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.     

Physiological levels of glutathione enhance Zn(II) binding by a Cys4 zinc finger

Using fluorescence and UV-vis spectroscopies and mass spectrometry, we demonstrated that the presence of physiological levels of reduced glutathione enhances the binding of Zn(II) to XPAzf, a Cys4 zinc finger peptide derived from the XPA protein, by means of formation of a ternary complex of a general formula ZnXPAzf[GSH]. Similar complexes were also indicated by ESI-MS for isostructural Co(II)- and Cd(II)-substituted XPAzf. The observed enhancement of the Zn(II) binding to XPAzf by a factor of 50 over the physiological range of GSH concentrations of 1-20 mM corresponds to a dissociation constant of GSH from the ZnXPAzf[GSH] complex of 0.05 μM. This effect may account for an apparent discrepancy between relatively low Zn(II) binding constants measured in vitro for many zinc fingers, and the requirement of tight Zn(II) binding enforced by intracellular zinc buffering by the thionein/metallothionein couple.     

Synthesis and DNA-binding ability of Spl protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Spl-ZF2), its mutant (Spl-ZF2/HT. E20→H, R23→T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoCl2 indicated that the formation of zinc finger structure was affected not only by the hy-drophobic amino acids but also by the change of the distance between Cys and His. Gel-retardation electrophoresis as-says indicated that the Grlu and Arg residues are very important for recognition. A single zinc finger like Spl-ZF2 isable to bind DNA sequence specifically.     

Structure based design of protein linkers for zinc finger nuclease

Zinc finger nucleases are a promising tool to edit DNA in many biological applications, in particular for gene knockout. Despite many efforts the number of genes that can be effectively targeted with ZFNs remains severely limited, as available constructs cannot address arbitrary gene sequences. Here, we develop a novel concept to significantly enhance the number of DNA sequences that can be targeted by ZFN. Using an efficient computational model, we provide an extensive library of possible linker molecules between individual zinc finger motifs in the construct that can skip up to 10 base pairs between adjacent zinc finger recognition sites in the DNA sequence, which increases the number of genes that can be efficiently targeted by more than an order of magnitude.     

大豆C2H2型锌指蛋白基因SCTF-1的克隆及功能分析

锌指蛋白是一类具有手指型结构的蛋白质,其中一些锌指蛋白是转录因子,对真核生物的生长发育及非生物逆境胁迫的耐受能力都有着重要作用。文章从大豆(Glycine max(L.)Merr.)中克隆了一个新的C2H2型锌指蛋白基因SCTF-1(GenBank登录号:JQ692081),该基因包含一个699 bp的开放阅读框,编码233个氨基酸,无内含子,有两个典型的C2H2型锌指结构。锌指结构中有植物锌指蛋白特有的保守氨基酸序列QALGGH。经软件预测分析,其等电点pI=8.33,分子量24.9 kDa。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验结果表明,SCTF-1蛋白能够定位到细胞核中。通过RT-PCR检测发现该基因在大豆叶和花中的表达量较高,在茎和根的表达量相对较低。在对大豆幼苗的低温胁迫中,SCTF-1基因的表达量明显增加。将SCTF-1基因转入烟草(Nicotiana tabacum L.)中,发现SCTF-1基因的过量表达能够明显提高转基因烟草的耐冷能力。     

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos

 The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation. Received: 19 October 1998 / Accepted: 16 February 1999