MediGene Aktiengesellschaft M25519PC BO Myocardium- and skeletal muscle-specific nucleic acid, its preparation and use The invention relates to a nucleic acid which is expressed in human myocardium and skeletal muscle, to its preparation and use as diagnostic aid, medicinal product and test for identifying functional interactors.
The heart is a muscular hollow organ which has the task of keeping the bloodstream in the vessels in motion by alternating contraction (systole) and relaxation (diastole) of atria and ventricles.
The muscle of the heart, the myocardium, is composed of specialized striped muscle cells between which there is connective tissue. Each cell has a central nucleus, is bounded by the plasma membrane, the sarcolemma, and contains numerous contractile myofibrils which are separated irregularly by sarcoplasm. The contractile substance of the heart is formed by long parallel myofibrils. Each myofibril is divided into several identical structural and functional units, the sarcomeres. The sarcomeres in turn are composed of the thin filaments which mainly consist of actin, tropomyosin and troponin, and the thick filaments which mainly consist of myosin.
The molecular mechanism of muscle contraction is based on a cyclic attachment and detachment of the globular myosin heads by the F actin filaments. On electrical stimulation of the myocardium, Ca 2 1 is released from the sarcoplasmic reticulum which influences, through an allosteric reaction, the troponin complex and tropomyosin, and thus opens the way for contact of the actin filament with the myosin L~ ~II_ II 2 head. The attachment causes a conformational change in the myosin which thus pulls the actin filament along on itself. ATP is needed to reverse this conformational change and return to the start of a contraction cycle.
Short-term adjustment of the activity of the myocardium to the particular perfusion requirement, that is to say blood flow requirement, of the body is possible by nervous and hormonal regulation measures.
It is thus possible to increase both the force of contraction and the rate of contraction. Long-term overstrain results in physiological transformation processes in the myocardium, which are characterized mainly by an increase in myofibrils (myocyte hypertrophy) If the myocardium is damaged, the originally physiological adaptation mechanisms often lead in the long term to pathophysiological states which develop into chronic cardiac insufficiency, that is to say cardiac weakness, and usually end with acute heart failure. In cases of severe chronic insufficiency, the heart may no longer respond appropriately to changed output requirements, and even slight physical exertion leads to exhaustion and shortness of breath.
Damage to the myocardium results from ischaemia, that is to say depletion of blood, caused by coronary disease, bacterial or viral infections, toxins, metabolic abnormalities, autoimmune diseases or genetic defects. Therapeutic measures are currently aimed at strengthening the force of contraction and controlling the compensatory neuronal and hormonal compensation mechanisms. Despite this treatment, the mortality from this disease remains high (35-50% in the first 5 years after diagnosis) Cardiac insufficiency is the main cause of death in the world. The only causal therapy is a heart transplant.
The molecular changes in chronic cardiac insufficiency are only inadequately known. In 3 particular, the genetic changes underlying cardiac insufficiency are substantially unknown. The question of why secondary damage by toxins or viruses leads to cardiac insufficiency in some people but not in others also remains unanswered.
The present invention is thus based on the object of identifying and isolating genes which are at least partly responsible for, if not in fact the causes of, genetically related cardiac disorders.
Surprisingly, a gene has now been found, in a human cardiac tissue cDNA bank, which is expressed more strongly in insufficient cardiac tissue than in healthy cardiac tissue and thus is causally connected with a genetically related cardiac insufficiency. A so-called EST (expressed sequence tag) already exists for this gene, although it is faulty and no function at all can be assigned to it (Tanaka, T. et al. (1996) Genomics, 231-235; EMBL AC:CO4498; clone 3NHC3467).
One aspect of the invention is therefore a nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, preferably at least 10 nucleotides, in particular at least 15 nucleotides, especially at least 20 nucleotides, except a nucleic acid having the sequence: 1 GCCAACACGC ANTCCGACGA CAGTGCAGCC ATGGTCATTG CAGAGATGCN TCAAAGTCAA 61 TGAGCACATC ACCAACGTAA ACGTCGAGTC CAACTTCATA ACGGGAAAGG GGATCCTGGC 121 CATCATGAGA GCTCTCCAGC ACAACACGGT GCTCACGGAG CTGCGTTTCC ATAACCAGAG 181 GCACATCATG GGCAGCCAGG TGGAAATGGA GATTGTCAAG CTNCTGAAGG AGAACACGAC 241 GCTNCTGAGG CTGGGNTACC ATTTTNAACT CCCAGGACC in which N denotes A, T, G or C.
The nucleic acid according to the invention was isolated from a human cardiac tissue cDNA bank and sequenced. For this, firstly complete RNA was isolated by standard methods from a healthy and insufficient cardiac sample and transcribed with the aid of a -4- 3'anchor primer mixture, for example a 5'-T 12 ACN-3' primer, in which N denotes any deoxyribonucleotide, and reverse transcriptase into c-DNA. The cDNA was then amplified with a method based on the so-called differential display method of Liang and Pardee (Liang, P. Pardee, A. (1992) Science 257, 967-970) under specific PCR conditions with the aid of a 3' primer, for example a T 12 ACN primer, and of an arbitrarily selected 5'-decamer primer, for example a 5'-CCTTCTACCC-3' decamer primer. It was possible thereby to amplify a 321 base pair (bp)-long DNA fragment which is surprisingly present not in the healthy heart sample but distinctly in the insufficient heart sample. This was surprising because the conventional methods such as the differential display method or else subtractive cDNA gene banks are associated with the problem of redundancy, of underrepresentation and of false-positive clones. In particular, it is possible to identify the gene products of weakly expressed genes only under special conditions. It is therefore also not astonishing that the hit rate is generally very low (10-20%) and, for example in the differential display method, also depends on the chosen PCR conditions, the primer length or, for example in the production of subtractive banks, on the hybridization temperature. The complete gene was then isolated from a cDNA gene bank with the aid of the found DNA fragment and sequenced.
In every case it is necessary to find out by further methods whether the found cDNA can be assigned to an active and/or tissue-specific gene. Hence mRNAs from various human tissues were hybridized with the found DNA fragment in a so-called Northern blot, and the amount of bound m-RNA was determined, for example, via the radiolabelling of the DNA fragment. This experiment led to detection of the corresponding RNA in particular in striped muscle, that is to say myocardial 5 and skeletal muscle tissue, and very weakly in prostate tissue. In a further experiment comparing between healthy and insufficient cardiac tissues, increased expression was detected, for example expression of the RNAs increased by about 35%, in insufficient tissue by comparison with healthy tissue. It was possible to demonstrate in particular that a relatively small RNA species preferentially shows increased expression in insufficient tissue by comparison with healthy tissue.
The increased expression of the relatively small RNA species is readily evident for example in the Northern blot in the form of a double band (see Fig. Comparison of the derived polypeptide sequence with a protein database additionally revealed a certain relationship (homology) with the protein tropomodulin (see Fig. Tropomodulin is known to be a polypeptide which in chicken cardiomyocytes has an influence on the development of the myofibrils and the contractility of the cells (Gregorio et al. (1995) Nature 377, 83-86) This protein binds on the one hand to tropomyosin, and on the other hand to the actin filaments, but is not itself regulated in its activity. The derived polypeptide according to the invention likewise has some of the structural features of tropomodulins, such as, for example, a tropomyosin binding domain. In contrast to tropomodulin, the polypeptide according to the invention has additional structural features indicating regulation of the activity of the polypeptide by so-called tyrosine kinases (see Fig. 4) The term "functional variant" therefore means for the purpose of the present invention polypeptides which are functionally related to the polypeptide according to the invention, that is to say can likewise be referred to as a regulable modulator of the contractility of myocardial cells, are expressed in striped muscle, preferably in myocardial, skeletal muscle and/or prostate tissue, especially in myocardial L-l 6 and/or skeletal muscle and, in particular, in myocardial cells, have structural features of tropomodulin, such as, for example, one or more tropomyosin binding domains, and/or whose activity can be regulated by tyrosine kinases. Examples of functional variants are the corresponding polypeptides derived from other organisms than humans, preferably from non-human mammals such as, for example, monkeys.
In the wider sense, the term ,,functional variant" includes polypeptides which have a sequence homology, in particular a sequence identity, of about preferably about 80%, in particular about especially about 95%, with the polypeptide having the amino acid sequence shown in Fig. 4. These include, for example, polypeptides encoded by a nucleic acid which is isolated from non-heart-specific tissue, for example skeletal muscle tissue, but which has, after expression in a heart-specific cell, the identified function(s).
These furthermore include deletions of the polypeptide in the region of about 1-60, preferably of about 1-30, in particular of about 1-15, especially of about amino acids. For example, the first amino acid methionine can be absent with negligible alteration in the function of the polypeptide. These also include fusion proteins which comprise the above-described polypeptides according to the invention, it being possible for the fusion proteins themselves to have -the function of a regulable modulator of the contractility of myocardial cells, or to acquire the specific function only after elimination of the fusion portion.
They particularly include fusion proteins with a content of, in particular, non-heart-specific sequences of about 1-200, preferably about 1-150, in particular about 1-100, especially about 1-50, amino acids.
Examples of non-heart-specific peptide sequences are prokaryotic peptide sequences which may be derived, for example, from the galactosidase of E. coli.
7 The nucleic acid according to the invention is generally a DNA or RNA, preferably a DNA. Preferred for expression of the relevant gene is in general a doublestranded DNA and for use as probe is a single-stranded DNA. Particular preference is given to a double- or single-stranded DNA having a nucleic acid sequence as shown in Fig. i, 2 or 3 and the parts thereof described in detail above, with the DNA region coding for the polypeptide being particularly preferred. This region starts with the nucleic acids "ATG" coding for methionine at position 89 to "TAG" coding for "amber" (stop) at position 1747.
The nucleic acid according to the invention can, for example, be chemically synthesized on the basis of the sequences disclosed in Figs. 1-3 or on the basis of the polypeptide sequence disclosed in Fig. 4 by use of the genetic code, for example by the phosphotriester method (see, for example, Uhlmann, E. Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. Another possibility for obtaining the nucleic acid according to the invention is isolation from a suitable gene bank, for example from a heart-specific gene bank, using a suitable probe (see, for example, J. Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual 2 nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Suitable as probe are, for example, single-stranded DNA fragments with a length of about 100-1000 nucleotides, preferably with a length of about 200-500 nucleotides, in particular with a length of about 300-400 nucleotides, whose sequence can be derived from the nucleic acid sequences shown in Figs. 1-3. One example of a probe is the DNA fragment of Example 1, which is 321 bp in size and corresponds to the underlined region in Fig. 1, using which the nucleic acid according to the invention has already been isolated successfully from human cardiac tissue (see Example 2).
8 The nucleic acid according to the invention is normally present in a vector, preferably in an expression vector or vector effective for gene therapy.
The vector effective for gene therapy preferably contains heart-specific regulatory sequences such as, for example, the troponin C (cTNC) promoter (see, for example, Parmacek, M.S. et al. (1990) J. Biol. Chem.
265 (26) 15970-15976 and Parmacek, M.S. et al. (1992) Mol. Cell Biol. 12(5), 1967-1976), which is functionally connected to the nucleic acid according to the invention.
The expression vectors may be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors for expression in E. coli are, for example, the vectors pGEM or pUC derivatives, and of eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for example, the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl.
Acids Res., 22, 5767-5768) for expression in insect cells are, for example, baculovirus vectors as disclosed in EP-B1 0 127 839 or EP-B1 0 549 721, and for expression in mammalian cells are, for example, the vectors Rc/CMV and Rc/RSV or SV40 vectors, which are all generally available.
The expression vectors generally also contain promoters suitable for the particular host cell, such as, for example, the trp promoter for expression in E.
coli (see, for example, EP-B1 0 154 133), the ADH2 promoter for expression in yeasts (Russell et al.
(1983), J. Biol. Chem. 258, 2674-2682), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1 0 127 839) or the SV40 early promoter or LTR promoters, for example of MMTV (mouse mammary tumour virus; Lee et al. (1981) Nature 214, 228-232).
Examples of vectors effective for gene therapy are virus vectors, preferably adenovirus vectors, in 9 particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, for example an adenoassociated virus vector which consists exclusively of two inverted terminal repeats (ITR).
An adenovirus vector and, in particular, a replication-deficient adenovirus vector are particularly preferred for the following reasons.
The human adenovirus belongs to the class of double-stranded DNA viruses with a genome of about 36 kilobase pairs The viral DNA codes for about 2700 different gene products, a distinction being made between early ("early genes") and late. ("late genes").
The "early genes" are divided into four transcriptional units El to E4. The late gene products code for the capsid proteins. It is possible to distinguish immunologically at least 42 different adenoviruses and subgroups A to F, all of which are suitable for the present invention. A precondition for transcription of the viral genes is expression of the El region which codes for a transactivator of adenoviral gene expression. This dependence of the expression of all subsequent viral genes on the El transactivator can be utilized to construct adenoviral vectors not capable of replication (see, for example, McGrory, W.J. et al.
(1988) Virol. 163, 614-617 and Gluzman, Y. et al.
(1982) in "Eukaryotic Viral Vectors" (Gluzman, Y. ed.) 187 192, Cold Spring Harbor Press, Cold Spring Harbor, New York). In adenoviral vectors, especially of type 5 (for sequence, see Chroboczek, J. et al. (1992) Virol. 186, 280-285) and especially of subgroup C, in general the El gene region is replaced by a foreign gene with its own promoter or by the nucleic acid construct according to the invention. Replacement of the El gene region which is a precondition for expression of the downstream adenoviral genes results in an adenovirus not capable of replication. These 10 viruses are then able to replicate only in a cell line which replaces the missing El genes.
Replication-deficient adenoviruses are therefore generally formed by homologous recombination in the so-called 293 cell line (human embryonic kidney cell line) which has a copy of the El region stably integrated into the genome. For this purpose, the nucleic acid according to the invention is cloned into recombinant adenoviral plasmids under the control of its own promoter, for example the troponin C promoter mentioned above. Homologous recombination then takes place with an El-deficient adenoviral genome such as, for example, d1327 or de11324 (adenovirus 5) in the 293 helper cell line. Where recombination is successful, viral plaques are harvested. The replication-deficient viruses produced in this way are employed in high titres (for example 109 to 1011 plaque forming units) for infecting the cell culture or for somatic gene therapy.
The exact site of insertion of the nucleic acid according to the invention into the adenoviral genome is in general not critical. It is, for example, also possible to clone the nucleic acid according to the invention in place of the deleted E3 gene (Karlsson, S.
et al. (1986), EMBO J. 5, 2377 2385).
However, it is preferred for the El region or parts thereof, for example the E1A or E1B region (see, for example, WO 95/00655), to be replaced by the nucleic acid according to the invention, especially when the E3 region is also deleted.
However, the present invention is not confined to the adenoviral vector system; on the contrary, adeno-associated virus vectors are also particularly suitable in combination with the nucleic acid according to the invention for the following reasons.
The AAV virus belongs to the family of parvoviruses. These are distinguished by an K- I- i 11 icosahedral, non-enveloped capsid which has a diameter of 18 to 30 nm and which contains a linear, singlestranded DNA of about 5 kb. For efficient replication of AAV, coinfection of the host cell with helper viruses is necessary. Examples of suitable helpers are adenoviruses (Ad5 or Ad2), herpesviruses and vacciniaviruses (Muzyczka, N. (1992) Curr. Top. Microbiol.
Immunol. 158, 97-129). In the absence of a helper virus, AAV passes into a latency state where the virus genome is able to integrate stably into the host cell genome. The property of AAV integrating into the host genome makes it particularly interesting as transduction vector for mammalian cells. Generally sufficient for the vector functions are the two inverted terminal repeats (ITR: see, for example, WO 95/23867) which are about 145 bp long. They carry the signals necessary in "cis" for replication, packaging and integration into the host cell genome. For packaging into recombinant vector particles, a vector plasmid which harbours the genes for non-structural proteins (rep proteins) and for structural proteins (cap proteins) is transfected into adenovirus-infected cells. After a few days, a cell-free lysate containing, besides the recombinant AAV particles, also adenoviruses is prepared. The adenoviruses can advantageously be removed by heating at 56°C or by banding in a caesium chloride gradient. It is possible with this cotransfection method to achieve rAAV titres of 105 to 106 IE/ml. Contamination by wild-type viruses is below the detection limit if the packaging plasmid and the vector plasmid have no overlapping sequences (Samulski, R.J. (1989) J. Virol. 63, 3822 3828) Transfer of the nucleic acid according to the invention into somatic cells can be effected by AAV into resting, differentiated cells, which is particularly advantageous for gene therapy of the heart. The ability to integrate which has been 12 mentioned also ensures long-lasting gene expression in vivo, which in turn is particularly advantageous.
A
further advantage of AAV is that the virus is not pathogenic for humans and is relatively stable in vivo.
Cloning of the nucleic acid according to the invention into the AAV vector or parts thereof takes place by methods known to the skilled person, as described, for example, in WO 95/23867, in Chiorini, J.A. et al.
(1995), Human Gene Therapy 6, 1531-1541 or Kotin, R.M.
(1994), Human Gene Therapy 5, 793-801.
Vectors effective for gene therapy can also be obtained by complexing the nucleic acid according to the invention with liposomes, because it is possible thereby to achieve a very high transfection efficiency, in particular of myocardial cells (Felgner, P.L. et al.
(1987), Proc. Natl. Acad. Sci USA 84, 7413-7417). In lipofection, small unilamellar vesicles of cationic lipids are prepared by ultrasound treatment of the liposome suspension. The DNA is bound ionically to the surface of the liposomes, specifically in a ratio such that a positive net charge remains and the plasmid DNA is 100% complexed by the liposomes. Besides the lipid mixtures DOTMA (1, 2 -dioleoyloxypropyl-3-trimethylammonium bromide) and DOPE (dioleoylphosphatidylethanolamine) employed by Felgner et al. (1987, supra), numerous new lipid formulations have now been synthesized and tested for their efficiency in transfecting various cell lines (Behr, J.P. et al.
(1989), Proc. Natl. Acad. Sci. USA 86, 6982-6986; Felgner, J.H. et al. (1994) J. Biol. Chem. 269, 2550-2561; Gao, X. Huang, L. (1991), Biochim.
Biophys. Acta 1189, 195-203) Examples of the novel lipid formulations are DOTAP dioleoyloxy)propyl]-N,N,N-trimethylammonium ethyl sulphate or DOGS (TRANSFECTAM; dioctadecylamidoglycylspermine). One example of the preparation of DNA-liposome complexes and successful use thereof in r lr; 13 heart-specific transfection is described in DE 44 11 402.
For use of the nucleic acid according to the invention in gene therapy, it is also advantageous if the part of the nucleic acid which codes for the polypeptide contains one or more noncoding sequences, including intron sequences, preferably between the promoter and the start codon of the polypeptide, and/or a polyA sequence, in particular the naturally occurring polyA sequence or an SV40 virus polyA sequence, especially at the 3' end of the gene, because this makes it possible to stabilize the mRNA in the myocardial cell (Jackson, R.J. (1993) Cell 74, 9-14 and Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478-482).
The present invention further relates to the polypeptide itself having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids, preferably having at least 12 amino acids, in particular having at least 15 amino acids and especially having at least 164 amino acids, except a polypeptide having the sequence:
PTRNPTTVQPWSLQRCIKVNEHITNVNVESNFITGKGILAIMRALQ
20 30 HNTVLTELRFHNQRHIMGSQVEMEIVKLLKENTTLLRLG
YHFKLPG
60 70 80 The polypeptide is prepared, for example, by expression of the nucleic acid according to the invention in a suitable expression system as described above using methods generally known to the skilled person. Examples of suitable host cells are the E. coli strains DH5, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, the lepidopteran insect cell line for example Spodoptera frugiperda, or the animal 14 cells COS, Vero, 293 and HeLa, all of which are generally obtainable.
The said parts of the polypeptide can also be synthesized by classical synthesis (Merrifield technique). They are particularly suitable for obtaining antisera which can be used to screen suitable gene expression banks in order thus to obtain further functional variants of the polypeptide according to the invention.
The present invention therefore relates also to antibodies which react specifically with the polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids, preferably having at least 12 amino acids, in particular having at least 15 amino acids and especially having at least 164 amino acids, the abovementioned parts of the polypeptide either themselves being immunogenic or being able to be made immunogenic, or have their immunogenicity increased, by coupling to suitable carriers such as, for example, bovine serum albumin.
The antibodies are either polyclonal or monoclonal. The preparation, to which the present invention also relates, takes place, for example, by generally known methods, by immunizing a mammal, for example a rabbit, with the said polypeptide or the said parts thereof, where appropriate in the presence of, for example, Freund's adjuvant and/or aluminium hydroxide gels (see, for example, Diamond, B.A. et al.
(1981) The New England Journal of Medicine, 1344-1349) The polyclonal antibodies raised in the animal on the basis of an immunological response can then easily be isolated from the blood by generally known methods and purified, for example, by column chromatography. It was -thus possible, for example, to produce in rabbits a polyclonal antiserum against a polypeptide which had amino acids 1-90 according to the invention, as shown 15 in Fig. 4, which was expressed as fusion protein in bacteria and purified by affinity chromatography. The antibodies according to the invention specifically recognized the corresponding protein of about 80 kD in extracts of human heart tissue.
Monoclonal antibodies can be prepared, for example, by the known method of Winter Milstein (Winter, G. Milstein, C. (1991) Nature, 349, 293-299) The present invention also relates to a medicinal product which contains a nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 8 nucleotides, or a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 6 amino acids and, where appropriate, suitable additives or excipients, and to a process for producing a medicinal product for treating cardiac disorders, in particular cardiac insufficiency, in which a said nucleic acid or a said polypeptide is formulated with a pharmaceutically acceptable carrier.
One example of the use of nucleic acid fragments as therapeutic agent is the use of DNA fragments in the form of antisense oligonucleotides (Uhlmann, E. Peyman, A. (1990) Chemical Reviews, 543-584, No. 4) A particularly suitable medicinal product for use for human gene therapy is one which contains the said nucleic acid in naked form or in the form of one of the vectors effective for gene therapy which are described above, or in a form complexed with liposomes.
The pharmaceutical carrier is, for example, a physiological buffer solution, preferably with a. pH of about 6.0-8.0, preferably of about 6.8-7.8, in particular of about 7.4 and/or an osmolarity of about 16 200-400 milliosmol/litre, preferably of about 290-310 milliosmol/litre. The pharmaceutical carrier may additionally contain suitable stabilizers such as, for example, nuclease inhibitors, preferably complexing agents such as EDTA and/or other excipients known to the skilled person.
The said nucleic acid is normally administered intravenously, for example with the aid of a catheter, where appropriate in the form of the virus vectors described in detail above or as liposome complexes. It is advantageous, for example, to infuse the nucleic acid according to the invention directly into the patient's coronary arteries (so-called percutaneous coronary gene transfer, PCGT), in particular in the form of recombinant adenovirus vectors or adenoassociated virus vectors. Administration with the aid of a balloon catheter is particularly preferred because it is possible thereby to confine the transfection not only to the heart but also to the injection site within the heart (see, for example, Feldman, L.J. et al.
(1994) JACC 235A, 906-934).
It is also possible to administer the polypeptide itself intravenously or with the aid of a catheter or balloon catheter, where appropriate with suitable additives or excipients, such as, for example, physiological saline, stabilizers, proteinase inhibitors etc., in order to influence the function of the heart immediately and directly.
The present invention further relates to a diagnostic aid containing a nucleic acid, a polypeptide or antibody according to the present invention and, where appropriate, suitable additives or excipients and to a process for producing a diagnostic aid for diagnosing cardiac disorders, in particular cardiac insufficiency, in.which a nucleic acid, a polypeptide or antibody according to the present invention is mixed with suitable additives or excipients.
;i l~i 17 It is possible, for example, according to the present invention to produce on the basis of the said nucleic acid a diagnostic aid based on the polymerase chain reaction (PCR diagnosis, for example as disclosed in EP-0 200 362) or on a Northern blot as described in detail in Example 3 using the 321 bp DNA fragment according to the invention as probe. These tests are based on the specific hybridization of said nucleic acids with the complementary strand, normally of the corresponding mRNA. The nucleic acid may also in this case be modified as described, for example, in EP 0 063 879. A DNA fragment, in particular the DNA fragment described in Example i, is preferably labelled using suitable reagents, for example radioactively with c~-32P-dCTP or non-radioactively with biotin, by generally known methods and incubated with isolated RNA, which has preferably been pre-bound to suitable membranes made of, for example, cellulose or nylon. It is additionally advantageous, before the hybridization and binding to a membrane, for the isolated RNA to be fractionated according to size, for example by agarose gel electrophoresis. With the same amount of investigated RNA from each tissue sample, it is thus possible to determine the amount of mRNA specifically labelled by the probe.
It is thus possible by using the diagnostic aid according to the invention also to measure a cardiac tissue sample in vitro specifically for the strength of expression of the corresponding gene in order to be able to diagnose reliably possible cardiac insufficiency (see Example A cDNA having a sequence as shown in Fig. 1 is particularly suitable for diagnosing a possible cardiac insufficiency (see Example 2).
A further diagnostic aid contains the polypeptide according to the present invention or the immunogenic parts thereof described above in detail.
I~ 18 The polypeptide or the parts thereof, which are preferably bound to a solid phase, for example made of nitrocellulose or nylon, can, for example, be brought into contact in vitro with the body fluid to be investigated, for example blood, in order to react for example with autoimmune antibodies. The antibodypeptide complex can then be detected for example by means of labelled antihuman IgG or antihuman IgM antibodies. The label is, for example, an enzyme such as peroxidase which catalyses a colour reaction. The presence and the amount of autoimmune antibody present can thus be detected easily and rapidly by the colour reaction.
Another diagnostic aid contains the antibodies according to the invention themselves. These antibodies can be used, for example, for investigating a cardiac tissue sample easily and quickly to find whether the relevant polypeptide is present in an increased amount, in order thus to obtain information about possible cardiac insufficiency. In this case, the antibodies according to the invention are labelled for example with an enzyme, as already described above. The specific antibody-peptide complex can thus be detected easily and equally quickly by an enzymatic colour reaction.
The present invention also relates to a test for identifying functional interactors containing a nucleic acid according to the invention coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 8 nucleotides, a polypeptide having the amino acid sequence as shown in Fig. 4 or a functional variant thereof, and the abovementioned parts thereof having at least 6 amino acids or the antibodies according to the invention and, where appropriate, suitable additives or excipients.
1.
19 A suitable test for identifying functional interactors is, for example, the so-called two-hybrid system (Fields, S. Sternglanz, R. (1994) Trends in Genetics, 10, 286-292) In this test, a cell, for example a yeast cell, is transformed or transfected with one or more expression vectors which express a fusion protein which contains a polypeptide according to the present invention and a DNA binding domain of a known protein, for example of Gal4 or LexA from E. coli, and/or expresses a fusion protein which contains an unknown polypeptide and a transcription activating domain, for example of Gal4, herpes virus VP16 or B42. The cell additionally contains a reporter gene, for example the lacZ gene from E. coli, green fluoresdence protein or the yeast amino acid biosynthesis genes His3 or Leu2, which is controlled by regulatory sequences, such as, for example, the LexA promoter/operator or by a socalled upstream activation sequence (UAS) of yeast. The unknown polypeptide is encoded, for example, by a DNA fragment which is derived from a gene bank, for example from a human cardiac tissue-specific gene bank.
Normally a cDNA gene bank is produced directly, using the expression vectors described, in yeast so that the test can be carried out immediately thereafter.
For example, a nucleic acid according to the present invention is cloned into a yeast expression vector in a functional unit with the nucleic acid coding for the LexA DNA binding domain, so that a fusion protein consisting of the polypeptide according to the invention and the LexA DNA binding domain is expressed in the transformed yeast. In another yeast expression vector, cDNA fragments from a cDNA gene bank are cloned in a functional unit with the nucleic acid coding for the Gal4 transcription activating domain, so that a fusion protein consisting of an unknown polypeptide and the Gal4 transcription activating 20 domain is expressed in the transformed yeast. The yeast which is transformed with the two expression vectors and is, for example, Leu2- additionally contains a nucleic acid which codes for Leu2, and is controlled by the LexA promoter/operator. In the event of a functional interaction between the polypeptide according to the invention and the unknown polypeptide, the Gal4 transcription activating domain binds via the LexA DNA binding domain to the LexA promoter/operator, whereby the latter is activated and the Leu2 gene is expressed. The result of this is that the Leu2 yeast is able to grow on minimal medium which contains no leucine.
On use of the lacZ or green fluorescence protein reporter gene in place of an amino acid biosynthesis gene, activation of transcription can be detected by the formation of blue or green-fluorescing colonies. The blue or fluorescent coloration can also be quantified easily in a spectrophotometer, for example at 585 nm in the case of a blue coloration.
Thus, it is possible to screen expression gene banks easily and quickly for polypeptides which interact with a polypeptide according to the present invention. It is then possible for the novel peptides found to be isolated and further characterized.
Another possible use of the two-hybrid system is the influence on the interaction between a polypeptide according to the present invention and a known or unknown polypeptide by other substances such as, for example, chemical compounds. Thus, it is also possible to find easily novel and valuable active substances which can be chemically synthesized and can be employed as therapeutic agent for treating a cardiac disorder. The present invention is therefore not restricted to a method for finding polypeptide-like interactors, but also extends to a method for finding substances which are able to interact with the protein- 7 7. 21 protein complex described above. Such polypeptide-like, as well as chemical interactors are therefore referred to as functional interactors for the purpose of the present invention.
The surprising advantage of the present invention is thus the possibility of using the subjectmatters according to the invention for specific and reliable diagnosis and therapy of cardiac disorders, especially cardiac insufficiency. However, other valuable therapeutic and diagnostic possibilities also emerge. For example, the functional interactors which can be easily identified using the described test methods are so advantageous because it is possible with their aid in the form of suitable medicinal products to influence deliberately the activity of the polypeptide according to the invention in its natural environment in the myocardium and thus also the contractility of the myocardial cells, in particular since the activity of this polypeptide can be regulated as already described in detail above.
The following figures and examples are intended to illustrate the invention in detail without restricting it.
Description of the figures Fig. 1 shows a 1936 nucleotide-long heartspecific DNA sequence. The region which codes for the corresponding polypeptide is shown in bold. The DNA fragment from Example 1 is underlined.
Fig. 2 shows a 2080 nucleotide-long heartspecific DNA sequence which has an extension at the end of the DNA sequence from Fig. i. The region which codes for the corresponding polypeptide is once again shown in bold.
Fig. 3 shows a 2268 nucleotide-long heartspecific DNA sequence which has an extension at the end of the DNA sequence from Fig. 1 or Fig. 2. The _11 o. :7_ 22 region which codes for the corresponding polypeptide is likewise shown in bold.
Fig. 4 shows a 552 amino acid-long polypeptide sequence encoded by one of the DNA sequences shown in Figs. 1-3. The regions homologous with human tropomodulin are shown in bold. The sequence motifs which indicate regulation of the polypeptide by tyrosine kinase signal transduction pathways are underlined.
Figs. 5a and 5b show Northern blots of mRNAs which correspond to the nucleic acid sequences shown in Figs. 1-3 for detecting expression in various human tissues (Fig. 5a) and for detecting expression in healthy and insufficient human cardiac tissue (Fig.
Examples 1. Isolation of a DNA fragment from human insufficient cardiac tissue Complete RNA was initially isolated by standard methods (Chomczynski Sacchi (1987), Anal. Biochem, 162 156-159) from a healthy and an insufficient cardiac tissue sample. The RNA was then treated with DNAse in order to remove DNA contamination. An aliquot of this RNA (0.2 pg) was then incubated in a 20 ul reaction mix with 1 x RT buffer (Gibco Y00121), 10 mM DTT, 20 pM dNTP mix, 1 U/pl RNAsin (Promega N2511), 1 pM 3' anchor primer mixture of the 5'-T 12 ACN-3' type, where N can be any deoxynucleotide, and 10 U/pl SuperScript RNAse H- reverser transcriptase at 37 0 C for min and thus transcribed into cDNA. A cDNA aliquot was then subjected to a 20 ul PCR in 1 x PCR buffer (Perkin-Elmer) which, besides 1 pm 3' primer T 12 AC and 1 pM 5'-decamer primer (5'-CCTTCTACCC-3'), contains 10 pCi of a- 3 2 P-dCTP, 2 pM dNTP mix and 1 U of AmpliTaq (Perkin Elmer). The mixture was incubated firstly at 94 0 C for 1 min, then 40 cycles each of 30 s at 94 0
C,
i- jl
I~
23 for 2 min and 72 0 C for 30 s and finally at 72 0
C
for 10 min. The resulting DNA fragment mixture was then fractionated on a 6% polyacrylamide gel and autoradiographed. A DNA fragment which is 321 bp in length and which is not present in the healthy heart sample but is distinctly present in the insufficient heart sample is thus prepared. This fragment was then cut out of the gel on the basis of the X-ray film and was reamplified by PCR under the conditions already described. The resulting fragment was then cloned into an appropriate vector, and the DNA sequence was determined. A fragment prepared in this way contains nucleotides 1627-1936 of the sequence according to Claim 1 and the 12 thymine nucleotides from the 3' anchor primer.
2. Isolation of heart-specific nucleic acids A plaque hybridization was carried with a cDNA gene bank from cardiac tissue under standard conditions (see Sambrook, Frisch, E.F. Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, ch. 8-10) using an a- 32 P-dCTP-labelled DNA fragment from Example 1 which comprises the nucleotides from position 1627-1936 in Fig. i. The cDNAs found were then isolated and sequenced. The sequences are shown in Figs. 1-3. It emerged from this that the cDNA having the sequence shown in Fig. 1 could be isolated with greater probability from insufficient cardiac tissue than the cDNA having the sequence shown in Fig. 2 or 3, which could be isolated with greater probability from healthy cardiac tissue.
3. Detection of the strength of expression of the heart-specific gene in various human tissues by means of Northern blots.
The DNA fragment 321 bp in length already described in Examples 1 and 2 and Fig. 1 was firstly 77.-- 24 radiolabelled with a- 32 P-dCTP by the random primer labelling method (Feinberg, A.P. Vogelstein,
B.
(1983) Anal. Biochem., 132, The RTS RadPrime DNA labelling system (GibcoBRL 10387-017) was used for this purpose. The hybridization of blots with poly A* RNA from human tissues (see Figs. 5a and 5b) took place at 68 0 C for 1 hour in accordance with the manufacturer's instructions (Multiple Tissue Northern Blots I II, Clontech Laboratories GmbH, Heidelberg, #7760-1, #7759-1). in ExpressHyb hybridization solution (Clontech #8015-1). The blots were then washed with 2 x SSC and 0.05% SDS for 30 minutes and thereafter with 0.1 x SSC and 0.1% SDS for 1 hour and autoradiographed. It emerged that the probe 321 bp in length hybridizes strongly with a polyA' RNA of about 2400 bp strongly in cardiac tissue and skeletal muscle, very weakly in prostate tissue and not in leucocytes, large intestinal, small intestinal, ovarian, testicular, thymus, splenic, renal, hepatic, lung, placental and brain tissue (Fig. Expression of the corresponding RNAs in healthy and insufficient cardiac tissue was also investigated.
Complete RNA was isolated from various human cardiac tissue samples for this purpose (Chomczynski Sacchi (1987), Anal. Biochem. 162, 156-159). Subsequently in each case 10 pg of RNA were fractionated using a 1% formaldehyde agarose gel and transferred by the capillary method to a charged nylon membrane (Zeta- Probe GT BioRad #162-0197). The membrane was briefly washed with 2 x SSC and then baked at 80 0 C for minutes. The membranes were incubated with prehybridization solution (0.5 M Na 2
HPO
4 pH 7.2; 7% SDS) at 65 0 C for at least 1 hour. The solution was then replaced by a fresh solution, and the radioactive, heat-denatured probe was added. The hybridization was carried out at 65 0 C for 15 hours. The membranes were then washed firstly with 40 mM Na 2
HPO
4 pH 7.2; 5% SDS Z, ,7 7-~S 25 at 65 0 C for 15 hours and then with 40 mM Na 2 HP0 4 pH 7.2; 1% SDS at 65 0 C for 2 x 30 minutes, and subsequently autoradiographed. It emerged that various RNA species having a length from about 2200 to 2400 bp were fractionated in 1% agarose gels. These different species correspond well with the sizes of the three cDNAs found, including an average polyA tail 150 bp long (see Figs. In particular, the smallest RNA species was more clearly detectable in diseased tissue than in healthy tissue. Quantification of the blot using a PhosphorImager and the ImageQuant software (Molecular Dynamics GmbH, Krefeld), taking into account a control hybridization with p4-thymosin and actin, revealed an approximately 35% increased expression of the detected RNAs in insufficient cardiac tissue by comparison with healty tissue.
26 SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: MediGene Aktiengesellschaft STREET: Lochhamer Str. 11 CITY: 82152 Martinsried COUNTRY: Germany POSTAL CODE: D-82152 TELEPHONE: 089-89 56 32 0 FAX: 089-89 56 32 (ii) TITLE OF INVENTION: Myocardium- and skeletal muscle-specific nucleic acid, its preparation and use (iii) NUMBER OF SEQUENCES: (iv) COMPUTER-READABLE
FORM:
MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Word Perfect 3.1 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1936 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 77 27 (iv) ANTISENSE:
YES
(vi) ORIGINAL
SOURCE:
TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 1: CAGCCTGCCA CTTGCCTCCC TGCCrGCTrC TGGCTGCCT GAATCCrG
TCCTTCAAGC
TCCTTCTGGG
TAAATACOAA
GGAGCTAGAG
GCAAAAGALGC
CTATTGGGA
GGTTGCAGAA
GGTITCTGAG
AGAAGACACT
A.AATTATGAT
GAACATAAAT
CCCTTGTGGA
TGACACCACA
CTTTGCTGAA
TGCCGACGAC
CAACGTAAAC
TCTCCAGCAC
CAGCCAGO
GGGATACCAT
TATGGATAAA
AGGACCCAAT
ATCTCCCAGG
GAGCCGTCCT
CCCTCTCT
GAAA-AAGCrZ: GrCATTACAA
TATT:-TAAA:;
T7CCCGACCT
AACCAGCATA
TZT-TAGAA23 T-ThAAAATA::
ATTCA.AGA
TTCTTTTrTA
TCTGACAAAG
TCCATCGACC
AGAGAGTTGG
CTGACAGAGA
AAGGAGTCCC
GACAAAGAGG
C-AAGTGTATA
GACCAAGAGG
AGTGTCA6ATT
TTGACCAATG
AATCCTACAG
GAAGTCAATT
GCCCTCAAGG
AGTGCACCCA
GTCGAGTCCA
AACACGGTGC
GAAATGGAGA
TTTGAACTCC
CAGAGGCAAA
CAGGGACCAT
AGGXI'CAACT
A6AGAcATTGA
AAACCCCCAC
A.AAAACTCT
AAAGTGAAGA
CAGAGGAGGA
AAAGAACAAT
CTGACAACTC
GCAGCAATGG
TGArGAGGA
TGAACAACAT
ACAACACTGT
TGGCCATTGC
ACTTCATAAC
TCACGGAGCT
TTGTCAAGCT
CAGGACCAAG
AACGTTTGCA
GTCTACT
cC'CTCaCrCT
ACCTCACCGC
AGGGACATC
GGAGAAGGAG
AGAGCTTATC
GGAGGAOGAG
TGAAACTGCA
TAAGCCAAAC
GAGGAACACA
CGCTrTGGAC
TGAGAACATC
GGTGAAGACG
AGAGATGCTC
GGGAAAGGGG
GCGTTTCCAT
CCTGAAGGAG
AATGAGCATG
GGAGCAAAAA
AAGAGGAACA
AAAALCTCCCC
TCCTCCTCCT
TCCTCCTCCT
AGTCATCAAA
AGGGA.AAG
GTCAGTGCAA
TCATC-GAPAT
AGTTCCAGAA
GGTATTACAT
CCCTGACTTT
CATGTTTCTT
GGCTACCGAA
CTGTCAGCCG
AACCr-rccc
AGCAGG
ACGCTGGGGG
TrACTGAAA TCcCAGGAGG
AAAGGGATTA
ATATTTAAAA
GAGTCCCCAG
AAGATTAAAA
ACAACACAGA
TTCAGTCTGG
AAAGCCAATG
ATCCTG.GCCA
AACCAGAGGC
AACACCACGC
ACGAGCATTT
CAGCAGGAGG
CCTAGCTCrT
AAAAAAGTCC
CCCCCTCCTC
CCecciCerCC
CAACAGGAGA
CTCAAGAAAC
GAC-AAGAA
CTCATGGAAG
GCCCTGCdAT
GAAATGCATT
AAAALZATAATC
CTGTAAATAT
GAGGACTCAG
AGGAGCTGAA
TO;GGCCTAAC
CACTGATGGC
AATGTGOAAA
GTAACAG=A
AAGAGGAGGA
ATGGAACTGT
GTCAAATAGA
CTGCCATTcA
GCAATGACCC
CCCTTACCCG
CCAACACGCA
AGCACATCAC
TCATGAGAGC
ACATCATGGG
TGCTGAGGCT
TGACAAGAAA
GATACGATGG
CACCTTATGT
AGACTGTGAG
CTCCTCCTCC
CACTCCCAGA
GTGCCCAACG
AGCCAAACAG
TGGAAGACAG
CAATTCGGGG
GGGAACATGA
GTGAGATGTT
TCACCCATTA
GAAAATAAAT
600 660 720 780 840 900 960 :.02 0 1080 1140 1200 1260 1320 1380 1440 1-740 la:o 1920 1936 CTTAGGACCA AAGTCTrGCA CACTCACCCT GGTCATCCCC CTGTCTCCTG TGGCCACACT TCCCAAAGGC TGCCACCACC ATTACCAGAA ACATTGCAGA A-l GG'CAAA AAAAGAAAAA SAAATAAA;A ATTCTCTOAG CT AC:-C C.AC AGAGATCAGC AAACAGCTAA AGCGGGTGGA AGGATGCAGfA ACTGTTCAGT CT7C1TlCAAT TCAAAATGAT C;AATCTTAAG. AAACAATCAG
TGTCGT
28 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 2080 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:
NO
(iv) ANTISENSE: YES (vi) ORIGINAL SOURCE: TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 2: r: ;ILL rr r. riS 1111~ _11 29
CAGCCTGCCA
TCCTTCTGQG
ThAAATACGAA
GGAGCTAGAG
GCAAAArGAGC
GGTTGCAGAA
GGTTTCTGAG
AGAAGACAGT
AAATTATGAT
GAACATAAAT
CCCTTGTGGA
TGACACCACA
CT=GSCTGAA
T3CCGA,-G.C CAACGT:JkAZ Tr:TCCAGCAC
CAGCCAGGTG
CGGATACCAtT 7AT-GA':AAA
ATCTCCCAG
GAGCCGTCCT
CCCTCCTTCT
GA.AAAACCTC
GGCATTACAA
TATTCTAAAG
TTCCCGACCT
AAGCAGCATA
TCTTTAGAAG
TCTAAAATAC
ATTCCAAAGA
TTCTT'TTTrA
CTATCTGTGG
TTTGTAATCT
CTGCCTCCC
TC~rGACAAAG
TCCATCGACG
AGAGAGTTGG
CTGACAGAG3A
AAGGAGTCCC
GACAAAGAGG
GAAGTGTATA
GACGAAGAGG
AGTGTCAATT
TGACCAATG
AATCCTACAG
GAAGTCA6ATT GCCCTCAA3G
AGTGCAGC:A
STCGAZTCCA
.kACACGGTGC
GAATOGAGA
TTTGA.7CTCC
CAGAGGCAAA
*Z-TTAGZACZA
CACTCACCZT
CTGTCTCCTG
TCCCAAAGGC
ATTACCAGAA
AATGGACAAA
GAAATAAA
rCTACCCCAC
AAACAGCTAA
AGGATGCAGA
CTTCTCAAT
GAATCTThAG
TGTCGTGAGA
ATGTGTTGGT
CAATAAATGT
TGCCTGCTTC
CAGGGACCAT
AGGATGAACT
AAGACATTGA
AAACCCCCAC
AAAAACTCT'r
AAAGTGNAAGA
CAGAGGAGGA
AAAGAACAAT
CTGACAACTC
GCAGCAATGG
TGATTGAGGA
TGAACAACAT
ACAACACTGT
TGGCCATTGC
ACTTCATAAC
TCACGGAGCT
T-GTCAAGCT
C;XGACCAAG
A67,GTTTGCA
A;,GTCTGGCA
GGTCATCCCC
TGGCCACACT
TGCCACCACC
ACATTGCAGA
AAAAGAAAAA
ArrCTCTGAG
AGAGATCAGC
AGCGGGTGGA
ACTGTTCAGT
TCAAAATGAT
AAACAATCAG
TTTGTATTGG
AACTCCGAGT
GGATTGAAGT
TGGCTGCCTT
GTCTACCTTT
CCTCGCCTCC
ACCTGACCGC
AGGGACATTC
GGAGAAGGAG
AGAGCTTATC
GGAGGAGGAG
TGAAACTGCA
TAAGCCAAAG
GAGGAACACA
CGCTGGAC
TGAGAACATC
GGTCAA(GACG
AGAGATGCTC
GGGAAAGGGG
GCG7TTZCAT
GCTGAAGGAG
7ATGAGZ:ATG
GGACAAAAA
AAC-AGGA.ACA
AAAAC-CCCC
'rCCTCCTCCT
TCCTCCTCCT
AGTCATCAAA
AGGGAAAAGC
GT:CAGTGCAA
TCATGAGAAT
AGTTCCAGAA
GTATTACAT
CCCTGACTTT
CATGTTTCTT
CAAGAAGCAG
TGTAATGAGT
TTTTTCCCTT
GAATGCCTGG
GGCTACCGAA
CTGTCA~CCG
AACCTTCCCG
AGCAGAGAGG
AGGCTGGGGG
TTACTGAAA
TCCCAGGAGG
AAAGGGATTA
ATATTTAAAA
GAGTCCCCAG
AAGATTAAAA
ACAACACAGA
TTCA6GTCTGG
AAAGCCAATO
ATCCTGGCCA
AACCAGAGGC
AACACGACGC
ACCAGCATTT
CAGCAGGAGG
CCTAGCTCTT
AAAA)UGTCC
CCCCCTCCTC
CCCCCTCCTC
CAACAGGAGA
GTCAAGAAAC
GAGAAGAAAA
CTCAITGGAAC
GCCCTGCGAT
GAAATGCATT
AAAA.ATAATC
CTGTAAATAT
TTAAT=AAA
TCATGAAATG
TCCTTCAAGC
GAGGACT1CAG
AGGAZCTOAA
TGGGGCTAAG
CACTGATGGC
AATGTGGAAA
GTAACAGTGA
AAGAGGAGGA
ATGGAACTGT
GTCAAATAGA
CTGCCATrCA
GCAATGACCC
CCCTTACCCG
CCAACACGCA
AGCACATCAC
TCATGAGAGC
ACATCATGGG
TGCTGAGGCT
TC-ACAAGAAA
GATACGATGG
CACCTTATGT
AGACTGTGAG
CTCCTCCTCC
CACTCCCAGA
GTGCCCAACG
AGCCAAACAG
TGGAAGACAG
CAATrCGGCG
GGGAACATGA
GTGAGATGTT
TCACCCATTA
GAAAATAAAT
GATGCTCTTC
TGCTCTTATT
620 240 300 350 420 480 540 660 lC2C 144:- 1-zc 1=9: 2-4C ::z INFORMATION FOR SEQ ID NO: 3:
SEQUENCE*CHARACTERISTICS:
LENGTH: 2268 base pairs 30 TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL:
NO
(iv) ANTISENSE:
YES
(vi) ORIGINAL
SOURCE:
TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 3: CAkGCCTGCCA
TCCTTCTGGG
T.-AA7rACGAA,
GGAGCTAGAG
G(CAAAAC-AG.:
CTATTGGGAA
GITT3CAAA GGTTTCTA-:3 AG3AASACAG7 AAATTATGA7
GAACATAAAT
CCCTZTGTGGA
TOACACCACA
CTTTOCTGA
TGCCGACGAC
CAACGTAAAC
TCTCCAGCAC
CAGCCAGGTG
GGGATACCAT
TATGGATAA6A
AGGACCCAAT
ATCTCCCAG
GAGCCGTCCT
CTTGCCTCCC
7CT1,ACAkkA
C.ZATCGACG
:-GA*3AGTTGG MTACAGAzA
.Z.AC-AGTC~C
3rGTA7A -AC -AAGAG -:3T5TCArTT 7TG.ACCA6A7G
AATCCTACAG
3AAGTCAArr 3CCCTCAAGG
A'GTGCAGCCA
GTCGAGTCCA
A6ACACGGTGC
GAAATGGAGA
.TTGAACTCC
CAGAGGCAAA
=TAGGACCA
CACTCACCCT
CTGTCTCCrG
TGCCTGCTTC
CAGGGACCAT
AGGATGAACT
AAGACATTGA
AAACCCCCAC
AAAAACTCTT
AXZGTGAA.GA
CAGAGGAGGA
AAAGAACAAT
CTGACAACTC
CCAGCAATGG
TGATTGAGGA
T1GAACAACAT
ACAACACTGT
TGGCCATTGC
ACTTCATA6AC
TCACGGAGCT
TGTCAAGCT
CAGGACCAAG
AACGII-rGCA
AAGTCTGCCA
GGTCATCCCC
TGGCCACACT
TGGCTGCCT
GTCTACCTTT
CCTCGCCTcC
AZCTGACCGC
GGAG-AAGGAG
AGAGCTTATC
GGAGGAGGAAG
TGAAACrGCA TA6AGCCAAAG
GAGC-ACACA
CGCTTTGGAC
TGAGA.AATC
GOTGA.AGACG
AGAGATGCTC
GGGAAAGGGG
GCGT-,TCCAT
GCTGAAGGAG
AATGAGCATG
GGAGCAAAAA
AAGAGGAACA
AAAACTCCCC
TCCTCCTCCT
GAATG.CCTGG
GGCTACCGAA
CGTCAGCCG
AACCrrCCG
AGCAGAGAGG
AGGCTGGGGG
TTTACTGAAA
TCCCAGGAGG
AAAGGGATTA
ATATTAAA
GA6GTCCCCAG
AAGATTAAAA
ACAACACAGA
TTCAGTCTGO
AALAGCCAATG
ATCCTGGCCA
AACCAGAGOC
AACACGACGC
ACGAGCATTT
CAGCAGOGAGG
CCTAOCTCTT
AAAAAAGTCC
CCCCCTCeCT
TCCT'TCAAGC
GAGGACTCAG
AGGAGCTGAA
TCGGGCTAAG
CACTGAkTGGc
AATGTGGAA,
GTAACAGTGA
AAGAGGAGGA
ATGGAAC7GT
GTCAAATAGA
CTGCCATTCA
GCA6ATGACCC
CCCTTACCCG
CCAACACGCA
AGCACA7CAC
TCATGAGAGC
ACATCATCGG
TGCTGAGGCT
TGACAAGAAA
GATACGATGG
CACCrTATGT
AGACTGTGAG
CTCCTCC'rCC 0 i~C 73C 1:2z Ica: -7-7 ,1 31 CCCTCCTTCT TCCCAAAGC
TGCCACCACC
GAAAAAGT ATTACCGA
ACATTGCAGA
GGCATTACAA AATGGACAAA
AAAAGAAAAA
TATTCTAAAG GAAATAAAAA
ATTCTCTGAG
TTCCCGACCT TCTACCCCAC
AGAGATCAGC
AAGCAGCATA AAACAGCTA
AGCGGGTGGA
TCTrTAGAAG AGGATGCAGA
ACTGTTCAGT
TCTAAAATAC CTTCTTCAAT
TCAAAATGAT
ATTCCAA6AGA GAATCTTAAG
AAACAATCAG
TTCTTTTTrA TGTCGTGAGA 1-rTGTATrG CTATCTGTGG ATGTGTTGGT AACTCCGAGr TTTGTAATCT CAArAAATGT
GGATTGAAGT
I'TT=TrGTG ACTTGATACA
TCTGTCAGAT
?TTTrTCCCT TTX74TTAAAA AGCCA6AACTA GGTGTGTATG TAACATTACT
GGACATTAAA
TCCTCCTCCT
AGTCATCAAA
AGGGAAAAAG
GTCAGTGCAA
TCATGAGAAT
AGTTCCAGA6A
GGTATTACAT
CCCTGACTTT
CATGTTTCTT
CAAGAAGCAG
TGTAATGAGT
TT-CCCTT
TTTTGTAATC
ATATTTTTCT
AAAAATTATT
CCCCCTCCTC
CAACALCAGA
OTCAAGAAAC
GAGAAGAAAA
CTCATIGGAAG
GCCCTGCGAT
GAAATGCATT
AAAAATAATC
CTGTAAATAT
TTAA?1"rAAA
TCATGAAATG
TTTTTAAAGC
TCGATAAATG
GTGAGTTAAT
ACATTCTC
CACTCCCAGA
GTCCCCAACC
AGCCAAACAG
TGGAAGC
CAATTCGGGG
GGGAACATGA
GTGAGATGTr
TCACCCATTA
GAA&LATAAAT
GATGCTC'rTC TGCTGTrATT
CAAACTAATA
TGTATTGAAG
ACATCTGTCA
144C isoc 156C 162C 16 Sc 174 C 186C 192C 204C 2:OC 222C INFORMATION FOR SEQ ID NO: 4: SEQUENCE
CHARACTERISTICS:
LENGTH: 552 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
NO
(iv) ANTISENSE: YES (vi) ORIGINAL
SOURCE:
TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 4: T 7 -32 Met Ser Thr Phe Gly Tyr Arg Arg Giy Leu Ser Lys Tyvr Glu Ser Ile 1 5 10 Asp Glu Asp Glu Leu Leu Ala Ser Leu Ser Ala Giu Glu Leu Lys Glu .2025 Leu Giu Arg, Giu Leu Glu Asp Ile Giu Pro Asp Arg Asn Leu Pro Val 40 Gly Leu Arc, Gin Lys Scr Leu Thr Glu Lys Thr Pro Thr Gly Thr Phe 55 7- 7-77-7777-7 33 Ser Arg Giu Ala Lcu Met Ala Tyr Trp Glu Lys Giu -ter t 6 1n Lys Lau 70 75 Lett Glu Lys Glu Arg Lcu Gly Glu Cys Gly Lys Val Ala Glu Asp Lys 90 Glu Glu Ser Glu Glu Glu Leu fie Phe Thr Glu Scr Asn Scr Glu Val 100 105 110 Ser Glu Glu Val Tyr Thr Glu Glu Glu Glu Giu Giu Ser Gin Glu Glu 115 120 125 Glu Glu Glu Glu Asp Ser Asp Glu Glu Glu Arg Thr lie Glu Thr Ala 130 135 140 Lys Gly fie Asn Gly Thr Val Asri Tyr Asp Scr Val Asn Ser Asp Asn 145 ISO 155 Ser Lys Pro Lys le Phe Lys 5cr Gin le Glu Asn le Asn Leu Thr 165 170 175 Asn Gly Ser Asn Gly Arg Asn 7%r Glu Ser Pro Ala Ala Ile His Pro 180 185 190 Cys Gly Asn Pro Thr Val lie Glu Asp Ala Leu Asp Lys lie Lys Scr 1195 200 205 Asn Asp Pro Asp Thr Thr Glu Val Asn Leu Asn Asn Ile Glu Asn lie 210 2115 220 Thr Thr CGI n Thr Leu Thr Arg Phe Ala Glu Ala Leu Lys Asp Asn Thr 225 230 235 240 Val Vaf Lvs Thr Phe Scr Leu Ala Asn Thr His Ala Asp Asp Ser Ala 245 250 '.33 Ali Met Ala fie Ala Gin Met Leu Lys Ala Asn Glu His Ilie Thr Asn 260 265 270 Val Asn Val Glu 5cr Asn Phe lie Thr Gly Lys Gly fie Leu Ala fie 275 280 285 Met Arg Ala Leu Gin His Asn Thr Val Leu Thr Glu Leu Arg Phe His 290 295 300 Asn Gin Arg His Ile Met Gly Ser Gin Val Glu Met Glu le Val Lys 305 310 315 320 Leu Leu Lys Glu Asn Thr Thr Leu Leu Arg Leu Gly Tyr His Phe Glu 325 330 335 Leu Pro Gly Pro Arg Met Ser Met Thr Ser le Lcu Thr Ariz Asn Met 340 345 350 Asp Lys Gin Arg Gin Lys Arg Leu Gin Glu Gin Lys Gin Gin Glu Gly 355 360 365 Tyr Asp Gly Gly Pro Asn Leu Arg Thr Lys Va! Trp Gin Arg Gly Thr 370 375 380 34 Pro Ser Ser Ser Pro Tyr Val Ser Pro Arg His Scr Pro Trp Scr Scr 385 39w 395 400 Pro Lys Leu Pro Lys Lys Val Gin Thr Val Arg Ser Arg Pro Leu Ser 405 410 415 Pro Val Ala Thr Leu Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 420 425 430 Pro Ser Ser Gin Arg Leu Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 435 440 445 Leu Pro'Glu' Lys Lys Lau lie Thr Arg Asn Ile Ala Glu Val lie Lys 450 455 460 Gin Gin Giu Ser Ala Gin Arg Ala Leu Gin Asn Gly Gin Lys Lys Lys 465 470 475 480 Lys Gly Lys Lys Val Lys Lys Gin Pro Asn Ser le Leu Lys Glu Ile 485 490 495 Lys Asn Ser Lcu Arg Ser Val Gin Glu Lys Lys Met Giu Asp Ser Ser 500 505 510 Arg Pro Ser Thr Pro Gin Arg Ser Ala His Gtu Asn Leu Met Glu Ala 515 520 525 Fe Arg Gly Set Ser Ile Lys Gin Leu Lys Arg Val Glu Val Pro Giu 530 535 540 Ala Leu Arg Trp Giu His Asp Leu 545 550 INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: 10 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 35 (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES (vi) ORIGINAL SOURCE: TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO:
CCTTCTACCC
INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 279 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES (vi) ORIGINAL SOURCE: TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 6: GCCAACACGC ANTCCGACGA CA=TCAGCC ATGGTCAITT CAGAGATGCN TCAAAGTCAA TGAGCACATC ACCAACGTAA ACOTCGAGTC CAACTTCAA ACGGGAAAGG GGATCCTGGC 120 CATCATGAGA GC-&CrCCAGC ACAACACGGT GCTCACGGAG CTGCGGI7TC ATAACCAGAG 180O GCACATCATG GGCAGCCAGG TCGAAATGGA GATTGTCAAG CTNCTGAAGG AGAACACGAC 240 GC-)NCTGAGG CT=G kTACC ArrTTNAAcT cccAG-.Acc 279 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 93 amino acids 36 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES (vi) ORIGINAL SOURCE: TISSUE TYPE: cardiac tissue (xi) SEQUENCE DESCRIPTION SEQ ID NO: 7: PTRNPTTVQPWSLQRCIKVNEHITNVNVESNFITGKGILA1MRALQ 20 30 43 HNTVLTELRFHNQRHI MGSQVEME IVKLLKENTTLLRLG YHF KLPG 60 70 8C 1 T