Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1996-06-05
2000-05-02
Mertz, Prema
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
5303871, 5303879, 5303881, 5303892, 4241301, 4241391, 4241451, C07K 1624, A61K 39395
Patent
active
060574275
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
DESCRIPTION OF THE BACKGROUND
Mammalian cell growth, differentiation, and migration are directed by hormones and specific protein ligands, often termed cytokines. In particular, cells comprising the neuroendocrine, hematopoietic and the immune/inflammatory systems are known to be governed by cytokines. Cytokines, like other ligands, interact with cells by means of specific receptors, usually expressed on the cell surface.
A fundamental problem confronting biomedical scientists is to discern how signals are transduced through ligand receptors and how these signals determine the response of the cell. Many ligands influence their target cells by stimulating the expression of specific genes. However, the genes signaled by most cytokines remain largely unknown owing to the complexity of cellular biochemistry. Moreover, the gene products that are vital for performing different cellular processes are often only expressed transiently, and/or in very low concentrations so that they are difficult to detect, isolate and characterize.
Interleukin-2 (IL-2) is a cytokine that is critical for the immune system: it directs the proliferation and differentiation of T lymphocytes (T-cells), B lymphocytes (B-cells), and natural killer (NK) cells. Just how IL-2 signals these cellular events in the various types of target cells remains unknown. A few genes have been identified that are expressed as a result of IL-2 stimulation of T cells. These include the cellular protooncogenes c-fos, c-myb, c-myc, pim-1, and c-raf-1. However, exactly how many and what other genes are expressed as a result of IL-2/IL-2 receptor interaction remains unknown.
Since the discovery of DNA cloning, methods have become available to isolate specific genes expressed by cells. However, it has been difficult to devise new methods to isolate and clone all or most of the genes expressed by a cell activated by a given ligand, a task that must be done before one can understand how the ligand directs the cell to perform specific functions. In addition, methods of identifying a particular gene or genes stimulated early on after ligand receptor activation have not been easily forthcoming as the number of genes expressed constitutively is usually quite large, while those genes induced by the ligand are usually quite small. i.e., about 100 genes out of a total of about 10,000 genes.
Therefore, what is needed are methods to select and enrich only for those genes stimulated by a given ligand. Ideally, these methods should detect those genes expressed in low concentrations, as well as those expressed at high concentrations.
SUMMARY OF THE INVENTION
This invention pertains to complementary deoxyribonucleic acid (cDNA) libraries enriched in clones containing genes induced by ligand stimulation of a cell having a corresponding receptor for the ligand, and to methods of producing the same. This invention also relates to the genes which are expressed immediately or early on as a consequence of such a ligand-receptor interaction, and to methods of identifying these genes. In accordance with the method of the invention, a cDNA library highly enriched in ligand-inducible genes is produced by activating a cellular receptor with a ligand to induce the expression of genes as a result of ligand-receptor binding, reverse transcribing these RNA, and differentially probing the cDNA and selecting clones that bind to induced cDNA, but not to uninduced cDNA. Useful ligands include any of those which can activate a specific cellular receptor, including natural or synthetic ligands for the receptor, e.g., cytokines such as the interleukins, cellular growth factors, colony stimulating factors, hormones, peptides, antibodies, and receptor-binding fragments thereof.
In one embodiment, the present invention relates to a cDNA library (ies) of nucleic acids induced by a specific ligand (s) and/or all redundant DNA sequences encoding the CR proteins, homologues and fragments, to a vector (s) carrying the library (ies), and to transfected cells carrying the h
REFERENCES:
Riyer et al. Glossary of Genetics 2 Cytogenetics, pp. 16-19, Springer-Verlag, Berlin Heidelberg N.Y. 4th edition, 1976.
PCT International Search Report.
Dickinson, L.A., et al., "A Tissue-Specific MAR/SAR DNA-Binding Protein with Unusual Binding Site Recognition", Cell, 70: 631-645 (Aug. 21, 1992).
Regan, J.W., et al., "Cloning of a Novel Human Prostaglandin Receptor with Characteristics of the Pharmacologically Defined EP.sub.2 Subtype", Molec. Pharmacol., 46: 213-220, (1994).
Abdollahi, A., et al.,"Sequence and expression of a cDNA Encoding MyD118: a novel myeloid differentiation primary response gene induced by multiple cytokines", Oncogene, 6: 165-167, (1991).
Siderovski, D.P., et al., "A Human Gene Encoding a Putative Basic Helix-Loop-Helix Phosphoprotein Whose mRNA Increses Rapidly in Cycloheximide-Treated Blood Mononuclear Cells", DNA and Cell Biology, 13(2):125-147, (1994).
Yoshimura, A., et al., "A novel cytokine-inducible gene CIS encodes an Shr-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors", The EMBO Journal, 14(12):2816-2826, (Nov. 1995).
Beadling Carol
Smith Kendall A.
Mertz Prema
Trustees of Dartmouth College
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