scholarly journals Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins

The Analyst ◽  
2015 ◽  
Vol 140 (12) ◽  
pp. 3947-3952 ◽  
Author(s):  
Soodong Noh ◽  
Dat Thinh Ha ◽  
Haesik Yang ◽  
Moon-Soo Kim
Keyword(s):  
Alkaline Phosphatase ◽  
Electrochemical Detection ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Direct Detection ◽  
Electrochemical Measurement ◽  
Zinc Finger Proteins ◽  
Double Stranded Dna ◽  
Ito Electrode ◽  
Finger Protein

We report sensitive and direct detection of specific bacterial dsDNA sequences using (i) the zinc finger protein labelled with alkaline phosphatase and (ii) electrochemical measurement at the ITO electrode.

Single zinc-finger extension: enhancing transcriptional activity and specificity of three-zinc-finger proteins

2005 ◽  
Vol 386 (2) ◽  
pp. 95-99 ◽  
Author(s):  
Alexander E.F. Smith ◽  
Farzin Farzaneh ◽  
Kevin G. Ford
Keyword(s):  
Transcription Factors ◽  
Fusion Protein ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Finger Protein ◽  
Finger Extension ◽  
Vp16 Fusion

AbstractIn order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.

scholarly journals Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Dongxin Zhao ◽  
Zhongxian Huang
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Zinc Finger Proteins ◽  
Hereditary Diseases ◽  
Expression Systems ◽  
His Tag ◽  
Finger Protein ◽  
Inclusion Body Formation ◽  
Structure And Functions

Zinc finger proteins are associated with hereditary diseases and cancers. To obtain an adequate amount of zinc finger proteins for studying their properties, structure, and functions, many protein expression systems are used. ZNF191(243-368) is a zinc finger protein and can be fused with His-tag to generate fusion proteins such as His6-ZNF191(243-368) and ZNF191(243-368)-His8. The purification of His-tag protein using Ni-NTA resin can overcome the difficulty of ZNF191(243-368) separation caused by inclusion body formation. The influences of His-tag on ZNF191(243-368) properties and structure were investigated using spectrographic techniques and hydrolase experiment. Our findings suggest that insertion of a His-tag at the N-terminal or C-terminal end of ZNF191(243-368) has different effects on the protein. Therefore, an expression system should be considered based on the properties and structure of the protein. Furthermore, the hydrolase activity of ZNF191(243-368)-His8has provided new insights into the design of biological functional molecules.

scholarly journals Identification, nuclear localization, and DNA-binding activity of the zinc finger protein encoded by the Evi-1 myeloid transforming gene.

1990 ◽  
Vol 10 (3) ◽  
pp. 1259-1264 ◽  
Author(s):  
T Matsugi ◽  
K Morishita ◽  
J N Ihle
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Nuclear Protein ◽  
Zinc Finger Protein ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Finger Protein ◽  
Myeloid Leukemias ◽  
Transforming Gene

Activation of the Evi-1 zinc finger gene is a common event associated with transformation of murine myeloid leukemias. To characterize the gene product, we developed antisera against various protein domains. These antisera primarily detected a 145-kilodalton nuclear protein that bound double-stranded DNA. Binding was inhibited by chelating agents and partially restored by zinc ions.

scholarly journals A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

2002 ◽  
Vol 22 (12) ◽  
pp. 4256-4267 ◽  
Author(s):  
Kazuhiro Tanaka ◽  
Noriyuki Tsumaki ◽  
Christine A. Kozak ◽  
Yoshihiro Matsumoto ◽  
Fumihiko Nakatani ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Promoter Activity ◽  
Expression Vector ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Limb Bud ◽  
Collagen Gene ◽  
Specific Expression ◽  
Finger Protein ◽  
Krab Domain

ABSTRACT Type XI collagen is composed of three chains, α1(XI), α2(XI), and α3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the −530-bp promoter segment of the α2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

The Regulation of Zinc Finger Proteins by Mirnas Enriched in ALL-Microvesicles

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1448-1448
Author(s):  
Huiyu Li ◽  
Xiaomei Chen ◽  
Wei Xiong ◽  
Fang Liu ◽  
Shiang Huang
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression Profiles ◽  
Zinc Fingers ◽  
Zinc Finger Proteins ◽  
Bioactive Molecules ◽  
Chromosomal Protein ◽  
Normal Peripheral Blood ◽  
Finger Protein

Abstract Abstract 1448 Microvesicles (MVs) are submicrometric membrane fragments and they can “hijack” membrane components and engulf cytoplasmic contents from their cellular origin. MVs are enriched in various bioactive molecules of their parental cells, such as proteins, DNA, mRNA and miRNAs. Microvesicles (MVs) released by leukemia cells constitute an important part of the leukemia microenvironment. As a cell-to-cell communication tool, MVs transfer microRNA (miRNA) between cells. MVs miRNAs may also provide an insight in the role of miRNAs playing in the underlying of pathophysiologic processes of various leukemia. We determined the miRNA expression profiles of ALL-derived MVs using Agilent miRNA microarray analysis. The five miRNAs obtained by microarray profiling were validated using real-time PCR. The putative target genes were predicted by bioformation software. We identified 182 and 166 dysregulated miRNAs in MVs derived from Nalm 6 cells and from Jurkat cells, respectively. Both up regulated (123/182 in Nalm 6-MVs and 114/166 in Jurkat- MVs) and down regulated (59/182 in Nalm 6-MVs and 52/166 in Jurkat- MVs) expressions were observed compared with MVs from normal peripheral blood the MVs normal control. When we analyzed those miRNA with bioinformatic tools (TargetScan), we found an interesting phenomenon that presence of 111 zinc fingers genes were regulated by 52 miRNAs, indicating that the ALL-microvesicles were enriched with miRNAs regulating zinc finger proteins. They encompassed zinc fingers and homeoboxes 2, zinc finger, ZZ-type containing 3, zinc finger, SWIM-type containing 1, zinc finger, RAN-binding domain containing 3, zinc finger, NFX1-type containing 1, zinc finger, MYM-type 4, zinc finger, FYVE domain containing 1 and their 5 subtypes; zinc finger, DHHC-type containing16, and other subtypes; zinc finger, CCHC domain containing 14 and 7A, zinc finger, BED-type containing 4; zinc finger protein, X-linked; zinc finger protein, multitype 2; zinc finger protein 81, and their 55 subtypes; zinc finger and SCAN domain containing 18, zinc finger and BTB domain containing 9. ALL-microvesicles were enriched with expression changes of distinct sets of miRNAs regulating zinc finger proteins. This provides clues that genes commonly function together. It is worth noting that 52 miRNA regulating above zinc finger protein genes were up-expressed, suggeting that miRNA regulating zinc fingers were active in ALL-MVs. Zinc finger proteins are important transcriptions in eukaryotes and play roles in regulating gene. Some members of the Zinc finger family have close relationaship with tumour. Zinc finger X-chromosomal protein (Zfx) is a protein that in humans is encoded by the ZFX gene. The level of Zfx expression correlates with aggressiveness and severity in many cancer types, including prostate cancer, breast cancer, gastric tumoural tissues, and leukemia. [1,2]. Zinc finger and homeoboxes 2 (ZHX2) was target gene of miRNA-1260. The role of miRNA are negatively regulated host gene expressions. ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors. Loss of nuclear ZHX2 might be an early step in the development of HCC[3]. In our study, the miRNA-1260 were 9 fold higher in ALL MVs. In leukeima microenvironment, ALL-MVs may transfer aberantly expressed miRNAs to their target cell lead to abnormally regulated the zinc finger proteins that may play roles in ALL. In this study, we demonstrated that ALL-microvesicles were enriched with expression changes of distinct sets of miRNAs regulating zinc finger proteins. Futhermore, Zinc fingers were active in ALL-MVs and commonly function together. Disclosures: No relevant conflicts of interest to declare.

Identification, nuclear localization, and DNA-binding activity of the zinc finger protein encoded by the Evi-1 myeloid transforming gene

1990 ◽  
Vol 10 (3) ◽  
pp. 1259-1264
Author(s):  
T Matsugi ◽  
K Morishita ◽  
J N Ihle
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Nuclear Protein ◽  
Zinc Finger Protein ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Finger Protein ◽  
Myeloid Leukemias ◽  
Transforming Gene

Activation of the Evi-1 zinc finger gene is a common event associated with transformation of murine myeloid leukemias. To characterize the gene product, we developed antisera against various protein domains. These antisera primarily detected a 145-kilodalton nuclear protein that bound double-stranded DNA. Binding was inhibited by chelating agents and partially restored by zinc ions.

Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA–RNA Hybrids

2017 ◽  
Vol 89 (3) ◽  
pp. 2024-2031 ◽  
Author(s):  
Chiew San Fang ◽  
Kwang-sun Kim ◽  
Byeongjun Yu ◽  
Sangyong Jon ◽  
Moon-Soo Kim ◽  
...  
Keyword(s):  
Electrochemical Detection ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Finger Protein

Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein

2014 ◽  
Vol 6 (14) ◽  
pp. 4991-4994 ◽  
Author(s):  
Jinhee Lee ◽  
Atsuro Tatsumi ◽  
Koichi Abe ◽  
Wataru Yoshida ◽  
Koji Sode ◽  
...  
Keyword(s):  
Electrochemical Detection ◽  
Zinc Finger ◽  
Genomic Dna ◽  
Pathogenic Bacteria ◽  
Zinc Finger Protein ◽  
Detection System ◽  
Glucose Dehydrogenase ◽  
Finger Protein ◽  
Pcr Products ◽  
Probe Hybridization

We developed an electrochemical detection system for pathogenic bacteria by utilizing a glucose dehydrogenase-fused zinc finger protein (ZF-GDH), which could detect PCR products electrochemically without the need for DNA probe hybridization. Using ZF-GDH, we could specifically detect 10 copies of genomic DNA derived fromEscherichia coliO157.

Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein

2018 ◽  
Vol 90 (7) ◽  
pp. 4776-4782 ◽  
Author(s):  
Chiew San Fang ◽  
Kwang-sun Kim ◽  
Dat Thinh Ha ◽  
Moon-Soo Kim ◽  
Haesik Yang
Keyword(s):  
Electrochemical Detection ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Finger Protein
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