Novel cytidine deaminase
AssigneeTakeda Chemical Industries, Ltd.
InventorsTasuku Honjo, Masamichi Muramatsu
Life of this patent
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Abstract
Genes encoding novel proteins named AID (Activation-Induced cytidine Deaminase), having structural relationship to APOBEC-1, one of RNA editing enzymes, and having a cytidine deaminase activity similar to APOBEC-1, has been found by preparing cDNA libraries for mouse B cell clone CH12F3-2, which undergoes class switch recombination from IgM to IgA at an extremely high rate together with activation of cells by stimulation with cytokines, with and without stimulating with cytokine, and performing subtraction cloning using the libraries.
Claims49
An isolated nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8 or a functional fragment thereof.
An isolated nucleic acid or a fragment thereof comprising the nucleotide sequence of SEQ ID NO:1 or 7.
An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:
- (a) nucleotides 93 to 689 of SEQ ID NO:1;
- and
- (b) nucleotides 80 to 676 of SEQ ID NO:7.
An isolated nucleic acid selected from the group consisting of:
- (a) a nucleic acid that (i) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:1, and (ii) encodes a polypeptide having a cytidine deaminase activity;
- (b) a nucleic acid that (i) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:7, and (ii) encodes a polypeptide having a cytidine deaminase activity;
- and
- (c) a fragment of (a) or (b).
A substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 8 or a fragment thereof that (a) has a cytidine deaminase activity activity or (b) is immunogenic.
A substantially pure polypeptide comprising substantially the same amino acid sequence as that of SEQ ID NO:2 or 8 and having a cytidine deaminase activity.
A substantially pure polypeptide selected from the group consisting of:
- (a) a mammalian polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the sequence of SEQ ID NO:1, wherein the polypeptide has a cytidine deaminase activity;
- and
- (b) a mammalian polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the sequence of SEQ ID NO:7, wherein the polypeptide has a cytidine deaminase activity.
An isolated nucleic acid comprising the nucleotide sequence of (a) SEQ ID NO:9, (b) SEQ ID NO:10, or (c) SEQ ID NO:35.
An isolated nucleic acid comprising the nucleotide sequence of (a) SEQ ID NO:11, (b) SEQ ID NO:12, (c) SEQ ID NO:13, (d) SEQ ID NO:14, or (e) SEQ ID NO:15.
An isolated nucleic acid comprising a sequence complementary to any one of:
- (a) SEQ ID NO:9,
- (b) SEQ ID NO:10,
- (c) SEQ ID NO:11,
- (d) SEQ ID NO:12,
- (e) SEQ ID NO:13,
- (f) SEQ ID NO:14,
- (g) SEQ ID NO:15, or
- (h) SEQ ID NO:35.
An isolated oligonucleotide comprising a sequence complementary to a fragment of any one of:
- (a) SEQ ID NO:9,
- (b) SEQ ID NO:10,
- (c) SEQ ID NO:11,
- (d) SEQ ID NO:12,
- (e) SEQ ID NO:13,
- (f) SEQ ID NO:14,
- (g) SEQ ID NO:15, or
- (h) SEQ ID NO:35.
A pair of isolated oligonucleotide primers selected from the group consisting of:
- (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:31 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:32,
- (b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:20 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:22,
- (c) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:21 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:30,
- (d) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:24 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:25,
- (e) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:27,
- (f) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28,
- (g) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29
- (h) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:27,
- (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28,
- (j) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29,
- (k) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:34 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28,
- (l) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:34 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29,
- (m) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:33 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29, or
- (n) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:18 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:19.
A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:
- (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein, and
- (b) (i) comparing the level of the AID protein produced by the cells cultured in the presence of the substance with the level of the AID protein produced by the cells cultured in the absence of the substance, or
- (ii) comparing the level of the AID protein-encoding mRNA transcribed in the cells cultured in the presence of the substance with the level of the AID protein-encoding mRNA transcribed in the cells cultured in the absence of the substance.
A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:
- (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and a second protein other than the AID protein, wherein transcription of a gene encoding the second protein into mRNA is dependent in the cells on the degree of transcription of the gene encoding the AID protein into mRNA, and
- (b) comparing the level of the second protein produced by the cells cultured in the presence of the substance with the level of the second protein produced by the cells cultured in the absence of the substance.
A method for identifying a substance that inhibits an enzyme activity of an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8, the method comprising the step of:
- (a) culturing, separately in the presence and the absence of the substance, mammal-derived B cells or tissues comprising the B cells, and comparing enzyme activities of the AID proteins in the B cells separately cultured, or
- (b) (i) administering the substance separately to an AID) gene knockout mouse whose endogenous AID gene is inactivated so that transcription of the endogenous AID gene into mRNA is inhibited, and to a normal mouse, and
- (ii) comparing enzyme activities of the AID proteins in the B cells isolated from the respective mice.
Dependencies are inferred from the claim text.