Thr Val His Cys
Ala 565 570 575Pro Gly Asp
Leu Ile Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys 580
585 590Phe Val Val Ser Gly Ser Leu Glu Val Ile
Gln Asp Asp Glu Val Val 595 600
605Ala Ile Leu Gly Lys Gly Asp Val Phe Gly Asp Val Phe Trp Lys Glu 610
615 620Ala Thr Leu Ala Gln Ser Cys Ala
Asn Val Arg Ala Leu Thr Tyr Cys625 630
635 640Asp Leu His Val Ile Lys Arg Asp Ala Leu Gln Lys
Val Leu Glu Phe 645 650
655Tyr Thr Ala Phe Ser His Ser Phe Ser Arg Asn Leu Ile Leu Thr Tyr
660 665 670Asn Leu Arg Lys Arg Ile
Val Phe Arg Lys Ile Ser Asp Val Lys Arg 675 680
685Glu Glu Glu Glu Arg Met Lys Arg Lys Asn Glu Ala Pro Leu
Ile Leu 690 695 700Pro Pro Asp His Pro
Val Arg Arg Leu Phe Gln Arg Phe Arg Gln Gln705 710
715 720Lys Glu Ala Arg Leu Ala Ala Glu Arg Gly
Gly Arg Asp Leu Asp Asp 725 730
735Leu Asp Val Glu Lys Gly Asn Ala Leu Thr Asp His Thr Ser Ala Asn
740 745 750His Gly Leu Ala Lys
Ala Ser Val Val Thr Val Arg Glu Ser Pro Ala 755
760 765Thr Pro Val Ala Phe Gln Ala Ala Ser Thr Ser Thr
Val Ser Asp His 770 775 780Ala Lys Leu
His Ala Pro Gly Ser Glu Cys Leu Gly Pro Lys Ala Gly785
790 795 800Gly Gly Asp Pro Ala Lys Arg
Lys Gly Trp Ala Arg Phe Lys Asp Ala 805
810 815Cys Gly Gln Ala Glu Asp Trp Ser Lys Val Ser Lys
Ala Glu Ser Met 820 825 830Glu
Thr Leu Pro Glu Arg Thr Lys Ala Ala Gly Glu Ala Thr Leu Lys 835
840 845Lys Thr Asp Ser Cys Asp Ser Gly Ile
Thr Lys Ser Asp Leu Arg Leu 850 855
860Asp Asn Val Gly Glu Ala Arg Ser Pro Gln Asp Arg Ser Pro Ile Leu865
870 875 880Ala Glu Val Lys
His Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu Gln 885
890 895Ala Thr Val Leu Glu Val Lys Tyr Glu Leu
Lys Glu Asp Ile Lys Ala 900 905
910Leu Asn Ala Lys Met Thr Ser Ile Glu Lys Gln Leu Ser Glu Ile Leu
915 920 925Arg Ile Leu Met Ser Arg Gly
Ser Ser Gln Ser Pro Gln Asp Thr Cys 930 935
940Glu Val Ser Arg Pro Gln Ser Pro Glu Ser Asp Arg Asp Ile Phe
Gly945 950 955 960Ala Ser
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