Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-25
2001-07-24
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S006120, C435S252300, C435S320100, C435S325000, C536S023200, C536S024500
Reexamination Certificate
active
06265194
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel serine-threonine kinase gene.
BACKGROUND OF ART
Fetal tissues are comprised of many undifferentiated cells that proliferate actively, highly activated cells, nascent vascular endothelial cells, and so on. Although the activity of these cells in fetal tissues is stringently regulated and inhibited as individuals mature, the state of fetal tissues can be considered similar to that of a solid tumor except that the activity is regulated. Therefore, some of the genes expressed specifically or more strongly in fetal tissues (fetal genes) can be genes involved in the phenomena characteristic of solid tumors such as abnormal growth, immortalization, infiltration, metastasis, and angiogenesis. In addition, some diseases other than tumors are also supposed to arise because fetal genes, which are repressed in a normal living body, are abnormally activated. Therefore, genes involved in various diseases such as tumors can be screened by isolating and analyzing fetal genes.
However, there are still few reports on systematic analysis focusing merely on fetal genes from these viewpoints, and at present there is a far from perfect understanding of these gene groups.
DISCLOSURE OF THE INVENTION
An objective of this invention is to isolate genes expressed specifically in fetal tissues and to screen genes related to diseases.
The present inventors thought that fetal tissue cells could be a model for solid tumor cells and that genes involved in diseases such as tumors could be screened by isolating and analyzing fetal genes. Furthermore, the present inventors thought it possible to develop a medicine with a novel action mechanism by designing drugs targeting the genes. Based on these thoughts, the present inventors have tried to isolate fetal genes.
Specifically, the present inventors prepared a subtraction library with many genes expressed specifically in fetal livers (or more strongly than adult livers) by the suppression subtractive hybridization method, extracted clones from this library at random, and analyzed their structure. By doing so, the present inventors succeeded in isolating a novel gene, VRK1, having the consensus sequence of a serine-threonine kinase active site. The present inventors also performed a data base search based on the amino acid sequence deduced from the isolated gene. The present inventors thus have found this gene product exhibits a significant homology with B1R kinase, which is presumably involved in DNA replication of vaccinia virus. In addition, the present inventors found human EST having a very high homology with this gene in the database and isolated its full-length cDNA, VRK2. Analyzing the expression of the two isolated genes in various cells by northern blot analysis showed that these genes are strongly expressed, especially in actively growing cells such as human fetal livers, testes, and various tumor cell lines. Furthermore, the present inventors have found that the VRK1 protein actually has protein kinase activity.
Thus, the present invention relates to novel serine-threonine kinase genes, VRK1 and VRK2. More specifically, the present invention relate to:
(1) a protein having the amino acid sequence of SEQ ID NO: 2, or a protein having the same amino acid sequence where one or more amino acids are added, deleted, or substituted and having serine-threonine kinase activity,
(2) a protein having the amino acid sequence of SEQ ID NO: 4, or a protein having the same amino acid sequence where one or more amino acids are added, deleted, or substituted and having serine-threonine kinase activity,
(3) a protein encoded by a DNA sequence that hybridizes with the DNA sequence of SEQ ID NO: 1 or its complementary sequence and having serine-threonine kinase activity,
(4) a protein encoded by a DNA sequence that hybridizes with the DNA sequence of SEQ ID NO: 3 or its complementary sequence and having serine-threonine kinase activity,
(5) a DNA encoding the protein of any one of (1) to (4),
(6) a vector comprising the DNA of (5),
(7) a transformant carrying the vector of (6),
(8) a method of producing the protein of any one of (1) to (4), wherein the method comprises cultivating the transformant of (7),
(9) an antibody binding to the protein of any one of (1) to (4),
(10) an antisense DNA against the DNA of (5) or part of it,
(11) a method of screening compounds having inhibitory activity of serine-threonine kinase activity of the protein of any one of (1) to (4), wherein the method is comprised of
(a) contacting the protein of any one of (1) to (4) with a substrate to be phosphorylated by this protein in the presence of a test compound to detect the kinase activity of the protein of any one of (1) to (4), and
(b) comparing the kinase activity detected in step (a) with that detected in the absence of the test compound and selecting a compound that lowers the kinase activity of the protein of any one of (1) to (4).
The present invention relates to novel serine-threonine kinases, “VRK1” and “VRK2.” The nucleotide sequence of the “VRK1” cDNA and the amino acid sequence of the protein are shown in SEQ ID NO: 1 and 2, respectively. In addition, the nucleotide sequence of the “VRK2” cDNA and the amino acid sequence of the protein are shown in SEQ ID NO: 3 and 4, respectively. “VRK1” cDNA has a significant homology with B1R kinase, which is presumably involved in DNA replication of vaccinia virus. The gene is also characterized by its strong expression in actively growing cells such as fetal livers, testes, and various tumor cell lines. In addition, overexpression of “VRK1” protein drastically increases the growing activity of NIH3T3 cells. These facts imply “VRK1” is involved in the regulation mechanism of cell growth. “VRK1” protein has protein kinase activity, which presumably plays an important roll in the regulation of cell growth. “VRK2” has a high homology with “VRK1,” especially in the serine-threonine kinase site. “VRK2,” like “VRK1,” has a significant homology with B1R kinase, and the gene is characterized by its strong expression in actively growing cells such as fetal livers, testes, and various tumor cell lines. These facts imply “VRK2” has the same function as that of “VRK1.”
“VRK1” and “VRK2” proteins can be prepared as recombinant proteins with recombinant DNA techniques or as natural proteins. The recombinant proteins can be prepared, for example, by cultivating cells transformed with the DNAs encoding these proteins, as will be described later. Natural proteins can be isolated from fetal livers, testes, or tumor cell strains such as HeLa S3, in which these proteins are highly expressed, by a method well-known to one skilled in the art, such as affinity chromatography with the antibodies of the present invention as described later. Either polyclonal or monoclonal antibodies can be used. The polyclonal antibodies can be prepared from, for example, serum from small animals such as rabbits immunized with these proteins by, for example, ammonium sulfate precipitation, protein A- or protein G-column chromatography, DEAE ion exchange chromatography, affinity chromatography using a column coupled with these proteins, etc. The monoclonal antibodies can be prepared as follows. First, a small animal such as a mouse is immunized with these proteins. The spleen is extracted from the mouse and dissociated to cells. The resulting cells are fused to mouse myeloma cells using a reagent such as polyethylene glycol, and the clone that produces antibodies against these proteins is screened from the fusion cells (hybridoma) thus generated. The hybridoma thus obtained is then transplanted into a mouse abdominal cavity. Ascites is collected from the mouse and purified by, for example, ammonium sulfate precipitation, protein A- or protein G-column chromatography, DEAE ion exchange chromatography, affinity chromatography using a column coupled with “VRK1” or “VRK2” protein, etc. If the antibodies obtained are to be used for administering to a human body (for antibody therapy or the like, etc.), humanized antibodies or human a
Nezu Jun-ichi
Oku Asuka
Chugai Research Institute for Molecular Medicine, Inc.
Fish & Richardson P.C.
Monshipouri Maryam
Prouty Rebecca E.
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