非洲爪蟾GATA-1b中与GATA-1a的差异位点突变后的功能  

Effects of Site-directed Mutagenesis at Amino Acid Residues of GATA-1b Different from That of GATA-1a in Xenopus

作　　者：冯湘玲[1] 刘卫东[1] 李虹[1] 王磊[1] 杨旭宇[1] 韩为农[1] 周文[1] 姚开泰[1] 

机构地区：[1]中南大学湘雅医学院肿瘤研究所,长沙410078

出　　处：《生物化学与生物物理学报》2003年第12期1105-1110,共6页

基　　金：国家自然科学基金资助项目 (No .3 9910 161994)~~

摘　　要：非洲爪蟾GATA 1因子分为GATA 1a和GATA 1b两种亚型。与其他种类的GATA 1因子相类似 ,GATA 1是红细胞分化成熟所必需的因子之一。二种亚型在造血方面具有同样的作用 ,但只有xGATA 1b具有抑制神经发生的功能。比较二者的结构 ,发现二者的核酸和蛋白质结构虽然都非常相似 ,但并不完全一致。为探讨这些差异结构与功能之间的关系 ,我们对xGATA 1b中与xGATA 1a差异的Ser1 6 8和His1 6 9位点进行缺失突变 ,并定点突变Thr30 4和Thr359位点。之后将体外制备好的野生型或突变型xGATA 1bmRNA单独或与DN BRmRNA共同导入非洲爪蟾 2细胞胚胎内 ,利用动物帽系统分析突变体在神经发生和造血方面的功能。结果发现 ,Ser1 6 8和His1 6 9以及Thr359位点突变后 ,xGATA 1b再不能抑制DN BR诱导的神经发生 ;但所有突变都未影响xGATA 1b在造血方面的功能。由此 ,我们首次证实了Ser1 6 8和His1 6 9以及Thr359位点可能是导致xGATA 1b和xGATA 1a之间功能分离的结构基础之一。The GATA-1 of Xenopus (xGATA-1), which has two subtypes xGATA-1a and xGATA-1b, is a necessary factor for erythroid differentiation and maturation as similar as that of other GATA-1s. Although both xGATa-1a and xGATA-1b are able to stimulate erythropoiesis, only xGATA-1b is capable of inhibiting neurogenesis in Xenopus embryos. Compared between their structures, xGATA-1a and xGATA-1b are very similar in nucleotide and amino acids composition, but not identical. Therefore, it is responsible for studying the role of the diverse codons between the two genes, so the desired mutations: S 168, H 169 double deletion and point mutation of T 304→A, T 359→A, were introduced into xGATA-1b gene through site-directed mutagenesis. Then, mRNA from each mutant as well as wtxGATA-1b was co-injected with DN-BR mRNA or separately injected into Xenopus stage 2 embryos, and the role of mutants in erythropoiesis and neurogenesis was analyzed by using animal cap culture system. The results showed that the neural-inhibiting activity of xGATA-1b, but not hematopoiesis-inducing activity, was aborted because of deletion of Ser 168 and His 169 or point mutation of T 359→A. So it is demonstrated for the first time that Ser 168 and His 169 or Thr 359 in xGATA-1b may be one of the structural basis for explanting the different function between xGATA-1b and xGATA-1a.

关 键 词：非洲爪蟾 GATA-1b 红细胞 分化 GATA-1a 核酸 蛋白质结构 突变 

分 类 号：Q75[生物学—分子生物学]