猪GATA-3基因的电子克隆、表达及其分子进化分析

基于猪的表达标签数据库电子克隆了猪的GATA-3基因,并通过RT-PCR验证了猪的GATA-3基因的核苷酸序列。测序结果显示猪的GATA-3基因的核苷酸长度由1,760bp个碱基组成,包括1,335bp的开放阅读框,编码产物为由444个氨基酸残基组成的多肽。通过半定量RT-PCR检测了GATA-3 mRNA在大白猪的各个组织的表达情况。GATA转录因子家族通常有2个Ⅳ型锌指蛋白结构,根据Ⅳ型锌指蛋白结构序列,使用Mega3．1软件构建了分子进化关系树。系统发育分析表明所有的脊椎动物的GATA转录因子都起源于共同的祖先,拓扑结构也表明进化过程中有多种事件发生,包括基因复制和Ⅳ型锌指蛋白结构域重组,根据所得数据有利于进一步了解GATA家族基因趋同和趋异的进化途径。     

Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.     

Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins.

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.