# Patent

## Title
YS68 gene involved in primitive hematopoiesis

## Abstract

A novel gene, dubbed “YS68”, involved in primitive hematopoiesis was successfully isolated from cDNA derived from mouse yolk sacs. In addition, a human gene corresponding to this gene was successfully isolated. Expression characteristics of these genes suggested their involvement in primitive hematopoiesis. The proteins of this invention and genes encoding the proteins may be utilized as tools for drug development against diseases, such as hematological disorders.

## Background
This application is a continuation of U.S. patent application Ser. No. 10/118,513, filed Apr. 8, 2002, now abandoned which is a continuation-in-part of International Patent Application PCT/JP00/05756, filed Aug. 25, 2000, which claims priority to Japanese Patent Application Nos. 11/288,738, filed Oct. 8, 1999; 11/288,739, filed Oct. 8, 1999; and 2000-123721, filed Apr. 19, 2000.


TECHNICAL FIELD
The present invention relates to novel proteins involved in primitive hematopoiesis and genes encoding the proteins. These molecules may be utilized, for example, in the field of drug development.
BACKGROUND
There are two kinds of hematopoiesis: one is the transient primitive hematopoiesis (embryonic hematopoiesis) that functions only during the embryonic stage, and the other is the definitive hematopoiesis (adult hematopoiesis) that contributes to lifelong hematopoiesis. Research by Medvinsky et al. (Medvinsky et al., Cell 86:897-906, 1996; Cumano et al., Cell 86:907-916) revealed that, in contrast to primitive hematopoiesis that develops within the yolk sac on around embryonic day 9, definitive hematopoiesis is initiated within the AGM (Aorta-Gonad-Mesonephros) region on around embryonic day 10. Furthermore, regarding the origin of hematocytes, various studies have suggested that definitive hematopoiesis originates from hemangioblasts, thought to be precursor cells common to hematopoietic cells and vascular endothelium cells.
While the mainly accepted view was that hemangioblasts, which are the origin of definitive hematopoiesis, exist in the AGM region, Yorder et al. argued against the existing theory and demonstrated that hemangioblasts, which may contribute to definitive hematopoiesis, also exist in the yolk sac (Yoder et al., Immunity 7:335-344, 1997). Therefore, it is now generally accepted that the surrounding environment is important for the differentiation of hemangioblasts to hematopoietic cells.
Thus, while the origin of hematopoietic cells and the site of development have been gradually elucidated by phenomenological research, the molecular mechanism of hematopoietic development remains unclear. The isolation of a novel molecule involved with primitive hematopoiesis is thought to be an important step for the development of unprecedented drugs associated with hematological disorders.
SUMMARY
The subject of the present invention is to provide novel proteins involved in primitive hematopoiesis and genes encoding the proteins, as well as production and use of the same.
Although the existence of hemangioblasts has been reported in the mouse AGM (Aorta-Gonad-Mesonephros) region on embryonic day 9 to day 12, Yorder and Nishikawa et al. have reported that hemangioblasts exist in embryonic day 9 yolk sacs, but no longer exist in embryonic day 13 yolk sacs (Yoder et al., Immunity 7:335-344, 1997; Nishikawa et al., Immunity 8:761-769, 1998). The present inventors conducted cloning of genes to identify molecules involved with primitive hematopoiesis by subtracting the cDNA derived from embryonic day 13 mouse yolk sac in which hemangioblasts are assumed to be absent, from the cDNA derived from embryonic day 9 mouse yolk sac in which hemangioblast is suggested to be present. Inventors succeeded in isolating a novel gene that was named “YS68”. In addition, a primer was constructed based on the nucleotide sequence of the mouse gene, and, by performing 5′-RACE and 3′-RACE using human fetal liver Marathon-Ready cDNA as a template, the corresponding human gene was successfully isolated.
Determination and comparison of the full-length human (SEQ ID NO: 13) and mouse (SEQ ID NO: 11) cDNA sequences showed a very high sequence homology of 87% in the N-terminal region (human 1-1137 of SEQ ID NO: 13, mouse 1-1137 of SEQ ID NO: 11); whereas the homology in the central region (human 1138-1683 of SEQ ID NO: 13, mouse 1138-1679 of SEQ ID NO: 11) was 57%; and the homology in the C-terminal region (human 1684-2266 of SEQ ID NO: 13, mouse 1680-2243 of SEQ ID NO: 11) was very low at 45%. Many nuclear transport signals were found to exist in the low-homology C-terminal region. On the other hand, two WD repeats that are known to be necessary for interaction with proteins were found to exist in the high-homology N-terminal region.
To investigate the role of “YS68” in hematopoiesis, RT-PCR analysis of the expression pattern of “YS68” in mouse hematopoietic tissue was performed; the results revealed that the expression pattern of “YS68” correlated with the transport of hematopoietic tissues during the embryonic stage. In addition, “YS68” was expressed in CD34-positive undifferentiated hematocytes. Therefore, “YS68” is suggested to have an important function in primitive hematopoiesis.
The “YS68” protein of this invention is useful as a tool for elucidating the mechanism of primitive hematopoiesis, furthermore, its application to drug development for various diseases related to hematopoietic system is anticipated.
This invention relates to novel proteins involved in primitive hematopoiesis and genes encoding the proteins, as well as the production and use of the same. More specifically, this invention provides the following:
(1) a DNA selected from the group of:

    
    
        (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:12 or 14;
        (b) a DNA comprising the coding region of the nucleotide sequence of SEQ ID NO:11 or 13;
        (c) a DNA encoding a protein comprising of the amino acid sequence of SEQ ID NO:12 or 14, in which one or more amino acids are modified by substitution, deletion, insertion and/or addition, wherein said protein is functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 12 or 14; and
        (d) a DNA hybridizing under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 11 or 13, and encoding a protein that is functionally equivalent to a protein consisting of the amino acid sequence of SEQ ID NO:12 or 14;
    
    


(2) a DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO:12 or 14;
(3) a protein or a peptide encoded by the DNA of any one of (1) or (2);
(4) a vector into which the DNA of any one of (1) or (2) is inserted;
(5) a host cell retaining the vector of (4);
(6) a method for producing the proteins or peptides of (3); comprising the step of culturing the host cells of (5), and recovering expressed protein from said host cell or the culture supernatant;
(7) a polynucleotide comprising at least 15 nucleotides that are complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 11 or 13 or to a complementary strand thereof;
(8) a method of screening for a compound that binds to the protein of (3), comprising the steps of:

    
    
        (a) contacting a test sample, containing at least one compound, with the protein or partial peptide of (3);
        (b) detecting the binding activity between the compound and the protein or partial peptide thereof; and
        (c) selecting the compound that has the activity to bind to the protein or partial peptide thereof;
    
    


(9) a compound binding to the protein of (3);
(10) the compound of (9), which is an antibody; and
(11) a compound binding to the protein of (3), which may be isolated by the method of (8).
The present invention provides novel proteins involved in primitive hematopoiesis and DNA encoding these proteins. The nucleotide sequence of the full-length cDNA of mouse “YS68” isolated by the present inventors is indicated in SEQ ID NO: 11, and the amino acid sequence of the protein encoded by this cDNA is indicated in SEQ ID NO:12. In addition, the nucleotide sequence of the full-length cDNA of human “YS68” isolated by the present inventors is indicated in SEQ ID NO: 13, and the amino acid sequence of the protein encoded by this cDNA is indicated in SEQ ID NO: 14.
Hematopoietic stem cells contributing to lifelong hematopoiesis are formed by the differentiation of hemangioblasts, the common mother cells of hematocytes and blood vessels. Several transcription factors thought to be important for primitive hematopoiesis have been reported according to recent gene disruption experiments. Not only angiogenesis but also hematopoiesis was not confirmed in mouse with disruption in SCL (Porcher et al., Cell 86:47-57, 1996; Visvader et al., Genes Dev. 12:473-479, 1998). In addition, AML-1 and c-Myb knockout mice did not show abnormalities in angiogenesis, but they completely lacked definitive hematopoiesis (Okuda et al., Cell 84:321-330, 1996; Lin et al., Curr. Top Microbiol. Immunol. 211:79-87, 1996). However, how these transcription factors interact with each other at the stage of primitive hematopoiesis and become involved in determining the fate of cells remains unknown.
The mouse “YS68” gene (SEQ ID NO: 11) identified by the present inventors was isolated by subtracting cDNA derived from embryonic day 13 mouse yolk sac, which is said to lack the hemangioblast, from cDNA derived from embryonic day 9 yolk sac, which is suggested to have a hemangioblast. The isolated “YS68” gene was expected to encode a protein of 1,265 amino acids, and showed an expression pattern with a high level expression in embryonic day 9 yolk sac followed by a gradual decrease. In addition, an expression of the gene was observed in the AGM region (considered to be the site of hematopoietic stem cell development) from day 10 embryos and in embryonic day 13 livers; the expression then shifted to strong expression at the thymus and spleen of day 16 embryos. Furthermore, expression in these regions considerably diminished in adult mice. Thus, the “YS68” cloned by the present inventors with such an expression pattern in the developmental stage can be considered as a new member of molecules involved in primitive hematopoiesis.
Although “YS68” is expected to be a nuclear protein because it has multiple nuclear transport signals in its C-terminal region, strong expression was observed not only in the nucleus but also around the nucleus in hepatocytes (Example 6). The finding that WD repeats necessary for binding to proteins existed in the N-terminus, and immunoprecipitation caused coprecipitation of multiple proteins (Example 4) suggested that transport of this protein to the nucleus is regulated by interactions with other proteins.
The idea that blood cells develop from the vascular endothelium has existed for a relatively long time, but was actually proven only recently. Jaffredo et al. stained the entire avian blood vessel with fluorescence-labeled LDL and revealed that the stained vascular endothelium differentiated into hematocytes (Jaffredo et al., Development 125:4575-4783, 1998). In addition, Hara et al. found that hemangioblasts can be concentrated by sorting the cells of the AGM region by PCLP-1 (podocalyxin-like protein 1). Localization of hemangioblasts in the vascular endothelium was suggested by the localized PCLP-1 expression in the AGM region in the vascular endothelium (Hara et al., Immunity 11:567-578, 1999). As shown in Example 5, the expression site of YS68 in the AGM region was the same vascular endothelium as PCLP-1. In addition, this expression pattern is the same as those of AML-1 and SCL, both of which are known to be important for primitive hematopoiesis. Considering that expression of YS68 in the hematocyte of CD34 positive cells, which are thought to be a group of relatively immature hematocytes (Example 6), is strong, YS68 is suggested to function in the process of differentiation from hemangioblasts to hematocytes.
The “YS68” proteins of this invention and DNAs encoding the proteins are useful as differentiation markers and as regulating factors of developmental differentiation and the hematopoietic function of hematopoietic stem cells. Additionally, they may be applicable for diagnosis, prevention, and treatment of diseases in which a protein of this invention is involved. In current medicine, means for artificial amplification of hematopoietic stem cells does not exist. Artificial in vitro proliferation of hematopoietic stem cells may be enabled by forced expression of YS68 using a virus vector in hemangioblasts that are the origin of hematopoietic cells, or by administration of cytokines or compounds that induces the expression of YS68. Therefore, YS68 may be applied to medical treatment, as a new alternative to bone marrow transplant.
In addition, many human blood cell tumors, such as myeloid leukemia and lymphoid leukemia, are often caused by abnormalities in transcription factors, and human “YS68” gene of this invention is likely to be one of the causative genes of these diseases. Therefore, human “YS68” may be particularly applied to genetic diagnosis or gene therapy of such diseases. Furthermore, drug development targeting the human “YS68” gene and protein themselves or molecules that regulate them, or molecules or genes that are regulated by the human “YS68” protein may be useful in the treatment and prevention of the above-mentioned diseases.
Furthermore, this invention includes proteins that are functionally equivalent to the “YS68” protein (SEQ ID NO:12 and 14). For example, mutant forms of the “YS68” protein are included in such proteins. The term “functionally equivalent” herein means that the protein of interest has the function of regulating the development and/or differentiation of hematopoietic cells or has the function of interacting with other proteins.
For example, the function of a protein to regulate the development and/or differentiation of hematopoietic cells can be evaluated using as an index the expression characteristics within the hematopoietic tissues, such as those described in Example 2. On the other hand, the function of a protein to interact with other proteins can be determined; for example, by utilizing immunoprecipitation, such as those described in Example 4.
As a method well known by a person skilled in the art for preparing a protein functionally equivalent to a given protein, methods for introducing mutations into proteins are known. For example, one skilled in the art can prepare proteins functionally equivalent to the “YS68” proteins (SEQ ID NO:12 and 14) by introducing an appropriate mutation in the amino acid sequence of the protein by site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-275, 1995; Zoller et al., Methods Enzymol. 100:468-500, 1983; Kramer et al., Nucleic Acids Res. 12:9441-9456, 1984; Kramer et al., Methods. Enzymol. 154:350-367, 1987; Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985; Kunkel, Methods Enzymol. 85:2763-2766, 1988). Mutation of amino acids can occur in nature, too. The proteins of the present invention include those proteins that comprise the amino acid sequences of the “YS68” protein (SEQ ID NO: 12 and 14), wherein one or more amino acids are mutated and yet are functionally equivalent to the protein comprising the sequence of “YS68” protein. It is considered that the number of amino acids to be mutated in such a mutant, is generally 100 amino acids or less, preferably 50 amino acids or less, more preferably 20 amino acids or less, and more preferably 5 amino acid or less.
As for the amino acid residue to be mutated, it is preferable that it is mutated into a different amino acid such that the properties of the amino acid side-chain are conserved. Examples of properties of amino acid side chains are, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W) (The parenthetic letters indicate the one-letter codes of amino acids).
It is well known that a protein having deletion, addition, and/or substitution of one or more amino acid residues in the sequence of a protein can retain the original biological activity (Mark et al., Proc. Natl. Acad. Sci. USA 81:5662-5666, 1984; Zoller et al., Nucleic Acids Res. 10:6487-6500, 1982; Wang et al., Science 224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA 79:6409-6413, 1982).
The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. For example, the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Accordingly, the invention includes a polypeptide having a sequence shown as SEQ ID NO:12 or 14. The invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ ID NO:12 or 14. The polypeptide can differ from the sequence of SEQ ID NO: 12 or 14 by having one or more amino acids substituted, deleted, inserted and/or added. For example, the polypeptide can be a fusion protein, having an additional amino acid sequence at the N- or C-terminus of SEQ ID NO:12 or 14. In preferred embodiments, the protein has no more than 50, 30, 20, 10 or 5 amino acids substituted, deleted, inserted and/or added. Preferably, the difference is a difference or change at one or more non-essential residues or one or more conservative amino acid substitutions, as defined above. In one embodiment, the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO:12 or 14, or a fragment thereof. Preferably, the polypeptide is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:12 or 14 and has at least one YS68 activity described herein, e.g., the protein can regulate development or differentiation of hematopoietic cells. Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO: 12 or 14 and have at least one YS68 activity described herein. Or alternatively, the fragment can be merely an immunogenic fragment.
A fusion protein comprising “YS68” protein is encompassed in the protein, wherein one or more amino acids residues are added to the amino acid sequence of “YS68”. Fusion proteins are fusions of the “YS68” protein and other peptides or proteins, and are included in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking the DNA encoding the “YS68” protein (SEQ ID NO: 12 and 14) with DNA encoding other peptides or proteins so as the frames match, inserting this linked DNA into an expression vector, and expressing it in a host. There is no restriction as to the peptides or proteins to be fused to a protein of the present invention.
Known peptides, for example, FLAG (Hopp et al., Biotechnology 6:1204-1210, 1988), 6× His consisting of six His (histidine) residues, 10× His, Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, and such, can be used as peptides to be fused to a protein of the present invention. Examples of proteins that may be fused to a protein of the present invention are, GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose-binding protein), and such. Fusion proteins can be prepared by fusing commercially available DNA encoding these peptides or proteins with a DNA encoding a protein of the present invention and expressing the fused DNA prepared.
Furthermore, a protein, in which multiple amino acid residues have been added to the amino acid sequence of the “YS68” protein, includes a protein encoded by the nucleotide sequence starting from “a” at position 98 to “g” at position 6922 of SEQ ID NO:15 (protein comprising the amino acid sequence, wherein an amino acid sequence comprising “Met-Ala-Ala-Glu-Arg-Arg-Cys-Gly-Ser” (SEQ ID NO:16) is added to the N terminus of the amino acid sequence of SEQ ID NO:14).
In addition, as a method well known to those skilled in the art for preparing proteins that are functionally equivalent to a known protein, methods that utilize hybridization techniques (Sambrook et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) can be mentioned. More specifically, those skilled in the art may readily isolate DNAs having high homology to the DNA sequences (SEQ ID NO: 11 and 13) encoding the “YS68” protein, based on the entire DNA sequence or parts thereof, and isolate DNA encoding proteins functionally equivalent to the “YS68” protein from these DNAs. The present invention includes proteins that are functionally equivalent to the “YS68” protein, and which are encoded by DNAs that hybridize under stringent conditions with DNA encoding the “YS68” protein. When isolating a cDNA that has high sequence homology to the DNA encoding the “YS68” protein, it is considered to be preferable to use embryonic stage hematopoietic tissues (for example; tissues such as the AGM region and yolk sac during early development; and thymus, spleen, and liver during mid to late development).
Hybridization conditions for isolating DNAs encoding proteins that are functionally equivalent to the “YS68” protein can be appropriately selected by those skilled in the art. Conditions for hybridization, for example, may be those with low stringency. Low stringency conditions means that the washing conditions after hybridization are, for example, 42° C., 2×SSC, and 0.1% SDS, or preferably 50° C., 2×SSC, and 0.1% SDS. Examples of hybridization conditions that are more preferable are conditions with high stringency. An example of high stringency conditions is 65° C., 0.1×SSC and 0.1% SDS. Under these conditions, the higher the temperature, the higher the homology of the obtained DNA will be. However, several factors such as temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can appropriately select such factors to accomplish a similar stringency.
In addition, instead of hybridization, DNA encoding functionally equivalent proteins to “YS68” protein can be isolated by gene amplification methods, for example, by polymerase chain reaction (PCR), which uses primers that are synthesized based on sequence information of DNA encoding the “YS68” protein (SEQ ID NO: 11 and 13).
A protein that is functionally equivalent to a “YS68” protein, encoded by a DNA that is isolated by such hybridization techniques and gene amplification techniques, will normally have a high amino acid sequence homology to the “YS68” protein (SEQ ID NO:12 and 14). The proteins of this invention also include proteins that are functionally equivalent to a “YS68” protein and at the same time have a high sequence homology to the amino acid sequence of SEQ ID NO:12 or 14. High sequence homology typically means a homology of 30% or more, preferably a homology of 50% or more, more preferably a homology of 70% or more, and even more preferably a homology of 90% or more (for example, homology of 95% or more). To determine the homology of a protein, an algorithm described in the literature (Wilbur et al., Proc. Natl. Acad. Sci. USA 80:726-730, 1983) can be used.
The proteins of this invention may have different amino acid sequences, molecular weights, and isoelectric points, as well as differences in the presence or absence of sugar chains and their forms, depending on the cells or hosts to produce the protein or production method, which will be described later. However, so long as the obtained protein has the same function as the “YS68” protein, it is included in this invention. For example, if a protein of this invention is expressed in a prokaryotic cell such as E. coli, a methionine residue will be added to the N terminus of the amino acid sequence of the original protein. The proteins of this invention will also include such proteins.
The proteins of the present invention can be prepared as recombinant proteins or naturally occurring proteins, by methods well known by those skilled in the art. A recombinant DNA can be prepared by inserting a DNA (for example, the DNA comprising the nucleotide sequence of SEQ ID NOs: 11 or 13) which encodes a protein of the present invention into an appropriate vector, collecting the recombinant obtained by introducing the vector into appropriate host cells, obtaining the extract, and purifying by subjecting the extract to chromatography such as ion exchange, reverse, gel filtration, or affinity chromatography in which an antibody against a protein of the present invention is fixed on column or by combining more than one of these columns.
Also when a protein of the present invention is expressed within host cells (for example, animal cells and E. coli) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column.
After purifying the fusion protein, it is also possible to exclude regions other than the objective protein by cutting with thrombin or factor-Xa as required.
A naturally occurring protein can be isolated by methods known by a person skilled in the art, for example, by using an affinity column in which the antibody binding to a protein of the present invention (described below) is bound against an extract of tissues or cells expressing a protein of the present invention is expressed. An antibody can be a polyclonal or a monoclonal antibody.
The present invention also contains partial peptides of the proteins of the present invention. A partial peptide of the present invention comprises at least 7 amino acids or more, preferably 8 amino acids or more, and more preferably 9 amino acids or more. The partial peptides can be used, for example, for preparing an antibody against a protein of the present invention, screening a compound binding to a protein of the present invention, and for screening accelerators or inhibitors of a protein of the present invention. The partial peptides can be also used as antagonists or a competitive inhibitors against a protein of the present invention.
A partial peptide of the invention can be produced by genetic engineering, known methods of peptide synthesis, or by digesting a protein of the invention with an appropriate peptidase. For peptide synthesis, for example, solid phase synthesis or liquid phase synthesis may be used.
As used herein, an “isolated nucleic acid” is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in random, uncharacterized mixtures of different DNA molecules, transfected cells, or cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
Accordingly, in one aspect, the invention provides an isolated or purified nucleic acid molecule that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated nucleic acid molecule includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 11 or 13. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97% 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID NO:11 or 13. In the case of an isolated nucleic acid molecule which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:11 or 13, the comparison is made with the full length of the reference sequence. Where the isolated nucleic acid molecule is shorter that the reference sequence, e.g., shorter than SEQ ID NO: 11 or 13, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
As used herein, “% identity” of two amino acid sequences, or of two nucleic acid sequences, is determined using the algorithm of Karlin and Altschul (Proc Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignment for comparison purposes GappedBLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.
A DNA encoding a protein of the present invention can be used for the production of the protein in vivo or in vitro as described above as well as for, for example, application to gene therapy for diseases attributed to genetic abnormality in the gene encoding the protein of the present invention. Any form of the DNA can be used, so long as it encodes a protein of the present invention. Specifically, cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA can be used. The present invention includes a DNA comprising a given nucleotide sequence based on degeneracy of genetic codons, as long as it encodes a protein of the present invention.
A DNA of the present invention can be prepared by methods known to those skilled in the art. For example, a DNA of the present invention can be prepared from a cDNA library from cells which express a protein of the present invention by conducting hybridization using a partial sequence of the DNA of the present invention (e.g., SEQ ID NO: 11 and 13) as a probe. A cDNA library can be prepared, for example, by the method described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989, or using commercially available cDNA libraries. A cDNA library can be also prepared by extracting RNA from cells expressing a protein of the present invention, synthesizing cDNA using reverse transcriptase, synthesizing an oligo DNA base on the sequence of the DNA of the present invention (for example, SEQ ID NOs: 11 and 13), conducting PCR by using these as primers, and amplifying cDNA encoding the protein of the present invention.
In addition, by sequencing the nucleotides of the obtained cDNA, a translation region encoded by the cDNA can be determined, and the amino acid sequence of a protein of the present invention can be obtained. Moreover, by screening the genomic DNA library using the obtained cDNA as a probe, genomic DNA can be isolated.
More specifically, mRNAs may first be prepared from a cell, tissue, or organ (for example, embryonic stage hematopoietic tissues such as AGM region and yolk sac of early development; thymus, spleen, and liver of mid to late development) in which a protein of the invention is expressed. Known methods can be used to isolate mRNAs; for instance, total RNA is prepared by the guanidine ultracentrifugation (Chirgwin et al., Biochemistry 18:5294-5299, 1979) or the AGPC method (Chomczynski et al., Anal. Biochem. 162:156-159, 1987), and mRNA is purified from total RNA using mRNA Purification Kit (Pharmacia) and such. Alternatively, mRNA may be directly purified by QuickPrep mRNA Purification Kit (Pharmacia).
The obtained mRNA is used to synthesize cDNA using reverse transcriptase. A cDNA may be synthesized using kits, such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo). Alternatively, a cDNA may be synthesized and amplified following the 5′-RACE method (Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988; Belyavsky et al., Nucleic Acids Res. 17:2919-2932, 1989) which uses a primer and such, described herein, the 5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction (PCR).
A desired DNA fragment is prepared from the PCR products and ligated with a vector DNA. The recombinant vectors are used to transform E. coli and such, and a desired recombinant vector is prepared from a selected colony. The nucleotide sequence of the desired DNA can be verified by conventional methods, such as the dideoxynucleotide chain termination method.
A DNA of the invention may be also designed to have a sequence that is expressed more efficiently by taking into account the frequency of codon usage in the host to be used for expression (Grantham et al., Nucleic Acids Res. 9:43-74, 1981). A DNA of the present invention may be altered by a commercially available kit or a conventional method. For instance, a DNA may be altered by digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, or insertion of the initiation codon (ATG) and/or the stop codon (TAA, TGA, or TAG).
The DNAs of this invention include a DNA that (a) hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 11 or 13 and (b) encodes a protein that is functionally equivalent to a protein of this invention mentioned above. Stringent conditions for hybridization can be selected appropriately by those skilled in the art, and those conditions specifically mentioned above may be used. Under these conditions, DNA having higher homology are obtained as the temperature is raised. The above-mentioned DNA to be hybridized is preferably a naturally occurring DNA, for example, a cDNA or chromosomal DNA.
The present invention also provides vectors into which a DNA of the present invention is inserted. The vectors of the present invention are useful to retain a DNA of the present invention in host cell, or to express a protein of the present invention.
When E. coli is used as the host cell and a vector is amplified therein to produce a large amount in E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), the vector should have an “ori” that may be amplified in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug-resistance gene selected by a drug (e.g., ampicillin, tetracycline, kanamycin, or chloramphenicol)). For example, the M13-series vectors, the pUC-series vectors, pBR322, pBluescript, pCR-Script, and so on can be used. In addition to the vectors described above, pGEM-T, pDIRECT, and pT7, for example can also be used for subcloning and extracting cDNA. When a vector is used to produce a protein of the present invention, an expression vector is especially useful. For example, an expression vector to be expressed in E. coli should have the above characteristics to be amplified in E. coli. When E. coli, such as JM109, DH5α, HB101, or XL1 Blue, are used as the host cell, the vector should, in addition to the above characteristics, have a promoter so that the vector is copied in the host, for example, the lacZ promoter (Ward et al., Nature 341:544-546, 1989; FASEB J. 6:2422-2427, 1992), the araB promoter (Better et al., Science 240:1041-1043, 1988), or the T7 promoter and such, that can efficiently express the desired gene in E. coli. As such a vector, for example, pGFX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP or pET (in this case, the host is preferably BL21 which expresses T7 RNA polymerase) can be used in addition to the above vectors.
A vector also may certain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei et al., J. Bacteriol. 169:4379; 1987) can be used in the case of producing proteins into the periplasm of E. coli. For introducing a vector into host cells, for example, the calcium chloride method, and the electroporation method can be used.
Besides E. coli, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids. Res. 18(17):5322, 1990), pEF, pCDM8); expression vectors derived from insect cells (for example, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8); expression vectors derived from plants (for example pMH1, pMH2); expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw); expression vectors derived from retroviruses (for example, pZIPneo); expression vector derived from yeast (for example, “Pichia Expression Kit” (Invitrogen), PNV11, SP-Q01); expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50) can be used as vectors for producing a protein of the present invention.
In order to express a vector in animal cells, such as CHO, COS, or NIH3T3 cells, the vector should have a promoter necessary for expression in such cells, for example, the SV40 promoter (Mulligan et al., Nature 277:108, 1979), the MMLV-LTR promoter, the EF1αpromotor (Mizushima et al., Nucleic Acids Res. 18:5322, 1990), or the CMV promoter, and such, and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by a drug (e.g., neomycin, G418)). Examples of vectors with these characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOp13, and so on.
In addition, for the purpose of stably expressing a gene and amplifying the copy number of the gene in cells, for example, a method wherein a vector comprising the complementary DHFR gene (for example pCHO I) is introduced into CHO cells in which the nuclei acid synthesizing pathway is deleted and amplified by methotrexate (MTX) can be used. On the other hand, in the case of transient expression of a gene, a method wherein a vector (e.g., pcD) comprising replication origin of SV40 is transformed using COS cells comprising the SV40 T antigen expressing gene on chromosomes can be used. The origin used for replication may be those of polyomavirus, adenovirus, bovine papilloma virus (BPV), and the like. In addition, the expression vector may include a selection marker gene for amplification of the gene copies in host cells. Examples of such markers include, but are not limited to, the aminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene, the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, and the dihydrofolate reductase (dhfr) gene.
On the other hand, a DNA of the present invention can be expressed in vivo in animals, for example, by inserting a DNA of the present invention into an appropriate vector and introducing it in vivo by a conventional method, such as the retrovirus method, the liposome method, the cationic liposome method, and the adenovirus method. By using these methods, gene therapy against diseases attributed to mutation of ‘YS68’ gene of the present invention can be effected. As a vector, for example, adenovirus vector (for example pAdexlcw), and retrovirus vector (for example, pZIPneo) can be used, but the present invention is not restricted thereto. Common gene manipulation, for example, insertion of a DNA of the present invention to a vector, can be performed according to any standard method (Molecular Cloning, 5.61-5.63). Administration into a living body can be either an ex vivo method, or in vivo method.
The present invention relates to a host cell into which a vector of the present invention has been introduced. The host cell into which a vector of the invention is introduced is not particularly limited. E. coli or various animal cells can be used. The host cells of the present invention can be used, for example, as production system for producing or expressing a protein of the present invention. The present invention provides methods of producing a protein of the invention both in vitro or in vivo. For in vitro production, eukaryotic cells or prokaryotic cells can be used as host cells.
Useful eukaryotic cells as host include animal, plant, or fungi cells. As animal cells, mammalian cells, such as CHO (J. Exp. Med. 108:945, 1995), COS, 3T3, myeloma, baby hamster kidney (BHK), HeLa, and Vero cells; amphibian cells, such as Xenopus oocytes (Valle et al., Nature 291:340-358, 1981); or insect cells, such as Sf9, Sf21, and Tn5 cells can be used. CHO cells lacking the DHFR gene (dhfr-CHO) (Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) or CHO K-1 (Proc. Natl. Acad. Sci. USA 60:1275, 1968) may be also used. In animal cells, CHO cells are particularly preferable for mass expression. A vector can be introduced into host cells by, for example, the calcium phosphate method, the DEAE dextran method, the cationic liposome DOTAP (Boehringer Mannheim), the electroporation method, or the lipofection method.
As plant cells, plant cells originating from Nicotiana tabacum are known as a protein-production system, and may be used as callus cultures. As fingi cells, yeast cells, such as Saccharomyces, including Saccharomyces cerevisiae, or filamentous fungi such as Aspergillus, including Aspergillus niger, are known and may be used herein.
Useful prokaryotic cells include bacterial cells, such as E. coli, for example, JM109, DH5α, HB101 are known. Regarding others, Bacillus subtilis is known.
These host cells are transformed by a desired DNA, and the resulting transformants are cultured in vitro to obtain a protein. Transformants can be cultured using known methods. Culture medium for animal cell, for example, DMEM, MEM, RPMI1640, or IMDM may be used with or without serum supplement such as fetal calf serum (FCS). The pH of the culture medium is preferably between about pH 6 to 8. Such cells are typically cultured at about 30 to 40° C. for about 15 to 200 hr, and the culture medium may be replaced, aerated, or stirred if necessary.
Animal and plant hosts may be used for in vivo production. For example, a desired DNA can be introduced into an animal or plant host. Encoded proteins are produced in vivo, and then recovered. These animal and plant hosts are included in the host cells of the present invention.
Animals to be used for the production systems described above include, but are not limited to, mammals and insects. Mammals, such as goat, porcine, sheep, mouse, and bovine, may be used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Alternatively, the mammals may be transgenic animals.
For instance, a desired DNA may be prepared as a fusion gene with a gene encoding a protein specifically produced into milk, such as goat β casein. DNA fragments comprising a fusion gene having the desired DNA are injected into goat embryos, which are then introduced back to female goats. Proteins are recovered from milk produced by the transgenic goats (i.e., those born from the goats that had received the modified embryos) or from their offspring. To increase the amount of milk containing the proteins produced by transgenic goats, appropriate hormones may be administered to them (Ebert et al., Bio/Technology 12:699-702, 1994).
Alternatively, insects, such as the silkworm, may be used. A desired DNA inserted into baculovirus can be used to infect silkworms, and a desired protein is then recovered from their body fluid (Susumu et al., Nature 315:592-594, 1985).
As plants, for example, tobacco can be used. In use of tobacco; a desired DNA is inserted into a plant expression vector, such as pMON530, which is then introduced into bacteria, such as Agrobacterium tumefaciens. Then, the bacteria is used to infect tobacco, such as Nicotiana tabacum, and a desired polypeptide is recovered from the leaves of the plant (Julian et al., Eur. J. Immunol. 24:131-138, 1994).
A protein of the present invention obtained as above may be isolated from the interior or exterior (e.g. medium) of the cells or hosts, and purified as a substantially pure homogeneous protein. The method for protein isolation and purification is not limited to any specific method; in fact, any standard method may be used. For instance, column chromatography, filter, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the protein.
For chromatography, for example, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and such may be used (Strategies for Protein Purification and Characterization: A Laboratory Course Manuals Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies may be performed by liquid chromatography, such as HPLC and FPLC. Thus, the present invention provides for highly purified proteins, produced by the above methods.
A protein of the present invention may be optionally modified or partially deleted by treating it with an appropriate protein modification enzyme before or after purification. Useful protein modification enzymes include, but are not limited to, trypsin, chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase.
The present invention provides an antibody that binds to a protein of the invention. The antibody of the invention can be used in any form, such as monoclonal or polyclonal antibodies, and includes antiserum obtained by immunizing a rabbit with a protein of the invention, all classes of polyclonal and monoclonal antibodies, human antibodies, and humanized antibodies produced by genetic recombination.
A protein of the invention used as an antigen to obtain an antibody may be derived from any animal species, but is preferably derived from a mammal such as a human, mouse, or rat, or more preferably from a human. A human-derived protein may be obtained from the nucleotide or amino acid sequences disclosed herein.
In the present invention, a protein to be used as an immunization antigen may be a complete protein or a partial peptide of a protein. A partial peptide may be, for example, an amino (N)-terminal or carboxy (C)-terminal fragment of the protein. Herein, “an antibody” is defined as an antibody that specifically reacts with either the full-length or a fragment of a protein.
A gene encoding a protein of the invention or its fragment may be inserted into a known expression vector, which is then used to transform a host cell as described herein. The desired protein or its fragment may be recovered from the exterior or interior of the host cells by any standard method, and may be used as an antigen. Alternatively, cells expressing the protein or their lysates, or a chemically synthesized protein may be used as an antigen. Short peptides are preferably bound with carrier proteins such as bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin to be used as the antigen.
Any mammalian animal may be immunized with the antigen, but preferably the compatibility with parental cells used for cell fusion is taken into account. In general, animals of the orders Rodentia, Lagomorpha, or Primate are used.
Rodents include, for example, mouse, rat, and hamster. Lagomorphs include, for example, rabbit. Primates include, for example, a monkey of catarrhine (old world monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon, or chimpanzee.
Methods for immunizing animals against antigens are known in the art. Intraperitoneal injection or subcutaneous injection of antigens is used as a standard method for immunization of mammals. More specifically, antigens may be diluted and suspended in an appropriate amount with phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals. Preferably, it is followed by several administrations of antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. An appropriate carrier may also be used for immunization. After immunization as above, serum is examined for increase of the amount of desired antibodies by a standard method.
Polyclonal antibodies against a protein of the present invention may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and separating serum from the blood by any conventional method. Polyclonal antibodies may be used as serum containing the polyclonal antibodies, or if necessary, a fraction containing the polyclonal antibodies may be isolated from the serum. Immunoglobulin G or M can be prepared by obtaining a fraction which recognizes only a protein of the present invention using an affinity column coupled with the protein of the present invention and further purifying this fraction by using protein A or protein G column.
To prepare monoclonal antibodies, immune cells are collected from the mammal immunized against an antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other parental cells can be fused with the above immunocyte; for example, preferably myeloma cells of mammalians, and more preferably myeloma cells which acquired the property for selecting fused cells by drugs can be used.
The above immunocyte and myeloma cells can be fused by known methods, for example, the method by Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981).
Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as the HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). The cell culture is typically continued in the HAT medium for several days to several weeks, a sufficient time to allow all the other cells, except desired hybridoma (non-fused cells), to die. Then, by the standard limiting dilution method, a hybridoma cell producing the desired antibody is screened and cloned.
In addition to the above method, in which a non human animal is immunized against an antigen for preparing hybridoma, human lymphocytes, such as that infected by EB virus, may be immunized with a protein, protein expressing cells, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody having binding ability to the protein can be obtained (Unexamined Published Japanese Patent Application (JP-A) No. Sho 63-17688).
Next, the monoclonal antibody, obtained by transplanting the obtained hybridomas into the abdominal cavity of a mouse and by extracting ascites, can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which a protein of the present invention is coupled. An antibody of the present invention can be used not only for purification and detection of a protein of the present invention, but also as a candidate for agonists and antagonists of a protein of the present invention. In addition, an antibody can be applied to antibody treatment for diseases associated with a protein of the present invention. When the obtained antibody is used for the administration to the human body (antibody treatment), a human antibody or a humanized antibody is preferable for reducing immunogenicity.
For example, transgenic animals having a repertory of human antibody genes may be immunized against a protein, protein expressing cells, or their lysates as an antigen. Antibody producing cells are collected from the animals, and fused with myeloma cells to obtain hybridoma, from which human antibodies against a protein can be prepared (see W092-03918, W093-2227, W094-02602, W094-25585, W096-33735, and W096-34096).
Alternatively, an immune cell, such as an immunized lymphocyte, producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.
Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). A DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. The present invention also provides recombinant antibodies prepared as described above.
Furthermore, an antibody of the present invention may be a fragment of an antibody or modified antibody, so long as it binds to one or more of the proteins of the invention. For instance, the antibody fragment may be Fab, F(ab′)2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). More specifically, an antibody fragment may be generated by treating an antibody with enzymes such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J. Immunol. 152:2968-2976, 1994; Better et al., Methods Enzymol. 178:476-496, 1989; Pluckthum et al., Methods Enzymoi. 178:497-515, 1989; Lamoyi, Methods Enzymol. 121:652-663, 1986; Rousseaux et al., Methods Enzymol. 121:663-669, 1986; Bird et al., Trends Biotechnol. 9:132-137, 1991).
An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. These modification methods are conventional in this field.
Alternatively, an antibody of the present invention may be obtained as a chimeric antibody, between a variable region derived from nonhuman antibody and the constant region derived from human antibody; or as a humanized antibody, comprising the complementarity determining region (CDR) derived from nonhuman antibody, the frame work region (FR) derived from human antibody, and the constant region.
Obtained antibodies may be purified into homogeneity. An antibody used in the present invention can be separated and purified by conventional methods used for separating and purifying usual proteins. For example, the separation and purification of a protein can be performed by an appropriately selected and combined use of column chromatography, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988); however, the present invention is not limited thereto. The concentration of antibodies obtained above can be determined by measuring absorbance, by the enzyme-linked immunosorbent assay (ELISA), and so on.
Examples of columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns using protein A column include Hyper D, POROS, Sepharose F. F. (Pharmacia), etc.
In addition to affinity chromatography, the chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). The chromatographic procedures can be carried out by liquid-phase chromatography such as HPLC, FPLC, or the like.
For example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence may be used to measure the antigen binding activity of an antibody of the invention. In ELISA, an antibody of the present invention is immobilized on a plate, a protein of the invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated. Next, after washing, an enzyme substrate, such as ρ-nitrophenyl phosphate, is added to the plate, and the absorbance is measured to evaluate the antigen binding activity of the sample. A fragment of a protein, such as a C-terminal fragment, may be used as a protein BIAcore (Pharmacia) may be used to evaluate the activity of an antibody according to the present invention.
The above methods allow for the detection or measurement of the proteins of the invention, by exposing an antibody of the invention to a sample assumed to contain a protein of the invention, and detecting or measuring the immune complex formed by the antibody and the protein. Because the method of detection or measurement of proteins according to the invention can specifically detect or measure proteins, the method may be useful in a variety of experiments in which the protein is used.
The present invention provides a polynucleotide having at least 15 nucleotides that is complementary to the DNA that encodes the “YS68” protein (SEQ ID NO: 11 or 13) or the complementary strand thereof.
Herein, the term “complementary strand” is defined as one strand of a double strand DNA composed of A:T and G:C base pairs to the other strand. In addition, “complementary” is defined as not only those completely matching within a continuous region of at least 15 nucleotides, but also having a homology of at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or higher within that region. The homology may be determined using the algorithm described herein.
Probes or primers for detection and amplification of a DNA encoding a protein of this invention, or nucleotides or nucleotide derivatives for suppressing protein expression (for example, antisense oligonucleotides and ribozymes, or DNA encoding-them) are included in these polynucleotides. In addition, such polynucleotides may be also used for preparing DNA chips.
When used as a primer, the region on the 3′ side is designed to be complementary to a DNA encoding a protein of the invention, and restriction enzyme recognition sequence and tags can be added to the 5′ side.
For example, an antisense oligonucleotide that hybridizes with a portion of the nucleotide sequence of SEQ ID NO: 11 or 13 is also included in the antisense oligonucleotides of the present invention. An antisense oligonucleotide is preferably one against at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 11 or 13. More preferably, it is an antisense oligonucleotide having at least 15 continuous nucleotides that contains the translation initiation codon.
Derivatives or modified products of antisense oligonucleotides can be used as antisense oligonucleotides. Examples of such modified products are, lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphothioate modifications and phosphoamidate modifications.
The term “antisense oligonucleotides” as used herein means, not only those in which the entire nucleotides corresponding to those constituting a specified region of a DNA or mRNA are complementary, but also those having a mismatch of one or more nucleotides, so long as DNA or mRNA and an oligonucleotide can specifically hybridize with the nucleotide sequence of SEQ ID NO:11 or 13.
An antisense oligonucleotide derivative of the present invention has inhibitory effect on the function of a protein of the present invention as a result that the derivative inhibits the expression of the protein of the invention by acting upon cells producing the protein of the invention and by binding to the DNA or mRNA encoding the protein to inhibit its transcription or translation or to promote the degradation of the mRNA.
An antisense oligonucleotide derivative of the present invention can be made into an external preparation, such as a liniment and a poultice, by mixing with a suitable base material which is inactive against the derivatives.
Also, as necessary, the derivatives can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops, and freeze-drying agents and such by adding excipients, isotonic agents, solubilizing agents, stabilizers, preservative substance, pain-killers and such. These can be prepared by following usual methods.
An antisense oligonucleotide derivative is given to a patient by directly applying onto the ailing site or by injecting into a blood vessel so that it will reach the site of ailment. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples are, liposome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives of these.
The dosage of an antisense oligonucleotide derivative of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
An antisense oligonucleotide of the invention inhibits the expression of a protein of the invention and thereby is useful for suppressing the biological activity of the protein of the invention. Also, expression-inhibitors comprising an antisense oligonucleotide of the invention are useful in that they can inhibit the biological activity of a protein of the invention. It is thought that it is possible to use an antisense oligonucleotides of this invention for the purpose of suppressing biological activities of a protein of the invention.
A protein of the invention may be used for screening compounds binding to the protein. Specifically, a protein may be used in methods of screening for compounds comprising the steps of: (1) exposing a protein of the present invention to a test sample in which a compound binding to the protein is expected to be contained; (2) detecting the binding activity of the protein to the test sample; and (3) selecting the compound having the binding activity to the protein.
A protein of the present invention to be used for screening may be a recombinant protein, a protein derived from the nature, or partial peptide thereof. Alternatively, the protein may be in a form expressed on a cell surface or in a form of cell membrane fraction. Any test sample, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular compounds and naturally occurring compounds, can be used. A protein of the present invention to be contacted with a test sample can be contacted, for example, as a purified protein, a soluble protein, a form bound to a carrier, a fusion protein with another protein, a form expressed on cell membrane, or a cell membrane fraction.
By using a protein of the present invention, for example, in a method for screening for proteins binding to the protein thereof, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, the immunoprecipitation method, specifically, in the following manner. A gene encoding a protein of the present invention is expressed in a host cell, such as an animal cell, by inserting the genie into an expression vector for foreign gene, such as pSV2neo, pcDNA I, pCD8. As a promoter to be used for the expression, any promoter which can be generally used can be selected; for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, p. 83-141, 1982), the EF-1α promoter (Kim et al., Gene 91:217-223, 1990), the CAG promoter (Niwa et al., Gene 108:193-200, 1991), the RSV LTR promoter (Cullen Methods in Enzymology 152:684-704, 1987), the SRα promoter (Takebe et al., Mol. Cell. Biol. 8:466, 1988), the CMV immediate early promoter (Seed et al., Proc. Natl. Acad. Sci. USA 84:3365-3369, 1987), the SV40 late promoter (Gheysen et al., J. Mol. Appl. Genet. 1:385-394, 1982), the Adenovirus late promoter (Kaufman et al., Mol. Cell. Biol. 9:946, 1989), the HSV TK promoter, and so on may be used.
To express a foreign gene by introducing the gene into animal cells, the electroporation method (Chu et al., Nucl. Acid Res. 15:1311-1326, 1987), the calcium phosphate method (Chen et al., Mol Cell. Biol. 7:2745-2752, 1987), the DEAE dextran method (Lopata et al., Nucl. Acids Res. 12:5707-5717, 1984; Sussman et al., Mol. Cell. Biol. 4:1642-1643, 1985), the Lipofectin method (Derijard, Cell 7:1025-1037, 1994; Lamb et al., Nature Genetics 5:22-30, 1993; Rabindran et al., Science 259:230-234, 1993), and such can be exemplified, and any method can be used.
A protein of the present invention can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose property has been revealed, to N or C terminus of the protein of the present invention. A commercially available epitope-antibody system can be used (Experimental Med. 13:85-90, 1995). Through a multiple cloning site, a vector which can express a fusion protein with, for example, β-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), is available in the market.
Methods have been reported in which fusion proteins are prepared by introducing only small epitopes comprising several to a dozen of amino acids, so that the properties of the proteins of the present invention may not change by making the proteins fusion proteins. Epitopes, for example, polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), epitope such as E-tag (an epitope on monoclonal phage), and monoclonal antibodies recognizing these can be used as an epitope-antibody system for screening a protein binding to a protein of the present invention (Experimental Med. 13:85-90, 1995).
In the immunoprecipitation, an immune complex is formed by adding these antibodies to cell eluate prepared by using an appropriate detergent. This immune complex comprises a protein of the present invention, a protein having a binding affinity for the protein, and an antibody. Immunoprecipitation can be conducted by an antibody against a protein of the present invention, besides using antibodies against the above epitopes. An antibody against a protein of the present invention can be prepared, for example, by introducing a gene encoding the protein of the present invention into an appropriate E. coli expression vector; expressing the gene in E. coli; purifying the expressed protein; and immunizing animals, for example, rabbits, mice, rats, goats, domestic fowls, and such, with such protein. The antibody can be prepared also by immunizing the above animals against a synthesized partial peptide of a protein of the present invention.
An immune complex can be precipitated, for example, by Protein A Sepharose or Protein G Sepharose when the antibody is mouse IgG antibody. When a protein of the present invention is prepared as a fusion protein with an epitope, for example GST, an immune complex can be formed by using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B, in the same manner as in the use of an antibody against a protein of the present invention.
Popular Immunoprecipitation can be performed by following or according to, for example, the reference (Harlow, E. and Lane, D.: Antibodies pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988)).
SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the binding protein can be analyzed depending on the molecular weight of the protein by using gel with an appropriate concentration. In general, because it is difficult to detect a protein binding to a protein of the present invention by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing in a culture medium containing radioactive isomer, 35S-methionine or 35S-cystein, labeling proteins in the cells, and detecting the proteins. The target protein can be purified from the SDS-polyacrylamide gel and its sequence can be determined directly alter the molecular weigh of the protein is determined.
The present inventors have detected multiple proteins that bind to a protein of this invention by immunoprecipitation in the Example (Example 4).
To isolate proteins that bind to a protein of the present invention by using the protein, for example, West western blotting (Skolnik et al., Cell 65:83-90, 1991) may be used. More specifically, it is conducted as follows: (1) constructing a cDNA library using a phage vector (λgt11, ZAP, etc.) from cells, tissues, and organs (for example, AGM region and yolk sac during early development; thymus, spleen, and liver during mid to late development, and such) that are expected to express binding proteins that bind to the protein of this invention; (2) expressing the cDNA library on LB-agarose and immobilizing the expressed protein onto a filter; (3) reacting the purified and labeled protein of this invention with the filter; and (4) detecting the plaque expressing the protein that binds to the protein of this invention by the label. Methods to label a protein of this invention may be a method that utilizes the binding characteristics of biotin and avidin; a method utilizing antibodies that bind specifically to the protein of this invention or to peptides or polypeptides fused to the protein of this invention (for example GST and such); a method that utilizes radioisotopes; a method that utilizes fluorescence; and such.
Further, another embodiment of the screening method of this invention is exemplified by a method utilizing the two-hybrid system using cells (Fields et al., Trends. Genet. 10:286-292, 1994; Dalton et al., Cell 68:597-612; “MATCHMAKER Two-Hybrid System”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (all manufactured by Clonetech); and “HybriZAP Two-Hybrid Vector System” (manufactured by Stratagene)). In the two-hybrid system, a protein of this invention or a partial peptide thereof may be fused to the DNA binding region of SRF or GAL4, and expressed in yeast. A cDNA library is constructed from cells predicted to express proteins that bind to the protein of this invention, wherein the cDNA library is constructed in such a way that the proteins are expressed as fusion proteins with transcription activation regions of VP16 or GAL4. The cDNA library is transfected into the above yeast, and then positive clones are detected to isolate the cDNA derived from the library (expression of a protein that binds to the protein of the invention in yeast leads to the binding of the two proteins, and results in the activation of the reporter gene, which allows to detect positive clones). The protein encoded by the isolated cDNA may be obtained by introducing the cDNA into E. coli and expressing it therein. Thus, it is possible to prepare proteins that bind to a protein of this invention and genes encoding them. The reporter gene used in the two-hybrid system may be such as Ade2 gene, Lac Z gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor type 1) gene, and such besides HIS3 gene, but are not limited to these examples.
A protein binding to a protein of the present invention can be screened using affinity chromatography. For example, a preferred method for screening of the present invention utilizes affinity chromatography. A protein of the invention is immobilized on a carrier of an affinity column, and a test sample, in which a protein capable of binding to the protein of the invention is supposed to be expressed, is applied to the column. A test sample herein may be, for example, cell extracts, cell lysates, etc. After loading the test sample, the column is washed, and proteins bound to the protein of the invention can be prepared.
The amino acid sequence of the obtained protein is analyzed, an oligo DNA was synthesized based on the sequence, and cDNA libraries are screened using the DNA as a probe to obtain a DNA encoding the protein.
A biosensor using the Surface Plasmon Resonance phenomenon may be used as a means for detecting or quantifying the bound compound in the present invention. When such a biosensor is used, the interaction between a protein of the invention and a test compound can be observed in real-time as a surface plasmon resonance signal, using only a minute amount of proteins without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between a protein of the invention and a test compound using a biosensor such as BIAcore.
Methods of screening molecules that bind when an immobilized protein of the present invention is exposed to synthetic chemical compounds, natural substance banks, or a random phage peptide display library, and methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273:458-64, 1996; Verdine, Nature 384:11-13, 1996; Hogan, Jr., Nature 384:17-9, 1996) are well known to those skilled in the art as methods for isolating not only proteins but also chemical compounds that bind to a protein of the present invention (including agonist and antagonist).
Compounds that bind to a protein of this invention serve as drug candidates for promoting or inhibiting the activity of the protein of this invention, and may be applied to treatment of diseases caused by expressional or functional abnormalities of the protein of this invention, or diseases that may be treated by regulating the activity of the protein of this invention. Compounds obtained by using the screening method of this invention, wherein the structure of compounds having binding activity toward a protein of this invention is partially altered by addition, deletion, and/or replacement, are also included as compounds that bind to a protein of this invention.
When a compound binding to a protein of the present invention is used as a pharmaceutical for humans and other mammals, such as, mice, rats, guinea pigs, rabbits, chicken, cats, dogs, sheep, pigs, bovines, monkeys, baboons, chimpanzees, the isolated compound can be administered not only directly, but also as dosage forms using known pharmaceutical preparation methods. For example, according to the need, the drugs can be taken orally as sugarcoated tablets, capsules, elixirs and microcapsules; or non-orally in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with pharmacologically acceptable carriers or medium, specifically, sterilized water, physiological saline, plant-oil, emulsifiers, suspending agent, surface-active agent, stabilizers, flavoring agents, excipients, vehicles, preservatives and binders, into a unit dose form required for generally accepted drug implementation. The amount of active ingredient in these preparations makes a suitable dosage within the indicated range acquirable.
Examples of additives which can be mixed to tablets and capsules are, binders such as gelatin, corn starch, tragacanth gum and gum acacia; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier such as oil can also be included in the above ingredients. Sterile composites for injection can be formulated following normal drug implementations using vehicles such as distilled water used for injections.
Physiological saline, glucose, and other isotonic liquids including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injections. These can be used in conjunction with suitable solubilizers, such as alcohol, specifically ethanol; polyalcohols such as propylene glycol and polyethylene glycol; and non-ionic surfactants such as Polysorbate 80 (TM) and HCO-50.
Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as solubilizers; they further may be formulated with a buffer such as phosphate buffer and sodium acetate buffer, a pain-killer such as procaine hydrochloride, a stabilizer such as benzyl alcohol and phenol, and an anti-oxidant. The prepared injection may be filled into a suitable ampule.
Methods well known to one skilled in the art may be used to administer the pharmaceutical compounds of the present invention to patients, for example as intraarterial, intravenous, percutaneous injections and also as intranasal, transbronchial, intramuscular percutaneous, or oral administrations. The dosage varies according to the body-weight and age of a patient and the administration method, but one skilled in the art can suitably select them. If the compound can be encoded by a DNA, the DNA can be inserted into a vector for gene therapy to perform the therapy. The dosage and method of administration vary according to the body-weight, age, and symptoms of a patient, but one skilled in the art can select them suitably.
Although there are some differences according to the symptoms, the dose of a compound that binds with a transcriptional regulatory factor of the present invention and inhibits its activity is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).
When administering parenterally in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also, in the case of other animals too, it is possible to administer an amount converted to 60 kgs of body-weight or an amount converted to body surface.
All publications and patents cited herein are incorporated by reference in their entirety.



DESCRIPTION OF DRAWINGS
FIG. 1 depicts photomicrographs indicating the localization of YS68 within cells. YS68 tagged with a flag epitope is expressed in COS7 cells, and upon staining with anti-Flag antibodies, the expression sites of YS68 were investigated (right). In addition, the same cells were treated with Hoechst to selectively stain the nucleus (left).
FIG. 2 depicts photographs demonstrating the result of electrophoresis showing the expression distribution of YS68 in tissues. RNA was prepared from liver, thymus, or spleen tissues of an embryonic day 14 (E14) or embryonic day 18 (E18) mouse embryo, respectively, or from the tissues of an adult mouse to perform Northern hybridization. The lower panel shows 18S ribosomal RNA before blotting as a control.
FIG. 3 depicts photographs demonstrating the result of electrophoresis showing the result of analyzing YS68 expression by RT-PCR in the yolk sac at each stage of a developing embryo.
FIG. 4 depicts photographs demonstrating the result of electrophoresis showing the result of analyzing YS68 expression by RT-PCR in the AGM region at each stage of a developing embryo is shown in (A); and in (B) the E10.5 AGM region was cultivated in the presence or absence of oncostatin M (OSM), and RNA was prepared on the 5th day of cultivation. Expression of YS 68 was then compared to those of uncultivated AGM region by RT-PCR.
FIG. 5 depicts photographs demonstrating the result of electrophoresis showing the result of comparison of the expression level of YS68 by RT-PCR upon extraction of RNA from liver, thymus and spleen of embryonic (E11.5 to E16.5), 7-day-old, and adult mice, respectively.
FIG. 6 depicts photographs showing the result of in situ hybridization on slices prepared from an E11.5 embryo. A is an autoradiogram, and B is an image obtained by staining the same slice by hematoxylin. Li: liver.
FIG. 7 depicts photographs showing the result of in situ hybridization on slices prepared from an E14.5 embryo. A and C are autoradiograms, while B and D are images obtained by staining the same slices by hematoxylin. Li: liver, Lu: lung, Th: thymus, and N: neural tube.
FIG. 8 depicts a comparison of the amino acid sequences between human (SEQ ID NO: 14) and mouse YS68 (SEQ ID NO: 12).
FIG. 9 depicts the comparison of the amino acid structures of human and mouse YS68.
FIG. 10 depicts a photograph showing the result of analysis on proteins that coprecipitate with YS68. After primary cultivation of E14.5 liver, cell lysate was prepared. Then, the lysate was subjected to immunoprecipitation with anti-YS68 antibody and protein A (Lane 1), rabbit IgG and protein A (Lane 2), and protein A alone (Lane 3). Following SDS-PAGE, the gel was visualized by silver staining. Arrow: YS68; and *: protein that coprecipitated with YS68.
FIG. 11 depicts photographs showing the result of immunostaining of YS68 in tissues. The dorsal aorta (A, B, C, D, and E), the umbilical artery (F) of an E11.5 mouse; and the blood vessels within an E9 yolk sac (H) were stained with erythroid marker TER119 (A, B, and G) and with anti-YS68 antibody (C, D, E, and H). B and D are enlargements of A and C, respectively, and E shows a different view of the aorta. The site where the hematocyte is budding from the vascular endothelium is indicated by an arrow.
FIG. 12 depicts photographs showing the result of staining primary culture cells of E14.5 liver with anti-YS68 antibodies (A), or with rabbit IgG (B). The expression of YS68 was strong at the nucleus and around the nucleus.
FIG. 13 depicts photographs showing the result of investigation on the expression of YS68 in hematocytes isolated from E14 liver. The Giemsa stained hematocytes of the liver (A); hematocytes of the E14.5 liver (B); CD34 negative cells (C); and CD34 positive cells (D) were stained with anti-YS68 antibodies. Whether the sorted cells are CD34 positive or not was confirmed (E-H). E-F and G-H are taken from the same views, E and G are fluorescence photographs, and F and H are visual photographs. Most of the cells sorted by CD34 were weakly CD34 positive to strongly positive (E and F). Cells that passed through the CD34 column were hardly expressing any CD34 (G and H).
FIG. 14 depicts photographs showing the localization of YS68 within cells. A slightly magnified photograph is shown on the left, and a largely magnified photograph is shown on the right. Cells derived from fetal liver were stained with anti-YS68 antibodies to investigate endogenous expression sites of YS68 (top row). In addition, pEFBOSE-F-YS68 (5-1148) that expresses the N-terminal region of YS68 (middle row), or pEFBOSE-F-YS68 (981-2243) that expresses the C-terminal region of YS68 (bottom row) were transfected to COS7 cells, and these cells were stained with anti-Flag antigens to investigate the localization within the cell.



DETAILED DESCRIPTION
The present invention will be described specifically by way of examples below, however this invention is not restricted in any way to these examples.
Example 1
Isolation of YS68 Gene
To obtain molecules that are expressed specifically in hemangioblasts, an experiment was carried out in which cDNA of an E14 yolk sac was subtracted from the cDNA of an E9 yolk sac Poly A RNAs were purified from each of the E9 and E14 yolk sacs, respectively; then PCR-Select cDNA Subtraction Kit (Clonetech) was used for the subtraction. The obtained cDNA fragments were subcloned into pGEM-T vectors (Promega), and then, after selecting highly expressed cDNAs in E9 yolk sacs by dot blotting, selected cDNA were sequenced. The clone #68 was a novel gene fragment that was not registered in the database. Thus, a primer was designed from the sequence of this gene fragment, and using mouse 15-day Embryo Marathon-Ready cDNA (Clonetech) as a template, a full-length cDNA was isolated by the 5′-RACE method. Mouse YS68 encodes 1,265 amino acids, but is expected to have further upstream sequence.
The obtained YS68 did not have a characteristic motif within its amino acid sequence. However, existence of multiple nuclear transport signals was confirmed. Consequently, YS68 was anticipated to be a protein that functions in the nucleus. Therefore, to confirm the hypothesis, a vector (pEFBOSE-Flag (Nakashima et al., FEBS Let. 403:79-82, 1997) that expresses the mouse YS68 protein (1265 amino acids) tagged with Flag was transfected to COS7 cells. After 24 hours, the cells were fixed with 4% formalin, and was treated with 0.1% Triton-X 100. Then, this was reacted with anti-Flag antibodies, followed by FITC-labeled anti-mouse IgG, and was observed through a fluorescence microscope. Consequently, expression of YS68 was strong in the nucleus, as expected (FIG. 1). Since the cell nucleus is the site where DNA transcription occurs, YS68 is anticipated to be a transcription factor involved with DNA transcription.
Human YS68 gene was isolated by 5′-RACE and 3′-RACE by designing a primer based on the genetic sequence of mouse YS68. More specifically, based on the genetic sequence of mouse YS68, EST fragments that are thought to be YS68 homologues in humans were searched in the EST database. Primers were designed based on this EST fragment, and using human fetal liver Marathon-Ready cDNA (Clonetech) as a template, the 5′ region and the 3′ region cDNA were isolated by 5′-RACE and 3′-RACE according to the instructed procedure. The isolated cDNA nucleotide sequence is described in SEQ ID NO:11, and the amino acid sequence of the protein encoded by this cDNA is described in SEQ ID NO:12. A comparison of human and mouse YS68 amino acid sequences is shown in FIG. 8.
Example 2
Expression Pattern Analysis of YS68
The expression distribution of YS68 within tissues was analyzed by Northern blotting. Total RNA was prepared from each tissues of embryonic or adult mice using ISOGEN (Wako). 25 μg/lane of these samples were electrophoresed. After blotting onto a nylon membrane, hybridization was performed with YS68 cDNA fragments labeled with 32P. Hybridization was performed in ExpressHyb solution (Clonetech) at 68° C. for 2 hours; then, after several washings with 2×SSC and 0.1% SDS at room temperature, followed by several washings with 0.1×SSC and 0.1% SDS at 65° C., autoradiography was performed.
The expression of YS68 in adult tissue was the strongest in testis, followed those in kidney and lung. Observation of YS68 expression in hematopoietic tissues showed that expression was very strong in liver, thymus and spleen that function as hematopoietic tissues during the embryonic stage. However, expression in these tissues rapidly decreased or was absent in those of adult (FIG. 2).
Further, the expression pattern in tissues known to be involved in primitive hematopoiesis was investigated in detail. The site of hematopoiesis is known to shift during the embryonic stage as described below from previous studies. First, primitive hematopoiesis starts in the yolk sac at E8, and definitive hematopoiesis begins later in the AGM region at E10.5. Hematocytes that developed in AGM are immediately transported to liver that is formed around E11.5, then differentiate and proliferate at this site until immediately after birth. Meanwhile, hematopoiesis begins to take place in thymus and spleen that are formed around E16.5. After birth, the site of hematopoiesis changes to bone marrow. Based on these facts, the expression pattern of YS68 in these tissues was analyzed in further detail by RT-PCR. Total RNA was extracted from each tissue of mouse embryos at each developmental stage, or an adult mouse; and 1 μg of each total RNA was reverse transcribed to cDNA using SUPERSCRIPT II preamplification system (Gibco). This was used as a template and a YS68-specific primer (68 3: 5′-CACCCGTGAAGAAACAAATAGGCA-3′/SEQ ID NO:3, 68 4: 5′-CCTTTGGTACATGAGCTTCTATTT-5′/SEQ ID NO:4) or a G3PDH-specific primer was used to perform PCR (25 cycles of 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C. for 30 seconds). Then was electrophoresed on 1% agarose gel, and the gel was stained with ethidium bromide.
Expression of YS68 decreased gradually in the yolk sac, as development proceeded (FIG. 3). Against expectations, expression of YS68 was low in the AGM region at E10.5, when definitive hematopoiesis begins (FIG. 5A). On the other hand, in liver, thymus, and spleen known to function as sites for hematopoiesis in the embryonic stage, expression of YS68 was very high (FIG. 4) and correlated to the period when these tissues function as hematopoietic organs.
Furthermore, the expression distribution of YS68 in mouse embryo was analyzed by in situ hybridization. A vector constructed by inserting a 545 bp cDNA of the 5′-region of YS68 (positions 898 to 1443) into pBluescript II was used as a template to perform in vitro transcription using T7 RNA polymerase or T3 RNA polymerase (Boeringer Mannheim), and to synthesize sense or antisense 35S-labeled RNA, respectively. The mouse embryo was removed and frozen to produce slices using a cryostat. After immobilization and acetylation with 4% paraformaldehyde/PBT, hybridization was performed overnight at 55° C. with the above-mentioned RNA probe. After treating the reaction solution with RNase A, it was washed several times and autoradiography was performed.
The expression of YS68 was the strongest in liver at E11.5 (FIG. 6). YS68 was mainly strongly expressed in liver and in the developing thymus, and expression was also confirmed in lungs and neural tube at E14.5 (FIG. 7).
These results indicate that the expression of YS68 is localized in tissues where active hematopoiesis takes place in a period-specific manner, and strongly suggests that YS68 is a molecule involved in primitive hematopoiesis. Its expression was low in the E10.5 AGM region, which is thought to be the site of development for hematopoietic cells. However this may be due to the absolute number of cells involved in hematopoiesis within the entire AGM region, which is not so high. In fact, Suda et al. revealed that the percentage of hemangioblasts in the AGM region at E10.5 is 5% or less using TEK as a marker for hemangioblasts (Hamaguchi et al., Blood 93:1549-1556, 1999). On the other hand, when E10.5 AGM region is dispersed and cultivated on a dish, the emergence of hematocytes can be confirmed around the 5th day of cultivation (Mukouyama et al., Immunity 8:105-114, 1998). Interestingly, the expression of YS68 had increased in AGM derived cells cultivated for 5 days (FIG. 4B). According to these results, the expression of YS68 is expected to rise in cells that have acquired hematopoietic ability, or in immature hematocytes.
Example 3
Full-Length Cloning of Mouse and Human YS68
Using primers constructed from the YS68 gene sequence obtained so far, 5′-RACE was performed using the mouse 15-day Embryo Marathon-Ready cDNA and human fetal liver Marathon-Ready cDNA (Clonetech) as templates, to clone the upstream 5′ region of mouse and human YS68 gene. Full-length human and mouse cDNA sequences were determined by repeating this 5′-RACE protocol.
Consequently, human and mouse YS68 were anticipated to encode 2,266 and 2,243 amino acids, respectively (FIG. 9). Comparing the human (SEQ ID NO: 14) and mouse (SEQ ID NO: 12) amino acid sequences, interestingly, the N-terminal region (human 1-1137 of SEQ ID NO: 14, mouse 1-1137 of SEQ ID NO: 12) had a very high homology of 87%; whereas the homology in the central region (human 1138-1683 of SEQ ID NO: 14, mouse 1138-1679 of SEQ ID NO: 12) was 57%, and that in the C-terminal region (human 1684-2266 of SEQ ID NO: 14, mouse 1680-2243 of SEQ ID NO: 12) was very low showing a homology of 45%. In the C-terminal region with low homology, many nuclear transport signals existed. On the other hand, in the N-terminal region with high homology, two WD repeats existed, which repeats are known to be necessary for interaction among proteins. Since the homology in this region is very high between humans and mice, this region is anticipated to be important for the function of YS68.
Example 4
Proteins Binding to YS68
It was expected that YS68 is bound to some protein in vivo because a protein-binding site (WD repeats) exists in the N-terminal region of YS68. Therefore, cell lysate was prepared from cultivated cells of embryonic liver and immunoprecipitation with anti-YS68 antibody was performed. Then, SDS polyacrylamide gel electrophoresis was performed to investigate whether a protein that coprecipitates with YS68 exists. Specifically, cultivated mouse liver cells at E14.5 were solubilized with lysis buffer (0.5% NP-40, 10 mM Tris-HCl pH7.6, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml aprotinin). After incubation overnight at 4° C. with anti-YS68 antibodies, protein G was added and was further incubated for 1 hour. SDS polyacrylamide gel electrophoresis was conducted after immunoprecipitation, and the gel was stained with silver.
Consequently, existence of multiple molecules that coprecipitate with YS68 within cells of embryonic liver was confirmed (FIG. 10). This suggested that YS68 functions by binding to several types of proteins within the cell.
Example 5
Expression Site of YS68 within Tissues
For detailed analysis of the YS68 expression site, the YS68 protein was used to immunize rabbits to produce polyclonal antibodies against YS68. The protein encoding the 1208-1482 amino acid region of mouse YS68 was expressed in E. coli, was purified according to standard procedures, and was used as the antigen in the production of YS68 polyclonal antibodies. Immunization was carried out on rabbits (New Zealand White, 2.5 kg, female) using 200 μg antigen for 1 immunization, with an interval of 10 days for 4 immunizations. Then upon collection of whole blood, antiserum was obtained. Furthermore, an affinity column with immobilized antigens was prepared, and anti-YS68 polyclonal antibodies were purified from the antiserum.
Using these antibodies, the expression site in the AGM region of E11.5 embryo was investigated by immunostaining. Immunostaining was conducted as follows. First, slices of frozen mouse embryo were prepared using a cryostat (Leica). This was immobilized with 4% formaldehyde and was treated with methanol. After treatment with 0.3% aqueous hydrogen peroxide, blocking was carried out with 3% BSA, then upon reaction with primary antibodies overnight at 4° C. and with secondary antibodies (HRP-labeled anti-rabbit IgG) at room temperature for 1 hour, washing was repeated 3 times with PBS, and visualization was accomplished by the addition of substrate (Metal Enhanced DAB substrate kit, Pierce).
Consequently, the hematocytes existing in the endothelium were stained using red blood cell marker TER119 (used as a control; FIG. 11A, B), whereas, the vascular endothelium was stained specifically using anti-YS68 antibody (FIGS. 11C, D, and E). Interestingly, YS68 was darkly stained in the hematocytes emerging from the endothelium cells (FIG. 11E, arrow). In addition, strong expression of YS68 was indicated in the vascular endothelium of the umbilical vein (FIG. 11F). In contrast to TER119, which selectively stained hematocytes in the blood vessel, YS68 expression was stronger in vascular endothelium than in hematocytes in E9.5 yolk sacs (FIGS. 11G and H).
Example 6
Expression of YS68 within Cells
A liver was surgically removed from an embryo (E14.5), cut into small pieces with tweezers, and incubated in cell dissociation buffer (Gibco) at 37° C. for 30 minutes. The cells were further treated with 0.1% collagenase at 37° C. for 1 hour, and were loosened by pipetting. After washing several times with PBS, the cells were suspended in DMEM containing 10% FCS, and were cultivated on a 10-cm dish.
To investigate the localization of endogenous YS68 within cells, cultured hepatic cells were stained with anti-YS68 antibodies. First, the cells were fixed with 4% formalin, and then treated with 0.1% Triton-X 100 for cell staining. Next, cells were reacted with the primary antibodies, and then with secondary antibodies. The cells were visualized in the same manner as in Example 5.
Consequently, although YS68 has multiple nuclear transport signals, strong expression was found not only in the nucleus, but also around the nucleus, which expression depended on cells (FIG. 12). Next, similar analysis for the expression in hematocytes was carried out. YS68 expression in hematocytes separated from embryonic liver was found to have varied strengths of expression depending on the cell type (FIG. 13B).
Therefore, the group of hematocytes was sorted using CD34, which is a marker for immature hematocytes, and YS68 expression in CD34-positive cells was investigated. To collect CD34-positive cells, embryonic liver (E14.5) was incubated in a dissociation buffer at 37° C. for 30 minutes, and then the cells were dissociated by pipetting in PBS. After passing through a nylon mesh filter (Falcon), the cells were suspended in a sample buffer (0.5% BSA, 2 mM EDTA in PBS). The cells were reacted with biotin labeled anti-CD34 antibodies (Pharmingen), followed by FITC labeled streptavidin at 4° C., and then were incubated with anti-FITC microbeads. CD34 positive cells were eluted using MACS (Magnetic Cell Sorting) column, according to the instructed protocol. The cells were centrifuged on a slide glass at 400 rpm for 5 minutes to fix them onto the slide glass. Cell staining was performed in the same manner as described above.
Consequently, hematocytes that were concentrated using anti-CD34 antibodies (FIG. 12D) showed a higher expression of YS68 compared to hematocytes that passed through the CD34 column (FIG. 12C). Therefore, YS68 expression is anticipated in less differentiated CD34 positive hematocytes.
Example 7
Localization of Each Domain of YS68 within Cells
Using cDNA prepared from mouse embryonic liver as a template, cDNA encoding the N-terminal region (amino acids 5-1148) and C-terminal region (amino acids 981-2243) of mouse YS68 (SEQ ID NO: 12) were amplified by PCR. The amplified cDNAs were inserted downstream of the Flag region of animal cell expression vector pEFBOSE-F to produce pEFBOSE-F-YS68(5-1148) and pEFBOSE-F-YS68(981-2243) that expresses the N-terminal region of YS68 and the C-terminal region of YS68 (SEQ ID NO: 12), respectively. The expression vectors were then transfected into COS-7 cells using lipofectamine 2000 (Gibco), and 24 hours later, the cells were immobilized with methanol. To investigate the localizations of each YS68 expressed within the cells, the cells were reacted with anti-Flag antibody, followed by peroxidase-labeled anti-mouse IgG, and finally substrate was added for visualization.
Due to the multiple nuclear transport signals in the YS68 C-terminal region (FIG. 9), localization of YS68 in the nucleus was anticipated; however, endogenous YS68 was localized not only in the nucleus but also around the nucleus (FIG. 12). Additionally, constructs lacking the YS68 N-terminal region or the C-terminal region were prepared and were expressed in COS cells, and their localizations were investigated. The results confirmed that YS68 lacking the C-terminal region had strong tendency to localize in the cytoplasm, and YS68 lacking the N-terminal region in the nucleus (FIG. 14). These results suggested the possibility that the N-terminal region is inhibiting the transfer of YS68 into the nucleus. Since two WD repeats necessary for protein interaction exist in the N-terminal region, it was speculated that binding of this region to some molecule might possibly inhibit the transfer into the nucleus.
INDUSTRIAL APPLICABILITY
The present invention provides novel “YS68” proteins predicted to be involved in primitive hematopoiesis and genes encoding the proteins. The genes may be utilized as markers for hematopoietic cells involved in primitive hematopoiesis and as factors regulating hematopoiesis. In addition, they may be utilized for purification and cloning of new factors involved in hematopoiesis, and even as tools for drug development for various diseases arising due to abnormalities in expression of the genes of this invention caused by abnormalities in expression regulation in vivo. Further, the “YS68” genes of this invention may be involved in blood tumors. Therefore, drug development against tumors utilizing the proteins of this invention is anticipated. By designing medicaments that target the genes of this invention, development of drugs that have new mechanisms of action may be enabled. Proteins and genes derived from humans are especially preferred in drug development compared to those derived from other organisms

## Claims

1. An isolated DNA comprising a sequence encoding a polypeptide that comprises the amino acid sequence of SEQ ID NO:14.

2. The DNA of claim 1, wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:14.

3. An isolated DNA comprising the coding sequence of SEQ ID NO: 13.

4. An isolated DNA comprising the nucleotide sequence of SEQ ID NO: 13.

5. A vector comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14.

6. The vector of claim 5, wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:14.

7. A vector comprising the coding sequence of SEQ ID NO:13.

8. A vector comprising the nucleotide sequence of SEQ ID NO:13.

9. An isolated host cell harboring the vector of claim 5.

10. An isolated host cell harboring the vector of claim 6.

11. An isolated host cell harboring the vector of claim 7.

12. An isolated host cell harboring the vector of claim 8.

13. A method of producing a polypeptide, the method comprising:
(a) culturing a host cell expressing a vector encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14; and
(b) recovering the polypeptide from the host cell or from the culture supernatant thereof.

14. The method of claim 13, wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:14.

15. A method of producing a polypeptide, the method comprising:
(a) culturing a host cell expressing a vector that comprises the coding sequence of SEQ ID NO:13; and
(b) recovering the polypeptide encoded by SEQ ID NO:13 from the host cell or from the culture supernatant thereof.

16. The method of claim 15, wherein the vector comprises the nucleotide sequence of SEQ ID NO:13.