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The GLI oncogene, discovered by virtue of its amplification in human tumors, encodes a sequence-specific DNA-binding protein containing five zinc fingers. We have now characterized one member of a family of GLI-related zinc finger genes. A previously identified fragment of GLI3 genomic DNA was used to localize GLI3 to chromosome 7p13 and to isolate cDNA clones. Sequence analysis of these clones and identification of the GLI3 protein by using polyclonal antisera demonstrated that GLI3 encodes a protein of 1,596 amino acids and an apparent molecular mass of 190 kilodaltons. Amino acid sequence comparison with GLI demonstrated seven regions of similarity (53 to 88% identity), with the zinc fingers representing the most similar region. Furthermore, when produced in vitro, the GLI3 protein bound specifically to genomic DNA fragments containing GLI-binding sites. Amino acid sequence comparison with the product of another member of the GLI family, the Drosophila segment polarity gene cubitus interruptus Dominant, revealed additional similarity that was not shared with GLI. These studies suggest that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities. In addition, sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.  相似文献   

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Protein-protein interactions often play a crucial role in stabilizing protein-DNA complexes and thus facilitate site-specific DNA recognition. We have worked to incorporate such protein-protein contacts into our design and selection strategies for short peptide extensions that promote cooperative binding of zinc finger proteins to DNA. We have determined the crystal structure of one of these fusion protein-DNA complexes. The selected peptide extension was found to mediate dimerization by reaching across the dyad axis and contacting a hydrophobic patch on the surface of the zinc finger bound to the adjacent DNA site. The peptide-zinc finger protein interactions observed in this structure are similar to those of some homeodomain heterodimers. We also find that the region of the zinc finger surface contacted by the selected peptide extension corresponds to surfaces that also make key interactions in the zinc finger proteins GLI and SWI5.  相似文献   

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The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.  相似文献   

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Multiple genes encoding zinc finger domains are expressed in human T cells   总被引:13,自引:0,他引:13  
Proteins containing zinc finger domains have been implicated in developmental control of gene expression in Drosophila, Xenopus, mouse, and humans. Multiple cDNAs encoding zinc (II) finger structures were isolated from human cell lines of T-cell origin to explore whether zinc finger genes participate in the differentiation of human hematopoietic cells. Initial restriction analysis, genomic Southern blotting, and partial sequence comparisons revealed at least 30 nonoverlapping cDNAs designated cKox(1-30) encoding zinc finger motifs. Analysis of cKox1 demonstrated that Kox1 is a single-copy gene that is expressed in a variety of hematopoietic and nonhaematopoietic cell lines. cKox1 encodes 11 zinc fingers that were shown to bind zinc when expressed as a beta-gal-Kox1 fusion protein. Further analysis of the predicted amino acid sequence revealed a heptad repeat of leucines NH2-terminal to the finger region, which suggests a potential domain for homo- or heterodimer protein formation. On the basis of screening results it was estimated that approximately 70 zinc finger genes are expressed in human T cells. Zinc finger motifs are probably present in a large family of proteins with quite diverse and distinct functions. However, comparisons of individual finger regions in cKox1 with finger regions of cKox2 to cKox30 showed that some zinc fingers are highly conserved in their putative alpha-helical DNA binding region, supporting the notion of a zinc finger-specific DNA recognition code.  相似文献   

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Two major protein-binding sites within the negative regulatory element of the human immunodeficiency virus type 1 long terminal repeat have been identified. One (site B) contained a palindromic sequence with homology to steroid/thyroid hormone response elements but was distinct from previously described binding sites of this class. A novel T-cell protein recognized the palindromic sequence within site B and also bound estrogen- or thyroid hormone-response elements with lower affinity. A 7-base-pair mutation in the site B palindrome, which destroyed protein binding, resulted in increased expression from the human immunodeficiency virus type 1 long terminal repeat in T cells.  相似文献   

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Abstract: To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI -binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.  相似文献   

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The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.  相似文献   

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C2H2锌指是真核细胞中最常见的DNA结合模体。由于C2H2锌指域靶位点特异性与结构和功能的模块性构成,使得C2H2锌指域成为构建特定的DNA结合蛋白的常用骨架。保持C2H2锌指的基本骨架不变,替换锌指特定位点的氨基酸残基,并融合表达其他功能域就可以得到具有靶向性的人造锌指蛋白(ZFP)。ZFP可以介导靶基因的转录调控,抑制或激活特定基因的表达与配体依赖的靶基因激活或抑制;对DNA进行修饰,如人造限制性内切酶,重组酶,整合酶;抗病毒感染等。因此,人造锌指蛋白应用前景广阔,研究价值显著,是未来人类基因治疗的革命性的工具。  相似文献   

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ZPT2-2 is a DNA-binding protein of petunia that contains two canonical TFIIIA-type zinc finger motifs separated by a long linker. We previously reported that ZPT2-2 bound to two separate AGT core sites, with each zinc finger making contact with each core site. Here we present our further characterization of ZPT2-2 by using selected and amplified binding sequence imprinting and surface plasmon resonance analyses; together, these assays revealed some unusual features of the interaction between ZPT2-2 and DNA. These experiments allowed us to conclude that 1) the optimal binding sequence for the N-terminal zinc finger is AGC(T), and that of the C-terminal one is CAGT; 2) multiple arrangements of the two core sites accommodate binding; and 3) the spacing between the two core sites affects the binding affinity. In light of these observations, we propose a new model for the DNA-ZPT2-2 interaction. Further, consistent with this model, a high affinity binding site for ZPT2-2 was found in the promoter region of the ZPT2-2 gene. This site may serve as a cis-element for the autoregulation of ZPT2-2 gene expression.  相似文献   

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