人工转录因子研究进展

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合结构域与效应结构域两部分组成,研究发现这两个结构域可以各自独立发生作用。基于转录因子的这种结构特点,可以人为地选择针对特定序列的DNA结合结构域与具有特定作用的效应结构域构建人工转录因子。目前人工转录因子的DNA结合结构域多为C₂H₂ 型锌指结构,每一个锌指单元由大约30个氨基酸组成,识别DNA双螺旋大沟中相连的3bp序列,并可通过氢键作用与相应的碱基结合;多个锌指可以串联成簇,从而识别并结合较长的DNA序列区域。常见的人工转录因子的效应结构域有激活结构域以及抑制结构域,不同的效应结构域赋予人工转录因子不同的功能。目前人工转录因子已经在基础研究、药物设计以及基因治疗等领域得到了广泛的应用。     

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features--nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.     

一种特异识别SV40启动子的人工转录因子的构建

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合域与效应域两部分组成,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术,筛选到与SV40启动子上9bp序列特异结合的锌指结构,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV40下游的报告基因的表达有很显著的抑制作用。     

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.     

水稻多逆境诱导基因OsMsr4的克隆与表达分析

为深入了解水稻逆境反应的分子机理和发现新的耐逆相关功能基因,采用Affymetrix水稻表达芯片分析超级稻两优培九母本培矮64S(Oryza sativa L.)不同生长发育时期、不同组织器官全基因组在低温、干旱、高温逆境胁迫下的表达水平,筛选出多个多因子诱导高表达特异基因(待另文发表).OsMsr4是其中一个在多种逆境条件,各生长发育时期与组织器官,其表达量均显著上调的基因,用实时定量PCR方法对其表达水平进行了进一步的分析,所得结果与基因芯片结果基本吻合.用 PCR方法扩增获得长为550 bp全长基因序列,其编码的144个氨基酸残基形成Cys2His2型双锌指结构蛋白,并且锌指结构的α-螺旋区含有植物锌指蛋白特定的保守序列QALGGH.因此,OsMsr4有可能是TFⅢA型锌指蛋白,作为转录因子参与各种环境胁迫应答反应,调控多个逆境相关基因表达.     

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.