Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-08
2002-09-03
Carlson, Karen Cochrane (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023500, C435S006120, C435S069100, C435S069700, C435S320100, C435S007230, C530S350000, C530S300000, C530S324000, C530S387700, C530S389700, C424S185100, C424S136100, C514S002600, C514S012200, C514S013800, C514S014800, C514S008100
Reexamination Certificate
active
06444801
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the field of genetic engineering. For example, the protein of the present invention can be used in the field of pharmaceuticals as a therapeutic agent.
BACKGROUND ART
A gene is transcribed by forming a transcription initiation complex including RNA polymerase bound to the promoter region upstream to the gene. Gene transcription is considered to be regulated mainly by transcriptional regulatory factors binding to the promoter and interactive with the transcription initiation complex. In general, a transcriptional regulatory factor comprises a DNA binding region and a transcriptional activation region. The transcriptional activation region is considered to be involved in the interaction.
The relation between the expression of specific genes and various diseases has been revealed in the field of medical treatment. For example, the expression of cytokine genes, such as TNF (Molecular Medicine 33: 1010-1020 (1996)), IL-1 (Clin. Immunol. 27: 18-28 (1995)), and IL-8 (Clin. Immunol. 27: 80-85 (1995)), are reported to be associated with various diseases, including inflammation. Hyperactivated transcription of IL-2 is involved in the immune diseases such as graft rejection.
Furthermore, diseases for which there is currently no effective treatment, such as virus infections including cancer and AIDS, can be controlled if expression of a responsible cancer gene (such as c-Myc) and virus gene is inhibited.
Such diseases can be treated if the transcriptional regulatory factor involved in the disease is isolated and its function is controlled. Therefore, transcriptional regulatory factors drew much attention as targets for developing new drugs.
An inhibitor of a transcriptional regulatory factor has been screened by a DNA binding inhibitory experiment in a chemical library or natural substances derived from bacteria or plants, or by drug design based on the structure of a gene or a transcriptional factor to be controlled (Peterson, M. G. et al., Trends Biotechnol. 11: 11-18 (1993).
The DNA binding inhibitory experiment requires screening numerous samples. However, there is no effective method of screening at present.
Furthermore, drug design requires that the structure of transcriptional regulatory factors must be known in detail. Therefore, its application is limited.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a transcriptional regulatory factor having transcription-inhibitory activity, more specifically, a transcriptional regulatory factor having transcription-inhibitory activity on a specific promoter.
Normal animal cells contain hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), thus, uptake 6-thioguanine (6-TG) in their nucleic acid synthetic pathway. Those cells die in a 6-TG-containing medium due to its toxicity. In contrast, HGPRT-deficient mutant cells survive and proliferate in the 6-TG-containing medium, unaffected by the toxicity because those cells do not uptake 6-thioguanine (6-TG) in the nucleic acid synthetic pathway. Therefore, in the HGPRT-deficient mutant cells, the survival of the cells in the presence of 6-thioguanine (6-TG) can be determined by controlling the expression of HGPRT (or a protein with a similar function). The present inventors constructed the system to search for a gene involved in intracellular signal transduction, using the above characteristics of the HGPRT-deficient mutant cells.
It is evident that a promoter of the interleukin 8 gene (IL-8) is activated in response to stimulation by a tumor necrosis factor (TNF). The present inventors prepared an expression plasmid carrying a xanthine-guanine phosphoribosyl-transferase (gpt) gene which causes cell death when expressed in the presence of 6-TG similarly to HGPRT, by inserting the gene downstream to the IL-8 promoter sequence in the plasmid vector, and introduced the resultant plasmid into HGPRT-deficient cells. The cells die in the presence of a suitable quantity of 6-TG in response to the TNF stimulation because of the expression of gpt gene. However, when a certain gene capable of inhibiting the signal transduction pathway from the TNF stimulation to the activation of the IL-8 promoter is introduced into the above cells, the expression of gpt can be inhibited and then the cells survive. Therefore, genes capable of inhibiting the intracellular signal transduction can be isolated by introducing various genes in said cells and selecting surviving cells.
The present inventors successfully isolated four genes (S1-15, S1-b2, S2-3, and S20-1) which were suppressive for IL-8 promoter activation from a cDNA library by using the above screening system. The present inventors specifically analyzed “S1-15” which consistently exhibited IL-8 promoter inhibitory activity.
First, the present inventors determined the sequence of “S1-15.” Homology searches based on the determined nucleotide sequence found two homologous genes which were reported as those encoding transcriptional regulatory factors.
The present inventors noticed that the transcriptional regulatory factors, which were supposed to enhance transcription inherently, were in fact inhibitory for IL-8 promoter. Then, the present inventors analyzed the amino acid sequence of S1-15 in more detail. They found that S1-15 comprised a DNA binding region within the above transcriptional regulatory factor but lacked most of the rest, including the regions that interact with other factors involved in transcription.
Finally, the present inventors found that the transcriptional regulatory factors can be modified into transcriptional inhibitory factors by deleting sequences other than regions having DNA binding activity of transcriptional regulatory factors.
Based on the fact that S1-15 inhibits the activation of IL-18 promoter, the present inventors subsequently analyzed whether S1-15 has anti-inflammatory activity. The result demonstrated that S1-15 strongly inhibits the human IL-8 production stimulated with IL-1 &bgr; in MRC-5 cells. It also inhibited the production by IL-1 &bgr;-stimulated MRC cells of a neutrophil chemotactic factor GRO &agr; at the level comparable to the inhibition of IL-8 production. Thus, the present inventors have found that S1-15 is a protein having a potent anti-inflammatory activity.
Specifically, the present invention relates to:
(1) a protein having transcription inhibitory activity and lacking at least a part of regions other than a region having DNA binding activity in a transcriptional regulatory factor,
(2) the protein of (1), wherein said protein lacks at least a part of the region interacting with other factors involved in transcription in the transcriptional regulatory factor,
(3) a protein having inhibitory activity on transcription of interleukin 8 gene and having the amino acid sequence of SEQ ID NO: 1, or said amino acid sequence in which one or more amino acid residues are substituted, deleted, or added,
(4) a protein having the amino acid sequence of SEQ ID NO: 1,
(5) a DNA encoding the protein of (1) to (4),
(6) a vector comprising the DNA of (5),
(7) a cell carrying the vector of (6),
(8) a method of inhibiting transcription of a specific gene, which comprises introducing the protein of (1) or (2) into cells,
(9) a method of inhibiting transcription of the interleukin 8 gene, which comprises introducing the protein of (3) or (4) into cells,
(10) a method of inhibiting transcription of a specific gene, which comprises introducing a vector comprising DNA encoding the protein of (1) or (2) into cells and allowing the protein of (1) or (2) to be expressed in the cells,
(11) a method of inhibiting transcription of the interleukin 8 gene, which comprises introducing a vector comprising DNA encoding the protein of (3) or (4) into cells and allowing the protein of (3) or (4) to be expressed in the cells.
The invention also includes a substantially pure polypeptide comprising (1) an amino acid sequence at least 60% (e.g., 70%, 80%, 90%, 95%, or 99%) identical to SEQ ID NO: 1, (2) an amino acid sequence that is SEQ ID NO: 1 containing at least on
Nagasawa Yasuo
Yoshida Hideaki
Carlson Karen Cochrane
Institute of Cytosignal Research Inc.
Robinson Hope A.
Shanks & Herbert
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