CA2335646A1 - Genes of the dead box protein family, their expression products and use - Google Patents

Abstract

The invention relates to the preparation of two novel nucleic acids -Hc1 and Hc2- which are homologous to known DEAD box proteins, which correspond to RN A helicases, and which can be obtained from ciliates. The invention also relat es to the insertion of the novel nucleic acids into suited target cells using recombinant DNA technologies, and to the use thereof for regulating transcription and translation. In addition, the invention relates to the in- vitro or in-vivo transcription of the novel nucleic acids for producing nove l proteins and to the use thereof in treatment and diagnosis.

Description

"u WO 00/05388 PCT/EP99/04892 Genes of the DEAD box protein family, their expression products and use Description The present invention relates to the preparation of novel nucleic acids from ciliates coding for expression products, preferably for RNA helicases from the family of DEAD box proteins, and the use thereof.
The modulation of the RNA structure has an essential function in cellular processes, such as, for example, in pre-mRNA splicing, in RNA transport or in protein translation, as the cellular RNA is present in the cell in different secondary and tertiary structures and, in addition, a large number of RNA-binding proteins provides for further structuring of the RNA. Proteins from the family of the so-called DEAD protein family, inter alia, are involved in these modulation processes. The members of this protein superfamily, which characteristically contain a number of homologous protein sequences, so-called "protein boxes", are named after the highly conserved tetrapeptide Asp-Glu-Ala-Asp, in the single-letter code DEAD, as a motif. This protein superfamily includes in particular RNA helicases.
The characteristic protein sequences of DEAD proteins are highly conserved in evolution (cf. Figure 1 ).
The DEAD superfamily is divided into various subfamilies, which according to their sequence motif are called DEAD, DEAH or DExH subfamily. All family members have an ATP-binding and RNA-binding function and also an ATP hydrolysis and for the most part an RNA helicase function (Fig. 2).
A conserved region comprising approx. 300 amino acids and flanked by nonconserved amino acid sequences of varying length is characteristic of the various members of the DEAD box protein family w WO 00/05388 2 PCT/EP99/04892 (Schmid S.R., Lindner P., Mol Cell Biol 1991 11: 3463-3471 ).
The so-called homology boxes (synonymously conserved motifs), one of which is the "DEAD box", are located within the conserved region. The homology boxes confer not only structural but also functional similarity on the members of the DEAD box family. Considering the homology boxes (see Figure 2), DEAD box proteins are putative ATP-dependent RNA
helicases which take part in a plurality of cellular processes and are connected with secondary structure modification of RNA molecules (Fuller-Pace F.V. Trends Cell Biol. 1994 4:271-274, Pause A., Sonenberg N., Curr Opin Struct Biol 1993 3:953-959). Helicase-dependent processes have been described likewise for: translation initiation, nuclear and mitochondrial RNA splicing, mRNA transport, ribosome assembly and spliceosome assembly, mRNA stabilization and mRNA degradation (lost I., Dreyfus M., Nature 1994 372:193-196).
The functions of RNA helicases corresponding to the DEAD homology boxes in cellular processes, preferably in the context of protein biosynthesis, allow specifically these enzymes to be employed with regard to pharmaceutical, agricultural or biotechnological and analytical applications.
Important pharmaceutical applications are the development of substances which inhibit specifically bacterial, parasitical and viral helicases or helicases originating from pathogenic fungi, but which have no inhibitory effect on human helicases. Since helicases are for the most part essential enzymes, it is possible to achieve destruction of the pathogen (bacterium, fungus, parasite/protozoon, virus) by specific inhibition of these enzymes.
According to Missel et al., switching off the gene for a DEAD box protein leads to decreased growth in particular protozoa (Trypanosoma, Leishmania, Crithidia) (Missel A., Souza A.E., Norskau G, Goringer H.U., Mol Cell Biol 1997 17:4895-903). In the malaria pathogen Plasmodium falsiparum helicases control protein translation, mitosis and DNA repair (Thelu J, Burnod J, Bracchi V, Ambroise-Thomas P, DNA Cell Biol 1994 13: 1109-1115). Helicases are essential for the initiation of translation, in the spliceosome, in the cell cycle and assembly of ribosomes in yeast. Thus for example, the DEAD box protein ROK1 is essential for the viability of yeast, for pre-rRNA processing and for mitotic growth. Switching off ROK1 blocks 18S rRNA synthesis (Venema J., Bousquet-Antonelli C., Gelugne J.P., Caizegues-Ferrer M., Tollenrey, Mol Cell Biol 1997 17: 3398-3407).
Approx. 80% of all "positive-stranded" RNA viruses whose genomes have been sequenced code for at least one putative helicase. Examples are NS3 of hepatitis C virus, helicases of human coronavirus and adeno-associated virus, and vaccinia virus helicase (Kadare G., Haenni A.L., J Virol 1997:
2583-2590). Possible roles for viral helicases are (i) proof reading during replication, (ii) transcription initiation by unwinding the RNA and preventing loop formation behind RNA polymerase, (iii) translation initiation. Vaccinia virus helicase is essential for the life cycle of the virus and is nucleic acid-specific.
There are indications that at least some helicases can be very specifically activated. Thus, for example, DpbA from E. coli (Fuller-Pace F.V., Nicol S.M., Reid A.D., Land D.P., EMBO J 1993 12:3619-3626) and SIt22 from yeast (Xu D., Nouraini S., Field D., Tang S.J., Friesen J.D., Nature 1996 381: 709-716) need specific RNA ligands for activation.
In addition, DEAD box proteins are described in association with diseases.
The amplification of a specific gene in cancer cells (N-myc) is linked to the fact that a DEAD box protein is coamplified with N-myc, which points to a role of this protein in the degeneration of cancer cells (George R.E., Kenyon R.M., McGuckin A.G., Malcolm A.J., Pearson A.D., Lunec J., Oncogene 1996 12: 1583-7). Mutations of an RNA helicase are connected with Werner's syndrome - premature aging - (Yu C., Oshima J., Wijsman E.M., Nakura J. et al., Am J Hum Genet 1997 60: 330-341 ) and with xeroderma pigmentosum (Kobayashi T., Kuraoka I., Sailo M., Nakatsu Y.
Tanaka A. et al., Huma Mut 1997 9: 322-331 ). Furthermore, a possible connection between DEAD box proteins and connective tissue diseases has been postulated (Valdez B.C., Henning D., Perlaky L., Busch R.K., Busch H., Biochem Biophis Res Commun 1997 234: 335-340). Additionally a connection is known between defective DNA repair and a mutation in the helicase domain of the XNP/ATR-X gene (Villard L., Lossi AM, Cardoso C, Proud V, Chiaroni P, Colleaux L, Schwartz C, Fontes M Genomics 1997 43: 149-155).
In addition to helicases from humans or from various pathogens, helicases from plants are also of the greatest interest. Some DEAD box proteins from plants have become known (Lorkovic Z.J., Hermann R.G., Oelmialler R., Mol Cell Biol 1997 17: 2257-2265 and Aubourg et al., Gene 1997 199(1-2):
241-253). Although these proteins are structurally similar to DEAD box proteins from organisms other than plants, they form nevertheless a subgroup (Fig. 3). Fig. 3 shows the phylogenetic relationship of various eIFA4, one of the best characterized members of the DEAD box protein family, from various organisms. The plant proteins are much more closely related to each other than to eIF4A from animal eukaryotes. An application which is of interest to agricultural production is the stimulation of the activity of plant-specific RNA helicases in order to increase protein expression of economically relevant proteins. In this case it is possible either to stimulate helicases intrinsic to plants (for example by overexpression) or to express helicases plant-like heterologously in useful plants.
It is therefore the object of the present invention to provide novel nucleic acids coding preferably for RNA helicases.
The object is achieved by providing nucleic acids which code for RNA
helicases and which are obtained preferably from ciliates, particularly preferably Tetrahymena thermophila. The nucleic acids of the invention code for expression products originating from the family of DEAD box proteins and thus also for RNA helicases.

Expression products, preferably proteins from the DEAD protein superfamily, in accordance with this invention are those having conserved motifs among which one conserved motif comprises the amino acid sequence DEAD. Preferably the proteins comprise an RNA helicase activity and ATPase activity.
The present invention therefore relates to nucleic acids coding for RNA
helicases having a nucleic acid sequence depicted in SEQ ID No. 13 or SEQ ID No. 15 or a functional variant thereof, and parts thereof having at least 8 nucleotides, preferably having at least 15 or 20 nucleotides, in particular having at least 100 nucleotides, especially having at least 300 nucleotides (called "nucleic acids of the invention" in the following).
The nucleic acid of the invention having the nucleic acid sequence depicted in SEQ ID No. 13 ("Hc1" in the following) codes for an amino acid sequence depicted in SEQ ID No. 14.
The nucleic acid of the invention having the nucleic acid sequence depicted in SEQ ID No. 15 ("Hc2" in the following) codes for an amino acid sequence depicted in SEO ID No. 16.
Expression of the nucleic acids of the invention in E. coli led to an expression product which shows similar enzymatic activities to those of an RNA helicase. Further experiments according to the present invention confirmed that the nucleic acid is a nucleic acid coding for an RNA
helicase, in particular due to the presence of the characteristic homology boxes such as in SEQ ID No. 14 and SEQ ID No. 16, which are represented in Figurel.
In a preferred embodiment the nucleic acid of the invention is a DNA or RNA, preferably a double-stranded DNA, and in particular a DNA having a nucleic acid sequence coding for RNA helicases.

The term "functional variant" means according to the present invention a nucleic acid which is functionally related to RNA helicases having the described homology boxes.
In a wider sense, the term "variants" means according to the present invention nucleic acids having a homology, in particular a sequence identity of approx. 60%, preferably of approx. 75%, in particular of approx. 90% and especially of approx. 95%.
The parts of the nucleic acid of the invention may be used, for example, for preparing individual epitopes, as probes for identifying further functional variants or as antisense nucleic acids. For example, a nucleic acid of at least approx. 8 nucleotides is suitable as an antisense nucleic acid, a nucleic acid of at least approx. 15 nucleotides is suitable as a primer in the PCR method, a nucleic acid of at least approx. 20 nucleotides is suitable for identifying further variants and a nucleic acid of at least approx. 100 nucleotides is suitable as a probe.
In particular it is possible to use the nucleic acids of the invention in order to construct complementary and/or antisense nucleic acids which hybridize with Hc1 or Hc2 themselves or with related nucleic acids. Introducing the complementary and/or antisense nucleic acid into the target cell prevents expression of related RNA helicases or related expression products.
Antisense nucleic acids obtainable from the nucleic acids of the invention may therefore be used for the specific regulation of gene expression. In this case either the target cell may be transfected according to known methods with the anti-gene which is then transcribed in the cell, or in vitro synthesized antisense RNA or DNA is introduced into the target cell by microinjection. It is known that antisense RNA complementary to the coding region of the target mRNA can inhibit gene expression.

The duplex strand formed by mRNA and antisense RNA is susceptible to fast degradation by RNAses.
Inhibition of transcription and translation by the antisense technique discussed has also been successfully carried out in plant cells (van der Krol A.R. et al. Nature 1988 333: 866).
In a further preferred embodiment the nucleic acid of the invention comprises one or more noncoding sequences and/or a poly (A) sequence.
The noncoding sequences are, for example, intron sequences or regulatory sequences, such as promoter sequences or enhancer sequences, for the controlled expression of expression products, preferably of RNA helicases.
In a further embodiment the nucleic acid of the invention is therefore included in a vector, preferably in an expression vector or a vector effective for gene therapy.
The expression vectors may be, for example, prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are for expression in E. coli, for example, the T7 expression vector pGMlO or pGEX-4T-1 GST (Pharmacia Biotech), which codes for an N-terminal Met-Ala-His6 tag, which facilitates advantageous purification of the expressed protein via a Ni2+-NTA column. Suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for example, the vectors p426Met25 or p426GAL 1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767), while suitable vectors for expression in insect cells are, for example, baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and suitable vectors for expression in mammalian cells are, for example, SV40 vectors which are generally available.
In general the expression vectors also include regulatory sequences suitable for the host cell such as, for example, the trp promoter for expression in E. coli (see e.g. EP-B1-0154133) in E. coli, the ADH-2 promoter for expression in yeasts (Russet et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see e.g. EP-B1-0127839) or the early SV40 promoter or LTR promoters, for example of MMTV (mouse mammary tumour virus; Lee et al. (1981 ) Nature, 214, 228).
The recombinant proteins obtained in this way are purified using suitable methods (e.g. affinity chromatography, HPLC, FPLC) and dissolved (guanidine, urea). Characterization of the proteins and determination of enzyme activity are carried out with the aid of established assays (RNA
binding, ATPase activity, helicase activity).
Examples of vectors effective for gene therapy are virus vectors, preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, for example an adeno-associated virus vector consisting exclusively of two inserted terminal repeats (ITR).
Examples of suitable adenovirus vectors are described in McGrory, W.J. et al. (1988) Virol. 163, 614; Gluzman, Y. et al. (1982) in "Eukaryotic Viral Vectors" (Gluzman, Y. ed.) 187, Cold Spring Harbor Press, Cold Spring Habor, New York; Chroboczek, J. et al. (1992) Virol. 186, 280; Karlsson, S.
et al. (1986) EMBO J.. 5, 2377 or W095/00655.
Examples of suitable adeno-associated virus vectors are described in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97; W095/23867;
Samulski, R.J. (1989) J. Virol, 63, 3822; W095/23867; Chiorini, J.A. et al.
(1995) Human Gene Therapy 6, 1531 or Kotin, R.M. (1994) Human Gene Therapy 5, 793.
It is also possible to obtain vectors effective for gene therapy by complexing the nucleic acid of the invention with liposomes. Suitable for this purpose are lipid mixtures as described in Felgner, P.L. et al. (1987) Proc. Natl. Acad. Sci, USA 84, 7413; Behr, J.P. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982; Felgner, J.H. et al. (1994) J. Biol. Chem. 269, 2550 or Gao, X. & Huang, L. (1991 ) Biochim. Biophys. Acta 1189, 195. The liposomes are prepared by binding the DNA ionically on the surface of the liposomes, in a ratio such that a positive net charge remains and that the DNA is completely complexed by the liposomes.
The nucleic acids of the invention can, for example, be synthesized chemically, e.g. by the phosphotriester method (see, e.g., Uhlman, E. &
Peyman, A. (1990) Chemical Reviews, 90, 543, No. 4), either on the basis of the sequence disclosed in SEQ ID No. 13 and SEQ ID No. 15 or on the basis of the peptide sequence disclosed in SEQ ID No. 14 and SEQ ID No.
16 and making use of the genetic code.
A further possibility of obtaining the nucleic acids of the invention themselves and variants is that of using a suitable probe (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning. A laboratory manual. 2nd Edition, Cold Spring Harbor, New York) to isolate them from a suitable gene library. Examples of suitable probes are single-stranded DNA
fragments which have a length of from approx. 100 to 1000 nucleotides, preferably of a length of from approx. 200 to 500 nucleotides, in particular of a length of from approx. 300 to 400 nucleotides, and whose sequence can be derived from the nucleic acid sequence depicted in Figures 4 and 6.
The invention further relates to the use of the nucleic acids of the invention for specifically affecting protein biosynthesis.
Experiments show that increased helicase activity leads to enhanced unwinding of the target RNA. This makes the target RNA available for binding partners such as proteins, in particular enzymes or enzyme complexes for ribosomal translation or for degradation by the degradosome. Depending on the target RNA, this leads to a) increased translation and therefore increased protein biosynthesis, b) faster degradation by the degradosome and thus decreased protein biosynthesis.
Inhibition of the helicase activity may inhibit degradation by the degradosome and lead to a decreased protein biosynthesis. This is based on the finding that important biosynthetic processes can be specifically regulated by selective inhibition or activation of helicases.
For this purpose, the nucleic acids are expressed in a recombinant manner in suitable target organisms as described.
The nucleic acids of the invention, preferably Hc1, are an outstanding model for various eukaryotic RNA helicases, preferably from humans and parasites (Fig. 3A). The genetic relationship of Hc1 with the relevant eukaryotic helicases is close enough in order to draw conclusions from experiments with Hc1 about structure and function of other eukaryotic helicases (e.g. human). Figure 3A shows the genetic relationship of some helicases from various organisms in comparison with Hc1. Particularly surprising are the great structural similarity between Hc1 and mammalian helicases from humans and mice and the great structural difference between Hc1 and known viral helicases.
On the basis of phylogenetic studies depicted in Figure 3B, the nucleic acids of the invention, preferably Hc2, prove to be an outstanding model for various eukaryotic RNA helicases, preferably from plants. Figure 3B shows the genetic relationship of some RNA helicases from various organisms in comparison with Hc2. Particularly surprising is the great structural similarity between Hc2 and RNA helicases from plants. The recombinant expression of Hc2 makes it possible to use this new enzyme as a model for investigating in particular helicases from plants and the structure and function thereof and for developing suitable inhibitors or activators of these important enzymes. Thus it has been postulated for PRH75 from spinach that this enzyme needs a very specific RNA ligand in order to be active (Lorkovic Z.J., Herrmann R.G., Oelmuller R., Mol Cell Biol 17(4): 2257-2265 (1997)).

The invention therefore further relates to the specific heterologous expression - by means of overexpression according to known methods - of the nucleic acids of the invention, preferably Hc2, in suitable useful plants for the potential increase in the biosynthesis of relevant proteins.
The nucleic acids of the invention, preferably Hc2, may be introduced into plants by recombinant DNA techniques. A method which may be used is the introduction of the foreign gene with the aid of Agrobacterium tumefaciens. This involves introducing the foreign gene into the genome of the bacterium in a known manner. Infecting the target plant leads to stable integration of the genes of the bacterium including the foreign gene into the genome of the plant (Chilton M.D. et al., Cell 1977 11:263, Barton K.A. et al., Cell 1983 32: 1033). It is preferred to employ this method for the transformation of dicotyledons. Known methods such as calcium phosphate precipitation, PEG treatment, electroporation or a combination of these methods may be employed for the transformation of monocotyledons (Potrykus I. et al., Mol Gen Genet 1985 199: 183; Lorz H. et al., Mol Gen Genet 1985 199: 178; Fromm M et al. Nature 1986 319:791; Uchimiya H. et al., Mol Gen Genet 1986 204: 204). It is also possible to introduce the foreign DNA into plant cells with the aid of the so-called gene gun (Klein T.M. et al. Nature 1987 327: 70).
The invention further relates to the use of the nucleic acids as selection markers in molecular biology. Conventionally, antibiotics are used as selection markers. Molecular-biologically modified organisms carry a gene which confers resistance to an antibiotic. The organisms are grown in antibiotic-containing medium such that only the carriers of the resistance gene are able to develop. Analogously, helicase genes may be used as "resistance genes". It has been shown (Mullner et al, patent application DPA 19545126.0) that overexpression of a helicase gene in murine cells confers on these cells tolerance of an otherwise toxic substance, leflunomide. It is thus possible to employ the nucleic acids of the invention as selection markers in molecular biology, with the nucleic acids of the invention having to be introduced into cells using a suitable vector as described, and to select using a suitable substance, such as leflunomide), of which the cells are tolerant due to overexpression of the helicase.
The present invention furthermore also relates to the expression products, preferably polypeptides and polypeptide fragments (encoded by Hc1 and Hc2) themselves, having amino acid sequences depicted in SEQ ID No. 14 and SEQ ID No. 16 or a functional variant thereof, and parts thereof having at least six amino acids, preferably having at least 12 amino acids, in particular having at least 65 amino acids and especially having 257 amino acids Hc1 and 255 amino acids Hc2 (subsequently called "polypeptides of the invention"). For example, a polypeptide which is approx. 6-12 amino acids in length, preferably approx. 8 amino acids in length, can contain an epitope which, after having been coupled to a carrier, is used for preparing specific polyclonal or monoclonal antibodies (in this regard, see, e.g., US 5,656,435). Polypeptides whose length is at least approx. 65 amino acids can also be used directly, without any carrier, for preparing polyclonal or monoclonal antibodies.
In accordance with the present invention, the term "functional variant"
means polypeptides which are functionally related to the peptides of the invention, i.e. which have an RNA helicase activity. Variants also mean allelic variants or polypeptides which are derived from other human cells or tissue.
In the broader sense, it also means polypeptides which possess a sequence homology, in particular a sequence identity, with the poly-peptides having the amino acid sequences depicted in Figures 5 and 7 of approx. 70%, preferably of approx. 80%, in particular of approx. 90%, especially of approx. 95%. They furthermore also include deletion of the polypeptide in the range of approx. 1 - 60, preferably of approx. 1 - 30, in particular of approx. 1 - 15, especially of approx. 1 - 5 amino acids. In addition, they also include fusion proteins which comprise the above-described polypeptides of the invention,with the fusion proteins already themselves possessing the function of an RNA
helicase or only being able to acquire the specific function after the fusion moiety has been eliminated. Especially, they include fusion proteins having a moiety of, in particular, nonhuman sequences of approx. 1 - 200, preferably approx. 1 - 150, in particular of approx. 1 - 100, especially of approx. 1 - 50 amino acids. Examples of nonhuman peptide sequences are prokaryotic peptide sequences, for example from E. coli galactosidase or a so-called histidine tag, e.g. a Met-Ala-His6 tag. A fusion protein containing a so-called histidine tag is particularly advantageously suitable for purifying the expressed protein via metal ion-containing columns, for example via a Ni2+-NTA column. "NTA" stands for the chelating agent nitrilotriacetic acid (Qiagen GmbH, Hilden).
The parts of the polypeptide of the invention represent, for example, epitopes which can be specifically recognized by antibodies.
By comparing with known helicases, it was found that the polypeptides of the invention is a member of the so-called DEAD superprotein family.
Figure 1 shows the conserved motifs which are characteristic of this class of RNA helicases. All these motifs are highly conserved within the family and are also found in the polypeptides of the invention.
The polypeptide of the invention is prepared, for example, by expressing the nucleic acid of the invention in a suitable expression system, as already described above, using methods which are generally known to the skilled worker. The present invention therefore also relates to a method for preparing a polypeptide of the invention, with a nucleic acid of the invention being expressed in a suitable host cell and being isolated, where appropriate.
In particular, said parts of the polypeptide can also be synthesized by classical peptide synthesis (Merrifield technique).They are particularly suitable for obtaining antisera which can be used for screening suitable gene expression libraries in order to gain access to other functional variants of the polypeptide of the invention.
The present invention furthermore also relates to antibodies which react specifically with the polypeptide of the invention, with the abovementioned parts of the polypeptide either themselves being immunogenic or being able to be made immunogenic, or to have their immunogenicity increased, by being coupled to suitable carriers such as, for example, bovine serum albumin.
The antibodies are either polyclonal or monoclonal. Their production, which is also an aspect of the present invention, is carried out, for example, according to generally known methods, by immunizing a mammal, for example a rabbit, with the polypeptide of the invention or said parts thereof, where appropriate in the presence of, for example, Freund's adjuvant and/or aluminum hydroxide gels (see, e.g., Diamond, B.A. et al. (1981 ) The New England Journal of Medicine, 1344). The polyclonal antibodies which have been generated in the animal due to an immunological reaction can then be readily isolated from the blood using generally known methods and purified, for example, via column chromatography.
Monoclonal antibodies can, for example, be prepared using the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991 ) Nature, 349, 293).
The present invention furthermore also relates to a pharmaceutical which comprises a nucleic acid of the invention or a polypeptide of the invention and, where appropriate, suitable additives or excipients, and to a process for producing a pharmaceutical for treating cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermatitis, type I allergies or type IV
allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes, and/or for affecting the cell metabolism, in particular in association with immunosuppression, especially in association with transplants and/or genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases, in which pharmaceutical a nucleic acid of the invention, for example a so-called antisense nucleic acid, or a polypeptide of the invention is formulated together with pharmaceutically acceptable additives and/or excipients.
A pharmaceutical which comprises the nucleic acid of the invention in naked form or in the form of one of the above-described vectors which are effective for gene therapy, or in the form in which it is complexed with liposomes, is especially suitable for gene-therapeutic application in humans.
Examples of suitable additives and/or excipients are a physiological sodium chloride solution, stabilizers, proteinase inhibitors, nuclease inhibitors, etc.
The present invention furthermore also relates to a diagnostic agent which comprises the nucleic acids of the invention, the polypeptides of the invention or antibodies of the invention and, where appropriate, suitable additives and/or excipients, and to a process for preparing a diagnostic agent for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes, and/or for analyzing the cell metabolism, in particular the immune status, especially in association with transplants and/or for analyzing genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases, in which diagnostic agent suitable additives and/or excipients are added to a nucleic acid of the invention, a polypeptide of the invention or antibodies of the invention. For example, according to the present invention, the nucleic acid of the invention can be used to prepare a diagnostic agent which is based on the polymerase chain reaction (PCR diagnostics, e.g. as described in EP-0200362) or on a Northern blot. These tests are based on the specific hybridization of the nucleic acid of the invention with the complementary opposite strand, usually of the corresponding mRNA. In this connection, the nucleic acid of the invention can also be modified, as described, for example, in EP 0063879. Preference is given to labeling a DNA fragment of the invention with suitable reagents, for example radioactively with a-P32-dATP or nonradioactively with biotin, using generally known methods, and incubating it with isolated RNA which has preferably been bound to suitable membranes composed, for example, of cellulose or nylon. It is furthermore advantageous to fractionate the isolated RNA
according to size, e.g. by means of agarose gel electrophoresis, before hybridization and binding to a membrane. In this way, when the quantity of RNA examined from each tissue sample is the same, it is then possible to determine the quantity of mRNA which has been labeled specifically by the probe.
Another diagnostic agent comprises the polypeptide of the invention or the immunogenic parts thereof which have been described in more detail above. The polypeptide, or the parts thereof, which are preferably bound to a solid phase, for example composed of nitrocellulose or nylon, can, for example, be brought into contact in vitro with the body fluid, e.g. blood, to be investigated in order thereby to be able to react, for example, with autoimmune antibody. The antibody-peptide complex can then, for example, be detected using labeled anti-human IgG or anti-human IgM
antibodies. The label is, for example, an enzyme, such as peroxidase, which catalyzes a color reaction. The presence of autoimmune antibodies, and the quantity of these antibodies which are present, can thus be detected readily and rapidly via the color reaction.
Another diagnostic agent comprises the antibodies of the invention themselves. These antibodies can be used, for example, to readily and rapidly investigate a human tissue sample to determine whether the poly-peptide in question is present. In this case, the antibodies of the invention are, for example, labeled with an enzyme as already described above. The specific antibody-peptide complex can thereby be detected readily and just as rapidly via an enzymatic color reaction.
The present invention also relates to an assay for identifying functional interactors, such as, for example, inhibitors or stimulators, comprising a nucleic acid of the invention, a polypeptide of the invention or the antibodies of the invention, and, where appropriate, suitable additives and/or excipients.
An example of a suitable assay for identifying functional interactors is the so-called two-hybrid system (Fields, S. & Sternglanz, R. (1984) Trends in Genetics, 10, 286). In this assay, a cell, for example a yeast cell, is transformed or transfected with one or more expression vectors which express a fusion protein comprising the polypeptide of the invention and a DNA-binding domain from a known protein, for example from Gal4 or LexA
from E. coli, and/or which express a fusion protein comprising an unknown polypeptide and a transcription-activating domain, for example from Gal4, herpesvirus VP16 or B42. In addition, the cell contains a reporter gene, for example the E. coli LacZ gene, "green fluorescence protein" or the yeast amino acid biosynthesis genes His3 or Leu2, which is controlled by regulatory sequences such as, for example, the IexA promoter/operator or by a so-called upstream activation sequence (UAS) which is present in the yeast. The unknown polypeptide is, for example, encoded by a DNA
fragment which is derived from a gene library, for example from a human gene library. Normally, the described expression vectors are used to prepare a cDNA gene library directly in yeast so that the assay can be performed immediately thereafter.
For example, the nucleic acid of the invention is cloned in a yeast expression vector in functional unity onto the nucleic acid encoding the IexA DNA-binding domain, such that the transformed yeast expresses a fusion protein composed of the polypeptide of the invention and the LexA
DNA-binding domain. In another yeast expression vector, cDNA fragments from a cDNA gene library are cloned in functional unity onto the nucleic acid encoding the Gal4 transcription-activating domain, such that the transformed yeast expresses a fusion protein composed of an unknown polypeptide and the Gal4 transcription-activating domain. The yeast which is transformed with the two expression vectors, and which is, for example, Leu2-, additionally contains a nucleic acid which encodes Leu2 and which is controlled by the LexA promoter/operator. If a functional interaction takes place between the polypeptide of the invention and the unknown polypeptide, the Gal4 transcription-activating domain then binds, via the LexA DNA-binding domain, to the LexA promoter/operator, resulting in the latter being activated and the Leu2 gene being expressed. This then enables the Leu2- yeast to grow on minimal medium which does not contain any leucine.
When the LacZ reporter gene or the green fluorescence protein reporter gene is used instead of an amino acid biosynthesis gene, activation of transcription can be detected by blue- or green-fluorescent colonies being formed. However, the blue color or fluorescence color can also be readily quantified in a spectrometer, for example at 585 nM in the case of a blue color.
In this way, it is possible to screen gene expression libraries readily and rapidly for polypeptides which interact with the polypeptides of the invention. The new polypeptides which have been found can then be isolated and subjected to further characterization.
Another possibility of applying the two-hybrid system is that of using other substances, such as, for example, chemical compounds, to influence the interaction between the polypeptide of the invention and a known or unknown polypeptide. In this way, it is also readily possible to find novel, valuable, chemically synthesizable active compounds which can be employed as therapeutic agents. The present invention is therefore not only directed toward a process for finding polypeptide-like interactors but also extends to a process for finding substances which are able to interact with the above-described protein-protein complex. In accordance with the present invention, such peptide-like interactors, and also chemical interactors, are therefore designated functional interactors which may have an inhibitory or stimulatory effect.
Description of the figures and the important sequences Figure 1 shows diagrammatically the conserved regions (homology boxes) of the proteins of the DEAD protein superfamily. The numbers between the regions indicate the distances in amino acids between the homo boxes.
Figure 2 diagrammatically describes the conserved regions and known functions thereof of the expressed proteins according to Fuller Pace F.V.
(1994), supra.
Figures 3A and 3B describe the phylogenetic trees of Hc1 and Hc2 and establishes the evolutionary connections. These figures were prepared using the program: Lasergene (Modul MegAlign 3.1.7) by DNASTAR Inc., with the aid of the Clustal algorithm (Higgins D.G., Sharp P.M., CABIOS
(1989), Vol. 5, no. 2, 151-153).
SEQ ID No. 13 shows the nucleic acid sequence of Hc1.
SEQ ID No. 14 shows the amino acid sequence corresponding to SEQ ID
No. 13.
SEQ ID No. 15 shows the nucleic acid sequence of Hc2.
SEQ ID No. 16 shows the amino acid sequence corresponding to SEQ ID
No. 15.

The following examples serve to further illustrate the invention without restricting said invention to the products and embodiments described in the examples.
Examples The practical work which led to the present invention is mainly based on established known methods in microbiology, molecular biology and recombinant DNA technology.
Example 1: Cultivation of Tetrahymena thermophila Tetrahymena thermophila, strain B18681V, was inoculated in PPYS
medium (10 g/I proteose peptone No. 3 DIFCO, 1 g/I yeast extract DIFCO, 10 mg sodium citrate, 24. 3 mg FeCl3) in a 500 ml flask (100,000 cells/ml PPYS) and incubated at 25°C and 100-150 rpm for 2-3 days, up to a cell density of approx. 1 million/ml.
Example 2: Isolation of mRNA
Total RNA was isolated from 200 ml of shaker culture of Tetrahymena thermophila, strain B18681V, according to Chomczynski & Sacchi, (1987).
For this purpose approx. 2 million cells were lysed in the presence of guanidine thiocyanate/sarkosyl/beta-mercaptoethanol. After adding sodium acetate and chloroform/isoamyl alcohol/phenol (25:24:1 ), the mixture is mixed well, incubated on ice for 15 min and centrifuged thereafter at 10,000 x g, 4°C for 20 min. 1 volume of isopropanol is added to the aqueous phase which is then left standing at -20°G for at least 30 min.
The RNA pellet is obtained by centrifugation (10 min, 10,000 x g, 4°C
). The pellet is then washed twice, dried and resuspended in DEPC water. After incubating at 55-60°C for 10 - 15 min, it is possible to store the RNA
at -80°C. mRNA is purified from total RNA via oligo(dT)-Sepharose (Clontech mRNA Separator Kit #K1040-2).

Example 3: Preparation of cDNA
mRNA was transcribed into cDNA according to CLONTECH (CapFinderT""
PCR cDNA Library Construction Kit #K1051-1 ).
Example 4: Amplification of specific gene fragments Specific gene fragments were amplified with the aid of the polymerase chain reaction (PCR). A standard PCR mixture contains 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.001 % gelatin, 75 NM dNTP, 0.3 ng of each primer, 0.5,u1 of cDNA, 0.5 U of Taq polymerase.
The primers (2) 5'-GTTCTACCnATTCTGTG-3' and (3) 5'-ACnGGTTCnGGTAAGAC-3' were used for amplifying the fragment Hc1, the primers (4) 5'-ATAGAATTCCCnACnAGAGAAnTnGCT-3' and (8) 5'-ATAGGATCCGTTCTACCnATTCTGTG-3' were used for amplifying the fragment Hc2, with n being any nucleotide. The PCR program was 5 min 95°C, 95°C/37s - 50°C/37s - 72°C/37s - 30 cycles.
Example 5: Cloning and sequencing of the fragments After PCR the PCR product is fractionated on a 1 % agarose gel. The specific fragments are excised and purified using OIAGEN Gel Extraction Kit. The purified fragments are directly employed for cloning (Invitrogen Original TA Cloning Kit #K2000-01 ). Positive clones are grown in shaker culture, and the plasmid DNA is purified using QIAGEN Maxi-Prep Kit.
Sequencing is performed using the AbiPrism Model 377 automated sequencer.
Recombinant expression The gene fragments Hc1 and Hc2 are cloned into a suitable vector, preferably pGEX-4T-1 GST fusion vector (Pharmacia Biotech). For this purpose Hc1 and Hc2 are prepared from Tetrahymena thermophila cDNA
by PCR using suitable primers. A standard PCR mixture contains 10 mM
Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.001 % gelatine, 75 ,uM
dNTP, 0.3 ng of each primer, 0.5 ,ul of cDNA, 0.5 U of Taq polymerase. The primers (2A) 5' ATAAGAATGCGGCCGCTGTTCTACCGATTCTGTGAATATA 3';
(3A) 5' CGCGGATCCTC ACT GGT TCG GGT AAG ACT GCT ACT TTC
TCT 3' were used for amplifying the fragment Hcl, the primers (7A) 5' TATAGAATTCCCCACTAGAGAACTCGCTATGCAAATCGAA 3' (8A) 5' ATAAGAATGCGGCCGCGTTCTACCGATTCTGTGGACATAG 3' were used for amplifying the fragment Hc2. The primer (2A) contains a Notl cleavage site, the primer (3A) contains a BamHl cleavage site, the primer (7A) contains an EcoRl cleavage site and the primer (8A) contains a Notl cleavage site. The PCR program was 5 min 95°C, 95°C/37s -50°C/37s -72°C/37s - 30 cycles.
The fragments to be cloned are purified via a 1 % agarose gel (QIAgen Gel Extraction Kit) and the ends to be cloned are prepared by digestion with Notl and BamHl (Hc1) or EcoRl and Notl (Hc2). The vector pGEX-4T-1 is prepared likewise by digestion with Notl and BamHl (Hc1 ) or EcoRl and Notl (Hc2). Vector and insert are ligated at 16°C overnight, the ligation mixtures are employed for transformation of competent TOP10F' (Invitrogen) E. coli cells. Positive clones are picked and used for protein expression. The construct pGEX-Hc1 or pGEX-Hc2 permits translation of a fusion protein consisting of 257 amino acids (28.3 kDa) of Hc1 and glutathione S-transferase (24 kDa) or of 255 amino acids (28.1 kDa) of Hc2 and glutathione S-transferase (24 kDa). The fusion protein contains all homology boxes (DEAD, SAT,...) which characterize the members of the protein family. For recombinant protein expression an overnight culture is induced with IPTG and the fusion protein is purified from the supernatant in a batch process using glutathione Sepharose 4B or via a glutathione Sepharose 4B column. The glutathione S-transferase is cleaved with thrombin. For this purpose, for example, 100 Ng of GST fusion protein are incubated with one unit of thrombin proteinase in 1 x PBS at 22°C for 16 h.
The gene product of Hc1 or Hc2 is removed via gel filtration using, for example, a Superdex 200 HR 10/30 column (Pharmacia Biotech). The BioRad gel filtration chromatography standard (Ref. 151-1901 ) can be used as a standard.

Example 6: ATPase activity The activity is determined as described in the literature, for example according to Jaramillo et al., Mol Cell Biol 1991 11:5992; Rozen et al., Mol Cell Biol 1990 10: 1134, Ladomery M. et al. Nucl. Acid Res. 1997, 25:965-973 or Dong F. et al. Proc. Natl. Acad. Sci. USA 1996, 93: 14456-14461 or patent PCT/US97/01614. A concrete example is described in the following.
The reaction mixture for measuring the ATPase activity contains 150 mM
NaCI, 5 mM KCI, 1.5 mM MgCl2, 20 mM Hepes/KOH, pH 7, 1 mM
dithiothreitol, 1 mM PMSF, lO,uM ATP and 0.2,u1 of 32P-ATP in a total volume of 50,u1. The reaction mixture is heated to 37°C and Hc1 or Hc2 is added. After 30 min at 37°C the reaction is stopped by adding 400,u1 of 7%
activated carbon in 50 mM HCI and 5 mM H3P04. The samples are mixed and centrifuged at 13,000 rpm for 15 min. The released radioactivity in the supernatant is measured in a scintillation counter.
Example 7: helicase activity The helicase activity of Hc1 or Hc2 can be monitored by means of dissociation of double-stranded RNA. The substrate can be any RNA
oligomer which is labeled on one strand, for example with 32P. The reaction mixture contains in a 10 NI mixture 32P-labeled helicase substrate, Hc1 or Hc2 at various concentrations, 2 mM ATP, 5 mM dithiothreitol and 50 Ng of bovine serum albumin in 20 mM Tris-HCI. The reaction is carried out at 37°C for 30 min and stopped by heating.
The reaction mixture is applied to a 16 cm x 18 cm 12% non-denaturing polyacrylamide gel and fractionated at a constant current of 25 mA. The gel is dried in vacuo and exposed to film (e.g. Kodak RPXRP-5 film, -70°C
).
Example 8: Antisense The RNA opposite strands of the DNA fragments Hc1, Hc2 or of DNA
sequences homologous to Hc1 or Hc2 or of part sequences of Hc1, Hc2 or homologs can be used as antisense strands. Normally a plasmid is constructed which carries the desired antisense sequence and selection markers, for example neomycin, a promoter, which controls the expression of antisense RNA, and RNA-stabilizing sequences. The transfected sequences are transcribed in the cell and the transcript is hybridized with the target DNA. On the other hand it is also possible to introduce in vitro synthesized sequences into the cell by microinjection.
It is also conceivable to employ oligonucleotides. These may either be prepared synthetically or generated by restriction digestion of Hc1, Hc2 or homologs. The oligonucleotides must be highly pure. This is achieved by 2 - 5 lyophilizations. Pure oligonucleotides are taken up, for example, in HEPES-buffered saline, pH 7.4.
Example 9: Gene probe for detecting novel members of the DEAD box protein family The fragments Hc1 and Hc2 are used in order to isolate novel DEAD box proteins from suitable organisms. For this purpose the specific gene fragments are amplified with the aid of the polymerase chain reaction (PCR) and simultaneously labeled with digoxigenin, according to Boehringer Mannheim PCR DIG Probe Synthesis Kit #1636 090. Plasmid DNA of the cloned fragments Hc1 or Hc2 is used as a template. A PCR
mixture contains ExpandT"" High Fidelity buffer (Boehringer Mannheim #1636 090), 20 NM dATP, 200 NM dGTP, 200,uM dCTP, 130 NM dTTP, 70 NM DIG-11-dUTP, 0.3 ng of each primer, 100 pg plasmid DNA, 2.6 U
Taq polymerase.
The primers (2) 5'-GTTCTACCnATTCTGTG-3' and (3) 5'-ACnGGTTCnGGTAAGAC-3' were used for amplifying the fragment Hc1, the primers (4) 5'-ATAGAATTCCCnACnAGAGAAnTnGCT-3' and (8) 5'-ATAGGATCCGTTCTACCnATTCTGTG-3' were used for amplifying the fragment Hc2.
The PCR program was 5 min 95°C, 95°C/37s - 50°C/37s -72°C/37s - 30 cycles.

The PCR reaction mixture containing the labeled fragments is directly employed for hybridization studies. For this purpose the PCR product is denatured at 95°C for 10 min and then the hybridization solution DIG
Easy Hyb (Boehringer Mannheim Ref. 1603558) is added (conc. 2 NI/ml). This hybridization solution is used for screening cDNA libraries of suitable organisms at low stringency (hybridization temperature 30 - 50°C).
Example 10: Antibodies Protein expressed in a recombinant manner is purified by a suitable method and used for the purpose of generating polyclonal antibodies in a suitable organism, for example a rat or a rabbit. For this purpose, the fusion protein is purified, for example, initially via glutathione Sepharose and then via SDS-PAGE. The band containing the fusion protein is excised from the gel, ground up and injected for example into a rabbit or a rat. The obtained antiserum is passed through an IgG column, and the antibodies are eluted at low pH in 1 M Tris-HCI pH 8. The antibodies are dialyzed against 25 mM
HEPES pH 7.9, 12 mM MgCl2, 0.5 mM EDTA, 2 mM dithiothreitol, 17%
glycerol, 100 mM KCI.
Example 11: Use of the antibodies for isolating novel DEAD box proteins The antibodies obtained as described in the previous section are used for the purpose of isolating novel DEAD box proteins from suitable organisms.
For this purpose, the antibodies are covalently coupled to a suitable matrix, for example Sephadex G50 (Pharmacia). The Sephadex is used to pack, for example, BioRad columns 1.5 x 10 cm or 2.5 x 10 cm and the columns are equilibrated with 20 ml of buffer A (0.05 M Tris-HCI, 0.15 M NaCI, 0.005 M EDTA, 0.1 % NP40, pH 8.0). Subsequently, 3.0 g of protein A-Sepharose beads (Pharmacia CL-4B) are applied to the column. Leave overnight at 4°C. The antibody solution is applied to the column and allowed to drip through with a flow rate of -100 ml/h at 4°C. Then the column is washed several times: with 250 ml of buffer A (plus 0.5% NP40), then with 125 ml of 0.1 M borate buffer, pH 9.0, then with 125 ml of borate buffer, pH 8.0, then with 125 ml of 0.2 M triethanolamine, pH 8.2. The Fc region of the antibody is coupled to protein A - Sepharose via crosslinking.
Then the column is washed again, specifically, once with buffer B (0.15 M

Tris-HCI, 0.15 M NaCI, 1 mM EDTA pH 8.0, 10% glycerol, 10% NP-40), once with buffer C (0.05 M Tris-HCI, 0.5 M LiCI, 1 mM EDTA, pH 8.0, 10%
glycerol), and once with buffer D (0.01 M Pipes, 5 mM NaCI, 1 mM EDTA, pH 8.0, 10% glycerol). The non-crosslinked antibody is eluted with citrate buffer. The column is stored in borate buffer, pH 8.0 with 0.02% Na N3.
In order to isolate novel DEAD box proteins, cell lysate from suitable organisms is applied to the antibody column. The column is washed several times and the bound proteins are eluted with a suitable buffer, for example glycine, pH 3, in Tris-HC1, pH 8.
Example 12: Overexpression in useful plants The gene fragments Hc1 and Hc2 can be heterologously expressed in useful plants. The gene transfer can be mediated, for example, by Agrobacterium tumefaciens. A typical A. tumefaciens vector (Ti plasmid) contains a replication origin (ori Agro) which permits replication in Agrobacterium, a replication origin on E. coli which ensures functional replication in E. coli, a plurality of resistance genes, for example against kanamycin and spectinomycin, insertion sites for introducing the foreign gene and directed T-DNA flanking sequences which ensure recognition of beginning and end of the foreign gene in the gene transfer. A. tumefaciens is transformed with the Ti plasmid.
To infect the useful plant, leaf disks are punched out of said plant and put into a shallow dish (Petri dish). Subsequently, a solution of recombinant agrobacteria is added and after a few minutes the leaf disks are transferred onto a medium with feeder cells (e.g. filter paper). Injured cells on the edges of the leaf disks release factors which lead to infection of the plant cells by the agrobacterium. After 2-3 days, the leaf disks are transferred onto a sprout-stimulating medium containing an antibiotic which destroys the agrobacteria (e.g. cefotaxime) and cultivated for 2-3 weeks.

The sprouts are transferred onto a root-inducing medium and after a further 2-3 weeks planted in soil.
Example 13: Diagnostic probes The gene fragments Hc1 or Hc2 or homologous gene fragments or parts thereof (at least 20 base pairs long) which comprise the homology boxes characteristic of RNA helicases can be employed as diagnostic probes. For this purpose, the DNA fragments are immobilized on a suitable matrix (e.g.
nylon membrane, chip). mRNA of a patient is purified and transcribed into cDNA via reverse transcription by, for example, MMLV reverse transcriptase, 2 h at 37°C. Simultaneously, the cDNA is labeled, for example with 32P or digoxigenin. The cDNA is diluted in a suitable hybridization buffer, for example DIG EasyHyb (Boehringer Mannheim Ref. 1603558) and hybridized with the immobilized DNA under stringent conditions.
Example 14: Selection markers The gene fragments Hc1 and Hc2 may be employed as selection markers in molecular biology. It has been shown that overexpression of an RNA
helicase in mouse cells confers tolerance of the substance leflunomide on said cells (Mullner patent). In order to use Hc1 or Hc2 as a selection marker, an expression vector is constructed which contains Hc1 or Hc2 and a gene to be expressed. The gene to be expressed can be located beside or within the helicase gene. The vector is used to transform suitable host cells (e.g. cloning into a pGEX vector and introduction into E. coli). When the gene to be expressed is located beside the He gene, transformants become tolerant of leflunomide in the case of successful introduction of the vector. The success of the ligation has to be checked, for example via blue/white screening. When the gene to be expressed is located within the He gene, the He gene is destroyed in the case of successful ligation and the transformants lose their tolerance of leflunomide in the case of successful introduction of the vector.

SEQUENCE LISTING
<110> Aventis Research 6 Technologies Deutschland GnsbH i Co KG
<120> Neue Gene dez DEAD Box Protein-Familie, deren Expressionsprodukte and VerHendung <150> DE 198 20 608.9 <151> 1998-07-22 <160> 16 <210> 1 <211> 16 <212> DNA
<223> Artiticiel sequence <220>
<223> cDNA
<400> 1 G~_'TCTACCAT TCTGTG 16 <210> 2 <211> 15 <212> DNA
<213> Artificiel sequence <220>
<223> cDNA
<400> 2 <210> 3 <211> 23 <212> DNA

<213> Artificiel sequence <220>

<223> cDNA

<400> 3 <210> 4 <211> 25 <212> DNA

<213> Artificiel sequence <220>

<223> cDNA

<400> 4 z _ _ . ..___ <210>5 <211>90 <212>DNA

<213>Artificiel sequence <220>

<223>cDNA

<400> 5 <210>6 <211>4I

<212>DNA

<213>Rrtificiel sequence <220>

<223>cDNA

<400> 6 <210> 7 <211> 91 <212> DNA
<213> Artificiel sequence <220>
<223> cDNA
<900> 7 CGCGGATCC~_' CACTGGTTCG GGTAAGACTG CTACTTTCTC T 41 <210> 8 <211> 40 <212> DNA
<213> Attificiel sequence <220>
<223> cDNA
<900> B
ATAAGAATGC GGCCGCGTTC TACCGATTCT GTGGACA':AG 40 J
<210> 9 <211> 16 <212> DNA
<213> Artificiel sequence .
<220>
<223> cDNA
<400> 9 <210> 10 <211> 15 <212> DNA

<213> Artificiel sequence <220>

<223> cDNA

<900> 10 <210> 1I

<211> 23 <212> DNA

<213> Artificiel sequence <220>

<223> cDNA

<400> 11 <2i0> 12 <211> 25 <212> DNA

<213> Artificiel sequence <220>

<223> cDNA

<400> 12 ATAGGATCCG '_"TCTACCATT CTGTG 25 <210> 13 <211> 771 <212> DNA

<213> Tetrahymena thermophila <220>

<223> cDNA

. ~ CA 02335646 2001-O1-19 <400> I3 CCCACTAGAG AACTCGCTAT GCAAATCGAA AGAGAGTCCG AAAGATTTGG TAAATCC_TCT60 <210>14 <211>257 <212>Protein <213>Tetrahymena thermophila <220>

<223>PRT

<400> 19 Pro Thr Arg Glu Leu Ala Met Gln :ie Glu Arg Glu Ser Giu Arg Phe Gly Lys Ser Ser Lys Leu Lys Cys Ala Cys Ile Tyr Gly Gly Ala Asp Lys Tyr Ser Gln Arg Ala Leu Leu Gln Gln Gly Val Asp Val Val Ile Ala Thr Pro Gly Arg Leu Ile Asp Phe Leu Glu Se. Glu Th: Thr Thr Leu Arg Arg Val Thr Tyr Leu Va_ Leu Asp Glu Ala Pap n=g Met Leu Asp Met Gly Phe G_u Ile Gln Ile Arg Lys :le Leu Gly Gln Ile Arg Pro Asp A=g Gln :'hr Leu Met Phe Ser Ala T:m Trp Pro Lys Asp Val 100 105 1.0 Gln Asn Leu Ala Gln Asp Tyt Cys Lys Asn Thr Pro Val Tyr Val Gln v'vI

Ile Gly Lys His Glu Leu Ala Ile Asn Glu Arg Ile Lys Gln Ile Val I30 135 140 .
Tyr Vai Thr Asp Gln Ser Lys Lys Ile Asn Gln Leu Iie Lys Gln Leu Asp Cys Leu Thr Gln Lys Asp Lys Val Leu '_le Phe Ala Gln Thr Lys Lys Gly Cys Glu Ser Met Ser Arg Ile Leu Asn Lys Glu Gly Phe Lys Cys Leu Ala Ile His Gly Asp Lys Ala Gln Lys Asp Arg Asp Tyr Val Met Asn Lys Phe Lys Ser Gly Glu Cys Arg Iie Leu Ile Ala Thr Asp Val Ala Ser Arg Gly Leu Asp Val Lys Asp Val Ser His Val Phe Asn Tyr Asp Phe Pro Lys Val Met Glu Asp Tyr Val His Arg Ile Gly Arg Thr <210> 15 <211> 765 <212> DNA

<213> Tetrahymena thermophila <220>

<223> cDNA

<400> 15 ATAAACTATC ACCGTTATTA
TGTACTTAGG TGAATTCTTG

AAAGAGATTA

AATC':AAAAG

AAGACTTTAG TCACCGATCAC~_'TAAAATTA TTCATTTTGG ACGAAGCCGA 240 TGAAATGTTA

CGATATCTAG

GTTGCTC':TT TCTCTGCTAC:ATGGCT.~.CC GAAAT'::.T'_"G AAATTACCAA360 GTAATTTATG

AGAGACCCCG CTA:.TATCCTTGTCAAGRAT GATGACTTGA CTTTGGACGG 420 TF.TTAAATAA

TTCTACATCG CCTTAGATAAGGAAGAATGG AAGTTTGACA CCTTAG':C~~A 4BO
ATTATACAAT

CGATGAP.TTA

AATGGACCAA

TAnAACAGAG ATCTTATTAT_GAAGGAATTC AGAACCGG'_A CCTCCAGAGT 660 TCTTATCACT

<210>16 <211>255 <212>Protein <213>Tetrahymeaa thermophila <220>

<223>PRT

<400> 16 Pro Thz Azg Glu Leu Ala Gln Gln Thr Ile Thr Val Ile Met Tyr Leu Gly Glu Phe Leu Lys Val Ser Ala Tyz Ala Cys Thr Gly Gly Thr Asp Pro Lys Glu Asp Arg Lys Arg Leu Arg Glu Gly Val Gln Val Val Val Gly Thr Pro Gly Arg Val Leu Rsp Leu Iie Gln Lys Lys Thr Leu Val Thr Asp His Leu Lys Leu Phe I1e Leu Asp Glu Ala Asp Glu Met Leu Gly Arg Gly Phe Lys Rsp Gln Ile Asn Lys Ile Phe Gln Asn Leu Pro 85 90 g5 H_s Asp Ile Gln Val Ala Leu Phe Ser Ala Th_ Met Ala Pro Glu Ile Leu Glu Ile Thr Lys Gln Phe Met Arg Asp Pro A:a Thr Ile Leu Val Lys Asn Asp Asp Leu Thr Leu Asp Gly Ile Lys G1n Phe Tyr '_le Ala Leu Asp Lys Glu Glu Trp Lys Phe Asp Thr Leu Val G'_u Leu Tyr Asn Asn Ile Giu Ile Ala Gin Ala Ile ile Tyz Cys Asn Thr Lys Lys Arg Val Asp Glu Leu Arg Asp Lys Leu 11e Glu Lys Asn Met Thr Val Ser A1 a Met His Gly Glu Met Asp Gl.~. Gl.~. As.~. A_g Psp Leu I:e Met Lys Glu Phe Arg Thr Gly Thr Sez Arg Vai Leu _'le Thr Thr Asp Leu Leu 210 2i5 220 Ser Gly Gly Ile Asp Ile His Gln Val Asn Leu Val :le Asn Tyr Asp Leu Pro Leu Lys Lys Glu Cys Tyr Ile His Arg Ile Gly Azg Thr

Claims (24)

claims

1. A nucleic acid coding for an RNA helicase having an amino acid sequence depicted in SEQ ID No. 14 and parts thereof having at least 20 nucleotides, SEQ ID No. 13 being part of the claim, and nucleic acids coding for functional variants of the RNA helicase depicted in SEQ ID No. 14, functional variants being proteins having a sequence homology of at least 70% or proteins having deletions of up to 60 amino acids or fusion proteins which comprise the amino acid sequence of SEQ ID No. 14.

2. A nucleic acid coding for an RNA helicase having an amino acid sequence depicted in SEQ ID No. 16 and parts thereof having at least 25 nucleotides, SEQ ID No. 15 being part of the claim, and nucleic acids coding for functional variants of the RNA helicase depicted in SEQ ID No. 16, functional variants being proteins having a sequence homology of at least 70% or proteins having deletions of up to 60 amino acids or fusion proteins which comprise the amino acid sequence of SEQ ID No. 16.

3. A nucleic acid as claimed in claim 1 and 2, wherein the nucleic acid is a DNA or RNA, preferably a double-stranded DNA.

4. A nucleic acid as claimed in claim 1 to 3 and obtainable from ciliates.

5. A nucleic acid as claimed in claim 4 and obtainable from Tetrahymena thermophila.

6. A DNA antisense strand and an RNA antisense strand obtainable from nucleic acids as claimed in claim 1 to 5.

7. A nucleic acid as claimed in one of claims 1 to 6, wherein the nucleic acid is included in a vector, preferably in an expression vector or a vector effective for gene therapy.

8. A method for preparing a nucleic acid as claimed in one of claims 1 - 6, wherein the nucleic acid is chemically synthesized or isolated from a gene library using a probe.

9. A polypeptide having an amino acid sequence depicted in SEQ ID No.
14 and parts thereof having at least 65 amino acids and functional variants of the polypeptide depicted in SEQ ID No. 14, a functional variant being a polypeptide having a sequence homology of at least 70% or a polypeptide having deletions of up to 60 amino acids or a fusion protein which includes the amino acid sequence of SEQ ID No.
14.

10. A polypeptide having an amino acid sequence depicted in SEQ ID No.
16 and parts thereof having at least 12 amino acids and functional variants of the polypeptide depicted in SEQ ID No. 16, a functional variant being a protein having a sequence homology of at least 70%
or a protein having deletions of up to 60 amino acids or a fusion protein which includes the amino acid sequence of SEQ ID No. 16.

11. A method for preparing a polypeptide as claimed in claim 9 or 10, which comprises expressing a nucleic acid as claimed in one of claims 1 to 6 in a suitable host cell.

12. An antibody against a polypeptide as claimed in claim 9 or 10.

13. A method for producing an antibody as claimed in claim 12, which comprises immunizing a mammal with a polypeptide as claimed in claim 9 or 10 and, where appropriate, isolating the generated antibodies.

14. A pharmaceutical comprising a nucleic acid as claimed in one of claims 1 to 6 or a polypeptide as claimed in claim 9 or 10 and, where appropriate, pharmaceutically acceptable additives and/or excipients.

15. A process for producing a pharmaceutical for treating cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes, and/or for affecting the cell metabolism, in particular in association with immunosuppression, especially in association with transplants and/or genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases, which comprises formulating a nucleic acid as claimed in one of claims 1-6 or a polypeptide as claimed in claim 9 or 10 or an antibody as claimed in claim 12 together with a pharmaceutically acceptable additive and/or excipient.

16. A diagnostic agent comprising a nucleic acid as claimed in one of claims 1-6 or a polypeptide as claimed in claim 9 or 10 or an antibody as claimed in claim 12 and, where appropriate, suitable additives and/or excipients.

17. A process for preparing a diagnostic agent for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetes, and/or for analyzing the cell metabolism, in particular the immune status, especially in association with transplants, and/or for analyzing genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases, which comprises adding a pharmaceutically acceptable carrier to a nucleic acid as claimed in one of claims 1-6 or a polypeptide as claimed in claim 9 or 10 or an antibody as claimed in claim 12.

18. An assay for identifying functional interactors, which comprises a nucleic acid as claimed in one of claims 1-6 or a polypeptide as claimed in claim 9 or 10 or an antibody as claimed in claim 12 and, where appropriate, suitable additives and/or excipients.

19. The use of a nucleic acid as claimed in one of claims 1-6 or of a polypeptide as claimed in claim 9 or 10 for identifying functional interactors.

20. The use of a nucleic acid as claimed in one of claims 1-6 for detecting variants of RNA helicase, which comprises screening a gene library using said nucleic acid and isolating the variant found.

21. The use of the nucleic acids as claimed in one of claims 1-6 or of a polypeptide as claimed in claim 9 or 10 for affecting protein biosynthesis.

22. The use of the nucleic acids and polypeptides as claimed in claim 21 for inhibiting mRNA degradation and/or stimulating mRNA
degradation and/or stabilizing mRNA.

23. The use of the nucleic acids and polypeptides as claimed in claim 21 for heterologous expression in useful plants.

24. The use of the nucleic acids as claimed in one of claims 1-6 or of a polypeptide as claimed in claim 9 or 10 as selection markers in molecular biology.