AU757920B2 - Genes of the dead box protein family, their expression products and use - Google Patents

Description

WO 00/05388 PCT/EP99/04892 Genes of the DEAD box protein family, their expression products and use Field 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.

Background 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 15 secondary and tertiary structures and, in addition, a large number of RNAbinding 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 20 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 WO 00/05388 2 PCT/EP99/04892 (Schmid Lindner 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 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 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 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 Souza 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 WO 00/05388 3 PCT/EP99/04892 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 Bousquet-Antonelli C., Gelugne Caizegues-Ferrer Tollervey, 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 Haenni J Virol 1997: 2583-2590). Possible roles for viral helicases are 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 acidspecific.

There are indications that at least some helicases can be very specifically activated. Thus, for example, DpbA from E. coli (Fuller-Pace Nicol Reid Land EMBO J 1993 12:3619-3626) and Sit22 from yeast (Xu Nouraini Field Tang Friesen 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 McGuckin Malcolm Pearson Lunec J., Oncogene 1996 12: 1583-7). Mutations of an RNA helicase are connected with Werner's syndrome premature aging (Yu Oshima Wijsman Nakura J. et al., Am J Hum Genet 1997 60: 330-341) and with xeroderma pigmentosum (Kobayashi Kuraoka Sailo Nakatsu Y., Tanaka A. et al., Huma Mut 1997 9: 322-331). Furthermore, a possible WO 00/05388 4 PCT/EP99/04892 connection between DEAD box proteins and connective tissue diseases has been postulated (Valdez Henning Perlaky Busch R.K., Busch 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 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 Hermann OelmOller R., Mol Cell Biol 1997 17: 2257-2265 and Aubourg et al., Gene 1997 199(1-2): i* 241-253). Although these proteins are structurally similar to DEAD box proteins from organisms other than plants, they form nevertheless a 15 subgroup (Fig. Fig. 3 shows the phylogenetic relationship of various elFA4, 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 elF4A from animal eukaryotes. An application which is of interest to agricultural production is the stimulation of the activity 20 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.

Accordingly, there may be considered a need in the art to provide novel nucleic acids coding preferably for RNA helicases.

Summary of the invention as claimed In one aspect, the invention as claimed hereinafter provides an isolated or purified nucleic acid coding for an RNA helicase having an amino acid P:\WPDOCS\CRN\Shlley\Spm\7563620.sd-07/ 1/02 -4asequence depicted in SEQ ID No. 14 and parts thereof having at least 100 nucleotides 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 amino acids or fusion proteins which comprise the amino acid sequence of SEQ ID No. 14.

In another aspect, the invention as claimed provides an isolated or purified nucleic acid coding for an RNA helicase having an amino acid sequence 10 depicted in SEQ ID No. 16 and parts thereof having at least 25 nucleotides 0 and nucleic acids coding for functional variants of the RNA helicase of 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 S" proteins which comprise the amino acid sequence of SEQ ID No. 16.

In another aspect, the invention as claimed provides a DNA antisense strand or an RNA antisense strand obtained from nucleic acids as herein before described.

20 In a further aspect, the invention as claimed provides a method for preparing a nucleic acid as herein before described, wherein the nucleic acid is chemically synthesized or isolated from a gene library using a probe.

In yet a further aspect, the invention as claimed provides an isolated or purified 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 0 the amino acid sequence of SEQ ID No. 14.

P:\WPDOCS\CRN\ShelleySpec\7563620.,pe.doc-07/I /02 -4b- In an additional aspect, the invention as claimed provides an isolated or purified 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.

In a further aspect, the invention as claimed provides a method for preparing 10 a polypeptide as herein before described which comprises expressing a nucleic acid as herein before described in a suitable host cell.

Additionally, the invention as claimed hereinafter provides: an isolated or purified antibody against a polypeptide as herein before described; and, a method for producing such an antibody which comprises immunizing a mammal with said polypeptide, and, where appropriate, isolating the se** generated antibodies.

Furthermore, the invention as claimed hereinafter provides: a pharmaceutical 20 comprising a nucleic acid as herein before described or a polypeptide as herein before described, and where appropriate, pharmaceutically acceptable additives and/or excipients; and 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 /mpigmentosa and connective tissue diseases, which comprises formulating a 9 cleic acid as herein before described or a polypeptide as herein before P:\WPDOCS\CRN\Shellcy'SpC\7563620.spc.doc-07/1/02 -4cdescribed, or an antibody as herein before described together with a pharmaceutically acceptable additive and/or excipient.

In yet a further, aspect, the invention as claimed hereinafter provides: a diagnostic agent comprising a nucleic acid as herein before described or a polypeptide as herein before described or an antibody as herein before described and, where appropriate, suitable additives and/or excipients; and a process for preparing a diagnostic agent for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's 10 disease, allergies, in p laticular 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 herein before described or a polypeptide as herein before described or an antibody as herein before described.

20 In still further aspects, the invention as claimed herein after provides: i) the use of a nucleic acid as herein before described or of a polypeptide as herein before described for identifying functional interactors; ii) the use of a nucleic acid as herein before described for detecting variants of RNA helicase, which comprises screening a gene library using said nucleic acid and isolating the variant found; iii) the use of the nucleic acids as herein before described or of a polypeptide as herein before described for affecting protein biosynthesis; iv) the use of the nucleic acids and polypeptides as herein before described for inhibiting mRNA degradation and/or stimulating mRNA degradation and/or stabilizing mRNA; v) the use of nucleic acids and polypeptides as herein before described for heterologous expression in useful plants; and vi) the use of the fucleic acids as herein before described or of a polypeptide as herein before P:\WPDOCS\CRN'ShdllcSp\7563620.,pd.07/1/02 -4ddescribed as selection markers in molecular biology.

In other aspects, the invention as claimed hereinafter provides: 1) the use of the nucleic acids as herein before described or of a polypeptide as herein before described 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 cell metabolism in particular association with immunosuppression, 10 especially in association with transplants and/or genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases; and 2) the use of nucleic acids as herein before described or of a polypeptide as herein before described in the manufacture of 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 cell metabolism in particular association with immunosuppression, especially in association with transplants and/or genetic diseases, in particular 20 Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases.

Detailed description The present invention provides 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.

WO 00/05388 5 PCT/EP99/04892 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 ("Hcl" 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 SEQ 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.

WO 00/05388 6 PCT/EP99/04892 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. 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 Hcl 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.

WO 00/05388 7 PCT/EP99/04892 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 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 pGM10 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 Ni 2 +-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, 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 WO 00/05388 8 PCT/EP99/04892 (see e.g. EP-B1-0154133) in E. coli, the ADH-2 promoter for expression in yeasts (Russel 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 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 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 Feigner, P.L. et al. (1987) ^%Nroc. Natl. Acad. Sci, USA 84, WO 00/05388 9 PCT/EP99/04892 7413; Behr, J.P. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982; Feigner, 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, Uhlman, E. Peyman, A. (1990) Chemical Reviews, 90, 543, No. 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 '4egradation by the degradosome and thus decreased protein biosynthesis.

nhibition of the helicase activity may WO 00/05388 10 PCT/EP99/04892 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 Hcl, are an outstanding model for various eukaryotic RNA helicases, preferably from humans and parasites (Fig. 3A). The genetic relationship of Hcl with the relevant eukaryotic helicases is close enough in order to draw conclusions from experiments with Hcl about structure and function of other eukaryotic helicases human). Figure 3A shows the genetic relationship of some helicases from various organisms in comparison with Hcl. Particularly surprising are the great structural similarity between Hcl and mammalian helicases from humans and mice and the great structural difference between Hcl 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 Herrmann Oelmuller Mol Cell Biol 17(4): 2257- 2265 (1997)).

WO 00/05388 11 PCT/EP99/04892 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: 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), WO 00/05388 12 PCT/EP99/04892 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 Hcl 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 Hcl 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 polypeptides having the amino acid sequences depicted in Figures 5 and 7 of approx. 70%, preferably of approx. 80%, in particular of approx. 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 abovedescribed polypeptides of the invention,with the WO 00/05388 13 PCT/EP99/04892 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 WO 00/05388 14 PCT/EP99/04892 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, 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, rthrosis, atherosclerosis, osteoporosis, acute and chronic infectious seases and/or diabetes, and/or for affecting the cell metabolism, in 3q,, articular in association with immunosuppression, especially in association WO 00/05388 15 PCT/EP99/04892 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 socalled 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 WO 00/05388 16 PCT/EP99/04892 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 polypeptide in question is present. In this case, the antibodies of the invention 3 are, for example, labeled with an enzyme as already described above. The r, specific WO 00/05388 17 PCT/EP99/04892 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 lexA 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 lexA 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 Sfrom a WO 00/05388 18 PCT/EP99/04892 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

B

extends to a process for finding substances which are able to interact with the above-described WO 00/05388 19 PCT/EP99/04892 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.

S: 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 Hcl 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 Sharp CABIOS (1989), Vol. 5, no. 2, 151-153).

SEQ ID No. 13 shows the nucleic acid sequence of Hcl.

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. WO 00/05388 20 PCT/EP99/04892 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/l proteose peptone No. 3 DIFCO, 1 g/l yeast extract DIFCO, 10 mg sodium citrate, 24. 3 mg FeCI 3 in a 500 ml flask (100,000 cells/ml 15 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).

SFor 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°C 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 55-600C 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).

WO 00/05388 21 PCT/EP99/04892 Example 3: Preparation of cDNA mRNA was transcribed into cDNA according to CLONTECH (CapFinderTM 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 MgCI2, 0.001% gelatin, 75 pM dNTP, 0.3 ng of each primer, 0.5 p1 of cDNA, 0.5 U of Taq polymerase.

The primers 5'-GTTCTACCnATTCTGTG-3' and 5'-ACnGGTTCnGGTAAGAC-3' were used for amplifying the fragment Hcl, the primers 5'-ATAGAATTCCCnACnAGAGAAnTnGCT-3' and 5'-ATAGGATCCGTTCTACCnATTCTGTG-3' were used for amplifying the fragment Hc2, with n being any nucleotide. The PCR program was min 95 0 C, 95 0 C/37s 50 0 C/37s 72 0 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 QIAGEN 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 Hcl and Hc2 are cloned into a suitable vector, preferably pGEX-4T-1 GST fusion vector (Pharmacia Biotech). For this purpose Hcl 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 MgCI2, 0.001% gelatine, 75 pM dNTP, 0.3 ng of each primer, 0.5 /l of cDNA, 0.5 U of Taq polymerase. The primers WO 00/05388 22 PCT/EP99/04892 (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 BamHI cleavage site, the primer (7A) contains an EcoRI cleavage site and the primer (8A) contains a Notl cleavage site. The PCR program was 5 min 950C, 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 BamHI (Hcl) or EcoRI and Notl (Hc2). The vector pGEX-4T-1 is prepared likewise by digestion with Notl and BamHI (Hcl) or EcoRI and Notl (Hc2). Vector and insert are ligated at 160C overnight, the ligation mixtures are employed for transformation of competent (Invitrogen) E. coli cells. Positive clones are picked and used for protein expression. The construct pGEX-Hcl or pGEX-Hc2 permits translation of a fusion protein consisting of 257 amino acids (28.3 kDa) of Hcl 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, 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 pg of GST fusion protein are incubated with one unit of thrombin proteinase in 1 x PBS at 220C for 16 h.

The gene product of Hcl 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.

WO 00/05388 23 PCT/EP99/04892 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 MgCI2, 20 mM Hepes/KOH, pH 7, 1 mM dithiothreitol, 1 mM PMSF, 10 pM ATP and 0.2 l of 32P-ATP in a total volume of 50 pl. The reaction mixture is heated to 370C and Hcl or Hc2 is added. After 30 min at 370C the reaction is stopped by adding 400 p/ 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 Hcl 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 pl mixture 32P-labeled helicase substrate, Hcl or Hc2 at various concentrations, 2 mM ATP, 5 mM dithiothreitol and pg of bovine serum albumin in 20 mM Tris-HCI. The reaction is carried out at 370C 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 Kodak RPXRP-5 film, -700C Example 8: Antisense The RNA opposite strands of the DNA fragments Hcl, Hc2 or of DNA sequences homologous to Hcl or Hc2 or of part sequences of Hcl, Hc2 or homologs can be used as antisense strands. Normally a plasmid is constructed which carries the desired antisense sequence and WO 00/05388 24 PCT/EP99/04892 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 Hcl, 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 Hcl 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 Hcl or Hc2 is used as a template. A PCR mixture contains Expand T M High Fidelity buffer (Boehringer Mannheim #1636 090), 20 /M dATP, 200 pM dGTP, 200 /M dCTP, 130 pM dTTP, pM DIG-11-dUTP, 0.3 ng of each primer, 100 pg plasmid DNA, 2.6 U Taq polymerase.

The primers 5'-GTTCTACCnATTCTGTG-3' and 5'-ACnGGTTCnGGTAAGAC-3' were used for amplifying the fragment Hcl, the primers 5'-ATAGAATTCCCnACnAGAGAAnTnGCT-3' and 5'-ATAGGATCCGTTCTACCnATTCTGTG-3' were used for amplifying the fragment Hc2.

The PCR program was 5 min 950C, 95°C/37s 50°C/37s 72°C/37s SCcycles.

WO 00/05388 25 PCT/EP99/04892 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 pl/ml). This hybridization solution is used for screening cDNA libraries of suitable organisms at low stringency (hybridization temperature 30 500C).

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 MgCI2, 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 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% then with 125 ml of 0.1 M borate buffer, pH 9.0, then with 125 ml of borate Suffer, pH 8.0, then with 125 ml of 0.2 M triethanolamine, pH 8.2. The Fc lregion of the antibody is coupled to protein A Sepharose via crosslinking.

Thn the column is washed again, specifically, once with buffer B (0.15 M WO 00/05388 26 PCT/EP99/04892 Tris-HCI, 0.15 M NaCI, 1 mM EDTA pH 8.0, 10% glycerol, 10% once with buffer C (0.05 M Tris-HCI, 0.5 M LiCI, 1 mM EDTA, pH 8.0, 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-HCI, pH 8.

Example 12: Overexpression in useful plants The gene fragments Hcl 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 ori 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 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 cefotaxime) and cultivated for 2-3 weeks.

WO 00/05388 27 PCT/EP99/04892 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 Hcl 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 370C. 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 Hcl 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 (MOllner patent). In order to use Hcl or Hc2 as a selection marker, an expression vector is constructed which contains Hcl 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 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.

P:\WPDOCS\CRN\ShellySpc\7563620.spc.doc-07/ll/02 -27a- The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

WO 00/05388 PCT/EP99/04892 SEQUENCE LISTING <110> Aventis Research Technologies Deutschland GmbH Co KG <120> Neue Gene der DEAD Box protein-Familie, deren Expressionsprodukte und Verwendung <150> DE 198 20 608.9 <151> 1998-07-22 <160> 16 <210> <211> <212> <213> <220> <223> 1 16

DNA

Artificiel sequence cDNA <400> 1 GTTCTACCAT TCTGTG <210> <211> <212> <213> <220> <223> is

DNA

Artificiel sequence cDNA <400> 2 ACGGTTCGGT AAGAC <210> <211> <212> <213> <220> <223> 23

DNA

Artificiel sequence cDNA <400> 3 ATAGAATTCC CACAGAGAAT GCT <210> 4 <211> <212> DNA <213> Artificiel sequence <220> <223> cDNA WO 00/05388 <400> 4 ATAGGA2TCCG TTCTACCATT CTGTG PCT/EP99/04892 <210> <211> <212> <213> <220> <223> Artificiel sequence cDNA <400> ATAAGAATGC GGCCGCTGTT CTACCGATTC TGTGAATATA <210> <211> <212> <213> <22 0> <223> 41

DNA

Artificiel sequence cDNA <400> 6 CGCGGATCCT CACTGGTTCG GGTAAGACTG CTACTTTCTC T <210> <211> <212> <213> <220> <223> 41

DNA

Artificiel sequence cDNA <400> 7 CGCGGATCCT CACTGGTTCG GGTAAGACTG CTACTTTCTC T <210> 8 <211> <212> DNA <213> Artificiel sequence <220> <223> cDNA <400> 8 ATAAGAATGC GGCCGCGTTC TACCGATTCT GTGGACATAG WO 00/05388 PCT/E P99/04892 <210> <211> <212> <213> <220> <223> 16

DNA

Artificiel sequence cDNA <400> 9 GTTCTACCAT TCTGTG <210> <211> <212> <213> <220> <223>

DNA

Artificiel sequence cDNA <400> ACGGTTCGGT AAGAC <210> 11 <211> 23 <212> DNA <213> Artificiel sequernce <220> <223> cDNA <400> 11 ATAGAATTCC CACAGAGAAT GCT <210> 12 <211> <212> DNA <213> Artificiel sequence <220> <223> cDNA <400> 12 ATAGGATCCG TTCTACCATT CTSTG <210> 13 <211> 771 <212> DNA <213> Tetrahymeria thermophila <220> <223> cDNA WO 00/05388 <400> 1 PCT/E P99/04892 CCCACTAGAG AACTCGCTAT GCAAATCGAA AGAGAGTCCG AAAGATTTGG TAAATCCTCT AAGCTTAA6AT

CAATAAGGTG

GAAACTACTA

GATATGGGTT

ACATTGATGT

AAGAATACTC

AAATAAAT'rG

GATTGTTTGA

AGCATGAGTC

GCCTAAAAAG

ATTGCTACAG

TACGATTTCC

GTGCTTGTAT

TAGATGTAGT

CTTTACGTAG

TTGAAATTTA

TTTCTGCTAC

CCGTTTATGT

TTTATGTTAC

CTTAGAAAGA

GTATTTTGAA

ACAGAGACTA

ACG'rAGCCAG

CAAAGGTTAT

CTATGGTGGT GCTGACAAAT ACTCCTAAAG AGCACTTCTC

TATTGCTACT

AGTTACTTAT

AATTAGAAAA

CTGGCCTAAG

TCAAATCGGA

AGATCAATCA

TAAAGTATTG

TAAAGAAGGA

TGTTATCAAC

TAGAGGTTTG

GGAAGACTAT

CCTGGTAGAC

CTCGTATTAG

ATCTTGGGTT

AATGTTTAG.A

AAACATGAAT

AAGAAAATCA

ATTTTCGCTT

TTTAAGTGTC

AAGTTCAAAA

TTATTGACTT

ATGAAGCAGA

AAATTAGACC

ATCTTGCTTA

TAGCTATTAA

ATCAACTTAT

AAAC.AAAGAA

TTGCTATCCA

GCGGAGAATG

TTTAGAAAGT

TAGAATGTTA

TGATCGTTAA

AGATTATTGC

CGAAAGAATT

CAAGCAATTA

GGGATGTCAA

TGG'TGACAAA

CAGAATCCTT

CGTATTTAAT

G

GATGTTAAGG ATGTCTCCCA GTCCACAGAA TCGGTAGAAC <210> <211> <212> <213> <220> <223> <400> Pro 1 Gly 14 257 Protein Tetrahymena thermophila

PRT

14 Thr Arg Glu Leu Ala Met Gin Ile Giu Arg Glu Ser Giu Arg Phe Lys Ser Ser Lys Leu Lys Cys Cys Ile Tyr Gly Gin Giy Val Asp Giy Ala Asp Val Vai Ile Lys Tyr 5cr Gin Arg Ala Leu Ala Thr Pro Gly Arg Leu Asp Phe Leu Glu Giu Thr Thr Thr Arg Arg Val Thr Leu Val Leu Asp Ala Asp Arg Met Asp Met Giy Phe Pro Asp Arg Gin 100 Ile Gin Ile Arg le Leu Giy Gin Ile Arg Asn Val Thr Le'u Met Phe Ser Ala Thr Tzp Pro 105 WO 00/05388 PCT/E P99/04892 Gin Asn Leu Al1a Gin Asp Tyr Cys Lys Asn Thr Pro Val Tyr Val Gin 120 His Giu Leu Ala Ile Asn 135 le Giy 130 Tyr Val 145 Asp Cys Thr Asp Gin Lys Lys Ile Asp Lys Val 125 Giu Arg le Lys 140 Asri Gin Leu Ile 155 Leu Ile Phe Ala 170 Leu Asn Lys Giu Gin Ile Val Lys Gin Leu 160 Gin Thr Lys 175 Giy Phe Lys Leu Thr Lys Gly Cys Cys Leu Ala 195 Met Asn Lys Met Ser Arg His Gly Asp Gin Lys Asp 190 Asp Tyr Vai Ala Thr Asp 210 Val Ala Sex Phe Lys Ser A-rg Gly Leu 230 Gi y 215 Asp Cys Arg Ile Val Lys Asp Ser His Val Phe Hi~s Arg Ile Gly 255 Tyr Asp Phe Pro Val Met Glu Asp Tyr <210O> <211> <212> <213> <220> <223> <400> 765

DNA

Tetrahyiena thermophila cDNA CCCACCAGAG AATTAGCCCA ATAAACTATC ACCGTTATTA TGTACTTAGG TGAATTCTTG

AAGGTCTCCG

AGAGAAGGTG

AAGACTTTAG

GGAAGAGGTT

GTTGCTCTTT

AGAGACCCCG

TTCTACATCG

AACATCGAAA

AGAGACAAGC

TAAAACAGAG

CCTATGCTTG

TCCAAGTCGT

TCACCGATCA

TCAAGGATCA

TCTCTGCTAc

CTACTATCCT

CCTTAGATAA

TCGCTTAAGC

TTATTGAAAA

ATCTTATTAT

CACTGGTGGT

TGTTGGTACC

CTTAAAATTA

AATTAACAAA

CAT GGCTC CC

TGTCAAGAAT

GGAAGAATGG

TATTATCTAT

GAATATGACC

GAAGGAATTC

ACTGATCCCA

CCTGGTAGAG

TTCATTTTGG

ATCTTCTAAA

GAAATT.CTTG

GATGACTTGA

AAGTTTGACA

TGCAACACCA

GTCTCTGCTA

AGAACCGGT-A

AGGAAGATAG

TTTTGGATTT

ACGAAGCCGA

ACTTACCCCA

AAATTACCAA

CTTTGGACGG

CCTTAGTCGA

AGAAGAGAGT

T GCACGGTGA

CCTCCAGAGT

AAAGAGATTA

AAT CTAAAAG

TGAAATGTTA

CGATATCTAG

GTAATTTATG

TATTAAATAA

ATTATACAAT

CGATGAATTA

AATGGACCAA

TCTTATCACT

WO 00/05388 6 PCT/EP99/04892 ACTGATTTGC TCTCCAGAGG TATTGATATC CATCAAGTCA AC=TGGTTAT CAACTACGAC 720 TTACCCCTTA AGAAGGAATG TTATATTCAC AGAATCGGTA GAACA 765 <210> 16 <211> 255 <212> Protein <213> Tetrahymena thermophila <220> <223> PRT <400> 16 Pro Thr Arg Glu Leu Ala Gin Gin Thr Ile Thr Val Ile Met Tyr Leu 1 5 10 Gly Pro Gly Thr Gly His Leu Lys Leu 145 Asn Vai Ala Glu I Ser C 225 Leu 1 Glu Lys Thr Asp Arg Asp Glu Asn 130 Asp Ile Asp ,et Phe 210 ;ly Phe Glu Pro His Gly Ile Ile 115 Asp Lys Glu Glu His 195 Azg Gly Leu Asp Gly Leu Phe Gin 100 Thr Asp Glu Ile Leu 180 Gly Thr Ile I Lys Arg Arg Lys Lys Val Lys Leu Glu Ala 165 Arg Glu Gly Asp Lys 245 Val Lys Val Leu 70 Asp Ala Gin Thr Trp 150 Gin Asp Met Thr Ile 230 Glu I Ala Tyr Ala 25 Leu Arg Glu 40 Asp Leu lie Ile Leu Asp Ile Asn Lys 90 Phe Ser Ala 105 Met Arg Asp 120 Asp Giy Ile Phe Asp Thr Ile le Tyr 170 Leu Ile Glu 185 Gin Gin Asn 200 Arg Val Leu Gin Vai Asn Tyr Ile His 250 Cys Thr Gly Val Gin Lys Glu Ala 75 Ile Phe Thr Met Pro Ala Lys Gin 140 Leu Val 155 Cys Asn Lys Asn Arg Asp Ile Thr 220 Leu Vai 235 Arg Ile Gly Gly Gin Vai Lys Thr Asp Glu Gin Asn Ala Pro 110 Thr Ile 125 Phe Tyr Glu Leu Thr Lys Met Thr 190 Leu Ile 205 Thr Asp Ile Asn Gly Arg Thr Val Leu Met Leu Glu Leu Ile Tyr Lys 175 Val Met Leu Tyr Thr 255 Asp Val Val Leu Pro Ile Val Ala As n 160 Arg Ser Lys Leu Asp 240 !ro Leu Lys

Claims (25)

1. An isolated or purified nucleic acid coding for an RNA helicase having an amino acid sequence depicted in SEQ ID No. 14 and parts thereof having at least 100 nucleotides and nucleic acids coding for functional variants for the RNA helicase depicted in SEQ ID No. 14, functional variants being proteins having a sequence homology of at least 70% or or proteins having deletions of up to 60 amino acids /fusion proteins which comprise the amino acid sequence of SEQ ID No. 14. 1

2. An nucleic acid as claimed in claim 1 having a nucleotide sequence as °depicted in SEQ ID No. 13 and parts thereof having at least nucleotides.

3. An isolated or purified 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 and nucleic acids coding for functional variants of the RNA helicase of SEQ ID No. 16, functional variants being S"proteins having a sequence homology of at least 70% or proteins having 20 deletions of up to 60 amino acids or fusion proteins which comprise the amino acid sequence of SEQ ID No. 16.

4. A nucleic acid as claimed in any one of claims 1 to 3, wherein the nucleic acid is a DNA or RNA, preferably a double-stranded DNA. A nucleic acid as claimed in any one of claims 1 to 4 and obtained from ciliates.

6. A nucleic acid as claimed in claim 5 and obtained from Tetrahymena thermophila. P:\WPDOCsCR}N\ShIkSp./763620spdOd-07/1 1/02 -29-

7. A DNA antisense strand or an RNA antisense strand obtained from nucleic acids as claimed in any one of claims 1 to 6.

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

9. A method for preparing a nucleic acid as claimed in any one of claims 1 10 to 7, wherein the nucleic acid is chemically synthesized or isolated from a gene library using a probe.

10. An isolated or purified 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 or of at least 70% pfa polypeptide having deletions of up to 60 amino acids or a fusion protein which includes the amino acid sequence of SEQ ID No. 14.

11. An isolated or purified 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.

12. A method for preparing a polypeptide as claimed in claim 10 or 11, which s comprises expressing a nucleic acid as claimed in one of claims 1 to 7 in P 30S a suitable host cell. P:\WPDOCS\CRN\Shlc~ySp.\7563620.spc.d-07/11/02

13. An isolated or purified antibody against a polypeptide as claimed in claim or 11.

14. A method for producing an antibody as claimed in claim 13, which comprises immunizing a mammal with a polypeptide as claimed in one of claim 10 or 11, and, where appropriate, isolating the generated antibodies. 9 10 15. A pharmaceutical comprising a nucleic acid as claimed in one of claims 1 to 7 or a polypeptide as claimed in claim 10 or 11 and, where 9 appropriate, pharmaceutically acceptable additives and/or excipients

16. 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, *l acute and chronic infectious diseases and/or diabetes, and/or for affecting the cell metabolism, in particular in association with 20 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 claims 1 to 7 or a polypeptide as claimed in claims 10 or 11 or an antibody as claimed in claim 13 together with a pharmaceutically acceptable additive and/or excipient.

17. A diagnostic agent comprising a nucleic acid as claimed in one of claims 1 to 7 or a polypeptide as claimed in claim 10 or 11 or an antibody as claimed in claim 13 and, where appropriate, suitable additives and/or s excipients P:\WPDOCS\CRN\Shelley\Sp \7563620.spc.doc-07/11/02 -31-

18. 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 10 pharmaceutically acceptable carrier to a nucleic acid as claimed in claims 1-7 or a polypeptide as claimed in claim 10 or 11 or an antibody as S. claimed in claim 13.

19. An assay for identifying functional interactors which comprises a nucleic acid as claimed in one of claims 1 to 7 or a polypeptide as claimed in claim 10 or 11 or an antibody as claimed in claim 13 and, where appropriate, suitable additives and/or excipients.

20. The use of a nucleic acid as claimed in one of claims 1 to 7 or of a 20 polypeptide as claimed in claim 10 or 11 for identifying functional interactors.

21. The use of a nucleic acid as claimed in one of claims 1 to 7 for detecting variants of RNA helicase, which comprises screening a gene library using said nucleic acid and isolating the variant found.

22. The use of the nucleic acids as claimed in one of claims 1 to 7 or of a polypeptide as claimed in claim 10 or 11 for affecting protein biosynthesis. P:\WPDOCS\CRN\Shelley\Spec\7563620.spc.dloc-07/1/02 -32-

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

24. The use of nucleic acids and polypeptides as claimed in claim 22 for heterologous expression in useful plants. The use of the nucleic acids as claimed in claims 1 to 7 or of a polypeptide as claimed in claim 10 or 11 as selection markers in 10 molecular biology.

26. The use of the nucleic acids as claimed in any one of claims 1 to 7 or of a polypeptide as claimed in claim 10 or 11 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 cell metabolism in particular association with immunosuppression, especially in association with transplants and/or 20 genetic diseases, in particular Werner's syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases.

27. The use of nucleic acids as claimed in any one of claims 1 to 7 or of a polypeptide as claimed in claim 10 or 11 in the manufacture of 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 cell metabolism in S particular association with immunosuppression, especially in association with transplants and/or genetic diseases, in particular Werner's P:\WPDOCS\CRN\Shclley\Spcc\7563620.spc.doc-07/11/02 -33- syndrome, Bloom's syndrome, xeroderma pigmentosa and connective tissue diseases.

28. Nucleic acid sequences, antisensense DNA or RNA obtained therefrom, a method for preparing said nucleic acids polypeptides, a method for preparing said polypeptides, antibodies against said polypeptides, a method of obtaining said antibodies, pharmaceutical compositions comprising said nucleic acids or polypeptides, a process for producing said pharmaceutical, a diagnostic agent comprising said nucleic acids, 10 polypeptides or antibodies, a process for preparing said diagnostic agents, an assay for identifying functional interactors, or use of nucleic acids, or use of polypeptides substantially as hereinbefore described with reference to the Examples and accompanying figures. DATED this 7th day of November, 2002 AVENTIS RESEARCH TECHNOLOGIES GMBH CO. KG By its Patent Attorneys DAVIES COLLISON CAVE