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  Pharmaceutical Patents  

 

Title:  Regulation of kinase, regulated in COPD kinase (RC kinase)
United States Patent: 
7,829,685
Issued: 
November 9, 2010

Inventors:
 Watanabe; Shinichi (Nara, JP), Encinas; Jeffrey A. (San Diego, CA), Kondo; Shinichi (Nara, JP), Bacon; Kevin (San Diego, CA)
Assignee:
  Axikin Pharmaceuticals, Inc. (San Diego, CA)
Appl. No.:
 10/561,570
Filed:
 June 16, 2004
PCT Filed:
 June 16, 2004
PCT No.:
 PCT/EP2004/006474
371(c)(1),(2),(4) Date:
 September 11, 2007
PCT Pub. No.:
 WO2005/001083
PCT Pub. Date:
 January 06, 2005


 

Patheon


Abstract

Reagents which regulate human RC Kinase activity and reagents which bind to human RC Kinase gene products can be used to regulate this protein for therapeutic effects. Such regulation is particularly useful for treating chronic obstructive pulmonary disease, asthma, cancer, and diseases in which cell signaling is defective.

Description of the Invention

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a human RC Kinase. These and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is an isolated polynucleotide encoding a RC Kinase polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a RC Kinase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12; and the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12. b) a polynucleotide comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Another embodiment of the invention is a substantially purified RC Kinase polypeptide encoded by a polynucleotide of the above.

The present invention further relates to novel preventive, predictive, diagnostic, prognostic and therapeutic compositions and uses for COPD. Since RC Kinase expression levels are increased in the disease state, its gene product is a particularly useful target for treatment methods as well as diagnostic and clinical monitoring methods.

The present invention further relates to novel preventive, predictive, diagnostic, prognostic and therapeutic compositions and uses for COPD based on derivatives, fragments, analogues and homologues of the RC Kinase gene.

The present invention further relates to methods for detecting the dysregulation of RC Kinase in COPD on the DNA and mRNA levels.

The present invention further relates to a method for the prediction, diagnosis or prognosis of COPD by the detection of RC Kinase gene or RC Kinase genomic nucleic acid sequence which is altered in COPD.

In one embodiment the expression of the RC Kinase gene can be detected with arrays.

In a further embodiment, the expression of the gene can be detected with bead based direct fluorescent readout techniques such as provided by Luminex corporation (U.S. Pat. No. 6,268,222).

In one embodiment, the invention pertains to a method of determining the phenotype of a cell or tissue, comprising detecting the differential expression, relative to a normal or untreated cell, of the polynucleotide comprising SEQ ID NO: 1, 2, 3, 4, 5, or 6, wherein the polynucleotide is differentially expressed by at least about 1.5 fold, at least about 2 fold or at least about 3 fold.

In yet another embodiment the invention provides the human genomic region on chromosome 2q21.3, specifically the genomic region found on the human genomic sequence contig with the Genbank accession number NT.sub.--005058, for use in prediction, diagnosis and prognosis as well as prevention and treatment of COPD. In particular not only the intragenic regions, but also intergenic regions, pseudogenes or non-transcribed genes of said chromosomal regions can be used for diagnostic, predictive, prognostic and preventive and therapeutic compositions and methods.

In yet another embodiment the invention provides methods of screening for agents which regulate the activity of a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. A test compound is contacted with a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. Binding of the test compound to the polypeptide is detected. A test compound which binds to the polypeptide is thereby identified as a potential therapeutic agent for the treatment of COPD.

In even another embodiment the invention provides another method of screening for agents which regulate the activity of a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. A test compound is contacted with a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. A biological activity mediated by the polypeptide is detected. A test compound which decreases the biological activity is thereby identified as a potential therapeutic agent for decreasing the activity of the polypeptide encoded by a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in COPD. A test compound which increases the biological activity is thereby identified as a potential therapeutic agent for increasing the activity of the polypeptide encoded by the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in COPD.

In another embodiment the invention provides a method of screening for agents which regulate the activity of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. A test compound is contacted with a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. Binding of the test compound to the polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 is detected. A test compound which binds to the polynucleotide is thereby identified as a potential therapeutic agent for regulating the activity of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in COPD.

The invention thus provides polypeptides if SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 which can be used to identify compounds which may act, for example, as regulators or modulators such as agonists and antagonists, partial agonists, inverse agonists, activators, co-activators and inhibitors of the polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. Accordingly, the invention provides reagents and methods for regulating a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in COPD. The regulation can be an up- or down regulation. Reagents that modulate the expression, stability or amount of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or the activity of the polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 can be a protein, a peptide, a peptidomimetic, a nucleic acid, a nucleic acid analogue (e.g. peptide nucleic acid, locked nucleic acid) or a small molecule. Methods that modulate the expression, stability or amount of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or the activity of the polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 can be gene replacement therapies, antisense, ribozyme, RNA interference and triplex nucleic acid approaches.

In one embodiment of the invention provides antibodies which specifically bind to a full-length or partial polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 for use in prediction, prevention, diagnosis, prognosis and treatment of COPD.

Yet another embodiment of the invention is the use of a reagent which specifically binds to a polynucleotide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in the preparation of a medicament for the treatment of COPD.

Still another embodiment is the use of a reagent that modulates the activity or stability of a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or the expression, amount or stability of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in the preparation of a medicament for the treatment of COPD.

Still another embodiment of the invention is a pharmaceutical composition which includes a reagent which specifically binds to a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and a pharmaceutically acceptable carrier.

Yet another embodiment of the invention is a pharmaceutical composition including a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or encoding a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12.

In one embodiment, a reagent which alters the level of expression in a cell of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or encoding a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12, or a sequence complementary thereto, is identified by providing a cell, treating the cell with a test reagent, determining the level of expression in the cell of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or encoding a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a sequence complementary thereto, and comparing the level of expression of the polynucleotide in the treated cell with the level of expression of the polynucleotide in an untreated cell, wherein a change in the level of expression of the polynucleotide in the treated cell relative to the level of expression of the polynucleotide in the untreated cell is indicative of an agent which alters the level of expression of the polynucleotide in a cell.

The invention further provides a pharmaceutical composition comprising a reagent identified by this method.

Another embodiment of the invention is a pharmaceutical composition which includes a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or which is encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6.

A further embodiment of the invention is a pharmaceutical composition comprising a polynucleotide including a sequence which hybridizes under stringent conditions to a polynucleotide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 and encoding a polypeptide exhibiting the same biological function as RC Kinase, or encoding a polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12. Pharmaceutical compositions, useful in the present invention may further include fusion proteins comprising a polypeptide comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a fragment thereof, antibodies, or antibody fragments

DETAILED DESCRIPTION OF THE INVENTION

"RC Kinase" as used herein refers to the polypeptide of SEQ ID NO 7, 8, 9, 10, 11 or 12, as well as it derivatives, fragments, analogs, and homologues thereof, or the polypeptides encoded by the polynucleotide of SEQ ID NO: 1 as well as derivatives, fragments, analogs and homologues thereof.

SEQ ID NO: 1 shows variant 1 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 3719 bp in length, with an open reading frame extending from bases 1-3679 of the sequence. SEQ ID NO: 2 shows variant 2 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 3338 bp in length, with an open reading frame extending from bases 1-3243 of the sequence. SEQ ID NO: 3 shows variant 3 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 3510 bp in length, with an open reading frame extending from bases 1-3415 of the sequence. SEQ ID NO: 4 shows variant 4 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 4058 bp in length, with an open reading frame extending from bases 1-4018 of the sequence. SEQ ID NO: 5 shows variant 5 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 1460 bp in length, with an open reading frame extending from bases 1-1420 of the sequence. SEQ ID NO: 6 shows variant 6 of the DNA-sequence encoding an RC Kinase polypeptide. This variant is 1604 bp in length, with an open reading frame extending from bases 1-1564 of the sequence. SEQ ID NO: 7 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 1. SEQ ID NO: 8 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 2.

SEQ ID NO: 9 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 3. SEQ ID NO: 10 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 4. SEQ ID NO: 11 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 5. SEQ ID NO: 12 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID NO: 6.

Furthermore, the activity of a novel RC Kinase, particularly a human RC Kinase, is a discovery of the present invention. Human RC Kinase contains a single S_TKc kinase domain (Serine/-Threonine protein kinases, catalytic domain), beginning approximately 268 amino acid residues from the carboxy terminal of SEQ ID NO: 7, 8, 10 or 12 and spanning approximately 256 residues. Two of the variants of Human RC Kinase, SEQ ID NO: 9 and 11, are missing part of this kinase domain. The kinase domain of Human RC Kinase is highly homologous to the kinase domains of other known kinase type enzymes. Human RC Kinase as shown in SEQ ID NO: 7, 8, 10 or 12 is 44% identical and 67% similar over 287 amino acids (kinase domain) to the slime mold Dictyostelium discoideum protein identified by GenBank Accession No. AAC97114 and annotated as a "MEK kinase alpha." Similarly, human RC Kinase as shown in SEQ ID NO: 7, 8, 10, or 12 is 47% identical and 67% similar over 276 amino acids (kinase domain) to the common tobacco Nicotiana tabacum protein identified by GenBank Accession No. A48084 and annotated as a "STE11 protein kinase homolog NPK1," and is 46% identical and 63% similar over. 291 amino acids (kinase domain) to the human protein identified by GenBank Accession No. NP.sub.--002392 and annotated as a "MAP/ERK kinase kinase 3; MAPKKK3."

The coding sequences for SEQ ID NOS: 7-12 are shown in SEQ ID NOS: 1-6, respectively. The gene containing these coding sequences is located within the human chromosome 2 genomic contig identified with GenBank accession no. NT.sub.--005058, and is divided into at least 11 exons spanning more than 61,000 bases of the genome. If the 11 3'-most exons are labeled as exons C, D, E, F, G, H, I, J, K, L, and M, respectively, reading in order along the gene from 5' to 3', then six alternative splice variants are described by the invention as follows. SEQ ID NO: 1 describes a splice variant which uses all exons except exons E, G, and H. SEQ ID NO: 2 describes a splice variant which uses all exons except a portion of exon L. SEQ ID NO: 3 describes a splice variant which uses all exons except exons E and a portion of L. SEQ ID NO: 4 describes a splice variant which uses all exons except exon E. SEQ ID NO: 5 describes a splice variant which uses all exons except exons E, J, and a portion of L. SEQ ID NO: 6 describes a splice variant which uses all exons except exons E, and J.

A single exon containing most of the kinase catalytic domain has previously been annotated as "hypothetical protein FLJ23074," a gene with protein product and function unknown.

RCKinase has the ability to phosphorylate other RC Kinase polypeptides, MKK4, and MKK6. Taken together with the fact of importance of MAPK signalling, the modification of RC kinase activity can give chance of remedy against COPD, asthma, cancer, Alzheimer's disease, inflammatory diseases, and cardiovascular diseases.

Polypeptides

RC Kinase polypeptides according to the invention comprise the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a biologically active variant thereof, as defined below. A RC Kinase polypeptide of the invention therefore can be a portion of a RC Kinase molecule, a full-length RC Kinase molecule, or a fusion protein comprising all or a portion of a RC Kinase molecule.

Biologically Active Variants

RC Kinase variants which are biologically active, i.e., retain a RC Kinase activity, also are RC Kinase polypeptides. Preferably, naturally or non-naturally occurring RC Kinase variants have amino acid sequences which are at least about 50, preferably about 70, 75, 90, 96, or 98% identical to an amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12. Percent identity between a putative RC Kinase variant and an amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, or 12 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The PASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff" value (calculated by a predetermined formula based upon the length of the sequence the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman & Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active RC Kinase polypeptide can readily be determined by assaying for fibronectin binding or for RC Kinase activity, as is known in the art and described, for example, in Example 2.

Fusion Proteins

Fusion proteins are useful for generating antibodies against RC Kinase amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a RC Kinase polypeptide, including its active site. Methods such as protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A RC Kinase fusion protein comprises two protein segments fused together by means of a peptide bond. Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or from a biologically active variant thereof, such as those described above. Preferably, a fusion protein comprises a kinase domain and/or an ATP binding site of human RC Kinase. The first protein segment also can comprise full-length RC Kinase.

The second protein segment can be a full-length protein or a protein fragment or polypeptide. Proteins commonly used in fusion protein construction include .beta.-galactosidase, .beta.-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the RC Kinase polypeptide-encoding sequence and the heterologous protein sequence, so that the RC Kinase polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises RC Kinase coding sequences disclosed herein in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human RC Kinase can be obtained using RC Kinase polynucleotides (described below) to make suitable probes or primers to screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of RC Kinase, and expressing the cDNAs as is known in the art.

Polynucleotides

A RC Kinase polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a RC Kinase polypeptide. A partial coding sequence of a RC Kinase polynucleotide is shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6; coding sequences of RC Kinase also are contained within the genomic sequence shown in SEQ ID NO: 3, from nucleotides 11885 to 12023 and from nucleotides 10564 to 10693.

Degenerate nucleotide sequences encoding human RC Kinase polypeptides, as well as homologous nucleotide sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the RC Kinase coding sequences nucleotide sequences shown in SEQ ID NOS: 1 and 3 also are RC Kinase polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of RC Kinase polynucleotides which encode biologically active RC Kinase polypeptides also are RC Kinase polynucleotides.

Identification of Variants and Homologs

Variants and homologs of the RC Kinase polynucleotides disclosed above also are RC Kinase polynucleotides. Typically, homologous RC Kinase polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known RC Kinase polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions-2.times.SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2.times.SSC, 0.1% SDS, 50.degree. C. once, 30 minutes; then 2.times.SSC, room temperature twice, 10 minutes each--homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the RC Kinase polynucleotides disclosed herein can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of RC Kinase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T.sub.m of a double-stranded DNA decreases by 1-1.5.degree. C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human RC Kinase polynucleotides or RC Kinase polynucleotides of other species can therefore be identified, for example, by hybridizing a putative homologous RC Kinase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or an ephrin-like serine protease coding sequence of SEQ ID NO: 3 to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising RC Kinase polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to RC Kinase polynucleotides or their complements following stringent hybridization and/or wash conditions are also RC Kinase polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20.degree. C. below the calculated T.sub.m of the hybrid under study. The T.sub.m of a hybrid between a RC Kinase polynucleotide having a coding sequence disclosed herein and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to that nucleotide sequence can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T.sub.m=81.5.degree. C.-16.6(log.sub.10[Na.sup.+])+0.41(%G+C)-0.63(%formamide)-600/l), where l=the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4.times.SSC at 65.degree. C., or 50% formamide, 4.times.SSC at 42.degree. C., or 0.5.times.SSC, 0.1% SDS at 65.degree. C. Highly stringent wash conditions include, for example, 0.2.times.SSC at 65.degree. C.

Preparation of Polynucleotides

A naturally occurring RC Kinase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or synthesized using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated RC Kinase polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise RC Kinase nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

RC Kinase cDNA molecules can be made with standard molecular biology techniques, using RC Kinase mRNA as a template. RC Kinase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of RC Kinase polynucleotides, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize RC Kinase polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a RC Kinase polypeptide having, for example, the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a biologically active variant of that sequence.

Obtaining Full-Length Polynucleotides

The partial sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or its complement can be used to identify the corresponding full length gene from which they were derived. The partial sequences can be nick-translated or end-labeled with .sup.32P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., eds., Elsevier Press, N.Y., 1986). A lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., 1989, pg. 1.20).

Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al., 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting radiographies are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected and expanded, and the DNA is isolated from the colonies for further analysis and sequencing.

Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.

Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al., Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.

Various PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human RC Kinase to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72.degree. C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations are used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991. Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
 

Claim 1 of 12 Claims

1. An isolated polynucleotide encoding a RC Kinase polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a RC Kinase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least 90% identical to the amino acid sequence shown in SEQ ID NO: 10; and the amino acid sequence shown in SEQ ID NO: 10; b) a polynucleotide comprising the sequence of SEQ ID NO: 4; and c) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) or (b) due to the degeneration of the genetic code.
 

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