Physiological function of adenylate kinase isozymes

腺苷酸激酶是一种普遍存在的酶，参与细胞内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）被鉴定。为了了解这些同工酶的生理功能，我们计划分离一个或两个AK基因被破坏的细胞系。通过测定酶活性、酶蛋白量和mRNA水平，我们发现HeLa细胞有三个功能性AK基因。构建了2个人AK1基因的靶向载体。质粒AK1-Bsr含有一个杀囊虫素S抗性基因和一个白喉毒素a片段基因。质粒AK-Neo含有一个新霉素耐药基因和一个白喉毒素a片段基因。利用这些质粒，我们将能够通过同源重组分离ak1破坏的HeLa细胞。为了构建人AK2基因的靶向载体，我们以牛AK2 cDNA为探针，从HeLa细胞中克隆AK2 cDNA。然后，我们克隆了一部分AK2的gDNA，为构建基因靶向载体提供了依据。目前正在研究分离破坏AK基因的细胞系。

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.