WO1994017189A2 - Proteine-kinases - Google Patents

Proteine-kinases Download PDF

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WO1994017189A2
WO1994017189A2 PCT/US1994/000795 US9400795W WO9417189A2 WO 1994017189 A2 WO1994017189 A2 WO 1994017189A2 US 9400795 W US9400795 W US 9400795W WO 9417189 A2 WO9417189 A2 WO 9417189A2
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lys
arg
gly
ser
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WO1994017189A3 (fr
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Merl F. Hoekstra
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Salk Institute for Biological Studies
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Salk Institute for Biological Studies
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Priority to CA002132452A priority Critical patent/CA2132452C/fr
Priority to EP94915331A priority patent/EP0632832A1/fr
Priority to JP6517227A priority patent/JPH07505057A/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel polynucleotides encoding polypeptides which correspond to the class of protein kinase isolates heretofore referred to as casein kinase I and which possess protein kinase and/or DNA recombination/repair promoting functional capabilities.
  • the protein kinases comprise an exceptionally large family of eukaryotic proteins which mediate the responses of cells to external stimuli and are related by amino acid sequence homology within the so-called "catalytic domain" of the enzymes.
  • catalytic domain within the so-called "catalytic domain" of the enzymes.
  • in excess of 100 unique members of the protein kinase family from a wide variety of eukaryotic organisms have been described and characterized at the amino acid sequence level. See, e.g., Hanks, et al. (Science, 241-42-52, 1988) which presents a sequence alignment of 65 protein kinase catalytic domains which range in size from about 250 to 300 amino acids and Hanks, et al.
  • Protein kinases are generally classified into a protein- serine/threonine subfamily or a protein-tyrosine subfamily on the basis of phosphorylation substrate specificity.
  • proteins within the protein-serine/threonine kinase subfamily are two distinct classes which have been designated casein kinase I and casein kinase II based on the order of their elution from DEAE-cellulose.
  • the casein kinases are distinguished from other protein kinases by their ability to phosphorylate serine or threonine residues within acidic recognition sequences such as found in casein. Tuazon, et al. , (Adv. in Second Messenger and Phosphoprotein Res.
  • casein kinase II is active as a heterotetramer and the complete amino acid sequences of human, rat, Drosophila and yeast species catalytic regions have been determined.
  • partially purified casein kinase I preparations have been obtained from cell nuclei, cytoplasm, and cell membranes of various plant and animal species, prior to the present invention, nothing was known concerning the primary structure of its enzymatically active monomeric subunit.
  • Chromosomes experience single-stranded or double-stranded breaks as a result of energy-rich radiation, chemical agents, as well as spontaneous breaks occurring during replication among others. Although genes present in the chromosomes undergo continuous damage, repair, exchange, transposition, and splicing, certain enzymes protect or restore the specific base sequences of the chromosome.
  • the repair of DNA damage is a complex process that involves the coordination of a large number of gene products. This complexity is in part dependent upon both the form of DNA damage and cell cycle progression. For example, in response to ultraviolet (UV) irradiation, cells can employ photoreactivation or excision repair functions to correct genetic lesions. The repair of strand breaks, such as those created by X-rays, can proceed through recombinational mechanisms. For many forms of DNA damage, the cell is induced to arrest in the G2 phase of the cell cycle. During this G2 arrest, lesions are repaired to ensure chromosomal integrity prior to mitotic segregation.
  • UV ultraviolet
  • Phenotypic complementation as a way of identifying homologous normal functional genes, is widely used.
  • the human homologue of the yeast cell cycle control gene, cdc 2 was cloned by expressing a human cDNA library in Schizosaccharomyces pombe and selecting those clones which could complement a mutation in the yeast cdc 2 gene (Lee, et al, Nature, 327:31. 1987).
  • a mammalian gene capable of reverting the heat shock sensitivity of the RAS2 va119 gene of yeast has also been cloned by using complementation (Colicelli, et al, Proc.Nat'l.Acad.Sci. USA, 86:3599, 1989).
  • a rat brain cDNA library was used to clone a mammalian cDNA that can complement the loss of growth control associated with the activated RAS2 gene in yeast.
  • the gene, DPD (dunce-like phosphodiesterase) encodes a high-affinity CAMP phosphodiesterase.
  • the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts thereof) encoding eukaryotic protein kinases of the casein kinase I class herein designated as "HRR25-like" proteins and characterized by greater than 35% amino acid sequence homology with the prototypical yeast enzyme HRR25 through the protein kinase catalytic domain thereof.
  • Polynucleotides provided by the invention include RNAs, mRNAs and DNAs, including antisense forms thereof.
  • Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences and biological replicas thereof.
  • Saccharomyces cerevisiae DNAs including those encoding HRR25 and NUFl, Schizosaccharomyces pombe DNAs including those encoding Hhpl + and Hhp2+, and human DNAs including those encoding CKI ⁇ lHu, CKIe ⁇ Hu, CKI ⁇ 3Hu, CKI ⁇ lHu, CKTy2Hu, and CKI ⁇ Hu.
  • autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating such sequences and especially vectors wherein DNA encoding an HRR2J-like casein kinase I protein is linked to an endogenous or exogenous expression control DNA sequence.
  • host cells especially unicellular host cells such as procaryotic and eukaryotic cells, are stably transformed with DNA sequences of the invention in a manner allowing the desired polypeptides to be expressed therein.
  • Host cells expressing such HRR25-like products can serve a variety of useful purposes. To the extent that the expressed products are "displayed" on host cell surfaces, the cells may constitute a valuable immunogen for the development of antibody substances specifically immunoreactive therewith.
  • Host cells of the invention are conspicuously useful in methods for the large scale production of HR#25-like proteins wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown.
  • antibody substances e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like
  • other binding proteins which are specific for HRR25-like proteins (i.e., non-reactive with protein kinase molecules which are not related by at least 35% homology with HRR25 through the protein kinase catalytic domain).
  • Antibody substances can be developed using isolated natural or recombinant HRR25-l ⁇ ke proteins or cells expressing such products on their surfaces.
  • the antibody substances are useful, in turn, for purifying recombinant and naturally occurring HRR25-like polypeptides and identifying cells producing such polypeptides on their surfaces.
  • the antibody substances and other binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting, or stimulating) ligand-receptor binding reactions involving HRR25-like proteins.
  • Anti idiotypic antibodies specific for anti-HRR25-like antibody substances are also contemplated.
  • Assays for the detection and quantification of HRR25-like proteins on cell surfaces and in fluids such as serum and cytoplasmic fractions may involve a single antibody substance or multiple antibody substances in a "sandwich" assay format.
  • Recombinant HR_?25-like protein products obtained according to the invention have been observed to display a number of properties which are unique among the eukaryotic protein kinases.
  • the HRR25 protein possesses both protein-tyrosine kinase and protein-serine/threonine kinase activities.
  • HRR25 operates to promote repair of DNA strand breaks at a specific nucleotide sequence and is the only protein kinase known to have such recombination/repair promoting activity.
  • HRR25-like proteins which is provided by the present invention makes possible the identification and isolation of DNAs encoding other
  • HRR25-ik& proteins by such well-known techniques as DNA/DNA hybridization and polymerase chain reaction (PCR) cloning.
  • Recombinant HRR25-like proteins and host cells expressing the same are useful in screening methods designed to examine the effects of various compositions on DNA break repair and protein kinase activities of the proteins. Protein kinase inhibitory effects may be assessed by well-known screening procedures such as described in ⁇ idaka, et al. (Methods in Enzymology, 201:328- 339, 1991).
  • Figure 1 (A) presents an alignment of the predicted amino acid sequence of HRR25 with the catalytic domains of the yeast CDC28, yeast KSS1 and human RAFl protein kinases.
  • Figure 1(B) shows a schematic representation of the structure of HRR25, and
  • Figure 2 presents an alignment of the predicted amino acid sequences of HRR25 with the sequences of three other Saccharomyces cerevisiae HRR25-lik& proteins (YCK1/CKI2, YCK2/CKI1, and NUF1), two HRR25-like proteins (Hhpl + and Hhp2+) from Schizosaccharomyces pombe and three putative isoforms (CKI ⁇ lHu, CKIo * 2Hu, and CKI ⁇ 3Hu) of a human HRR2J-like protein.
  • the present invention relates to a DNA encoding a recombination/repair promoting polypeptide which can be used in an assay system to examine the effects of various compositions on DNA integrity.
  • These functional sequences which can be characterized by their ability to promote restoration of DNA strand breaks, permit the screening of compositions to determine whether a particular composition has an effect on the restoration of such repair activity.
  • the invention also provides a DNA sequence encoding a polypeptide which promotes normal mitotic recombination, but is defective in protein kinase activity and essentially unable to repair DNA strand breaks. This defective DNA sequence is highly useful for identifying other DNA sequences which encode proteins with functional protein kinase activity.
  • the present invention relates to the polypeptide encoded by the defective DNA sequence, as well as the polypeptide encoded by the functional wild-type DNA.
  • a method is provided whereby a DNA library is screened for nucleotide sequences capable of restoring DNA strand break repair in a mutant lacking such activity.
  • a method is further provided for identifying a composition which affects the activity of a mammalian polypeptide having protein kinase activity, wherein the polypeptide is capable of restoring DNA double-strand break repair activity in a mutant lacking such activity.
  • the defective protein kinase can be characterized by its ability to promote normal mitotic recombination, while being essentially unable to repair DNA double-strand break including that which occurs at the cleavage site:
  • the DNA double-strand breaks which the defective protein kinase is essentially unable to repair can be induced by various means, including endonucleases, x- rays, or radiomimetic agents including alkylating agents.
  • Preferred endonucleases are those which recognize the same nucleotide cleavage site as endonuclease HO.
  • Radiomimetic alkylating agents having methylmethane sulfonate activity are preferred.
  • the present invention specifically discloses mutants sensitive to continuous expression of the DNA double-strand endonuclease HO, which codes for a 65 kDa site-specific endonuclease that initiates mating type interconversion (Kostriken, et al , Cold Spring Harbor Symp. Quant. BioL , 49:89, 1984). These mutants are important to understanding the functions involved in recognizing and repairing damaged chromosomes.
  • This invention also discloses a yeast wild-type DNA recombination and repair gene called HRR25 (HO and/or radiation repair). ⁇ omozygous mutant strains, hrr25-l, are sensitive to methylmethane sulfonate and X-rays, but not UV irradiation.
  • the wild-type gene encodes a novel protein kinase, homologous to other serine/threonine kinases, which appears critical in activation of DNA repair functions by phosphorylation.
  • the HRR25 kinase is important for normal cell growth, nuclear segregation, DNA repair and meiosis, and deletion of HRR25 results in cell cycle defects.
  • These phenotypes coupled with the sequence similarities between the HRR25 kinase and the Raf/c-mos protein kinase subgroup suggest that HRR25 might play a similar role in S. cerevisiae growth and development.
  • DNA sequences encoding protein kinase polypeptides of the invention can be accomplished using a variety of techniques.
  • methods which can be employed include (1) isolation of a double-stranded DNA sequence from the genomic DNA of the eukaryote; (2) chemical synthesis of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double stranded DNA complement of MRNA is eventually formed which is generally referred to as CDNA.
  • novel DNA sequences of the invention include all sequences useful in providing for expression in prokaryotic or eukaryotic host cells of polypeptides which exhibit the functional characteristics of the novel protein kinase of the invention.
  • These DNA sequences comprise: (a) the DNA sequences as set forth in SEQ. I.D. No. 1 or their complementary strands; (b) DNA sequences which encode an amino acid sequence with at least about 35% homology in the protein kinase domain with the amino acid sequences encoded by the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences defined in (a) and (b) above.
  • genomic DNA sequences which encode allelic variant forms.
  • Part (c) specifically embraces the manufacture of DNA sequences which encode fragments of the protein kinase and analogs of the protein kinase wherein the DNA sequences thereof may incorporate codons which facilitate translation of mRNA. Also included in part (c) are DNA sequences which are degenerate as a result of the genetic code.
  • conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • polypeptide denotes any sequence of amino acids having the characteristic activity of the mutant or wild-type protein kinase of the invention, wherein the sequence of amino acids is encoded by all or part of the DNA sequences of the invention.
  • polypeptide resulting from expression of the DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the protein kinase in its natural cellular environment.
  • Isolation and purification of microbially expressed polypeptides provided by the invention may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation.
  • recombinant expression vectors useful in the present invention contain a promotor sequence which facilitates the efficient transcription of the inserted eukaryotic genetic sequence.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the polypeptides of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
  • DNA sequences of the present invention can be expressed in vt ' vo in either prokaryotes or eukaryotes. Methods of expressing DNA sequences containing eukaryotic coding sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors used to incorporate DNA sequences of the invention, for expression and replication in the host cell are well known in the art. For example, DNA can be inserted in yeast using appropriate vectors and introducing the product into the host cells. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, et al , Nature, 340:205, 1989; Rose, et al , Gene, 60:237, 1987). Those of skill in the art will know of appropriate techniques for obtaining gene expression in both prokaryotes and eukaryotes, or can readily ascertain such techniques, without undue experimentation.
  • Hosts include microbial, yeast, insect and mammalian host organisms.
  • the term "host” is meant to include not only prokaryotes, but also such eukaryotes such as yeast, filamentous fungi, as well as plant and animal cells which can replicate and express an intron-free DNA sequence of the invention.
  • the term also includes any progeny of the subject cell. It is understood that not all progeny are identical to the parental cell since there may be mutations that occur at replication. However, such progeny are included when the terms above are used.
  • Transformation with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
  • MgCl 2 or RbCl could be used in the reaction. Transformation can also be performed after forming a protoplast of the host cell.
  • the host is a eukaryote, various methods of DNA transfer can be used.
  • the rearranged sequences must be taken out of the bacteria in which they were cloned and reintroduced into a eukaryotic organism. Since there are still many functions in eukaryotic cells which are absent in prokaryotes, (e.g., localization of ATP-generating systems to mitochondria, association of DNA with histones, mitosis and meiosis, and differentiation of cells), the genetic control of such functions must be assessed in a eukaryotic environment. Cloning genes from other eukaryotes in yeast has been useful for analyzing the cloned eukaryotic genes as well as other yeast genes.
  • yeast vectors have been constructed for this purpose. All vectors replicate in E. coli, which is important for amplification of the vector DNA. All vectors contain markers, e.g., LEU 2, HIS 3, URA 3, that can be selected easily in yeast. In addition, these vectors also carry antibiotic resistance markers for use in E. coli. Many strategies for cloning human homologues of known yeast genes are known in the art. These include, but are not limited to: 1) low stringency hybridization to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features; and 3) complementation of mutants to detect genes with similar functions.
  • protein kinases which are homologous can be identified by structural as well as functional similarity.
  • Structural similarity can be determined, for example, by assessing amino acid homology or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the protein kinases of the invention.
  • amino acid homology is used as criteria to establish structural similarity, those amino acid sequences which have homology of at least about 35 % in the protein kinase domain with the prototypical HRR25 protein are considered to uniquely characterize polypeptides. conserveed regions of amino acid residues in HRR25 can be used to identify HRR25-like genes from other species.
  • conserved regions which can be used as probes for identification and isolation of HRR25-like genes include the nucleotides encoding amino acid sequences GPSLED (amino acids 86 to 91 in SEQ ID NO: 2), RDIKPDNFL (amino acids 127 to 135 in SEQ ID NO: 2), ⁇ IPYRE (amino acids 164 to 169 in SEQ ID NO: 2), and SVN (amino acids 181 to 183 in SEQ ID NO: 2), for example.
  • GPSLED amino acids 86 to 91 in SEQ ID NO: 2
  • RDIKPDNFL amino acids 127 to 135 in SEQ ID NO: 2
  • ⁇ IPYRE amino acids 164 to 169 in SEQ ID NO: 2
  • SVN amino acids 181 to 183 in SEQ ID NO: 2
  • a homologous amino acid sequence is considered equivalent to an amino acid sequence of the invention when the homologous sequence is essentially unable to repair (in the case of the repair defective mutant gene) or able to repair (in the case of the natural gene), DNA double-strand breaks, including that which occurs at a nucleotide cleavage site I CAACAG
  • This invention provides screening methods whereby genes are cloned from plasmid libraries by complementation of a recessive marker.
  • a recipient strain such as Saccharomyces cerevisiae is constructed that carries a recessive mutation in the gene of interest.
  • This strain is then transformed with a plasmid, for example, pYES2 (Invitrogen, San Diego, CA) containing the wild- type genomic DNA or cDNA.
  • the clone carrying the gene of interest can then be selected by replica plating to a medium that distinguishes mutant from wild- type phenotypes for the gene of interest.
  • the plasmid can then be extracted from the clone and the DNA studied.
  • yeast vectors allow the application of complementation systems to go beyond isolation of yeast genes.
  • Genes from a wide variety of species can be isolated using these vectors.
  • DNA sequences from any source are cloned into a vector and can be screened directly in yeast for activities that will complement specific yeast mutations.
  • the present invention uses a mutation in yeast, the hrr25 mutation, which was identified by sensitivity to DNA double- strand breaks induced by the HO endonuclease.
  • the genomic DNA which complements this mutation was isolated by transforming the hrr25 strain with a DNA library and subsequently screening for methylmethane sulfonate (MMS) resistance.
  • MMS methylmethane sulfonate
  • functional genes from a variety of mammalian species can now be cloned using the system described.
  • Yeast genes can be cloned by a variety of techniques, including use of purified RNA as hybridization probes, differential hybridization of regulated RNA transcripts, antibody screening, transposon mutagenesis, cross suppression of mutant phenotypes, cross hybridization with heterologous CDNA or oligonucleotide probes, as well as by complementation in E. coli.
  • Minor modifications of the primary amino acid sequence may result in proteins which have substantially equivalent or enhanced activity as compared to the sequence set forth in SEQ. I.D. NO. 2.
  • the modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous by HRR25 producing organisms. All of these modifications are included in the invention as long as HRR25 activity is retained. Substitution of an aspartic acid residue for a glycine acid residue at position 151 in the sequence shown in SEQ. I.D. NO. 2 identifies the mutant hrr25.
  • Antibodies provided by the present invention are immunoreactive with the mutant polypeptides and/or the naturally occurring protein kinase.
  • Antibody which consist essentially of numerous monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibody is made from antigen containing fragments of the polypeptide by methods well known in the art (Kohler,G. et al. , Nature 256:495, 1975; Current Protocols in Molecular Biology , Ausubel, F. et al, ed.,1989).
  • the invention also discloses a method for identifying a composition which affects the activity of a polypeptide having tyrosine kinase activity.
  • the polypeptide is capable of promoting restoration of DNA double-strand break repair activity in host cells containing the hrr25 gene.
  • the composition and the polypeptide are incubated in combination with host cells for a period of time and under conditions sufficient to allow the components to interact, then subsequently monitoring the change in protein kinase activity, for example, by decreased repair of DNA double-strand breaks.
  • the DNA strand breaks are induced, for example, by a radiomimetic agent, such as methylmethane sulfonate, x-rays, or by endonuclease like HO. Other means of inducing double-strand breaks that are well known in the art may be employed as well.
  • One embodiment of the invention provides a method of treating a cell proliferative disorder associated with or HRR25 or an HRR25-like protein comprising administering to a subject with the disorder, a therapeutically effective amount of reagent which modulates an HRR25 -like protein activity.
  • cell proliferative disorder denotes malignant as well as non-malignant cell populations which differ from the surrounding tissue both morphologically and/or genotypically. Such disorders may be associated, for example, with abnormal expression of HRR25-like protein genes.
  • “Abnormal expression” encompasses both increased or decreased levels of expression as well as expression of mutant forms such that the normal function of HRR2J-like genes is altered. Abnormal expression also includes inappropriate temporal expression during the cell cycle or expression in an incorrect cell type.
  • Antisense polynucleotides of the invention are useful in treating malignancies of the various organ systems. Essentially, any disorder which is etiologically linked to altered expression of HRR25-like genes is a candidate for treatment with a reagent of the invention. "Treatment" of cell proliferative disorder refers to increasing or decreasing populations of malignant or non-malignant cells.
  • the term "modulate” envisions the suppression of HRR25-like protein expression or the augmentation of expression.
  • appropriate reagents such as antisense or binding antibody can be introduced to a cell.
  • This approach utilizes, for example, antisense nucleic acid and ribozymes to block translation of a specific HRR25-like protein mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it with a ribozyme.
  • a sense polynucleotide sequence (the DNA coding strand) or HRR25-like polypeptide can be introduced into the cell by methods known in the art.
  • the term “therapeutically effective” refers to that amount of polynucleotide, antibody or polypeptide that is sufficient to ameliorate the HRR25-associated disorder.
  • “Ameliorate” denotes a lessening of the detrimental effect of the HRR25-associated disorder in the subject receiving therapy.
  • Antisense nucleic * acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. This interferes with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause non ⁇ specific interference with translation than larger molecules when introduced into the target HRR25 producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem. , 172:289, 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn. , 260:3030, 1988). A major advantage of this approach is that, because ribosomes are sequence-specific, only mRNAS with particular sequences are inactivated. There are two basic types of ribozymes namely, tetrahymena-type and "hammerhead"-type.
  • Tetrahymena-ty ⁇ e ribozymes recognize sequences which are four bases in length, while "hammerhead "-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-ty_pe ribozymes for inactivating a specific mRNA species and longer recognition sequences are preferable to shorter recognition sequences.
  • the present invention also provides gene therapy for the treatment of cell proliferative disorders which are mediated by HRR25-like polypeptides.
  • Such therapy comprises introducing into cells of subjects having the proliferative disorder, the HRR25-like antisense polynucleotide. Delivery of antisense polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Disorders associated with under-expression of HRR25 can similarly be treated using gene therapy with nucleotide coding sequences.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include but are not limited to 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al. Trends Biochem. Scl, 6:77, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotechniques, 6:682, 1988).
  • the targeting of liposomes has been classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in” on the desired cells.
  • a ligand may be any compound of interest which will bind to another compound, such as a receptor.
  • Example 1 addresses isolation of hrr25 mutant strains of Saccharomyces cerevisiae
  • Example 2 describes the isolation of HRR25 DNA by complementation screening
  • Example 3 is drawn to characterization of the DNA and putative amino acid sequence of HRR25
  • Example 4 addresses microscopic analysis of HRR25 wild type and hrr25 mutant yeast morphology
  • Example 5 addresses the relationship of the amino acid sequence of HRR25 and three exemplary protein kinases which are not HRR25- like
  • Example 6 describes the isolation of DNAs encoding two Schizosaccharomyces pombe HRR25-like protein kinases
  • Example 7 is directed to isolation of DNA encoding another Saccharomyces cerevisiae protein, NUF1
  • Example 8 is drawn to isolation of DNAs encoding various eukaryotic species HRR25-like proteins including three human isoforms, CKIcdHu, CKI ⁇ 2Hu, and CKI ⁇ Hu; Examples 9 and
  • S. cerevisiae strain K264-5B (MAT ho ura3 canl R tyrl his7 lys2 ade5 metlS trp5 leul ade5) was employed for the mutant isolation.
  • the yeast were transformed according to standard procedures with a Z7R_43-based integrating plasmid that contained a GAL1, 10-regulated HO endonuclease and a transformant was mutagenized to approximately 50% survival with ethyl methanesulfonate (EMS), as described (Current Protocols in Molecular Biology, supra).
  • EMS ethyl methanesulfonate
  • the culture was spread onto glycerol-containing rich medium (YPG, to avoid colonies), colonies were allowed to form at 30°C, and plates were replicated to glucose (HO repressing) and galactose (HO inducing) media. Mutants were identified by their inability to grow on galactose. Approximately 200 mutants were chosen for initial characterization and 62 maintained the gal- phenotype through repeated single colony purification. Among these, many were not complemented by various gal mutants. The remainder (25 mutants) were surveyed for overlapping DNA repair defects by determining sensitivity to ultraviolet (UV) irradiation and to methyl methane sulfonate (MMS) . This screening method identified five alleles of known rod mutations and one new mutation. This new mutation hrr25-l (HO and/or radiation repair), presented severe defects and was studied further.
  • UV ultraviolet
  • MMS methyl methane sulfonate
  • hrr25-l that includes sensitivity to MMS.
  • Hrr25-1 strains also show sensitivity at 5-20 Krad X- irradiation similar to that observed with mutations in the radiation repair genes RAD50 and RAD52 (Cole, et al , Mol. Cell.Biol. , 9:3101, 1989).
  • the hrr25-l strains are no more sensitive to UV irradiation than wild type and are not temperature sensitive for growth at 37 °C.
  • hypo- and hyper-rec rod mutants which have several of the hrr25-l phenotypes, hrr25-l strains undergo normal mitotic recombination (Cole, et al, Mol.
  • the hrr25-l mutation could be complemented by a number of radiation sensitive mutations (rad ⁇ , 50, 52, 54, and 57) that present some of the hrr25 phenotypes, suggesting that hrr25-l is a newly uncovered rad-l ⁇ ke mutation and not one of these previously described genes.
  • HRR25 plays a role in DNA repair and meiosis, but is not specifically required for the repair of spontaneous mitotic lesions by recombination.
  • HRR25 Isolation of HRR25
  • the HRR25 gene was obtained by complementing for MMS sensitivity using a yeast genomic library constructed in the plasmid YCp50 (Rose, et al. , Gene, 60:237, 1987).
  • An hrr25-l strain, MHML 3-36d (ura3 hrr25) was transformed by standard methods (Nickoloff, et al, J.Mol.BioL, 207:527, 1989) to uracil prototrophy, transformants were amplified on media without uracil and replicated to media containing 0.01 % MMS. Among 1200 transformants, a single MMS resistant isolate was identified.
  • Complementation for MMS sensitivity was found to segregate with the plasmid as determined by methods known in the art.
  • a 12 kb genomic fragment was identified and complementing activity was localized to a 3.1 kb Bar ⁇ l-Sa . fragment by transposon mutagenesis and subcloning. This region complemented DNA repair defects as well as meiotic deficiencies.
  • Gene targeting experiments linked this cloned region to hrr25-l.
  • Transposon insertion mutations within the BamHI-Sali fragment replaced into the cognate HRR25 genomic locus did not complement hrr25-l for MMS sensitivity, whereas adjacent chromosomal insertions outside the complementing region segregated in repulsion when crossed against hrr25-l .
  • the DNA sequence of the 3.1 kb fragment revealed a centrally located open reading frame of 1482 nucleotide.
  • a transposon insertion mutation in this open reading frame inactivated HRR25 complementation whereas insertions elsewhere in the 12 kb clone did not affect HRR25 complementation.
  • Transposon- mediated disruption of HRR25 also revealed several phenotypes not seen with hrr25-l.
  • a Tn70-based LUK transposon insertion (Huisman, et al. , Genetics, 116: 191, 1987) into the middle of plasmid-borne HRR25 coding region inactivated complementation for MMS sensitivity.
  • the entire HRR25 coding sequence was deleted. Briefly, deletion of the HRR25 coding sequence employed a hisG::URA3::hisG cassette (Alani, et al, Genetics, 116:541, 1988). The 3.1 kb HRR25 Sail fragment was cloned into pBluescript (Stratagene, La Jolla, CA). This plasmid was digested with BgRl and the two BglR fragments that span the entire HRR25 gene and its flanking sequences were deleted.
  • hrr25::LUK and hrr25A alleles behaved identically.
  • Yeast strain MFH14 MATa/MATot ura3/ura3
  • JSgfll-linearized YCp50-HRR25: :LUK uracil prototrophy
  • heterozygous disruption of HRR25 was verified by Southern blot analysis, the diploid was sporulated by starvation for nitrogen and fermentable carbon sources, tetrads dissected and cells allowed to germinate at 30 °C for 7 days. After a normal germination period of 2 days, the severe growth defect of hrr25: :LUK suggested that the deletion of HRR25 was lethal.
  • MFH14 is ade5/ADE5 ade2/ade2.
  • An ade5/ade2 strain was white, while an ADE5/ade2 strain was red.
  • FIG. 1A shows the alignment of the amino acid sequences for
  • FIG. 1B shows a schematic representation of the structure of HRR25.
  • the protein kinase homology is represented by a shaded region while the P/Q rich region is indicated by cross-hatchings.
  • the mutant, hrr25 can be distinguished from HRR25 by one amino acid substitution.
  • an aspartic acid is substituted for glycine.
  • HRR25 contains the hallmark signatures of sequence homology with the catalytic domain of serine/threonine protein kinase superfamily members (Hanks, et al , Science, 241:42. 1988).
  • HRR25 translation product was aligned with the catalytic domains for two subgroups of yeast protein kinases, the CDC28/cdc2 group and the KSS1/FUS3 group.
  • Located between amino acids 15 and 30 is a region that contains the conserved GXGXXG region. Just C-terminal to this region is a conserved lysine and glutamic acid present in most known kinases.
  • Lys 38 in HRR25 corresponds to the lysine found in all known protein kinases, and this subdomain is involved in ATP binding. Mutations at the conserved lysine in protein kinases such as v-src, v-mos, and DBF2 inactivate these proteins. The mutant /zrr25-Lys 38 allele was incapable of complementing hrr25-l , hrr25::LUK, and hrr25A alleles for all properties examined, an indication that the HRR25 kinase domain is required for in vivo function of HRR25.
  • the predicted HRR25 translation product (SEQ. I.D. NO. 2) has a number of notable features outside the region of homology to protein kinase catalytic domains. For example, the last 100 amino acids is proline and glutamine rich, containing 50 of these residues. Other proteins with regions rich in these two amino acids include the transcription factors Spl, jun, and HAP2, steroid hormone receptors, the S. pombe rani kinase, and mak-m ⁇ & germ cell-associated kinase (Courey, et al , Cell, 55:887, 1988; Bohmann, et al , Science, 238: 1386, 1987; Roussou, et al, Mol.
  • the glutamine richness of this region is similar to the opa or M-repeat seen in the Drosophila and Xenopus Notch/Xotch proteins (Wharton, et al , Cell, 40:55, 1985; Coffman, et al, Science, 249: 1438, 1990).
  • the function of the opa repeat is not certain, but it is found in several Drosophila genes.
  • the sequence TKKQKY at the C-terminal end of the region homologous to protein kinases is similar to the nuclear localizing signal of SV40 large T antigen and yeast histone H2B (Silver, et al , J. Cell.Biol , 109:983, 1989; Moreland, et al , Mol. Cell.Biol , 7:4048, 1987).
  • Plating efficiency was measured from mid-log phase cells by comparing the efficiency of colony formation on rich medium relative to the total number of cells determined by hemocytometer count.
  • Cell populations were analyzed for DNA content distribution by flow cytometric analysis following staining with propidium iodide as described (Hutter, et al. J.Gen.Microbiol , 113:369, 1979).
  • Cell sorting analysis showed that a large number of the cells in a haploid hrr25::LUK population were delayed in the cell cycle and exhibited G2 DNA content, but the population was not arrested uniformly in the cell cycle.
  • CDC28 and KSSl represent members of two subgroups of serine/threonine protein kinases in yeast. CDC28 is involved in cell cycle regulation while KSSl acts in the regulation of the yeast mating pathway.
  • HRR25 shows 21 % identity and 41 % similarity to CDC28 and 19% identity and 43% similarity to KSSl ( Figure 1A). HRR25 shows highest similarity to members of the Ra ⁇ /PKS/Mos family of protein kinases. Through the catalytic domain, HRR25 shows 30% identity and 49% similarity to Rafl.
  • the clones were isolated by a two-pronged approach: i) DNA- based screening methods; and ii) direct complementation in S. cerevisiae hrr25 mutant strains. Two genes were identified (Hhpl + and Hhp2+ - so named for HRR25 Homologue from Schizosaccharomyces pombe). Expression of Hhpl + in S. cerevisiae hrr25 mutants fully rescued all mutant defects. Expression of Hhp2+ in S. cerevisiae also rescued, to varying degrees, the defects associated with hrr25 mutations.
  • Hhpl+ 1 clones were identified (6 partial and 1 full length clone).
  • Hhp2+ 2 full length clones were identified. Both Southern and Northern analysis confirmed that these clones were from separate genes. These genes were sequenced using standard dideoxy method (Maniatis, et al , supra).
  • the nucleotide and deduced amino acid sequences for Hhpl + are set out in SEQ. ID. NOS. 3 and 4; the nucleotide and deduced amino acid sequences for Hhp2+ are set out in SEQ. ID. NOS. 5 and 6.
  • ADH1 promoter in a URA3-based vector pDB20 to allow for expression in S. cerevisiae (Fikes, et al , supra). These resulting clones were analyzed for their ability to alter/modify the suppress phenotypes associated with the hrr25-l mutation and the hrr25t. mutation following transformation into appropriate yeast strains by standard methods (Ito, et al , J. Bacteriol 153: 163, 1983). Transformants were analyzed for their ability to overcome defects associated with the hrr25 mutations (Hoekstra, et al , Science, 253: 1031, 1991).
  • Hhpl -Y expression fully complemented /zrr25-associated defects and was indistinguishable from wild type HRR25 in all analyses. Complementation was analyzed for the effect on DNA repair, cell cycle progression, cellular morphology, and sporulation. Hhp2+ complemented to a lesser degree than Hhpl + (its complementation level was 50% -75% that of bonafide HRR25). The alteration of zrr25-associated phenotypes was dependent upon the transformed yeast strains containing both a complementing Sc. pombe Hhp plasmid and having hrr25 mutations.
  • the degree of amino acid homology between HRR25 protein and Hhpl + protein is 73% through the kinase domain.
  • the degree of similarity which considers the presence of similar as well as identical amino acids, is greater than 85 % .
  • the amino acid identity of HRR25 protein and Hhp 2 + protein is 63 % with a percent similarity score of 80% .
  • the intraspecies comparison of Hhpl + protein to Hhp2+ protein is 72% identity. This structural and complementation analysis clearly indicates that these Sc. pombe clones are functional homologues of the S. cerevisiae HRR25. Such a high degree of relatedness is not seen with any other group of protein kinases.
  • bonafide functional homologues i.e., cdc2 protein kinases from S. cerevisiae, Sc. pombe, and humans
  • cdc2 protein kinases from S. cerevisiae, Sc. pombe, and humans
  • Mutations that inactivate or reduce the protein kinase activity of HRR25 in S. cerevisiae result in a wide variety of phenotypes including: sensitivity to various forms of DNA damage, severe cell cycle delay, sensitivity to drugs that affect cell cycle progression (e.g., caffeine), sensitivity to agents that affect microtubule integrity (e.g., benomyl), and sensitivity to agents that affect the integrity of replicating DNA (e.g., hydroxyurea).
  • pombe vector pHSS19 Hoekstra et al , Meth. Enzymol , 194:329. 1991
  • the Sc. pombe URA4 gene was inserted resulting in deletion of the Hhpl + kinase domain.
  • Sc. pombe was transformed by standard methods (Moreno, et al , Meth. Enzymol , 194:795. 1991) with the linearized DNA from the resulting plasmid construction. Stable transformants were identified and haploid hhplh. strains were verified by standard methods (Moreno, et al , Maniatis, et al).
  • the Hhp2+ gene was disrupted as follows: the Hhp2+ CDNA was cloned into the Sc. pombe based vector, plasmid pHSS19, and was disrupted by transposon shuttle mutagenesis using the mini-Tn3 transposon mTn3Leu2 (Hoekstra, et al , Meth. Enzymol supra.). Sc. pombe was transformed by standard methods with the linearized DNA from the resulting plasmid construction. Stable transformants were identified and haploid hhp2t. strains were verified by standard methods (see above). Standard physiological methods as described for S. cerevisiae
  • HRR25 (Hoekstra, et al , Science 253:1031, 1991) were employed to characterize hhp mutant strains. Phenotypic analysis revealed that both hhpl and hhp2 mutants showed defects previously seen in hrr25 mutants, including sensitivity to various DNA damaging treatments that include MMS treatment and X-ray treatment. The foregoing substantiates that Hhpl + and Hhp2+ are isoforms of S. cerevisiae HRR25 protein kinase. These three protein kinases show high levels of sequence identity. In addition, mutations that inactivate these kinases result in very similar defects in widely divergent organisms.
  • Sc. pombe hhp mutants prepared as described above were identical to S. cerevisiae hrr25 mutants and to show that HRR25-like protein kinases with greater than 35 % amino acid identity are functional homologues
  • the S. cerevisiae HRR25 gene was introduced into a Sc. pombe expression vector and transformed into Sc. pombe hhp mutants.
  • the DNA sequence at the HRR25 initiating methionine was changed into an Ndel site, (a silent coding alteration that maintains the reading frame but allows the HRR25 gene to be introduced into appropriate Sc. pombe plasmids). This was done by a site-directed DNA change was made in the S.
  • HRR25 gene by standard methods using a commercially available system (Bio-Rad, Cambridge, MA).
  • the altered HRR25 gene was ligated into the Sc. pombe expression plasmid, pREP 1 (Maundrell, K. J., Biol. Chem. 265: 10857, 1990), at an Ndel site and the resulting construction was transformed by standard methods into Sc. pombe hhp mutants.
  • Expression of HRR25 in Sc. pombe mutant strains resulted in complementation of the mutant defects as evaluated by physiological methods described by Hoekstra, et al. (Science, supra).
  • NUFl for Number Four.
  • the amplified products corresponding to NUFl were radiolabelled as described in Example 6 and used to screen a yeast YCp50- based genomic library (ATCC, Rockville, MD). Eight clones were identified and one of these clones included approximately 4 Kb Hindlll fragment containing the NUFl hybridizing gene.
  • NUFl is a separate gene from HRR2J, YCK1/CKI2, and YCK2/CKI1.
  • the Hindlll fragment was sequenced and revealed a protein kinase with about 65% identity to HRR25 through its protein kinase domain.
  • the DNA and deduced amino acid sequences for NUFl are set out in SEQ. ID. NOS. 23 and 24.
  • the Hindlll fragment was subcloned into the yeast plasmid YEplacll2 [Gietz and Sugino, Gene 74:521-541 (1988)].
  • the resulting construct was transformed into the hrr25 ⁇ deletion strain 7d and NUFl was found to complement for 1__T25 ⁇ mitotic defects (e.g., NUFl complemented for slow growth defect, aberrant morphology defect, DNA damaging agent sensitivities). Furthermore, a null mutant allele of NUFl was constructed by transposon shuttle mutagenesis and strains lacking the NUFl gene product were found to have hrr25 ⁇ mutant-like defects. In particular, like hrr25 ⁇ mutants, NUFl mutants showed slower mitotic growth rates and increased sensitivity to DNA damaging agents like MMS, UV, and X-irradiation.
  • 1__T25 ⁇ mitotic defects e.g., NUFl complemented for slow growth defect, aberrant morphology defect, DNA damaging agent sensitivities.
  • a null mutant allele of NUFl was constructed by transposon shuttle mutagenesis and strains lacking the NUFl gene product were found to have hrr25 ⁇ mutant-like defects.
  • cDNAS Human HRR25-like Genes Oligonucleotides derived from amino acid sequences described above in Example 6 A were used to amplify cDNAS from the following sources: Arabidopsis thaliana, Drosophila melanogaster, Xenopus, chicken, mouse, rat, and human ⁇ eLa cells. These cDNAS were obtained from reverse transcribed mRNA (Maniatis, et al , supra) or from commercially-available cDNA libraries (Stratagene, La Jolla, CA, and Clonetech, Palo Alto, CA) Amplification products of similar migration size to those obtained from S. cerevisiae HRR25 and Sc. pombe, Hhpl - and Hhp2+ genes were observed in 1.0% Agarose gels (Maniatis, et al. , supra). This result indicated that HRR25-like genes exist in all species examined.
  • Primer JH21 SEQ. ID. NO. 17 representing bovine top strand DNA bases 47-67;
  • Primer JH29 (SEQ. ID. NO. 19) representing bovine top strand DNA bases 604-623;
  • Primer JH30 (SEQ. ID. NO. 20) representing bovine top strand DNA bases 623-604;
  • Primer JH31 (SEQ. ID. NO. 21) representing bovine top strand DNA bases 835-817.
  • DNA amplification with combination of oligonucleotides JH21/JH30, JH22/JH30, and JH29/JH31 were carried out for 30 cycles with denaturation performed at 94 °C for 4 min for the first cycle and for 1 min for the remaining cycle annealing at 50°C for 2 min and extension at 72°C for 4 min.
  • Products of the expected size from the three amplifications were purified on preparative acrylamide gels and labeled with 32 P using random nick translation (to a specific activity between 7 x 10 6 cpm/ ⁇ g and 1.4 x 10 7 cpm/ ⁇ g.
  • the labelled probes were employed as a group to screen a commercial human fetal brain cDNA library (Stratagene).
  • Hybridization was carried out for 16 hours at 65°C in a hybridization buffer containing 3 x SSC, 0.1 % Sarkosyl, 10 x Denhart's solution and 20 mM sodium phosphate (Ph 6.8). Three washes at 65 °C in 2 x SSC, 0.1 % SDS were performed. Approximately 1.5 x 10 6 plaques were screened on 30 plates using duplicate filters. Six strong positive clones were isolated, purified and converted to plasmid form according to procedures recommended by the supplier of the library.
  • Clones 42A1, 51A1, and 47 A 1 were redesignated as CKI ⁇ lHu, CKI ⁇ 2Hu, and CKI ⁇ Hu.
  • the DNA and deduced amino acid sequences of the inserts are set out in SEQ. ID. NOS. 7 and 8; 9 and 10; and 11 and 12, respectively.
  • the deduced amino acid sequence for CKI ⁇ lHu was identical to the reported bovine CKI ⁇ sequence. Table 1, below sets out differences in nucleotides between the bovine and human DNAs, numbered from the first base in the initiation codon, ATG.
  • the CKI ⁇ 3Hu DNA also includes an insertion of 84 bases at position +454 in the coding sequence providing an intermediate extension of the CKI ⁇ 2Hu expression product by 28 amino acids.
  • This DNA insert is not present in the bovine gene, but it encodes an amino acid sequence insert which Rowles, et al. designated as CKI-alpha-L.
  • the last two codons of the CKI ⁇ Hu sequences differ from any of the bovine sequences or the sequences of CKI ⁇ lHu and CKI ⁇ 2Hu, causing the CKI ⁇ 3Hu expression product to terminate with a lysine, rather than a phenylalanine as found in all the other bovine and human casein kinase I sequences.
  • the 3' flanking sequence of CKI ⁇ Hu DNA differs significantly from that of CKI ⁇ lHu and CKI ⁇ 2Hu.
  • FIGURE 2 provides an alignment of the catalytic domain amino acid sequences of HRR25-like proteins whose DNAs were isolated in the above illustrative examples, including HRR25, Hhpl + , Hhp2 + , CKI ⁇ l ⁇ u, CKI ⁇ *2 ⁇ u, and CKI ⁇ 3Hu as well as YCK1/CKI2, and YCK2/CKI1. Note that with the exception of the CKI ⁇ 3Hu intermediate insert and the carboxy terminal region inserts of CKI ⁇ 2Hu and CKI ⁇ 3Hu, the sequences of the three human products are identical. "Common" residues are indicated in the Figure where at least 3 of the seven residues are identical at the corresponding position (the human sequences being taken as a single sequence).
  • the three human HRR25-like protein kinases showed very high degrees of amino acid identity to the HRR25 gene product (68%), establishing that these human clones were enzymatic isoforms of the yeast HRR25 gene.
  • the alignment of HRR25, Hhpl+, Hhp2+, and the human complementing-like kinase isoforms show that these enzymes share a number of primary structural features that indicate that these enzymes provide comparable activities in different species. This conclusion is reached based on several lines of evidence. First, all enzymes share the common primary sequence identifiers characteristic of protein kinases.
  • the enzymes share high degrees of amino acid identity in regions of the protein kinase domain that are not conserved in unrelated protein kinases.
  • these enzymes share regions of identity in the kinase domain which regions differ in primary sequence from other protein kinases, but are identical among the members of this isoform grouping. For example, greater than 95 % of all known protein kinases have a so-called A-P- E sequence (Alanine-Proline-Glutamate) approximately two-thirds of the way through the kinase domain.
  • A-P- E sequence Alignine-Proline-Glutamate
  • HRZ?25-like protein kinases lack the A-P-E sequence and have instead a S-I/V-N sequence (Serine-Isoleucine or Valine-Asparagine). Based on this primary sequence comparison, between known protein kinases and the protein kinases of the invention from evolutionarily divergent organisms, these enzymes of the invention are isoforms
  • HRR25 gene product might function as a casein kinase
  • HR/?2J-containing immunoprecipitates from yeast were incubated with casein and phosphorylated proteins were examined.
  • Yeast extracts were prepared by physical disruption. Equal volumes of a cells were suspended in lysis buffer and acid-washed 0.5 mm beads were mixed, 30 second bursts were interspersed with 1 min on ice, and the extent of disruption was followed microscopically.
  • Lysis buffer contained 10 Mm sodium phosphate (Ph 7.2), 150 Mm NaCl, 1% Nonidet P-40, 1 % Trasylol, 1 Mm DTT, 1 Mm benzamidine, 1 Mm phenylmethyl sulfonyl fluoride, 5 Mm EDTA, pepstatin (1 ug/ml), Pepstatin A (2 ug/ml), leupeptin (1 ug/ml), lOOmM sodium vanadate, and 50 Mm NaF. Extracts were clarified by a 100,000 x g centrifugation for 30 min. , made to 50% (vol/vol) with glycerol, frozen in liquid nitrogen, and stored at -70 degrees C. Little loss in protein kinase activity was seen in frozen extracts over several months.
  • Immune complex protein kinase assays were performed on the extracts according to the methods described in Lindberg, et al. (Mol. Cell. Biol 10:6316, 1991). Frozen extracts were diluted to 25% glycerol with lysis buffer or fresh extracts were used directly. Extracts were precleared with preimmune serum and protein A-Sepharose, and then treated with immune serum (obtained as described in Example 11, infra, from immunization of rabbits with E. coli- derived type-HRR2J fusion products). HRR25 kinase-containing immune complexes were precipitated with protein A-Sepharose. Immune complexes were washed four times with lysis buffer and twice with kinase buffer containing 15 Mm ⁇ epes (Ph 7.4), 100 Mm NaCl, and 10 Mm MgCl 2
  • Reaction mixtures of HRR25 immunoprecipitates and heat-treated casein were incubated at 30 degrees C for 5-10 min and contained 10 uCi of gamma- 32 P-ATP per 20 ul reaction volume. Reactions were stopped by the addition of SDS and EDTA, boiled in SDS/PAGE sample buffer and resolved in 10 % gels. Phosphoamino acid analysis was as described (Hunter et al , Proc. Natl.Acad. Sci. USA 77:1311, 1980).
  • HRR25 immunoprecipitates from yeast could phosphorylate casein several experiments were performed. HRR25 immunoprecipitated from E. coli strains expressing HRR25 (See Example 11) also showed casein kinase activity, whereas E. coli extracts lacking HRR25 protein did not phosphorylate casein. HRR25-containing baculovirus constructs produced casein kinase activity in immunoprecipitates. Wild-type baculovirus-infected cells showed (0.5 % casein kinase activity under comparable conditions. The protein kinase activity from S19 cells expressing HRR25 protein was sensitive to the same conditions that reduced or inactivated the HRR25 protein activity from yeast extracts.
  • HRR25- ⁇ ike Proteins Because the predominant protein kinase activity in E. coli is histidine kinase, rather than serine/threonine or tyrosine kinase, those procaryotic cells provide a system for examination of HRR25-like protein kinase activities which is not compromised by presence of endogenous kinases. Both HRR25 and ⁇ hpl + DNAs were, therefore, expressed in the IPTG-inducible T7 gene 10-based commercial expression system (Invitrogen, San Diego, CA) using E. coli strain BL21 (DE3) which contains an IPTG-inducible T7 RNA polymerase and T7 lysozyme gene.
  • HRR25 and Hhpl-Y expressing cells contained novel tyrosine phosphorylated proteins not observed in control cells (transformed with the vector alone or with kinase inactive mutants).
  • HRR25 and Hhpl-Y -containing E. coli strains were examined for tyrosine- phosphorylated protein by a sensitive and accurate radiolabelling and phosphoamino acid procedure.
  • cells were induced with IPTG and grown in the presence of 32 P-orthophosphate. Radiolabelled extracts were prepared by the freeze-thaw method, electrophoresed in polyacrylamide gels, and the gels were examined by autoradiographic methods.
  • Novel phosphoproteins were observed in the strains expressing HRR25 and Hhpl + , but not in the above controls. Phosphoproteins were examined by extracting and hydrolyzing the proteins from the gels using standard methods (Boyle, et al , Meth. Enzymol, 201: 110, 1991). These experiments verified that HRR25 and Hhpl+ could phosphorylate tyrosine, serine, and threonine residues on protein substrates.
  • the first plasmid construction involved plasmid pATH according to Koerner et al , Meth. Enzymol. , 194:477-491 (1991).
  • An approximately [606] base pair DNA fragment was isolated from the HRR25 open reading frame by Bgl II digestion and this fragment (which encodes amino acid residues 275-476) was ligated into pATH which had been digested with BamBI.
  • the resulting plasmid encoded a fusion protein comprising the E. coli TrpE gene product at its amino terminus and a carboxy terminal fragment of HRR25 at its carboxyl terminus.
  • Inclusion bodies were isolated from E. coli DH5 ⁇ (Bethesda
  • HRR25-like protein kinase encoding DNAs were isolated by combined DNA amplification and library screening methods. Oligonucleotides based on conserved regions in HRR25-like protein kinases were used to amplify DNA segments for use as probes in screening human a cDNA library. Redundant oligonucleotides of the sequence
  • Amplification conditions used 200 Mm Tris.Hcl (Ph 8.2), 100 Mm KC1, 60 Mm (NH4)2SO4, 15 Mm MgC12, 1 % Triton X-100, 0.5 ⁇ M of each primer, 100 ng library DNA template, 200 ⁇ M dNTPs and 2.5 U polymerase. The reactions were performed for 30 cycles.
  • Reactions were started with a 4 minute treatment at 94 °C and all cycles were 1 minute at 94 °C, 2 minutes at 5°C for annealing, and 4 minutes at 72 °C for extension.
  • the amplification reaction was electrophoresed through a 1 % agarose gel and the region corresponding to approximately 540 base pairs was excised and DNA was eluted using a Nal extraction and glass powder binding (GeneClean, BiolOl, La Jolla, CA).
  • the gel-purified fragment was ligated into Smal-digested Bluescript II SK(+) and the resulting plasmid contained a partial protein kinase domain that was used as a source of cDNA for library screening.
  • RNA was prepared from 2.8 x 10 8 cells of the B-cell lymphoma Manca using the "Fast Track" kit (Invitrogen). 5 ⁇ g of RNA was used for oligo d(T) primed cDNA synthesis with the cDNA Synthesis System (Gibco BRL, Burlington, Ontario, Canada); the resulting cDNA was size selected by agarose gel electrophoresis and ligated to EcoRI adapters with the Ribo Clone kit (Promega, Madison, WI).
  • Varying amounts of the adapted cDNA were ligated to Ec ⁇ RI-digested ⁇ gtlO with 1 unit of T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN) in a commercially prepared buffer supplied by the manufacturer with the enzyme.
  • the ligations were packaged with Gigapack packaging extracts (Stratagene) and the resulting phage pool (1.5 x 10 6 phage) was amplified in the C600 Hfi strain.
  • a total of 1 x 10 6 phage plaques were screened by standard hybridization methods (Maniatis, et al , supra).
  • Hybridizations were at 65°C for 18 hours in 6X SSPE (20X SSPE is 175.3 g/1 NaCl, 27.6 g/1 NaH 2 PO 4 .H 2 O), 7.4 g/1 EDTA, pH 7.4), 100 ⁇ g/ml salmon sperm carrier DNA, 5X Denhardt Reagent (50X Denhardts is 5% ficoll, 5 % polyvinyl pyrolidone, 5% bovine serum albumin), 0.1 % SDS and 5% sodium dextran sulfate. Filters were washed four times in 0.1X SSPE, 1 % SDS. Each wash was at 65 °C for 30 minutes. Five clones were chosen for further analysis.
  • DNA from these phage clones was prepared using a Qiagen lambda DNA preparation kit (Qiagen, Chatsworth, CA) and human cDNA inserts were excised by Ec ⁇ RI digestion. These inserts were subcloned into EcoRI-digested plasmid Bluescript II SK(+) (Stratagene) and the inserts were sequenced using an ABI 373A automated DNA sequencer. Two of the five cDNA contained near full-length cDNAS with a polyA tail and a protein kinase open reading frame. These protein kinases were most closely related to isoforms of casein kinase I were designated CKI7IHU and CKI 2HU.
  • Human CKI ⁇ was subcloned by first isolating the human gene from a human fetal brain library constructed in ⁇ ZAPII (Stratagene). A 2.2 kb Ec ⁇ RI fragment containing rat CKI ⁇ was gel purified through 1 % agarose, isolated from the gel by Nal extraction with glass powder (BiolOl, La Jolla, CA), and radiolabelled by random primer methods (Boehringer Mannheim) using 32 P-dCTP.
  • This probe was used to screen 1 X 10 6 plaques containing human fetal brain cDNA library. Plaque hybridization conditions were 3X SSC, 0.1% Sarkosyl, 10X Denhardts reagent, 50 ⁇ g/ml salmon sperm DNA carrier. Hybridization was allowed to proceed for 18 hours at 65 °C after which time the filters were washed
  • CKI ⁇ Hu One clone was determined to encode a full length CKI ⁇ isoform and was designated CKI ⁇ Hu.
  • the nucleotide sequence for CKI ⁇ Hu is set out in SEQ ID NO: 1
  • CKI ⁇ Hu mRNA levels were highest in kidney, liver and placenta cells, in contrast to the testes-specific expression of rat CKI ⁇ demonstrated by Graves, et al, [supra].
  • CKI7IHU was an isoform of yeast HRR25- like protein
  • the gene was expressed in yeast protein kinase mutants.
  • the cDNA was expressed under control of the yeast GALl promoter.
  • the expression plasmid was a derivative of plasmid pRS305 (Stratagene) that contains the yeast GALl promoter.
  • the parental plasmid with the GALl promoter was previously described [Davis et al , Cell 57:965-978 (1990)] and contained a Bglll site adjacent to the GALl promoter as well as Bam ⁇ l and S ⁇ cl sites adjacent to the BgRl site.
  • This plasmid was modified by site-directed mutagenesis to contain a unique Nc ⁇ l site between the GALl promoter and the BgRl site.
  • the Nc ⁇ l site was adjacent to the GALl promoter such that the order of genetic elements was GALl promoter-Nc ⁇ I-_3g ⁇ i-j3flmHI-S ⁇ cI.
  • Site-directed mutagenesis (MutaGene kit, BioRad) employed the oligonucleotide
  • the CKI7IHU cDNA was amplified from cDNA with oligonucleotides that would introduce an Ncol site at the initiating ATG and a BamHl site in the 3' untranslated region.
  • the sequence of the mutagenic oligonucleotide (with the Ncol site underlined) for the amino terminus was
  • the oligonucleotide M13rev purchased from Stratagene (Stratagene, La Jolla, CA) was used to introduce the BamRl site in the 3' untranslated region.
  • Amplification conditions used 200 Mm Tris-HCl (Ph 8.2), 100 Mm KC1, 60 mM (NH 4 ) 2 SO 4 , 15 mM MgCl 2 , 1 % Triton X-100, 0.5 ⁇ M of each primer, 100 ng template, 200 ⁇ M of each dNTP and 2.5 units polymerase. The reactions were performed for 30 cycles.
  • yeast CKI mutants employed yeast strains 7D (hrr 25 A, ura3-l, trpl-1, leu2-3, 112, his3-ll,15, canl-100, ade2-l) [DeMaggio, et al , (1992) supra] and YI227 (ckilD, cki2D, FOA R , ade2-l, canl- 100, his3-ll,15, leu2-3,12, trpl-1, ura3-l, pRS415::Ckilts)
  • Strain 7D l acked the HRR25 isoform of yeast CKI and strain YI227 contained a temperature sensitive allele of yeast CKII .
  • Plasmid pRS315::HRR25 was constructed by ligating a SaR/Ec ⁇ KL genomic fragment of HRR25 into S ⁇ /I/£coRI-digested pRS315.
  • Both HRR25 and CKJ ylHu when expressed in yeast mutants, are capable of fully complementing for the temperature-sensitive growth defect of CKI.
  • CKI7IHU partially suppressed a severe growth rate defect associated with HRR25 mutants.
  • the partial suppression of HRR25 growth defects by CKI7IHU was detected by a 10-20 fold greater plating efficiency relative to pRS305(N) 2 ⁇ GALl.
  • Human CKIcdHu was subcloned into plasmid pRS305(N) 2 ⁇ GALl by first introducing an Ncol site at the initiating methionine by site-directed mutagenesis.
  • the mutagenic oligonucleotide (with the Ncol site underlined) was
  • the mutagenized cDNA was digested with Ncol and BgRl and the CKIc Hu fragment was ligated into pRS305(n) 2 ⁇ GALl.
  • Plasmid pEC7B (containing CKI ⁇ Hu cD ⁇ A) was used as a template for site-directed mutagenesis (MutaGene, BioRad).
  • One plasmid construction employed _3gZII/StzcI-digested CKI DNA from the mutagenized cDNA that was ligated into 5g/II/S cI-digested pRS305(N) 2 ⁇ GALl to produce pRS305(CKI ⁇ ).
  • the second plasmid construct employed NcoI/S ⁇ cI-digested CKI ⁇ Hu cD ⁇ A from unmutagenized pEC7B cD ⁇ A that was ligated into NcoI/S ⁇ cI-digested pRS305( ⁇ ) 2 ⁇ GALl to produce pRS305(N)(CKI ⁇ ).
  • Plasmid pRS305(CKI ⁇ ) contained the nucleotides
  • Plasmid pRS305(N)(CKI ⁇ ) had a near-perfect fusion between the initiating methionine of CKI ⁇ Hu and the 3' end of GALl . Near perfect fusion indicates that the promoter and initiating methionine codon have few or no intervening nucleic acid sequences, and therefore are approximately abutting.
  • CKI ⁇ lHu and CKI ⁇ Hu-containing plasmids were transformed into yeast strains 7D and YI227 and were examined for their ability to complement for their mutant defects.
  • CKI ⁇ lHu partially complemented the growth defect associated with HRR25 mutations.
  • CKI ⁇ Hu was able to complement for the growth defect of temperature-conditional CKI strains, for the growth defect of HRR25 mutants, and for the DNA repair defect of HRR25.
  • the ability of CKI ⁇ Hu to complement for mutant defects in these yeast strains was indistinguishable from yeast HRR25 or CKI genes only when the appropriate plasmid construct was employed.
  • Plasmid pRS305(CKI ⁇ ), which contained the additional 21 bases was unable to complement for any mutant phenotypes, while the near-perfect fusion in pRS305(N)(CKI ⁇ ) was fully functional. This difference was attributed to the inability of yeast to translate extended and/or CG rich leader sequences.
  • SEQ ID NO: 41 was derived from the common amino terminus of CKI ⁇ lHu, CKI ⁇ 2Hu, and CKI ⁇ 3Hu
  • SEQ ID NO: 42 was derived from an internal alternative splice region in CKI ⁇ 3Hu.
  • peptides were initially each coupled to bovine gamma globulin (Sigma, St Louis, MO). Five mg of gamma globulin and 5 mg of peptide were resuspended in 0.4 ml 100 mM K 2 HPO 4 (pH 7.2) and to this mixture, 35 mg l-ethyl-3(3- dimethylamino propyl)-carbodiimide-HCl (EDC, Pierce) previously dissolved in 50 ⁇ l K 2 HPO 4 (pH 7.2) was added. The reaction was allowed to proceed for 16 hr at 4°C and was quenched by addition of 0.25 ml 2 M ethanolamine and 0.25 ml acetic acid.
  • the reaction mixture was then diluted to a final volume of 2.5 ml with PBS and desalted using Sephadex G-25M (Pharmacia) chromatography. Protein containing fractions were concentrated by centrifugal microconcentration (Amicon). Mice were then injected with 50 ⁇ g of the coupled peptide nine times over a period of 8 months. Antibody production was measured against the respective peptides by ELISA.
  • Fusions were performed by standard methods. Briefly, a single-cell suspension was formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640 media, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (RPMI) (Gibco). The cell suspension was filtered through a sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, NJ), and washed twice by centrifuging at 200 g for 5 minutes and the pellet resuspended in 20 ml serum free RPMI.
  • RPMI 1640 media supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (RPMI) (Gibco).
  • RPMI streptomycin
  • Thymocytes taken from 3 naive Balb/c mice were prepared in a similar manner.
  • NS-1 myeloma cells kept in log phase in RPMI with 11 % fetal bovine serum (FBS) (Hyclone, Laboratories, Inc., Logan,Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed twice as described in the foregoing paragraph. After washing, each cell suspension was brought to a final volume of 10 ml in serum free RPMI, and 10 ⁇ l was diluted 1:100.
  • FBS fetal bovine serum
  • the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 ⁇ M sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Mallinckrodt, St. Louis, MO) and 1.5 x 10 6 thymocytes/ml.
  • the suspension was dispensed into ten 96-well flat bottom tissue culture plates (Corning, Essex, United Kingdom) at 200 ⁇ l/well.
  • Cells in the plates were fed 2-3 times between fusing and screening by aspirating approximately half of the medium from each well with an 18 gauge needle (Becton Dickinson), and replenishing plating medium described above except containing 10 units/ ml IL-6 and lacking thymocytes.
  • Fusions were screened when cell growth reached 60-80% confluency (usually 7-9 days). Fusion 75 was screened by ELISA on either the common amino terminal peptide (SEQ ID NO: 41) or the internal peptide (SEQ ID NO: 42), and fusion 80 was screened on the amino terminal peptide (SEQ ID NO: 41) only. Immulon 4 plates (Dynatech, Cambridge, MA) were coated at 4°C overnight with 100 ng/well peptide in 50 mM carbonate buffer, Ph 9.6. Plates were washed three times with PBS containing 0.05 % Tween 20 (PBST) and 50 ⁇ l culture supernatant was added.
  • PBST 0.05 % Tween 20
  • Clones 75D3G, 75C10H, 75C2G, and 80G10H were isotyped to be IgGl, clone 80H4F IgG3, and 80J9E IgG2a.
  • Expression plasmids were constructed in order to express the CKIHu isoforms as fusion proteins with thioredoxin. Specifically, the coding sequence for each isoform was amplified by PCR with primers which created a 5 'Xbal restriction site and a 3 'BamHl site. The primer used to create the Xbal site for the CKI ⁇ Hu isoforms is set out in SEQ ID NO: 43 with the Xbal site underlined.
  • the Xbal and B ⁇ mHI sites were created in the CKI ⁇ Hu coding sequences with primers set out in SEQ ID NOS: 46 and 47, respectively.
  • the primers used to create the Xbal and JS ⁇ mHI sites in the coding regions of the CKI7HU isoforms are set out in SEQ ID NO: 48 and 49.
  • E.coli XL-1 Blue cells (Stratagene) were transformed with the individual expression plasmids by standard methods and grown at 37 °C to mid-log phase. Samples were collected to serve as controls for uninduced cells and the remaining cells were induced for four hours with 0.25 mM IPTG at 37°C. Cells were then lysed and inclusion bodies in the insoluble extract from cleared lysate were used to inject mice.
  • mice were injected with 50 ⁇ g of the peptide/gamma globulin complex on a varying schedule over a period of eight months.
  • Robinson, et al. Proc. Natl. Acad. Sci. USA, 89:28-32, 1992
  • YCKl and YCK2 which encode yeast casein kinase 1 homologues and also describes purification and partial sequencing of a rabbit casein kinase I from a rabbit reticulocyte lysate preparation.
  • HRR25 was noted to be 50% homologous to YCKl and YCK2 and 60% homologous to the partial rabbit CKI sequence.
  • Wang, et al. (Molecular Biology of the Cell, 3:275-286, 1992) describes the isolation of a 54 kDa CKI from S.
  • Example 7 employing information concerning the DNA sequence of HRR25, the procedures of Example 7 allowed the isolation partial cDNA clones of expected length from cDNA libraries derived from Arabidopsis thaliana, Drosophila melanogaster, Xenopus, chicken, mouse, rat, and human species. These partial cDNAs may, in turn, be employed in the manner of Examples 6 and 7 to isolate full length DNA clones encoding H_?R25-like proteins from these species. Each of these may be employed in the large scale production of the corresponding proteins by recombinant methods or for the generation of other useful polynucleotides such as antisense RNAs.
  • Recombinant expression products of such HRR25-like DNAs may be employed for generation of antibodies and in screens for compounds which modulate the protein kinase and/or recombination/repair functions of these enzymes.
  • multiple H7y_25-like isozymes are expected to exist in a variety of eukaryotic species as both membrane bound and cytoplasmic proteins. It appears reasonable to expect that a number of genes and gene products exist in human species, all of which are functionally related as well as structurally related to each other and to HRR25.
  • SEQ ID NO: 1 is the nucleic acid sequence and the deduced amino acid of a genomic fragment encoding a yeast-derived protein kinase, HRR25 of the present invention.
  • SEQ ID NO: 2 is the deduced amino acid sequence of a yeast- derived protein kinase HRR25 of the present invention.
  • SEQ ID NO: 3 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding Hhpl + of the present invention.
  • SEQ ID NO: 4 is the deduced amino acid sequence of Hhpl + of the present invention.
  • SEQ ID NO: 5 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding Hhp2+ of the present invention.
  • SEQ ID NO: 6 is the deduced amino acid sequence of Hhp2+ of the present invention.
  • SEQ ID NO: 7 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKl ⁇ lHu of the present invention.
  • SEQ ID NO: 8 is the deduced amino acid sequence of CKI ⁇ lHu of the present invention.
  • SEQ ID NO: 9 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKl ⁇ 2Hu of the present invention.
  • SEQ ID NO: 10 is the deduced amino acid sequence of CKl ⁇ 2Hu of the present invention.
  • SEQ ID NO: 11 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKl 3Hu of the present invention.
  • SEQ ID NO: 12 is the deduced amino acid sequence of CKl ⁇ 3Hu of the present invention.
  • SEQ ID NO: 13 is the primer, 4583, representing top strand DNA encoding residues 16-23 of HRR25.
  • SEQ ID NO: 14 is the primer, 4582, representing top strand DNA encoding residues 126-133 of HRR25.
  • SEQ ID NO: 15 is the primer, 4589, representing bottom strand DNA encoding residues 126-133 of HRR25.
  • SEQ ID NO: 16 is the primer, 4590, representing bottom strand DNA encoding residues 194-199 of HRR25.
  • SEQ ID NO: 17 is the primer JH21 , representing bovine top strand DNA bases 47-67.
  • SEQ ID NO: 18 is the primer JH22, representing bovine top strand DNA bases 223-240.
  • SEQ ID NO: 19 is the primer JH29, representing bovine top strand
  • SEQ ID NO: 20 is the primer JH30, representing bovine bottom strand DNA bases 623-604.
  • SEQ ID NO: 21 is the primer JH31, representing bovine bottom strand DNA bases 835-817.
  • SEQ ID NO: 22 is the mutated HRR25 kinase domain primer found on p. 33, Example 3.
  • SEQ ID NO: 23 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding NUFl of the present invention.
  • SEQ ID NO: 24 is the deduced amino acid sequence of NUFl of the present invention.
  • SEQ ID NOS: 25, 26 and 27 are the conserved motifs found on page 18.
  • SEQ ID NOS: 28 and 29 are redundant oligonucleotides, based on conserved regions of HRR25-like proteins, used to amplify a probe from a human cDNA library.
  • SEQ ID NO: 30 is the nucleotide sequence of the CKI7IHU gene.
  • SEQ ID NO: 31 is the deduced amino acid sequence of the CKI7IHU protein.
  • SEQ ID NO: 32 is the nucleotide sequence of the CKI72HU gene.
  • SEQ ID NO: 33 is the deduced amino acid sequence of the CKI72HU protein.
  • SEQ ID NO: 34 is the nucleic acid sequence for CKI ⁇ Hu.
  • SEQ ID NO: 35 is the deduced amino acid sequence for CKI ⁇ Hu.
  • SEQ ID NO: 36 is the mutagenic oligonucleotide used to generate an Ncol restriction site in expression plasmid pRS305.
  • SEQ ID NO: 37 is the mutagenic oligonucleotide used to generate an Ncol restriction site in CKI7I.
  • SEQ ID NO: 38 is the mutagenic oligonucleotide used to create an Ncol * restriction site in human CKI ⁇ a.
  • SEQ ID NO: 39 is the mutagenic oligonucleotide used to introduce a BgRl restriction site in CKI ⁇ .
  • SEQ ID NO: 40 is the intervening nucleic acids sequence between the GALl promoter and initiating methionine codon in the CKI ⁇ expression plasmid.
  • SEQ ID NOS: 41 and 42 are amino terminal and internal peptide fragments of CKI ⁇ isoforms to generate monoclonal antibodies.
  • SEQ ID NO: 43 is the primer used to create a Xbal restriction site in CKI ⁇ Hu coding sequences.
  • SEQ ID NO: 44 is the primer used to create a BamHl restriction site in the CKI lHu coding sequence.
  • SEQ ID NO: 45 is the primer used to create a BamHl restriction site in the CKI ⁇ 2Hu and CKI ⁇ 3Hu coding sequences.
  • SEQ ID NO: 46 is the primer used to create a Xbal restriction site in the CKI ⁇ Hu coding sequence.
  • SEQ ID NO: 47 is the primer used to create a BamHl restriction site in the CKI ⁇ Hu coding sequence.
  • SEQ ID NO: 48 is the primer used to create a Xbal restriction site in the CKI7IHU and CKI coding sequences.
  • SEQ ID NO: 49 is the primer used to create a BamHl restriction site in the CKI7IHU and CKI 2Hu coding sequences.
  • SEQ ID NO: 50 is an amino terminal peptide fragment of CKI7HU coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 51 is an amino terminal peptide fragment of CKI7HU coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 52 is an amino terminal peptide fragment of bovine CKI/3 coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 53 is an amino terminal peptide fragment of bovine
  • SEQ ID NO: 54 is an amino terminal peptide fragment of CKI ⁇ Hu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 55 is a carboxy terminal peptide fragment of CKI ⁇ Hu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 56 is an carboxy terminal peptide fragment of CKI ⁇ 2Hu and CKI ⁇ 3Hu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • SEQ ID NO: 57 is an internal terminal peptide fragment common to all human CKI isoforms coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.
  • MOLECULE TYPE DNA (genomic)
  • GGA AAA TTT CGT ATT GGC AGG AAG ATT GGG AGT GGT TCC TTT GGT 941 Gly Arg Lys Phe Arg lie Gly Arg Lys lie Gly Ser Gly Ser Phe Gly 10 15 20
  • AGA CCA AAT TAT TAT CCT GAA CCG TTA CTA CAG CAG CAA CAA AGA GAT 2189 Arg Pro Asn Tyr Tyr Pro Glu Pro Leu Leu Gin Gin Gin Gin Arg Asp 425 430 435
  • TCT GTA CCT CCA CAA ATG AGA TCT AAT CCA CAA CAG CCG CCT CAA GAT 2333 Ser Val Pro Pro Gin Met Arg Ser Asn Pro Gin Gin Pro Pro Gin Asp 470 475 480 485
  • AAACGCACAA AAATAAACAT ATGTATATAT AGACATACAC ACACACATAT ATATATATAT 2440
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • TGATTTTTTG ACTTTACTTT TCATCAATTC CTCTCTTACA CTACGTCTTT TAGTCTTAAA 1309 TTCCAAACCA TCTGTTGACG TTTTAAAGTT CCACAAATAT CTTTAATAAT TCCTGGCTTT 1369 CTTTTTTGTC TATGGATGGC CGGATTGCTA CACTAATACA CTTTGAGGTT TAGCTATTGT 1429 TTTGAGCTAT TCCATTTTGC CTAGAAGTTG AGTTTTAATG CCTTCTTTTTTT AAATAGACAT 1489 ATTGTGTAAA CCTCATACAT GCTTTACTGA AAAGACATAA TTAGAGGACA AAATTTAAAT 1549
  • MOLECULE TYPE DNA (genomic)
  • GAT AAA AAC CTC ACT GGC ACT GCC CGA TAT GCT AGC ATC AAT GCA CAT 751 Asp Lys Asn Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala His 180 185 190
  • MOLECULE TYPE DNA (genomic)
  • AAG ATT CTT CAA GGT GGG GTT GGC ATC CCC CAC ATA CGG TGG TAT GGT 536 Lys He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly 65 70 75 80
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • IMMEDIATE SOURCE
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • IMMEDIATE SOURCE
  • MOLECULE TYPE DNA (genomic)

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Abstract

Gènes mutants et de phénotype sauvage de protéine-kinase, codant des polypeptides de la classe désignée ici 'caséine-kinase I', et étant utilisables dans l'analyse de compositions susceptibles d'intervenir sur l'activité de réparation de ruptures d'ADN bicaténaire. Des procédés faisant appel aux polynucléotides dans le domaine des troubles à prolifération cellulaire sont également décrits.
PCT/US1994/000795 1993-01-21 1994-01-21 Proteine-kinases Ceased WO1994017189A2 (fr)

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CA002132452A CA2132452C (fr) 1993-01-21 1994-01-21 Proteine kinase agissant comme une caseine kinase i
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019993A1 (fr) * 1994-01-21 1995-07-27 The Salk Institute For Biological Studies Proteine-kinases
WO1998030902A1 (fr) * 1997-01-13 1998-07-16 Kudos Pharmaceuticals Limited Dosages, agents, therapie et diagnostic lies a la modulation de l'activite de reparation de l'adn cellulaire
WO1998030903A1 (fr) * 1997-01-13 1998-07-16 Kudos Pharmaceuticals Limited Procedes et moyens lies a l'integration de retrovirus et de retrotransposon
US5969117A (en) * 1995-08-17 1999-10-19 Hybridon, Inc. Modified protein kinase a-specific oligonucleotide
WO2001053493A3 (fr) * 2000-01-18 2002-03-07 Lexicon Genetics Inc Nouvelle proteine kinase humaine et polynucleotides codant pour cette proteine
US6624293B1 (en) 1995-08-17 2003-09-23 Hybridon, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
WO2002033099A3 (fr) * 2000-10-20 2003-10-09 Incyte Genomics Inc Kinases humaines
US7074768B2 (en) 1995-08-17 2006-07-11 Idera Pharmaceuticals, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
WO2005056802A3 (fr) * 2003-12-12 2007-04-19 Cancer Rec Tech Ltd Materiels et methodes de commande du cycle cellulaire

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993001205A1 (fr) * 1991-07-03 1993-01-21 The Salk Institute For Biological Studies Tyrosine-kinase

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019993A1 (fr) * 1994-01-21 1995-07-27 The Salk Institute For Biological Studies Proteine-kinases
US5969117A (en) * 1995-08-17 1999-10-19 Hybridon, Inc. Modified protein kinase a-specific oligonucleotide
US6624293B1 (en) 1995-08-17 2003-09-23 Hybridon, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
US7074768B2 (en) 1995-08-17 2006-07-11 Idera Pharmaceuticals, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
WO1998030902A1 (fr) * 1997-01-13 1998-07-16 Kudos Pharmaceuticals Limited Dosages, agents, therapie et diagnostic lies a la modulation de l'activite de reparation de l'adn cellulaire
WO1998030903A1 (fr) * 1997-01-13 1998-07-16 Kudos Pharmaceuticals Limited Procedes et moyens lies a l'integration de retrovirus et de retrotransposon
AU729066B2 (en) * 1997-01-13 2001-01-25 Kudos Pharmaceuticals Limited Methods and means relating to retrotransposon and retroviral integration
US6242175B1 (en) 1997-01-13 2001-06-05 Kudos Pharmaceuticals Limited Methods and means relating to retrotransposon and retroviral integration
US6753158B1 (en) 1997-01-13 2004-06-22 Kudos Pharmaceuticals Limited Assays, agents, therapy and diagnosis relating to modulation of cellular DNA repair activity
WO2001053493A3 (fr) * 2000-01-18 2002-03-07 Lexicon Genetics Inc Nouvelle proteine kinase humaine et polynucleotides codant pour cette proteine
WO2002033099A3 (fr) * 2000-10-20 2003-10-09 Incyte Genomics Inc Kinases humaines
WO2005056802A3 (fr) * 2003-12-12 2007-04-19 Cancer Rec Tech Ltd Materiels et methodes de commande du cycle cellulaire

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JPH07505057A (ja) 1995-06-08
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WO1994017189A3 (fr) 1994-10-13

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