Physiological function of adenylate kinase isozymes

腺苷酸激酶同工酶的生理功能

• 批准号: 05454160
• 负责人: NAKAZAWA Atsushi
• 金额: $ 3.78万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454160/
• 关键词: adenylate kinase; adenine nucleotide; gene targeting; isozyme; HeLa cell; ジーンターゲッティング; アイリザイム; 細胞内局在

Adenylate kinase is a ubiquitous enzyme which is involved in the interconversion of adenine nucleotides in the cells. In vertibrates, three isozymes (AK1, AK2, AK3) have been characterized. To understand the phisiological function of these isozyme, we have planned to isolate cell lines in which one or two AK genes were disrupted.We showed HeLa cells have three functional AK genes by measuring enzyme activities, enzyme protein amounts, and mRNA levels.Two targeting vectors for human AK1 gene were constructed. Plasmid AK1-Bsr contains a blasticidin S resistant gene and a diphtheria toxin A fragment gene. Plasmid AK-Neo contains a neomycin resistant gene and a diphtheria toxin A fragment gene. Using these plasmid, we shall be able to isolate AK1-disrupted HeLa cells by homologouse recombination.To make a targeting vector for the human AK2 gene, we cloned cDNA of AK2 from HeLa cells using bovine AK2 cDNA as a plobe. Then, we cloned a part of gDNA of AK2, which will be useful to construct a gene targeting vector.Isolation of cell lines which have disrupted AK genes are under investigation.

腺苷酸激酶是一种普遍存在的酶，参与细胞中腺嘌呤核苷酸的相互转化。在脊椎动物中，有三种同工酶(AK1、AK2、AK3)已被鉴定。为了了解这些同工酶的生理功能，我们计划分离出一个或两个AK基因突变的细胞系。通过对酶活性、酶蛋白含量和mRNA水平的测定，我们证明HeLa细胞具有三个具有功能的AK基因。AK1-bsr含有一个S抗性基因和一个白喉毒素A片段基因。AK-Neo含有一个新霉素抗性基因和一个白喉毒素A片段基因。为了构建人AK2基因的靶向载体，我们以牛AK2基因为倍体，从HeLa细胞中克隆了AK2基因的全长cDNA。然后，我们克隆了AK2基因的一部分gDNA，为构建基因打靶载体奠定了基础。我们正在对AK基因打乱的细胞系进行分离。

项目成果

S.C.Song: "Cloning and chracterization of a cDNA encoding human adenylate kinase isozyme 2." (In preparation.).

S.C.Song：“编码人腺苷酸激酶同工酶 2 的 cDNA 的克隆和表征。”

Song., S.C., Inouye.S.and Nakazawa, A.: "Clonng and characterization of human cDNA encoding adenylate kinase isozyme 2." (In preparation).

Song., S.C.、Inouye.S. 和 Nakazawa, A.：“编码腺苷酸激酶同工酶 2 的人类 cDNA 的克隆和表征。”

T.Tanabe: "Tissue-specific and developmentally regulated expression of the gene encoding adenylate kinase isozymes." J.Biochem.113. 200-207 (1993)

T.Tanabe：“编码腺苷酸激酶同工酶的基因的组织特异性和发育调节表达。”

Tanabe, T., Yamada, M., Noma, T., Kajii, T.and Nakazawa, A.: "Tissue-specific and developmentally regulated expression of the gene encoding adenylate kinase isozymes." J.Biochem.113. 200-207 (1993)

Tanabe, T.、Yamada, M.、Noma, T.、Kajii, T. 和 Nakazawa, A.：“编码腺苷酸激酶同工酶的基因的组织特异性和发育调节表达。”

Adachi, N.: "Characterization of the 5'-flanking region of the gene encoding adenylate kinase isozyme 3." Yamaguchi Igaku. 43 (2). 1-12 (1994)

Adachi, N.：“编码腺苷酸激酶同工酶 3 的基因 5 侧翼区域的特征。”