WorldWideScience

Sample records for chimaeric transposase zinc-finger

  1. Zinc finger proteins in cancer progression

    OpenAIRE

    Jen, Jayu; Wang, Yi-Ching

    2016-01-01

    Zinc finger proteins are the largest transcription factor family in human genome. The diverse combinations and functions of zinc finger motifs make zinc finger proteins versatile in biological processes, including development, differentiation, metabolism and autophagy. Over the last few decades, increasing evidence reveals the potential roles of zinc finger proteins in cancer progression. However, the underlying mechanisms of zinc finger proteins in cancer progression vary in different cancer...

  2. Zinc finger structure-function in Ikaros

    Institute of Scientific and Technical Information of China (English)

    Marvin; A; Payne

    2011-01-01

    The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function.The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo.Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers;four of which are alternately incorporated in the production of the various Ikaros isoforms.Although no complete structures are available for the Ikaros protein or any of its family members,considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins.This review summarizes the structural aspects of Ikaros zinc fingers,individually,and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.

  3. Genome Engineering With Zinc-Finger Nucleases

    OpenAIRE

    Carroll, Dana

    2011-01-01

    Zinc-finger nucleases (ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools. ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. This article briefly reviews the history of ZFN development and summarizes applications that have been made to genome editing in many different organisms and situations. Considerable progress has been mad...

  4. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  5. Speed-stability paradox in DNA-scanning by zinc-finger proteins

    OpenAIRE

    Iwahara, Junji; Levy, Yaakov

    2013-01-01

    Extensive contact with DNA via multiple zinc fingers allows highly specific DNA-binding of zinc-finger-class transcription factors, but can also slow the target search process. Here we introduce recent insights into how zinc-finger proteins can rapidly scan DNA. Potential application of the new knowledge to the zinc-finger-based technology is also discussed.

  6. Editing the Plasmodium vivax Genome, Using Zinc-Finger Nucleases

    OpenAIRE

    Moraes Barros, Roberto R.; Straimer, Judith; Sa, Juliana M; Salzman, Rebecca E.; Melendez-Muniz, Viviana A.; Mu, Jianbing; David A Fidock; Thomas E. Wellems

    2014-01-01

    Plasmodium vivax is a major cause of malaria morbidity worldwide yet has remained genetically intractable. To stably modify this organism, we used zinc-finger nucleases (ZFNs), which take advantage of homology-directed DNA repair mechanisms at the site of nuclease action. Using ZFNs specific to the gene encoding P. vivax dihydrofolate reductase (pvdhfr), we transfected blood specimens from Saimiri boliviensis monkeys infected with the pyrimethamine (Pyr)–susceptible Chesson strain with a ZFN ...

  7. Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

    OpenAIRE

    Hwang, Cheol Kyu; D'Souza, Ursula M.; Eisch, Amelia J.; Yajima, Shunsuke; Lammers, Claas-Hinrich; Yang, Young; Lee, Sang-Hyeon; Kim, Yong-Man; Nestler, Eric J.; Mouradian, M. Maral

    2001-01-01

    Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in ...

  8. Phylogenetic Analysis of the Plant-specific Zinc Finger-Homeobox and Mini Zinc Finger Gene Families

    Institute of Scientific and Technical Information of China (English)

    Wei Hu; Claude W.dePamphilis; Hong Ma

    2008-01-01

    Zinc finger-homaodomain proteins (ZHD) are present in many plants;however,the evolutionary history of the ZHD gene family remains largely unknown.We show here that ZHD genes are plant-specific,nearly all intronless,and related to MINI ZINC FINGER (MIF) genes that possess only the zinc finger.Phylogenetic analyses of ZHD genes from representative land plants suggest that non-seed plant ZHD genes occupy basal positions and angiosperm homologs form seven distinct clades.Several clades contain genes from two or more major angiosperm groups,including eudicots,monocots,magnoliids,and other basal angiosperms,indicating that several duplications occurred before the diversification of flowering plants.In addition,specific lineages have experienced more recent duplications.Unlike the ZHD genes,&fiFs are found only from seed plants,possibly derived from ZHDs by loss of the homeodomain before the divergence of seed plants.Moreover,the MIF genes have also undergone relatively recent gene duplications.Finally,genome duplication might have contributed substantially to the expansion of family size in angiosperms and caused a high level of functional redundancy/overlap in these genes.

  9. Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs

    Directory of Open Access Journals (Sweden)

    Christmann Alexander

    2005-06-01

    Full Text Available Abstract Our experiments describe an alternative method of dsDNA recognition using zinc finger (ZF molecules which bind DNA specifically and with high affinity. Our aim was to develop zinc finger probes which are able to bind to dsDNA molecules at predetermined sites. In our basic approach we used pairs of complementary oligonucleotides to form dsDNAs, containing one of the three SP1-transcription factor motifs as a zinc finger recognition site. Two zinc finger probes of the SP1 motif were chemically synthesized and modified with a Dy-633 fluorophore. The SP1 peptides were folded into functional zinc fingers using zinc chloride. The addressable dsDNAs were immobilized on optical fibres, and the kinetics and binding rates of the artificial zinc finger probes were measured by a fluorescence detecting device (photomultiplying tube. The two zinc fingers and their corresponding DNA recognition sites served as specific probes and controls for the matching site and vice versa. Our experiments showed that a variety of dsDNA-binding probes may be created by modification of the amino acid sequence of natural zinc finger proteins. Our findings offer an alternative approach of addressing dsDNA molecules, for example for use in a nanoarray device.

  10. The interaction of DNA with multi-Cys2His2 zinc finger proteins

    Science.gov (United States)

    Liu, Lei; Heermann, Dieter W.

    2015-02-01

    The multi-Cys2His2 (mC2H2) zinc finger protein, like CTCF, plays a central role in the three-dimensional organization of chromatin and gene regulation. The interaction between DNA and mC2H2 zinc finger proteins becomes crucial to better understand how CTCF dynamically shapes the chromatin structure. Here, we study a coarse-grained model of the mC2H2 zinc finger proteins in complexes with DNA, and in particular, we study how a mC2H2 zinc finger protein binds to and searches for its target DNA loci. On the basis of coarse-grained molecular dynamics simulations, we present several interesting kinetic conformational properties of the proteins, such as the rotation-coupled sliding, the asymmetrical roles of different zinc fingers and the partial binding partial dangling mode. In addition, two kinds of studied mC2H2 zinc finger proteins, of CG-rich and AT-rich binding motif each, were able to recognize their target sites and slide away from their non-target sites, which shows a proper sequence specificity in our model and the derived force field for mC2H2-DNA interaction. A further application to CTCF shows that the protein binds to a specific DNA duplex only with its central zinc fingers. The zinc finger domains of CTCF asymmetrically bend the DNA, but do not form a DNA loop alone in our simulations.

  11. Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating ESTs

  12. Editing the Plasmodium vivax genome, using zinc-finger nucleases.

    Science.gov (United States)

    Moraes Barros, Roberto R; Straimer, Judith; Sa, Juliana M; Salzman, Rebecca E; Melendez-Muniz, Viviana A; Mu, Jianbing; Fidock, David A; Wellems, Thomas E

    2015-01-01

    Plasmodium vivax is a major cause of malaria morbidity worldwide yet has remained genetically intractable. To stably modify this organism, we used zinc-finger nucleases (ZFNs), which take advantage of homology-directed DNA repair mechanisms at the site of nuclease action. Using ZFNs specific to the gene encoding P. vivax dihydrofolate reductase (pvdhfr), we transfected blood specimens from Saimiri boliviensis monkeys infected with the pyrimethamine (Pyr)-susceptible Chesson strain with a ZFN plasmid carrying a Pyr-resistant mutant pvdhfr sequence. We obtained Pyr-resistant parasites in vivo that carried mutant pvdhfr and additional silent mutations designed to confirm editing. These results herald the era of stable P. vivax genetic modifications.

  13. Comparative analysis of zinc finger proteins involved in plant disease resistance.

    Science.gov (United States)

    Gupta, Santosh Kumar; Rai, Amit Kumar; Kanwar, Shamsher Singh; Sharma, Tilak R

    2012-01-01

    A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R(2) =0.95) and the aliphatic (R(2) =0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-k(h)-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54. PMID:22916136

  14. Metal Coupled Folding of Cys2His2 Zinc-Finger

    OpenAIRE

    Li, Wenfei; Zhang, Jian; Wang, Jun; Wang, Wei

    2008-01-01

    Zinc-fingers, which widely exist in eukaryotic cell and play crucial roles in life processes, depend on the binding of zinc ion for their proper folding. To computationally study the zinc coupled folding of the zinc-fingers, charge transfer and metal induced protonation/deprotonation effects have to be considered. Here, by attempting to implicitly account for such effects in classical molecular dynamics and performing intensive simulations with explicit solvent for the peptides with and witho...

  15. Dopamine receptor regulating factor, DRRF: a zinc finger transcription factor.

    Science.gov (United States)

    Hwang, C K; D'Souza, U M; Eisch, A J; Yajima, S; Lammers, C H; Yang, Y; Lee, S H; Kim, Y M; Nestler, E J; Mouradian, M M

    2001-06-19

    Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain. PMID:11390978

  16. Research Progress of PR Domain Zinc Finger Protein 14

    Directory of Open Access Journals (Sweden)

    Yudong HAN

    2016-02-01

    Full Text Available PR domain zinc finger protein 14 (PRDM14 is an important member of the PRDM family, PRDM14 plays a key role in the maintenance of cell integrity and differentiation, growth and apoptosis of the cell. It also plays an critical role in the formation of primordial germ cells, the maintenance of the totipotency of stem cells and the formation of tissues and organs. PRDM14 bears a single PR domain and six tandemly repeated zinc fingers, which is involved in the process of the deacetylation and methylation of the histone, and is involved in the formation of tumor trough the change level of methylation in the promoter region. The abnormal methylation of PRDM14 can change the chromatin structure, DNA conformation and the interaction mode of DNA and protein, it can suppress transcription and expression of the gene, which caused the occurrence, development and metastasis of tumor. The research progress of PRDM14 is reviewed based on the relevant literatures published in China and abroad.

  17. Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    YANG Yu-wen; NI Wan-chao; ZHANG Bao-long; SHEN Xin-lian

    2008-01-01

    @@ The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating ESTs to GenBank.In this study,a full length of one Cys2/His2 type zinc finger protein (GZFP) and its 5 flanking sequence were obtained by RT-PCR and tail PCR.

  18. ZifBASE: a database of zinc finger proteins and associated resources

    Directory of Open Access Journals (Sweden)

    Punetha Ankita

    2009-09-01

    Full Text Available Abstract Background Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. Description ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones, the number of finger units in each of the zinc finger proteins (with multiple fingers, the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public

  19. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    Science.gov (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  20. Zinc-finger antiviral protein inhibits XMRV infection.

    Directory of Open Access Journals (Sweden)

    Xinlu Wang

    Full Text Available BACKGROUND: The zinc-finger antiviral protein (ZAP is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV, HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

  1. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Böhm Siegfried

    2004-07-01

    Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

  2. An expanded binding model for Cys2His2 zinc finger protein–DNA interfaces

    International Nuclear Information System (INIS)

    Cys2His2 zinc finger (C2H2-ZF) proteins comprise the largest class of eukaryotic transcription factors. The 'canonical model' for C2H2-ZF protein–DNA interaction consists of only four amino acid–nucleotide contacts per zinc finger domain, and this model has been the basis for several efforts for computationally predicting and experimentally designing protein–DNA interfaces. Here, we perform a systematic analysis of structural and experimental binding data and find that, in addition to the canonical contacts, several other amino acid and base pair combinations frequently play a role in C2H2-ZF protein–DNA binding. We suggest an expansion of the canonical C2H2-ZF model to include one to three additional contacts, and show that computational approaches including these additional contacts improve predictions of DNA targets of zinc finger proteins

  3. Stress Responsive Zinc-finger Protein Gene of Populus euphratica in Tobacco Enhances Salt Tolerance

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The Populus euphratica stress responsive zinc-finger protein gene PSTZ, which encodes a protein including typical Cys2/His2 zinc finger structure, was isolated by reverse transcription-polymerase chain reaction from P. euphratica.Northern hybridization revealed that its expression was induced under drought and salt stress conditions. To examine its function, cDNA of the PSTZ gene, driven by the cauliflower mosaic virus 35S promoter, was cloned into a plant expression vector pBin438 and introduced into tobacco plants. Transgenic tobacco showed an enhanced salt tolerance, suggesting that PSTZ may play a role in plant responsiveness to salt stress.

  4. Metal binding and folding properties of a minimalist Cys2His2 zinc finger peptide.

    OpenAIRE

    Michael, S.F.; Kilfoil, V J; Schmidt, M.H.; Amann, B T; Berg, J M

    1992-01-01

    A minimalist Cys2His2 zinc finger peptide, Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-Lys-Ala-Ala-Leu-Ala- Ala-His-Ala-Ala-Ala-His-Ala-Lys, has been synthesized. Metal binding studies using Co2+ as a probe indicated that this peptide forms a 1:1 peptide/metal complex with a dissociation constant comparable to that observed for other zinc finger peptides. At high peptide concentrations, a 2:1 peptide/metal complex also forms, with four cysteinates coordinated to Co2+. Additional studi...

  5. Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato

    Science.gov (United States)

    While C2H2 zinc finger transcription factors are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum) upon infesta...

  6. 人工锌指蛋白随机库的构建及其在锌指核酸酶筛选中的应用%Construction of Artificial Zinc Finger Libraries and Application in Screening Zinc Finger Nucleases

    Institute of Scientific and Technical Information of China (English)

    李战伟; 王昕; 任刚; 王令; 杨涵江; 张智英

    2012-01-01

    利用开源式(Oligomerized pool engineering,OPEN)方法筛选具有较高特异性和亲和性的锌指蛋白(Zinc finger protein,ZFP).以已知三锌指蛋白为框架,使单个锌指关键位点氨基酸编码序列产生随机突变,构建3个人工锌指蛋白随机库,建立和完善应用人工锌指蛋白随机库筛选特异性锌指蛋白的技术平台.以人DYRK1A基因为例,测试和验证该技术平台的可行性和效率,分别获得对DYRK1A基因靶序列中左侧和右侧位点具有较高亲和性的50个三锌指蛋白;然后将得到的锌指蛋白与非限制性核酸内切酶FokⅠ连接,构建DYRK1A锌指核酸酶(Zinc finger nucleases,ZFN),应用酵母验证系统测试DYRK1A锌指核酸酶的活性,结果表明,90%的组装锌指核酸酶能够在靶位点进行切割.%The oligomerized pool engineering (OPEN) approach was applied to select specific zinc finger proteins (ZFP) with high affinity. We constructed three zinc finger random libraries, each based on a synthetic "framework" ZFP. In each library, recognition helix residues -1, 1, 2, 3, 4, 5, and 6 from a single finger were randomized by cassette mutagenesis. The three zinc finger random libraries were used to screen specific zinc finger proteins. Human DYRK1A gene was exemplified to examine the feasibility and efficiency of the technology platform for screening of zinc finger proteins. As a result 5o zinc finger proteins with higher affinity to the recognition sites of DYRK1A gene were obtained. The zinc finger proteins were fused to the non-specific endonuclease domain of Fok I to form zinc finger nucleases. Then the activity of zinc finger nucleases was examined in yeast validation system. The result showd that 90% zinc finger nucleases worked well in yeast cells. This research provided the technique support for the application of zinc finger nucleases to the editing of the genome.

  7. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping;

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

  8. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Science.gov (United States)

    K, Muhammed Jamsheer; Laxmi, Ashverya

    2014-01-01

    Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  9. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Muhammed Jamsheer K

    Full Text Available Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  10. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN.

    Directory of Open Access Journals (Sweden)

    Jonathan E Foley

    Full Text Available BACKGROUND: Customized zinc finger nucleases (ZFNs form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering to generate high quality ZFN pairs that function in human and plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. CONCLUSIONS AND SIGNIFICANCE: In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight and suggests that OPEN

  11. Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins.

    Science.gov (United States)

    Zhou, Xixi; Sun, Xi; Mobarak, Charlotte; Gandolfi, A Jay; Burchiel, Scott W; Hudson, Laurie G; Liu, Ke Jian

    2014-04-21

    Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV-vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.

  12. Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

    Institute of Scientific and Technical Information of China (English)

    YAN Xiuying; SUN Xiuqin

    2009-01-01

    An open reading frame (lcn61) of iymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector.Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.

  13. Lead neurotoxicity: exploring the potential impact of lead substitution in zinc-finger proteins on mental health.

    Science.gov (United States)

    Ordemann, Jacqueline Michelle; Austin, Rachel Narehood

    2016-06-01

    Childhood lead poisoning is a costly and largely preventable public health problem that lowers IQs, decreases attention spans, and leads to the development of other childhood intellectual disabilities. Furthermore, recent evidence links developmental lead poisoning with the etiology of disorders that appear much later in life, such as Alzheimer's disease, Parkinson's disease, and schizophrenia. Little is known about how lead influences the onset of these disorders. This paper reviews the evidence that lead substitution for zinc in zinc-finger proteins contributes to the development of Alzheimer's disease, Parkinson's disease, and schizophrenia. The zinc-finger proteins potentially impacted by lead include DNA methyltransferase 1 (DNMT1) and Presenilin 1 and 2 (PSEN1/2) in Alzheimer's disease, the dopamine receptor in Parkinson's disease, and the NMDA receptor, zinc-finger protein 804A (ZNF804A), and disrupted-in-schizophrenia 1 (DISC1)-binding zinc-finger (DBZ) in schizophrenia. PMID:26745006

  14. Toward a Code for the Interactions of Zinc Fingers with DNA: Selection of Randomized Fingers Displayed on Phage

    Science.gov (United States)

    Choo, Yen; Klug, Aaron

    1994-11-01

    We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few α-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.

  15. Can Co(II) or Cd(II) substitute for Zn(II) in zinc fingers?

    Indian Academy of Sciences (India)

    P Rabindra Reddy; M Radhika

    2001-02-01

    Zinc finger domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. The role of zinc in a DNA binding finger was considered purely structural due to the absence of redox chemistry in zinc. However, whether other metals e.g. Co(II) or Cd(II) can substitute Zn(II) is not settled. For an answer the detailed interaction of Co(II) and Cd(II) with cysteine methylester and histidine methylester has been investigated as a model for the zinc core in zinc fingers. The study was extended to different temperatures to evaluate the thermodynamic parameters associated with these interactions. The results suggest that zinc has a unique role.

  16. Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

    Directory of Open Access Journals (Sweden)

    Wenwen Wang

    2012-08-01

    Full Text Available This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.

  17. Site-Specific Editing of the Plasmodium falciparum Genome Using Engineered Zinc-Finger Nucleases

    OpenAIRE

    Straimer, Judith; Lee, Marcus CS; Lee, Andrew H.; Zeitler, Bryan; Williams, April E.; Pearl, Jocelynn R.; Zhang, Lei; Rebar, Edward J.; Gregory, Philip D.; Llinás, Manuel; Urnov, Fyodor D; David A Fidock

    2012-01-01

    Malaria afflicts over 200 million people worldwide and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum pathogenesis, including drug resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite, using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger hom...

  18. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

    OpenAIRE

    Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

    2013-01-01

    The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases th...

  19. The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

    Science.gov (United States)

    D'Abrosca, Gianluca; Russo, Luigi; Palmieri, Maddalena; Baglivo, Ilaria; Netti, Fortuna; de Paola, Ivan; Zaccaro, Laura; Farina, Biancamaria; Iacovino, Rosa; Pedone, Paolo Vincenzo; Isernia, Carla; Fattorusso, Roberto; Malgieri, Gaetano

    2016-08-01

    The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties. PMID:27238756

  20. Transcripts from a novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments

    Energy Technology Data Exchange (ETDEWEB)

    Baban, S.; Freeman, J.D.; Mager, D.L. [Univ. of British Columbia, Vancouver, British Columbia (Canada)

    1996-05-01

    During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5{prime} termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C{sub 2}H{sub 2} type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5{prime} putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types. 41 refs., 8 figs.

  1. Metal Coupled Folding of Cys2His2 Zinc-Finger

    CERN Document Server

    Li, Wenfei; Wang, Jun; Wang, Wei

    2008-01-01

    Zinc-fingers, which widely exist in eukaryotic cell and play crucial roles in life processes, depend on the binding of zinc ion for their proper folding. To computationally study the zinc coupled folding of the zinc-fingers, charge transfer and metal induced protonation/deprotonation effects have to be considered. Here, by attempting to implicitly account for such effects in classical molecular dynamics and performing intensive simulations with explicit solvent for the peptides with and without zinc binding, we investigate the folding of the Cys2His2 type zinc-finger motif and the coupling between the peptide folding and zinc binding. We find that zinc ion not only stabilizes the native structure, but also participates in the whole folding process. It binds to the peptide at early stage of folding, and directs or modulates the folding and stabilizations of the component beta-hairpin and alpha-helix. Such a crucial role of zinc binding is mediated by the packing of the conserved hydrophobic residues. We also f...

  2. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tatsuo [Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505 (Japan); Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko [Department of Molecular Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Doshisha Women' s University, Kodo, Kyotanabe City, Kyoto 610-0395 (Japan); Itoh, Kohji, E-mail: kitoh@ph.tokushima-u.ac.jp [Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505 (Japan); Kuwahara, Jun, E-mail: jkuwahar@dwc.doshisha.ac.jp [Department of Molecular Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Doshisha Women' s University, Kodo, Kyotanabe City, Kyoto 610-0395 (Japan)

    2010-12-10

    Research highlights: {yields} Sp1 zinc fingers themselves interact with importin {alpha}. {yields} Sp1 zinc finger domains play an essential role as a nuclear localization signal. {yields} Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin {alpha} in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin {alpha} including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin {alpha}. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  3. Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C{sub 2}H{sub 2} zinc finger protein

    Energy Technology Data Exchange (ETDEWEB)

    Han, D.; Zhang, C. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); Fan, W.J. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province (China); Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China)

    2014-10-31

    Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C{sub 2}H{sub 2} motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

  4. Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia

    DEFF Research Database (Denmark)

    Mitchelmore, Cathy; Kjaerulff, Karen M; Pedersen, Hans C;

    2002-01-01

    BTB/POZ (broad complex tramtrack bric-a-brac/poxvirus and zinc finger) zinc finger factors are a class of nuclear DNA-binding proteins involved in development, chromatin remodeling, and cancer. However, BTB/POZ domain zinc finger factors linked to development of the mammalian cerebral cortex......, cerebellum, and macroglia have not been described previously. We report here the isolation and characterization of two novel nuclear BTB/POZ domain zinc finger isoforms, designated HOF(L) and HOF(S), that are specifically expressed in early hippocampal neurons, cerebellar granule cells, and gliogenic...

  5. The Electronic Behavior of Zinc-Finger Protein Binding Sites in the Context of the DNA Extended Ladder Model

    Science.gov (United States)

    Oiwa, Nestor; Cordeiro, Claudette; Heermann, Dieter

    2016-05-01

    Instead of ATCG letter alignments, typically used in bioinformatics, we propose a new alignment method using the probability distribution function of the bottom of the occupied molecular orbital (BOMO), highest occupied molecular orbital (HOMO) and lowest unoccupied orbital (LUMO). We apply the technique to transcription factors with Cys2His2 zinc fingers. These transcription factors search for binding sites, probing for the electronic patterns at the minor and major DNA groves. The eukaryotic Cys2His2 zinc finger proteins bind to DNA ubiquitously at highly conserved domains. They are responsible for gene regulation and the spatial organization of DNA. To study and understand these zinc finger DNA-protein interactions, we use the extended ladder in the DNA model proposed by Zhu, Rasmussen, Balatsky & Bishop (2007) te{Zhu-2007}. Considering one single spinless electron in each nucleotide π-orbital along a double DNA chain (dDNA), we find a typical pattern for the bottom of BOMO, HOMO and LUMO along the binding sites. We specifically looked at two members of zinc finger protein family: specificity protein 1 (SP1) and early grown response 1 transcription factors (EGR1). When the valence band is filled, we find electrons in the purines along the nucleotide sequence, compatible with the electric charges of the binding amino acids in SP1 and EGR1 zinc finger.

  6. ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

    Directory of Open Access Journals (Sweden)

    Voytas Daniel F

    2011-01-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms. Description ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s. Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence. Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the

  7. Zinc finger protein TTP interacts with CCL3 mRNA and regulates tissue inflammation*

    OpenAIRE

    Kang, Ju-Gyeong; Amar, Marcelo J.; Remaley, Alan T.; Kwon, Jaeyul; Blackshear, Perry J.; Wang, Ping-yuan; Hwang, Paul M.

    2011-01-01

    Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. Here, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CC chemokine ligand 3 (CCL3) mRNA as the most abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to the wild-type cells, TTP−/− macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 i...

  8. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains.

    OpenAIRE

    Vallee, B L; Coleman, J E; Auld, D S

    1991-01-01

    We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have be...

  9. Zinc finger protein 278, a potential oncogene in human colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Xiaoqing Tian; Danfeng Sun; Yanjie Zhang; Shuliang Zhao; Hua Xiong; Jingyuan Fang

    2008-01-01

    Zinc finger protein 278 (ZNF278) is a novel Krueppel Cys2-His2-type zinc finger protein that is ubiquitously distributed in human tissues. Whether ZNF278 is related to the development of colorectal cancer is still unclear. The transcriptional level of ZNF278 was studied in colorectal cancer by real-time polymerase chain reaction. The results showed that ZNF278 expression was increased in 53% of colorectal cancer tissues compared to corresponding non-cancerous tissues. The transcriptional down-regulation of ZNF278 was detected in only three (6%) human colorectal cancer tissues compared to corresponding non-cancer tissues. No significant difference was detected in 19 (41%) pairs of samples.However, we failed to find a significant association between the up-regulation of ZNF278 transcription and age, sex, the degree of infiltration, or the tumor size of colorectal cancer.To study the function of ZNF278 in colorectal carcinogenesis,the colon cancer cell line SW1116 was stably transfected with a wild-type ZNF278 plasmid to construct an overexpression system, and was transiently transfected with the small interfering RNA of ZNF278 to construct a ZNF278 knockdown system. Cell proliferation was assessed with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye and a cell counter. The results show that ZNF278 promotes cell growth, and its knockdown suppresses cell proliferation. ZNF278 could be a potential proto-oncogene in colorectal cancer.

  10. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  11. Predicting success of oligomerized pool engineering (OPEN for zinc finger target site sequences

    Directory of Open Access Journals (Sweden)

    Goodwin Mathew J

    2010-11-01

    Full Text Available Abstract Background Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable. Results Analysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88% and specificity+ (92%, but with reduced ROC AUC (0.77. Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT. Conclusion ZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function

  12. Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis.

    Science.gov (United States)

    Xu, Fengqin; Wang, Weiping; Lei, Chen; Liu, Qingmei; Qiu, Hao; Muraleedharan, Vinaydhar; Zhou, Bin; Cheng, Hongxia; Huang, Zhongkai; Xu, Weian; Li, Bichun; Wang, Minghua

    2012-04-01

    Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

  13. Modular system for the construction of zinc-finger libraries and proteins.

    Science.gov (United States)

    Gonzalez, Beatriz; Schwimmer, Lauren J; Fuller, Roberta P; Ye, Yongjun; Asawapornmongkol, Lily; Barbas, Carlos F

    2010-04-01

    Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.

  14. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases.

    Science.gov (United States)

    Straimer, Judith; Lee, Marcus C S; Lee, Andrew H; Zeitler, Bryan; Williams, April E; Pearl, Jocelynn R; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Llinás, Manuel; Urnov, Fyodor D; Fidock, David A

    2012-10-01

    Malaria afflicts over 200 million people worldwide, and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum-induced pathogenesis, including drug-resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene-deletion parasites with unprecedented speed (2 weeks), both with and without direct selection. ZFNs engineered against the parasite gene pfcrt, responsible for escape under chloroquine treatment, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. This method will enable a diverse array of genome-editing approaches to interrogate this human pathogen.

  15. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    Science.gov (United States)

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  16. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos.

    Science.gov (United States)

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-10-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos. PMID:27189642

  17. Functional promoter variant in zinc finger protein 202 predicts severe atherosclerosis and ischemic heart disease

    DEFF Research Database (Denmark)

    Frikke-Schmidt, R.; Nordestgaard, Børge; Grande, Peer;

    2008-01-01

    Objectives This study was designed to test the hypotheses that single nucleotide polymorphisms ( SNPs), in zinc finger protein 202 ( ZNF202), predict severe atherosclerosis and ischemic heart disease ( IHD). Background ZNF202 is a transcriptional repressor controlling promoter elements in genes...... involved in vascular maintenance and lipid metabolism. Methods We first determined genotype association for 9 ZNF202 SNPs with severe atherosclerosis ( ankle brachial index >0.7 vs. ...,998 controls. Finally, we determined whether g. -660A>G altered transcriptional activity of the ZNF202 promoter in vitro. Results Cross-sectionally, ZNF202 g. -660 GG versus AA homozygosity predicted an odds ratio for severe atherosclerosis of 2.01 ( 95% confidence interval [CI]: 1.34 to 3.01). Prospectively...

  18. The functional significance of common polymorphisms in zinc finger transcription factors.

    Science.gov (United States)

    Lockwood, Sarah H; Guan, Anna; Yu, Abigail S; Zhang, Chi; Zykovich, Artem; Korf, Ian; Rannala, Bruce; Segal, David J

    2014-09-01

    Variants that alter the DNA-binding specificity of transcription factors could affect the specificity for and expression of potentially many target genes, as has been observed in several tumor-derived mutations. Here we examined if such trans expression quantitative trait loci (trans-eQTLs) could similarly result from common genetic variants. We chose to focus on the Cys2-His2 class of zinc finger transcription factors because they are the most abundant superfamily of transcription factors in human and have well-characterized DNA binding interactions. We identified 430 SNPs that cause missense substitutions in the DNA-contacting residues. Fewer common missense SNPs were found at DNA-contacting residues compared with non-DNA-contacting residues (P = 0.00006), consistent with possible functional selection against SNPs at DNA-contacting positions. Functional predictions based on zinc finger transcription factor (ZNF) DNA binding preferences also suggested that many common substitutions could potentially alter binding specificity. However, Hardy-Weinberg Equilibrium analysis and examination of seven orthologs within the primate lineage failed to find evidence of trans-eQTLs associated with the DNA-contacting positions or evidence of a different selection pressure on a contemporary and evolutionary timescales. The overall conclusion was that common SNPs that alter the DNA-contacting residues of these factors are unlikely to produce strong trans-eQTLs, consistent with the observations by others that trans-eQTLs in humans tend to be few and weak. Some rare SNPs might alter specificity and remained rare due to purifying selection. The study also underscores the need for large-scale eQTLs mapping efforts that might provide experimental evidence for SNPs that alter the choice of transcription factor binding sites. PMID:24970883

  19. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan); Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki [System Instruments Co., Ltd., 776-2 Komiya-cho, Hachioji, Tokyo 192-0031 (Japan); Noda, Mamoru; Igimi, Shizunobu [Division of Biomedical Food Research, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Ikebukuro, Kazunori, E-mail: ikebu@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan)

    2013-11-01

    Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10{sup 6} copies.

  20. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

    International Nuclear Information System (INIS)

    Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 106 copies

  1. Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed Metallopeptides

    International Nuclear Information System (INIS)

    Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biological activity. The thermodynamic contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorimetry, potentiometry, and calorimetry to determine the thermodynamics of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation constants for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6 x 1016, 1.5 x 1015, or 2.5 x 1013 M-1, respectively. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, respectively, in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equivalent at physiological pH, ?G = -16.8 kcal/mol or a Ka = 2.0 x 1012 M-1. Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to determine the free energy required to fold the latter

  2. Involvement of C2H2 zinc finger proteins in the regulation of epidermal cell fate determination in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    An Yan; Minjie Wu; Yongqin Zhao; Aidong Zhang; Bohan Liu; John Schiefelbein; Yinbo Gan

    2014-01-01

    Cell fate determination is a basic developmental process during the growth of multicellular organisms. Trichomes and root hairs of Arabidopsis are both readily accessible structures originating from the epidermal cells of the aerial tissues and roots respectively, and they serve as excellent models for understanding the molecular mecha-nisms controlling cell fate determination and cell morphogen-esis. The regulation of trichome and root hair formation is a complex program that consists of the integration of hormonal signals with a large number of transcriptional factors, including MYB and bHLH transcriptional factors. Studies during recent years have uncovered an important role of C2H2 type zinc finger proteins in the regulation of epidermal cell fate determination. Here in this minireview we briefly summarize the involvement of C2H2 zinc finger proteins in the control of trichome and root hair formation in Arabidopsis.

  3. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins.

    Science.gov (United States)

    Segal, David J; Beerli, Roger R; Blancafort, Pilar; Dreier, Birgit; Effertz, Karin; Huber, Adrian; Koksch, Beate; Lund, Caren V; Magnenat, Laurent; Valente, David; Barbas, Carlos F

    2003-02-25

    In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

  4. Zinc finger nucleases and their application%锌指核酶技术的原理及应用

    Institute of Scientific and Technical Information of China (English)

    邓珊珊; 王颖芝; 马端

    2010-01-01

    锌指核酶(zinc finger nucleases,ZFNs)是将锌指蛋白的DNA识别域和非特异性核酸内切酶FokⅠ人工连接而构成的一种核酶.1对ZFNs能在DNA上产生双链断裂(double-strand breaks,DSB),诱导细胞发生同源重组(homology recombination,HR)或非同源末端连接(nonhomologous end joining,NHEJ).最近锌指核酶技术在基因功能的研究中受到重视.作者就ZFNs的作用原理、关键技术及其应用领域进行介绍.%Zinc finger nuclease (ZFN), which is a chimeric fusion structure between a Cys2-His2 zinc-finger protein (ZFP) and the cleavage domain of Fok Ⅰ endonuclease,can be used to introduce targeted double-stranded breaks (DSBs). ZFN-mediated cleavage leads to mutations when double-stranded breaks are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). In recent years, ZFNs are widely used in the fields of genetic research. In this review, the methodology and technical advantages of ZFNs were briefly discussed.

  5. Zinc finger protein 521 overexpression increased transcript levels of Fndc5 in mouse embryonic stem cells

    Indian Academy of Sciences (India)

    Motahere-Sadat Hashemi; Abbas Kiani Esfahani; Maryam Peymani; Alireza Shoaraye Nejati; Kamran Ghaedi; Mohammad Hossein Nasr-Esfahani; Hossein Baharvand

    2016-03-01

    Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human hematopoietic cells. Zfp521 triggers the cascade of neurogenesis inmouse embryonic stemcells through inducing expression of the early neuroectodermal genes Sox1, Sox3 and Pax6. Fndc5, a precursor of Irisin has inducing effects on the expression level of brain derived neurotrophic factor in hippocampus. Therefore, it is most likely that Fndc5 may play an important role in neural differentiation. To exhibit whether the expression of this protein is under regulation with Zfp521, we overexpressed Zfp521 in a stable transformants of mESCs expressing EGFP under control of Fndc5 promoter. Increased expression of Zfp521 enhanced transcription levels of both EGFP and endogenous Fndc5. This result was confirmed by overexpression the aforementioned vectors in HEK cells and indicated that Zfp521 functions upstream of Fndc5 expression. It is most likely that Zfp521 may act through the binding to its response element on Fndc5 core promoter. Therefore it is concluding that an enhanced expression of Fndc5 in neural progenitor cells is stimulated by Zfp521 overexpression in these cells.

  6. Identification of SCAN domain zinc-finger gene ZNF449 as a novel factor of chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Keita Okada

    Full Text Available Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6. We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

  7. Origin and evolution of new exons in the rodent zinc finger protein 39 gene

    Institute of Scientific and Technical Information of China (English)

    PENG Lixin; ZHENG Hongkun; LI Xin; YANG Shuang; CHEN Hong; WANG Wen

    2005-01-01

    The origin of new structures and functions is an important process in evolution. In the past decades, we have obtained some preliminary knowledge of the origin and evolution of new genes. However, as the basic unit of genes, the origin and evolution of exons remain unclear. Because young exons retain the footprints of origination, they can be good materials for studying origin and evolution of new exons. In this paper, we report two young exons in a zinc finger protein gene of rodents. Since they are unique sequences in mouse and rat genome and no homologous sequences were found in the orthologous genes of human and pig, the young exons might originate after the divergence of primates and rodents through exonization of intronic sequences. Strong positive selection was detected in the new exons between mouse and rat, suggesting that these exons have undergone significant functional divergence after the separation of the two species. On the other hand, population genetics data of mouse demonstrate that the new exons have been subject to functional constraint, indicating an important function of the new exons in mouse. Functional analyses suggest that these new exons encode a nuclear localization signal peptide, which may mediate new ways of nuclear protein transport. To our knowledge, this is the first example of the origin and evolution of young exons.

  8. Zinc finger protein 148 is dispensable for primitive and definitive hematopoiesis in mice.

    Directory of Open Access Journals (Sweden)

    Anna Nilton

    Full Text Available Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148 interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 (gt/gt mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 (gt/gt mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.

  9. Zinc finger peptide based optic sensor for detection of zinc ions.

    Science.gov (United States)

    Verma, Neelam; Kaur, Gagandeep

    2016-12-15

    In the present work, polyacrylamide gel has been used as a matrix for the immobilization of zinc finger peptide and fluorescent dye acrydine orange on the micro well plate to fabricate the fluorescence based biosensor for the detection of zinc ions in milk samples. The fluorescent dye moves in the hydrophobic groove formed after folding of the peptide in the presence of zinc ions. Under optimized conditions, linear range was observed between 0.001µg/l to 10µg/l of Zinc ions, with a lowest detection limit of 0.001µg/l and response time of 5min. Presented biosensor has shown 20% decrease in fluorescent intensity values after 5 regenerations and stable for more than one month, stored at 4°C. Interference study with other metal ions like lead, cadmium and copper showed a negligible change in fluorescence intensity in comparison to zinc ions. Developed bio sensing system was found to be novel, quick, reliable, miniaturized, stable, reproducible and repeatable and specific for zinc ion, which has been applied to various milk samples. PMID:27424265

  10. Classification of the treble clef zinc finger: noteworthy lessons for structure and function evolution.

    Science.gov (United States)

    Kaur, Gurmeet; Subramanian, Srikrishna

    2016-01-01

    Treble clef (TC) zinc fingers constitute a large fold-group of structural zinc-binding protein domains that mediate numerous cellular functions. We have analysed the sequence, structure, and function relationships among all TCs in the Protein Data Bank. This led to the identification of novel TCs, such as lsr2, YggX and TFIIIC τ 60 kDa subunit, and prediction of a nuclease-like function for the DUF1364 family. The structural malleability of TCs is evident from the many examples with variations to the core structural elements of the fold. We observe domains wherein the structural core of the TC fold is circularly permuted, and also some examples where the overall fold resembles both the TC motif and another unrelated fold. All extant TC families do not share a monophyletic origin, as several TC proteins are known to have been present in the last universal common ancestor and the last eukaryotic common ancestor. We identify several TCs where the zinc-chelating site and residues are not merely responsible for structure stabilization but also perform other functions, such as being redox active in C1B domain of protein kinase C, a nucleophilic acceptor in Ada and catalytic in organomercurial lyase, MerB. PMID:27562564

  11. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

    Science.gov (United States)

    Merlin, Christine; Beaver, Lauren E; Taylor, Orley R; Wolfe, Scot A; Reppert, Steven M

    2013-01-01

    The development of reverse-genetic tools in "nonmodel" insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into "one nucleus" stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and "nonmodel" insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

  12. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    Science.gov (United States)

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  13. Mutagenesis of Genes for Starch Debranching Enzyme Isoforms in Pea by Zinc-Finger Endonucleases

    International Nuclear Information System (INIS)

    Starch debranching enzymes in plants are divided into two groups based on their ability to hydrolyze different substrates. The first group, pullulanases, hydrolyze α-1,6-glucosidic linkages in substrates such as pullulan, amylopectin and glycogen. The second group of debranching enzymes, isoamylases, hydrolyze glycogen and amylopectin and are not active on pullulan. Three isoforms of isoamylase and a pullulanase have been isolated from a cDNA library of Pisum sativum. These isoamylases have been characterized following their heterologous expression in E. coli. Based on the DNA sequence that encodes these debranching enzymes, a specific mutagenesis targeting these enzymes will be attempted. The technique involves the homologous recombination of DNA mediated by zinc-finger endonucleases. Vectors will be constructed to include a fragment that will modify these genes. Using this technique, it is hoped that null mutants for each enzyme will be created and the exact role of these enzymes for the synthesis and degradation of starch in plants will be elucidated. (author)

  14. 15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation.

    Science.gov (United States)

    Sumanas, Saulius; Zhang, Bo; Dai, Rujuan; Lin, Shuo

    2005-07-01

    A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

  15. Zinc finger peptide based optic sensor for detection of zinc ions.

    Science.gov (United States)

    Verma, Neelam; Kaur, Gagandeep

    2016-12-15

    In the present work, polyacrylamide gel has been used as a matrix for the immobilization of zinc finger peptide and fluorescent dye acrydine orange on the micro well plate to fabricate the fluorescence based biosensor for the detection of zinc ions in milk samples. The fluorescent dye moves in the hydrophobic groove formed after folding of the peptide in the presence of zinc ions. Under optimized conditions, linear range was observed between 0.001µg/l to 10µg/l of Zinc ions, with a lowest detection limit of 0.001µg/l and response time of 5min. Presented biosensor has shown 20% decrease in fluorescent intensity values after 5 regenerations and stable for more than one month, stored at 4°C. Interference study with other metal ions like lead, cadmium and copper showed a negligible change in fluorescence intensity in comparison to zinc ions. Developed bio sensing system was found to be novel, quick, reliable, miniaturized, stable, reproducible and repeatable and specific for zinc ion, which has been applied to various milk samples.

  16. Classification of the treble clef zinc finger: noteworthy lessons for structure and function evolution

    Science.gov (United States)

    Kaur, Gurmeet; Subramanian, Srikrishna

    2016-01-01

    Treble clef (TC) zinc fingers constitute a large fold-group of structural zinc-binding protein domains that mediate numerous cellular functions. We have analysed the sequence, structure, and function relationships among all TCs in the Protein Data Bank. This led to the identification of novel TCs, such as lsr2, YggX and TFIIIC τ 60 kDa subunit, and prediction of a nuclease-like function for the DUF1364 family. The structural malleability of TCs is evident from the many examples with variations to the core structural elements of the fold. We observe domains wherein the structural core of the TC fold is circularly permuted, and also some examples where the overall fold resembles both the TC motif and another unrelated fold. All extant TC families do not share a monophyletic origin, as several TC proteins are known to have been present in the last universal common ancestor and the last eukaryotic common ancestor. We identify several TCs where the zinc-chelating site and residues are not merely responsible for structure stabilization but also perform other functions, such as being redox active in C1B domain of protein kinase C, a nucleophilic acceptor in Ada and catalytic in organomercurial lyase, MerB. PMID:27562564

  17. An elastic-network based local molecular field analysis of zinc-finger proteins

    CERN Document Server

    Dixit, Purushottam D

    2011-01-01

    We study two designed and one natural zinc-finger peptide each with the Cys2His2 (CCHH) type of metal binding motif. In the approach we have developed, we describe the role of the protein and solvent outside the Zn(II)-CCHH metal-residue cluster by a molecular field represented by generalized harmonic restraints. The strength of the field is adjusted to reproduce the binding energy distribution of the metal with the cluster obtained in a reference all-atom simulation with empirical potentials. The quadratic field allows us to investigate analytically the protein restraints on the binding site in terms of its eigenmodes. Examining these eigenmodes suggests, consistent with experimental observations, the importance of the first histidine (H) in the CCHH cluster in metal binding. Further, the eigenvalues corresponding to these modes also indicate that the designed proteins form a tighter complex with the metal. We find that the bulk protein and solvent response tends to destabilize metal-binding, emphasizing tha...

  18. Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

    Directory of Open Access Journals (Sweden)

    Carina Frauer

    Full Text Available Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

  19. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

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    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  20. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

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    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  1. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Science.gov (United States)

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  2. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  3. Establishment of transgenic mice carrying gene encoding human zinc finger protein 191

    Institute of Scientific and Technical Information of China (English)

    Jian-Zhong Li; Ji-Liang Fu; Xia Chen; Hua Yang; Shui-Liang Wang; Xue-Lian Gong; Hao Feng; Bao-Yu Guo; Long Yu; Zhu-Gang Wang

    2004-01-01

    AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Kruppel-like transcription factor, which might be relevant to many diseases such as liver cancer,neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF191 transgenic mouse model, which would promote the functional study of ZNF191.METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination.RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF191gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice.CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

  4. Zinc finger protein A20 protects rats against chronic liver allograft dysfunction

    Institute of Scientific and Technical Information of China (English)

    Jie Yang; Ming-Qing Xu; Lu-Nan Yan; Xiao-Bo Chen; Jiao Liu

    2012-01-01

    AIM:To investigate the effect of zinc finger protein A20 on chronic liver allograft dysfunction in rats.METHODS:Allogeneic liver transplantation from DA rats to Lewis rats was performed.Chronic liver allograft dysfunction was induced in the rats by administering low-dose tacrolimus at postoperative day (POD) 5.Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)-mediated gene transfer administered intravenously every 10 d starting from POD 10.The recipient rats were injected with physiological saline,rAdEasy-A20 (1 x 109 pfu/30 g weight) or rAdEasy (1 x 109 pfu/30 g weight) every 10 d through the tail vein for 3 mo starting from POD 10.Liver tissue samples were harvested on POD 30 and POD 60.RESULTS:Liver-transplanted rats treated with only tacrolimus showed chronic allograft dysfunction with severe hepatic fibrosis.A20 overexpression ameliorated the effects on liver function,attenuated liver allograft fibrosis and prolonged the survival of the recipient rats.Treatment with A20 suppressed hepatic protein production of tumor growth factor (TGF)-β1,interleukin1β,caspase-8,CD40,CD40L,intercellular adhesion molecule-1,vascular cell adhesion molecule-1 and E-selectin.A20 treatment suppressed liver cell apoptosis and inhibited nuclear factor-κB activation of Kupffer cells (KCs),liver sinusoidal endothelial cells (LSECs)and hepatic stellate cells (HSCs),and it subsequently decreased cytokine mRNA expression in KCs and LSECs and reduced the production of TGF-β1 in HSCs.CONCLUSION:A20 might prevent chronic liver allograft dysfunction by re-establishing functional homeostasis of KCs,LSECs and HSCs.

  5. Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

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    Sarah L Maguire

    2014-01-01

    Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

  6. ZFN-Site searches genomes for zinc finger nuclease target sites and off-target sites

    Directory of Open Access Journals (Sweden)

    Iseli Christian

    2011-05-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence. Results ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases. Conclusions ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.

  7. Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

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    Lingyan Xing

    Full Text Available foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd coding exon: a 17 base-pair (bp deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

  8. Hydrogen sulfide represses androgen receptor transactivation by targeting at the second zinc finger module.

    Science.gov (United States)

    Zhao, Kexin; Li, Shuangshuang; Wu, Lingyun; Lai, Christopher; Yang, Guangdong

    2014-07-25

    Androgen receptor (AR) signaling is indispensable for the development of prostate cancer from the initial androgen-dependent state to a later aggressive androgen-resistant state. This study examined the role of hydrogen sulfide (H(2)S), a novel gasotransmitter, in the regulation of AR signaling as well as its mediation in androgen-independent cell growth in prostate cancer cells. Here we found that H(2)S inhibits cell proliferation of both androgen-dependent (LNCaP) and antiandrogen-resistant prostate cancer cells (LNCaP-B), with more significance on the latter, which was established by long term treatment of parental LNCaP cells with bicalutamide. The expression of cystathionine γ-lyase (CSE), a major H(2)S producing enzyme in prostate tissue, was reduced in both human prostate cancer tissues and LNCaP-B cells. LNCaP-B cells were resistant to bicalutamide-induced cell growth inhibition, and CSE overexpression could rebuild the sensitivity of LNCaP-B cells to bicalutamide. H(2)S significantly repressed the expression of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes. In addition, H(2)S inhibited AR binding with PSA promoter and androgen-responsive element (ARE) luciferase activity. We further found that AR is post-translationally modified by H(2)S through S-sulfhydration. Mutation of cysteine 611 and cysteine 614 in the second zinc finger module of AR-DNA binding domain diminished the effects of H(2)S on AR S-sulfhydration and AR dimerization. These data suggest that reduced CSE/H2S signaling contributes to antiandrogen-resistant status, and sufficient level of H(2)S is able to inhibit AR transactivation and treat castration-resistant prostate cancer.

  9. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  10. The LSD1-type zinc finger motifs of Pisum sativa LSD1 are a novel nuclear localization signal and interact with importin alpha.

    Directory of Open Access Journals (Sweden)

    Shanping He

    Full Text Available BACKGROUND: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD. Arabidopsis thaliana LSD1 (AtLSD1 contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. METHODOLOGY/PRINCIPAL FINDINGS: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1, which is a homolog of AtLSD1. Subcellular localization analysis of green fluorescent protein (GFP-tagged PsLSD1 indicates that PsLSD1 is localized in the nucleus. Using a series of GFP-tagged PsLSD1 deletion mutants, we found that the three LSD1-type zinc finger motifs of PsLSD1 alone can target GFP to the nucleus, whereas deletion of the three zinc finger motifs or any individual zinc finger motif causes PsLSD1 to lose its nuclear localization, indicating that the three zinc finger motifs are necessary and sufficient for its nuclear localization. Moreover, site-directed mutagenesis analysis of GFP-tagged PsLSD1 indicates that tertiary structure and basic amino acids of each zinc finger motif are necessary for PsLSD1 nuclear localization. In addition, yeast two-hybrid, pull-down, and BiFC assays demonstrate that the three zinc finger motifs of PsLSD1 directly bind to importin α in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that the LSD1-type zinc finger motifs of PsLSD1 are a novel nuclear localization signal and directly bind to importin α, and suggest that the nuclear import of LSD1 may rely on the interaction between its zinc finger motifs and importin α. Moreover, the nuclear localization of PsLSD1 suggests that LSD1 may function as a transcription regulator involved in negatively regulating PCD.

  11. Molecular cloning and characterization of a novel RING zinc-finger protein gene up-regulated under in vitro salt stress in cassava.

    Science.gov (United States)

    dos Reis, Sávio Pinho; Tavares, Liliane de Souza Conceição; Costa, Carinne de Nazaré Monteiro; Brígida, Aílton Borges Santa; de Souza, Cláudia Regina Batista

    2012-06-01

    Cassava (Manihot esculenta Crantz) is one of the world's most important food crops. It is cultivated mainly in developing countries of tropics, since its root is a major source of calories for low-income people due to its high productivity and resistance to many abiotic and biotic factors. A previous study has identified a partial cDNA sequence coding for a putative RING zinc finger in cassava storage root. The RING zinc finger protein is a specialized type of zinc finger protein found in many organisms. Here, we isolated the full-length cDNA sequence coding for M. esculenta RZF (MeRZF) protein by a combination of 5' and 3' RACE assays. BLAST analysis showed that its deduced amino acid sequence has a high level of similarity to plant proteins of RZF family. MeRZF protein contains a signature sequence motif for a RING zinc finger at its C-terminal region. In addition, this protein showed a histidine residue at the fifth coordination site, likely belonging to the RING-H2 subgroup, as confirmed by our phylogenetic analysis. There is also a transmembrane domain in its N-terminal region. Finally, semi-quantitative RT-PCR assays showed that MeRZF expression is increased in detached leaves treated with sodium chloride. Here, we report the first evidence of a RING zinc finger gene of cassava showing potential role in response to salt stress.

  12. Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins.

    Science.gov (United States)

    Kedar, Vishram P; Zucconi, Beth E; Wilson, Gerald M; Blackshear, Perry J

    2012-02-17

    Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ∼5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.

  13. The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

    Directory of Open Access Journals (Sweden)

    Colasanti Joseph

    2006-06-01

    Full Text Available Abstract Background The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD, which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. Results Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. Conclusion Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein

  14. Characterization of the contradictory chromatin signatures at the 3' exons of zinc finger genes.

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    Kimberly R Blahnik

    Full Text Available The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3' exons of zinc finger genes (ZNFs are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3' exons of ZNFs are promoters. We further characterized the histone modifications at the 3' ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3' exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3' ZNF exon was removed from its natural genomic location, the isolated ZNF 3' exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3' exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3' exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3' exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.

  15. Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum

    Indian Academy of Sciences (India)

    Xiuhong Yang; Chao Sun; Yuanlei Hun; Zhongping Lin

    2008-03-01

    A RING zinc finger ankyrin protein gene, designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4-type RING finger domain was found at the C-terminal region of the AdZFP1 protein, and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root, stem and leaf of the plant. Semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity, cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

  16. A novel genetic system based on zinc finger nucleases for the identification of interactions between proteins in vivo.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available Yeast two-hybrid (Y2H methods are powerful tools for detecting protein-protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs. ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP and a non-specific nuclease domain (FokI. In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109, the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research.

  17. The DnaJ-like zinc finger domain protein PSA2 affects light acclimation and chloroplast development in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yan-Wen eWang

    2016-03-01

    Full Text Available The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development.

  18. Genetic dissection of photoreceptor subtype specification by the Drosophila melanogaster zinc finger proteins elbow and no ocelli.

    Directory of Open Access Journals (Sweden)

    Mathias F Wernet

    2014-03-01

    Full Text Available The elbow/no ocelli (elb/noc complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the 'dorsal rim area' (DRA of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease.

  19. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.

    Science.gov (United States)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

    2013-11-01

    An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies.

  20. Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

    Science.gov (United States)

    Wilen, Craig B.; Wang, Jianbin; Tilton, John C.; Miller, Jeffrey C.; Kim, Kenneth A.; Rebar, Edward J.; Sherrill-Mix, Scott A.; Patro, Sean C.; Secreto, Anthony J.; Jordan, Andrea P. O.; Lee, Gary; Kahn, Joshua; Aye, Pyone P.; Bunnell, Bruce A.; Lackner, Andrew A.; Hoxie, James A.; Danet-Desnoyers, Gwenn A.; Bushman, Frederic D.; Riley, James L.; Gregory, Philip D.; June, Carl H.; Holmes, Michael C.; Doms, Robert W.

    2011-01-01

    HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals. PMID:21533216

  1. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  2. The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery.

    Science.gov (United States)

    Bassler, Jochen; Klein, Isabella; Schmidt, Claudia; Kallas, Martina; Thomson, Emma; Wagner, Maria Anna; Bradatsch, Bettina; Rechberger, Gerald; Strohmaier, Heimo; Hurt, Ed; Bergler, Helmut

    2012-12-01

    The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

  3. The RNA Binding Zinc Finger Protein Tristetraprolin Regulates AU-Rich mRNAs Involved in Breast Cancer-Related Processes

    OpenAIRE

    Al-Souhibani, Norah; Al-Ahmadi, Wijdan; Hesketh, John E.; Blackshear, Perry J.; Khabar, Khalid S.A.

    2010-01-01

    Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc finger RNA binding protein that regulates the stability of certain AU-rich mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared to normal cell types. Here we found that TTP expression was lower in invasive breast cancer cells (MDA-MB-231) compared to normal breast cell lines, MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc finger TTP mutant that act as dominant negati...

  4. The SLEEPER genes: a transposase-derived angiosperm-specific gene family

    Directory of Open Access Journals (Sweden)

    Knip Marijn

    2012-10-01

    Full Text Available Abstract Background DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little is known about the presence of DAYSLEEPER orthologs in other species, or how and when it was domesticated. We studied the presence of DAYSLEEPER orthologs in plants and propose a model for the domestication of the ancestral DAYSLEEPER gene in angiosperms. Results Using specific BLAST searches in genomic and EST libraries, we found that DAYSLEEPER-like genes (hereafter called SLEEPER genes are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae and dicotyledonous plants possess such putative orthologous genes, but SLEEPER-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one SLEEPER gene. All SLEEPERs contain a C2H2 type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the SLEEPER family. Although SLEEPER genes are structurally conserved between species, constructs with SLEEPER genes from grapevine and rice did not complement the daysleeper phenotype in Arabidopsis, when expressed under control of the DAYSLEEPER promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the RICESLEEPER1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of SLEEPER genes, such as an event in the rice genome, which gave rise to the RICESLEEPER1 and 2 genes. Conclusions We propose the ancestral SLEEPER gene was formed after a process of retro

  5. Characterization of the target DNA sequence for the DNA-binding domain of zinc finger protein 191

    Institute of Scientific and Technical Information of China (English)

    Haoyue Wang; Ruilin Sun; Guoxiang Liu; Minghui Yao; Jian Fei; Hebai Shen

    2008-01-01

    Studies on the DNA-binding properties of transcription factors are important in searching for the downstream genes regulated by these factors. In the present study, we report on the DNA-binding property of a Cys2His2-type transcription factor, zinc finger protein 191 (Zfp191), which has been newly found to play a significant role in mice.By constructing a fusion protein containing the DNA-binding domain of Zfp191,we characterized target DNA by determining the protein's binding specificity and dependence on zinc.The data showed that the DNA-binding domain of Zfp191can specifically bind to the TCAT repeat motif and that there is a cooperative effect among the target DNA's multiple binding sites.Furthermore,the binding reaction is dependent on zinc.This work provides a foundation for further studies on the role of Zfp191 in gene regulation and development.

  6. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, Agnethe; Jenkins, Tom;

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  7. Arabidopsis VARIEGATED 3 encodes a chloroplasttargeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, A.; Jenkins, T.;

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  8. The Role of Cdkn1A-Interacting Zinc Finger Protein 1 (CIZ1 in DNA Replication and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2016-02-01

    Full Text Available Cdkn1A-interacting zinc finger protein 1 (CIZ1 was first identified in a yeast-2-hybrid system searching for interacting proteins of CDK2 inhibitor p21Cip1/Waf1. Ciz1 also binds to CDK2, cyclin A, cyclin E, CDC6, PCNA, TCF4 and estrogen receptor-α. Recent studies reveal numerous biological functions of CIZ1 in DNA replication, cell proliferation, and differentiation. In addition, splicing variants of CIZ1 mRNA is associated with a variety of cancers and Alzheimer’s disease, and mutations of the CIZ1 gene lead to cervical dystonia. CIZ1 expression is increased in cancers and rheumatoid arthritis. In this review, we will summarize the biological functions and molecular mechanisms of CIZ1 in these physiological and pathological processes.

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

    Science.gov (United States)

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  10. Structural and dynamical characterization of the Miz-1 zinc fingers 5-8 by solution-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, David; Bedard, Mikaeel; Bilodeau, Josee; Lavigne, Pierre, E-mail: pierre.lavigne@usherbrooke.ca [Universite de Sherbrooke, Departement de Biochimie, Faculte de Medecine et des Sciences de la Sante, Institut de Pharmacologie de Sherbrooke (Canada)

    2013-10-15

    Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15{sup INK4B} or p21{sup CIP1}. The C-terminus of Miz-1 contains 13 consensus C{sub 2}H{sub 2} zinc finger domains (ZF). ZFs 1-4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical {beta}{beta}{alpha} fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5-8 (Miz 5-8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical {beta}{beta}{alpha} fold for C{sub 2}H{sub 2} ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using {sup 15}N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the {mu}s-ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1-4 away from DNA.

  11. Evolutionary expansion and divergence in a large family of primate-specific zinc finger transcription factor genes

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, A T; Huntley, S; Tran-Gyamfi, M; Baggott, D; Gordon, L; Stubbs, L

    2005-09-28

    Although most genes are conserved as one-to-one orthologs in different mammalian orders, certain gene families have evolved to comprise different numbers and types of protein-coding genes through independent series of gene duplications, divergence and gene loss in each evolutionary lineage. One such family encodes KRAB-zinc finger (KRAB-ZNF) genes, which are likely to function as transcriptional repressors. One KRAB-ZNF subfamily, the ZNF91 clade, has expanded specifically in primates to comprise more than 110 loci in the human genome, yielding large gene clusters in human chromosomes 19 and 7 and smaller clusters or isolated copies at other chromosomal locations. Although phylogenetic analysis indicates that many of these genes arose before the split between old world monkeys and new world monkeys, the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. The paralogous loci are distinguished by sequence divergence within their zinc finger arrays indicating a selection for proteins with different DNA binding specificities. RT-PCR and in situ hybridization data show that some of these ZNF genes can have tissue-specific expression patterns, however many KRAB-ZNFs that are near-ubiquitous could also be playing very specific roles in halting target pathways in all tissues except for a few, where the target is released by the absence of its repressor. The number of variant KRAB-ZNF proteins is increased not only because of the large number of loci, but also because many loci can produce multiple splice variants, which because of the modular structure of these genes may have separate and perhaps even conflicting regulatory roles. The lineage-specific duplication and rapid divergence of this family of transcription factor genes suggests a role in determining species-specific biological differences and the evolution of novel primate traits.

  12. C. elegans PAT-9 is a nuclear zinc finger protein critical for the assembly of muscle attachments

    Directory of Open Access Journals (Sweden)

    Liu Qian

    2012-05-01

    Full Text Available Abstract Background Caenorhabditis elegans sarcomeres have been studied extensively utilizing both forward and reverse genetic techniques to provide insight into muscle development and the mechanisms behind muscle contraction. A previous genetic screen investigating early muscle development produced 13 independent mutant genes exhibiting a Pat (paralyzed and arrested elongation at the two-fold length of embryonic development muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9. Results Positional cloning, reverse genetics, and plasmid rescue experiments were used to identify the predicted C. elegans gene T27B1.2 (recently named ztf-19 as the pat-9 gene. Analysis of pat-9 showed it is expressed early in development and within body wall muscle lineages, consistent with a role in muscle development and producing a Pat phenotype. However, unlike most of the other known Pat gene family members, which encode structural components of muscle attachment sites, PAT-9 is an exclusively nuclear protein. Analysis of the predicted PAT-9 amino acid sequence identified one putative nuclear localization domain and three C2H2 zinc finger domains. Both immunocytochemistry and PAT-9::GFP fusion expression confirm that PAT-9 is primarily a nuclear protein and chromatin immunoprecipitation (ChIP experiments showed that PAT-9 is present on certain gene promoters. Conclusions We have shown that the T27B1.2 gene is pat-9. Considering the Pat-9 mutant phenotype shows severely disrupted muscle attachment sites despite PAT-9 being a nuclear zinc finger protein and not a structural component of muscle attachment sites, we propose that PAT-9 likely functions in the regulation of gene expression for some necessary structural or regulatory component(s of the muscle attachment sites.

  13. Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

    Full Text Available Abstract Background Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively. However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i to assemble and construct zinc finger arrays and nucleases (ZFN with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii to advance a model for pre-clinically testing lentiviral vectors (LV that deliver and transduce either ZFN genotype. Methods and Results First, we computationally generated the consensus sequences of (a 114 dsDNA-binding zinc finger (Zif arrays (ZFAs or ZifHIV-pol and (b two zinc-finger nucleases (ZFNs which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN. Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN. Second, a model for constructing lentiviral vectors (LVs that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively is proposed. Third, two preclinical models for controlled testing of

  14. Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

    Science.gov (United States)

    D'Astolfo, Diego S.; Cardoso, Fernanda C.; Rajão, Matheus A.; Mourão, Marina M.; Gava, Elisandra; Oliveira, Sérgio C.; Macedo, Andréa M.; Machado, Carlos R.; Pena, Sérgio D. J.; Kitten, Gregory T.; Franco, Glória R.

    2009-01-01

    Background During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. Methodology/Principal Findings In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present. Conclusion Taken together, these results strongly support the hypothesis

  15. Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21-p22

    DEFF Research Database (Denmark)

    Lanfrancone, L; Pengue, G; Pandolfi, P P;

    1992-01-01

    We report the structural and functional characterization of the HF.10 zinc finger gene (ZNF35) in normal human cells, as well as a processed pseudogene. The HF.10 gene spans about 13 kb and it is interrupted by three introns. All 11 zinc finger DNA-binding domains are contiguously encoded within...... and partial nucleotide sequencing of the HF.10 pseudogene indicated that it has arisen by retroposition of spliced HF.10 mRNA. In situ hybridization experiments revealed that both the functional locus and the pseudogene map to chromosome 3p21p22, a region that is frequently deleted in small cell lung...... and renal carcinomas. Hybridization of the HF.10 gene and the HF.10 pseudogene DNA probes to metaphases from a small cell lung carcinoma cell line with the 3p deletion revealed that both loci are part of the deleted chromosome region....

  16. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco

    OpenAIRE

    Mukhopadhyay, Arnab; Vij, Shubha; Tyagi, Akhilesh K

    2004-01-01

    Stress perception and signal transduction leading to tolerance involve a complex interplay of different gene products. We describe here the isolation and characterization of an intronless gene (OSISAP1) from rice encoding a zinc-finger protein that is induced after different types of stresses, namely cold, desiccation, salt, submergence, and heavy metals as well as injury. The gene is also induced by stress hormone abscisic acid. Overexpression of the gene in transgenic tobacco conferred tole...

  17. Localization and Differential Expression of the Krüppel-Associated Box Zinc Finger Proteins 1 and 54 in Early Mouse Development

    DEFF Research Database (Denmark)

    Albertsen, Maria; Teperek, Marta; Elholm, Grethe;

    2010-01-01

    -fused reporter gene into zygotes demonstrated the intracellular distribution of ZFP1-green fluorescent protein (GFP) and ZFP54-GFP colocalized with a DNA marker in the two-cell embryo. The KRAB domain was essential to colocalize with DNA, and deletion of the KRAB domain in ZFP1-GFP and ZFP54-GFP localized...... transcriptional repressors, zinc finger protein (ZFP1) and ZFP54, belonging to the Krüppel-associated box (KRAB) zinc finger family, were isolated. ZFP1 and ZFP54 contain an N-terminally located KRAB repressor domain followed by 8 and 12 repeats of Krüppel zinc-finger motifs, respectively. Reverse transcription...... (RT) and quantitative (q) PCR show that maternally contributed Zfp1 and Zfp54 mRNA are detected throughout preimplantation development. α-Amanitin-treated zygotes revealed that maternal Zfp1 and Zfp54 are fully degraded at the two-cell stage. Microinjections of in vitro-transcribed mRNA encoding a gfp...

  18. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations

    Science.gov (United States)

    Ahlawat, Sonika; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; De, Sachinandan

    2016-01-01

    PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF) domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species (cattle, yak, mithun, sheep and goat). We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12). We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species. PMID:27203728

  19. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations.

    Science.gov (United States)

    Ahlawat, Sonika; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; De, Sachinandan

    2016-01-01

    PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF) domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species (cattle, yak, mithun, sheep and goat). We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12). We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species. PMID:27203728

  20. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations.

    Directory of Open Access Journals (Sweden)

    Sonika Ahlawat

    Full Text Available PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species (cattle, yak, mithun, sheep and goat. We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12. We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species.

  1. Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

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    Sophiya Karki

    Full Text Available The zinc finger antiviral protein (ZAP is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV, the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

  2. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

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    Ebrahim Shahbazi

    2016-04-01

    Full Text Available Direct conversion of somatic cells into neural stem cells (NSCs by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

  3. Mutagenesis of genes for starch debranching enzyme isoforms in pea by means of zinc-finger endonucleases

    International Nuclear Information System (INIS)

    Starch debranching enzymes in plants are divided into two groups based on their ability to hydrolyse different substrates. The first group, pullulanases, hydrolyses α-1,6-glucosidic linkages in substrates such as pullulan, amylopectin and glycogen. The second group of debranching enzymes, isoamylases, hydrolyse glycogen and amylopectin and are not active on pullulan. Three isoforms of isoamylase and a pullulanase have been isolated from cDNA library of Pisum sativum. These isoamylases have been characterised based on the their heterologous expression in E coli. Based on the DNA sequence that encodes these debranching enzyme, a specific mutagenesis targeting at these DNA will be attempted. The method that will be employed are based on the techniques developed by Wright et al. (2005). This technique involves the homologous recombination of DNA that is mediated by zinc-finger endonucleases. Vectors will be constructed to include a fragment that will modify these genes. Microinjection technique will be used to insert these vectors into pollen which then will be fertilized. Using this technique, it is hoped that null mutant for each enzyme will be created and the exact role of these enzymes for the synthesis and degradation of starch in plants will be elucidate. (author)

  4. Suppression of zinc finger protein 467 alleviates osteoporosis through promoting differentiation of adipose derived stem cells to osteoblasts

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    You Li

    2012-01-01

    Full Text Available Abstract Osteoblast and adipocyte are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. In this study, a comparative analysis of gene expression profiling using cDNA microarray and realtime-PCR indicated that Zinc finger protein 467 (Zfp467 involved in adipocyte and osteoblast differentiation of cultured adipose derived stem cells (ADSCs. Our results showed that RNA interference for Zfp467 in ADSCs inhibited adipocyte formation and stimulated osteoblast commitment. The mRNA levels of osteogenic and adipogenic markers in ADSCs were regulated by si-Zfp467. Zfp467 RNAi in ADSCs could restore bone function and structure in an ovariectomized (OVX-induced osteoporotic mouse model. Thus Zfp467 play an important role in ADSCs differentiation to adipocyte and osteoblast. This has relevance to therapeutic interventions in osteoporosis, including si-Zfp467-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

  5. The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28

    Science.gov (United States)

    Murphy, Kristin E.; Shylo, Natalia A.; Alexander, Katherine A.; Churchill, Angela J.; Copperman, Cecilia; García-García, María J.

    2016-01-01

    KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity. PMID:27658112

  6. Zinc Finger Homeodomain Factor Zfhx3 Is Essential for Mammary Lactogenic Differentiation by Maintaining Prolactin Signaling Activity.

    Science.gov (United States)

    Zhao, Dan; Ma, Gui; Zhang, Xiaolin; He, Yuan; Li, Mei; Han, Xueying; Fu, Liya; Dong, Xue-Yuan; Nagy, Tamas; Zhao, Qiang; Fu, Li; Dong, Jin-Tang

    2016-06-10

    The zinc finger homeobox 3 (ZFHX3, also named ATBF1 for AT motif binding factor 1) is a transcription factor that suppresses prostatic carcinogenesis and induces neuronal differentiation. It also interacts with estrogen receptor α to inhibit cell proliferation and regulate pubertal mammary gland development in mice. In the present study, we examined whether and how Zfhx3 regulates lactogenic differentiation in mouse mammary glands. At different stages of mammary gland development, Zfhx3 protein was expressed at varying levels, with the highest level at lactation. In the HC11 mouse mammary epithelial cell line, an in vitro model of lactogenesis, knockdown of Zfhx3 attenuated prolactin-induced β-casein expression and morphological changes, indicators of lactogenic differentiation. In mouse mammary tissue, knock-out of Zfhx3 interrupted lactogenesis, resulting in underdeveloped glands with much smaller and fewer alveoli, reduced β-casein expression, accumulation of large cytoplasmic lipid droplets in luminal cells after parturition, and failure in lactation. Mechanistically, Zfhx3 maintained the expression of Prlr (prolactin receptor) and Prlr-Jak2-Stat5 signaling activity, whereas knockdown and knock-out of Zfhx3 in HC11 cells and mammary tissues, respectively, decreased Prlr expression, Stat5 phosphorylation, and the expression of Prlr-Jak2-Stat5 target genes. These findings indicate that Zfhx3 plays an essential role in proper lactogenic development in mammary glands, at least in part by maintaining Prlr expression and Prlr-Jak2-Stat5 signaling activity. PMID:27129249

  7. Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

    Institute of Scientific and Technical Information of China (English)

    Dongshan Yang; Jiangtian Tian; Feng Li; Jifeng Zhang; Lin Chang; Duanqing Pei; Y Eugene Chen; Liangxue Lai; Huaqiang Yang; Wei Li; Bentian Zhao; Zhen Ouyang; Zhaoming Liu; Yu Zhao; Nana Fan; Jun Song

    2011-01-01

    @@ Dear Editor, Gene targeting in mouse embryonic stem (ES) cells has revolutionized the field of mouse genetics and allowed for the analysis of diverse aspects of gene function in vivo.For more than two decades,researchers have been actively searching for ES cells from large animals such as pigs and cattle.Unfortunately,to date,no ES cell lines from large animals have passed the crucial test of germ line contribution.The sole method of gene targeting to date in these species remains somatic cell nuclear transfer (SCNT) combined with DNA homologous recombination (HR).Due to the limited proliferation competency and extremely low frequency of HR in somatic cells (less than 10-6),this process is highly inefficient and only a few successful examples have been achieved,even though enrichment strategies such as positivenegative marker selection,promoter-trap and adenoassociated viral delivery were previously used [1-3].The low efficiency of gene targeting in cultured somatic cells is the main barrier for gene targeting in large animals.Recently,zinc-finger nuclease (ZFN) technology has emerged as a powerful tool for genome editing.The success of ZFN technology for gene targeting in fruit flies,zebra fish,rodents as well as human cell lines encouraged us to establish a high-efficiency gene-targeting platform in large animals such as pigs [4-8].

  8. Structural Determinants of Sleeping Beauty Transposase Activity.

    Science.gov (United States)

    Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

    2016-08-01

    Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

  9. Structural Determinants of Sleeping Beauty Transposase Activity.

    Science.gov (United States)

    Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

    2016-08-01

    Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold. PMID:27401040

  10. Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

    Science.gov (United States)

    Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

    2016-08-01

    Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. PMID:27260399

  11. Promyelocytic leukemia zinc finger protein activates GATA4 transcription and mediates cardiac hypertrophic signaling from angiotensin II receptor 2.

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    Ning Wang

    Full Text Available BACKGROUND: Pressure overload and prolonged angiotensin II (Ang II infusion elicit cardiac hypertrophy in Ang II receptor 1 (AT(1 null mouse, whereas Ang II receptor 2 (AT(2 gene deletion abolishes the hypertrophic response. The roles and signals of the cardiac AT(2 receptor still remain unsettled. Promyelocytic leukemia zinc finger protein (PLZF was shown to bind to the AT(2 receptor and transmit the hypertrophic signal. Using PLZF knockout mice we directed our studies on the function of PLZF concerning the cardiac specific transcription factor GATA4, and GATA4 targets. METHODOLOGY AND PRINCIPAL FINDINGS: PLZF knockout and age-matched wild-type (WT mice were treated with Ang II, infused at a rate of 4.2 ng·kg(-1·min(-1 for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in PLZF knockout and WT mice (140 mmHg. WT mice developed prominent cardiac hypertrophy and fibrosis after Ang II infusion. In contrast, there was no obvious cardiac hypertrophy or fibrosis in PLZF knockout mice. An AT(2 receptor blocker given to Ang II-infused wild type mice prevented hypertrophy, verifying the role of AT(2 receptor for cardiac hypertrophy. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that PLZF bound to the GATA4 gene regulatory region. A Luciferase assay verified that PLZF up-regulated GATA4 gene expression and the absence of PLZF expression in vivo produced a corresponding repression of GATA4 protein. CONCLUSIONS: PLZF is an important AT(2 receptor binding protein in mediating Ang II induced cardiac hypertrophy through an AT(2 receptor-dependent signal pathway. The angiotensin II-AT(2-PLZF-GATA4 signal may further augment Ang II induced pathological effects on cardiomyocytes.

  12. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

    Science.gov (United States)

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong; Xiao, Han

    2015-03-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.

  13. Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.

    Science.gov (United States)

    Schjoldager, Katrine T-B G; Vakhrushev, Sergey Y; Kong, Yun; Steentoft, Catharina; Nudelman, Aaron S; Pedersen, Nis B; Wandall, Hans H; Mandel, Ulla; Bennett, Eric P; Levery, Steven B; Clausen, Henrik

    2012-06-19

    Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by zinc finger nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific O-glycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.

  14. The rice B-box zinc finger gene family: genomic identification, characterization, expression profiling and diurnal analysis.

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    Jianyan Huang

    Full Text Available BACKGROUND: The B-box (BBX -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX gene family until now. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97. In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. CONCLUSIONS/SIGNIFICANCE: The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the Os

  15. A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.

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    Xiaodong Deng

    Full Text Available Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38 harboring an arylsulfatase (ARS reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7, ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/TTTGG(G/T(G/TT was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.

  16. The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells

    International Nuclear Information System (INIS)

    Highlights: ► Zic transcription factors expressed early retinal progenitor cells. ► Zics sustain proliferation activity of retinal progenitor cells. ► Overexpression of Zic in retinal progenitors perturbed rod differentiation. ► Fate determination to rod photoreceptor was not affected. -- Abstract: Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

  17. Canine uterine bacterial infection induces upregulation of proteolysis-related genes and downregulation of homeobox and zinc finger factors.

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    Ragnvi Hagman

    Full Text Available BACKGROUND: Bacterial infection with the severe complication of sepsis is a frequent and serious condition, being a major cause of death worldwide. To cope with the plethora of occurring bacterial infections there is therefore an urgent need to identify molecular mechanisms operating during the host response, in order both to identify potential targets for therapeutic intervention and to identify biomarkers for disease. Here we addressed this issue by studying global gene expression in uteri from female dogs suffering from spontaneously occurring uterine bacterial infection. PRINCIPAL FINDINGS: The analysis showed that almost 800 genes were significantly (p2-fold in the uteri of diseased animals. Among these were numerous chemokine and cytokine genes, as well as genes associated with inflammatory cell extravasation, anti-bacterial action, the complement system and innate immune responses, as well as proteoglycan-associated genes. There was also a striking representation of genes associated with proteolysis. Robust upregulation of immunoglobulin components and genes involved in antigen presentation was also evident, indicating elaboration of a strong adaptive immune response. The bacterial infection was also associated with a significant downregulation of almost 700 genes, of which various homeobox and zinc finger transcription factors were highly represented. CONCLUSIONS/SIGNIFICANCE: Together, these finding outline the molecular patterns involved in bacterial infection of the uterus. The study identified altered expression of numerous genes not previously implicated in bacterial disease, and several of these may be evaluated for potential as biomarkers of disease or as therapeutic targets. Importantly, since humans and dogs show genetic similarity and develop diseases that share many characteristics, the molecular events identified here are likely to reflect the corresponding situation in humans afflicted by similar disease.

  18. The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Watabe, Yui [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan); Department of Ophthalmology, Teikyo University School of Medicine, Tokyo (Japan); Division of Orthoptics, Teikyo University School of Medical Care and Technology, Tokyo (Japan); Baba, Yukihiro [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan); Nakauchi, Hiromitsu [Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo (Japan); Mizota, Atsushi [Department of Ophthalmology, Teikyo University School of Medicine, Tokyo (Japan); Watanabe, Sumiko, E-mail: sumiko@ims.u-tokyo.ac.jp [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Zic transcription factors expressed early retinal progenitor cells. Black-Right-Pointing-Pointer Zics sustain proliferation activity of retinal progenitor cells. Black-Right-Pointing-Pointer Overexpression of Zic in retinal progenitors perturbed rod differentiation. Black-Right-Pointing-Pointer Fate determination to rod photoreceptor was not affected. -- Abstract: Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

  19. KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading.

    Directory of Open Access Journals (Sweden)

    Anna C Groner

    2010-03-01

    Full Text Available Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB-mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB-containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3, and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB-mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 beta (HP1beta between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1-dependent transcriptional repression at an endogenous KRAB-ZFP gene cluster, where KAP1 binds to the 3' end of genes and mediates propagation of H3K9me3 and HP1beta towards their 5' end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB-ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB-ZFPs and KAP1.

  20. The ZNF75 zinc finger gene subfamily: Isolation and mapping of the four members in humans and great apes

    Energy Technology Data Exchange (ETDEWEB)

    Villa, A.; Strina, D.; Frattini, A. [Consiglio Nazionale delle Ricerche, Milan (Italy)] [and others

    1996-07-15

    We have previously reported the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain on ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and organutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existance of at least two genes in cows and horses, suggests a relatively high conservation of this gene family. 20 refs., 5 figs., 1 tab.

  1. Comprehensive analysis of CCCH-type zinc finger gene family in citrus (Clementine mandarin) by genome-wide characterization.

    Science.gov (United States)

    Liu, Shengrui; Khan, Muhammad Rehman Gul; Li, Yongping; Zhang, Jinzhi; Hu, Chungen

    2014-10-01

    The CCCH-type zinc finger proteins comprise a large gene family of regulatory proteins and are widely distributed in eukaryotic organisms. The CCCH proteins have been implicated in multiple biological processes and environmental responses in plants. Little information is available, however, about CCCH genes in plants, especially in woody plants such as citrus. The release of the whole-genome sequence of citrus allowed us to perform a genome-wide analysis of CCCH genes and to compare the identified proteins with their orthologs in model plants. In this study, 62 CCCH genes and a total of 132 CCCH motifs were identified, and a comprehensive analysis including the chromosomal locations, phylogenetic relationships, functional annotations, gene structures and conserved motifs was performed. Distribution mapping revealed that 54 of the 62 CCCH genes are unevenly dispersed on the nine citrus chromosomes. Based on phylogenetic analysis and gene structural features, we constructed 5 subfamilies of 62 CCCH members and integrative subfamilies from citrus, Arabidopsis, and rice, respectively. Importantly, large numbers of SNPs and InDels in 26 CCCH genes were identified from Poncirus trifoliata and Fortunella japonica using whole-genome deep re-sequencing. Furthermore, citrus CCCH genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various stress conditions. Our comprehensive analysis of CleC3Hs is a valuable resource that further elucidates the roles of CCCH family members in plant growth and development. In addition, variants and comparative genomics analyses deepen our understanding of the evolution of the CCCH gene family and will contribute to further genetics and genomics studies of citrus and other plant species.

  2. A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Jin-Bon Hong

    Full Text Available PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB. Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

  3. A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

    Science.gov (United States)

    Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

    2014-01-01

    PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

  4. UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation

    Directory of Open Access Journals (Sweden)

    Onori Annalisa

    2013-01-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible. Results We have previously shown that the zinc finger-based artificial transcriptional factor “Jazz” corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named “UtroUp”, engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus. Conclusions This novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.

  5. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA.

    Science.gov (United States)

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-04-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P Ia, as compared with normal littermates, at 8 months following vector administration (P Ia.

  6. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function

    OpenAIRE

    EFSA Panel on Genetically Modified Organisms (GMO)

    2012-01-01

    The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3) which allows the integration of gene(s) in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3) is used to describe the technique ra...

  7. NF-κB activation and zinc finger protein A20 expression in mature dendritic cells derived from liver allografts undergoing acute rejection

    Institute of Scientific and Technical Information of China (English)

    Ming-Qing Xu; Wei Wang; Lan Xue; Lv-Nan Yan

    2003-01-01

    AIM: To investigate the role of NF-κB activation and zinc finger protein A20 expression in the regulation of maturation of dendritic cells (DCs) derived from liver allografts undergoing acute rejection. METHODS: Sixty donor male SD rats and sixty recipient male LEW rats weighing 220-300 g were randomly divided into whole liver transplantation group and partial liver transplantation group. Allogeneic (SD rat to LEW rat) whole and 50 % partial liver transplantation were performed. DCs from liver grafts 0 hour and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics and phenotypical features of DCs propagated for 10 days were analyzed by electron microscopy and flow cytometry, respectively. NF-κB binding activity, IL-12p70 protein and zinc finger protein A20expression in these DCs were measured by EMSA and Western blotting, respectively. Histological grading of rejection was determined. RESULTS: Allogeneic whole liver grafts showed no signs of rejection on day 4 after the transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate to severe rejection on day 4 after the transplantation. After propagation for 10 days in the presence of GM-CSF in vitro,DCs from allogeneic whole liver grafts exhibited features of immature DC with absence of CD40 surface expression,these DCs were found to exhibit detectable but very low level of NF-κB activity, IL-12 p70 protein and zinc finger protein A20 expression. Whereas, DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high level of CD40 surface expression, and as a consequence, higher expression of IL-12p70 protein, higher activities of NF-κB and higher expression of zinc finger protein A20 compared with those of DCs from whole liver grafts (P<0.001). CONCLUSION: These results suggest that A20expression is up-regulated in response to NF-κB activation in mature DCs derived from

  8. The LSD1-Type Zinc Finger Motifs of Pisum sativa LSD1 Are a Novel Nuclear Localization Signal and Interact with Importin Alpha

    OpenAIRE

    Shanping He; Kuowei Huang; Xu Zhang; Xiangchun Yu; Ping Huang; Chengcai An

    2011-01-01

    BACKGROUND: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD). Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. METHODOLOGY/PRINCIPAL FINDINGS: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1), which is a homolog ...

  9. Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

    Science.gov (United States)

    Mashimo, Tomoji

    2014-01-01

    The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed.

  10. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

    DEFF Research Database (Denmark)

    Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael;

    2014-01-01

    Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant...

  11. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

    DEFF Research Database (Denmark)

    Jensen, R B; Lykke-Andersen, K; Frandsen, G I;

    2000-01-01

    Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2...... and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins...

  12. Chorioallantoic Fusion Defects and Embryonic Lethality Resulting from Disruption of Zfp36L1, a Gene Encoding a CCCH Tandem Zinc Finger Protein of the Tristetraprolin Family

    OpenAIRE

    Stumpo, Deborah J.; Byrd, Noah A.; Phillips, Ruth S.; Ghosh, Sanjukta; Maronpot, Robert R.; Castranio, Trisha; Meyers, Erik N.; Mishina, Yuji; Blackshear, Perry J.

    2004-01-01

    The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3′-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities sim...

  13. Heme regulates the expression in Saccharomyces cerevisiae of chimaeric genes containing 5'-flanking soybean leghemoglobin sequences

    DEFF Research Database (Denmark)

    Jensen, E O; Marcker, K A; Villadsen, IS

    1986-01-01

    The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene. Expression of the chimaeric...... CAT gene is controlled specifically by heme at a post-transcriptional level, most likely by regulating the efficiencies of translation. Expression of another chimaeric gene consisting of the neomycin phosphotransferase (NPTII) gene fused to only the 5'-flanking region of the Lbc3 gene is regulated...

  14. Overexpression of myeloid zinc finger 1 suppresses matrix metalloproteinase-2 expression and reduces invasiveness of SiHa human cervical cancer cells.

    Science.gov (United States)

    Tsai, Su-Ju; Hwang, Jin-Ming; Hsieh, Shu-Ching; Ying, Tsung-Ho; Hsieh, Yi-Hsien

    2012-08-24

    Myeloid zinc finger 1 (MZF1) gene belongs to the Kruppel family of zinc finger transcription factors. MZF1 has been suggested to play an important role in the tumorigenesis, invasion, and apoptosis of various tumor cells. However, the role of MZF1 in human cervical cancer remains unclear. To investigate the molecular mechanisms of MZF1 and its functional role in human cervical cancer cell migration and invasion, we experimented on stable SiHa cells overexpressing MZF1. We found that MZF1 overexpression inhibits the migratory and invasive abilities of SiHa cervical cancer cells. In addition, the overexpression of MZF1 significantly reduces MMP-2 protein and mRNA levels. Luciferase and ChIP assays suggested that MZF1 directly binds to MMP-2 gene regulatory sequences in vivo and suppresses MMP-2 promoter activity in vitro. This study shows that MZF-1 represses MMP-2 transcription and suggests that this repression may be linked to inhibition of human cervical cancer cell migration and metastasis.

  15. An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa).

    Science.gov (United States)

    Zhang, Ye; Lan, Hongxia; Shao, Qiaolin; Wang, Ruqin; Chen, Hui; Tang, Haijuan; Zhang, Hongsheng; Huang, Ji

    2016-01-01

    The plant hormones gibberellins (GA) and abscisic acid (ABA) play important roles in plant development and stress responses. Here we report a novel A20/AN1-type zinc finger protein ZFP185 involved in GA and ABA signaling in the regulation of growth and stress response. ZFP185 was constitutively expressed in various rice tissues. Overexpression of ZFP185 in rice results in a semi-dwarfism phenotype, reduced cell size, and the decrease of endogenous GA3 content. By contrast, higher GA3 content was observed in RNAi plants. The application of exogenous GA3 can fully rescue the semi-dwarfism phenotype of ZFP185 overexpressing plants, suggesting the negative role of ZFP185 in GA biosynthesis. Besides GA, overexpression of ZFP185 decreased ABA content and expression of several ABA biosynthesis-related genes. Moreover, it was found that ZFP185, unlike previously known A20/AN1-type zinc finger genes, increases sensitivity to drought, cold, and salt stresses, implying the negative role of ZFP185 in stress tolerance. ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential. Our study suggests that ZFP185 regulates plant growth and stress responses by affecting GA and ABA biosynthesis in rice.

  16. ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers.

    Directory of Open Access Journals (Sweden)

    Christian B Gocke

    Full Text Available Histone modifications in chromatin regulate gene expression. A transcriptional co-repressor complex containing LSD1-CoREST-HDAC1 (termed LCH hereafter for simplicity represses transcription by coordinately removing histone modifications associated with transcriptional activation. RE1-silencing transcription factor (REST recruits LCH to the promoters of neuron-specific genes, thereby silencing their transcription in non-neuronal tissues. ZNF198 is a member of a family of MYM-type zinc finger proteins that associate with LCH. Here, we show that ZNF198-like proteins are required for the repression of E-cadherin (a gene known to be repressed by LSD1, but not REST-responsive genes. ZNF198 binds preferentially to the intact LCH ternary complex, but not its individual subunits. ZNF198- and REST-binding to the LCH complex are mutually exclusive. ZNF198 associates with chromatin independently of LCH. Furthermore, modification of HDAC1 by small ubiquitin-like modifier (SUMO in vitro weakens its interaction with CoREST whereas sumoylation of HDAC1 stimulates its binding to ZNF198. Finally, we mapped the LCH- and HDAC1-SUMO-binding domains of ZNF198 to tandem repeats of MYM-type zinc fingers. Therefore, our results suggest that ZNF198, through its multiple protein-protein interaction interfaces, helps to maintain the intact LCH complex on specific, non-REST-responsive promoters and may also prevent SUMO-dependent dissociation of HDAC1.

  17. A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

    OpenAIRE

    Lien, Huang-Wei; Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Hung, Chin-Chun; Liao, Wei-Hao; Hwang, Pung-Pung; Han, Yu-San; Huang, Chang-Jen

    2013-01-01

    The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly e...

  18. Genetic analysis of Kruppel-like zinc finger 11 variants in 5864 Danish individuals: potential effect on insulin resistance and modified signal transducer and activator of transcription-3 binding by promoter variant -1659G>C

    DEFF Research Database (Denmark)

    Gutiérrez-Aguilar, Ruth; Froguel, Philippe; Hamid, Yasmin H;

    2008-01-01

    CONTEXT: The transcription factor Krüppel-like zinc finger 11 (KLF11) has been suggested to contribute to genetic risk of type 2 diabetes (T2D). Our previous results showed that four KLF11 variants, in strong linkage disequilibrium (LD block including +185 A>G/Gln62Arg and -1659 G>C) were associa...

  19. Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatibility complex class I genes

    NARCIS (Netherlands)

    Veer, L.J. van 't; Lutz, P.M.; Isselbacher, K.J.; Bernards, R.A.

    1992-01-01

    We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative

  20. A Family of Zinc Finger Proteins Is Required forChromosome-specific Pairing and Synapsis during Meiosis in C.elegans

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Carolyn M.; Dernburg, Abby F.

    2006-06-07

    Homologous chromosome pairing and synapsis are prerequisitefor accurate chromosome segregation during meiosis. Here, we show that afamily of four related C2H2 zinc-finger proteins plays a central role inthese events in C. elegans. These proteins are encoded within a tandemgene cluster. In addition to the X-specific HIM-8 protein, threeadditional paralogs collectively mediate the behavior of the fiveautosomes. Each chromosome relies on a specific member of the family topair and synapse with its homolog. These "ZIM" proteins concentrate atspecial regions called meiotic pairing centers on the correspondingchromosomes. These sites are dispersed along the nuclear envelope duringearly meiotic prophase, suggesting a role analogous to thetelomere-mediated meiotic bouquet in other organisms. To gain insightinto the evolution of these components, wecharacterized homologs in C.briggsae and C. remanei, which revealed changes in copy number of thisgene family within the nematode lineage.

  1. The human zinc-finger protein-7 gene is located 90 kb 3' of MYC and is not expressed in Burkitt lymphoma cell lines.

    Science.gov (United States)

    Feduchi, E; Gallego, M I; Lazo, P A

    1994-09-15

    The zinc-finger gene-7 (ZNF7) was located 90 kb 3' of MYC on human chromosome 8 band q24 by pulsed-field gel electrophoresis (PFGE). This position lies between the MLV14 and BVR1 loci, 2 variant translocation breakpoints in Burkitt lymphomas. The structure of the ZNF7 gene was not altered by translocations in Burkitt-lymphoma cell lines as shown by its germline-restriction map configuration. The chromosomal region surrounding the ZNF7 gene was extensively methylated. The ZNF7 gene was not expressed in 19 BL cell lines. Expression was detected only in the BL41 and BL47 cell lines and in the SW756 cervical-carcinoma cell line. The RNA in each was of a different size. We postulate that the lack of ZNF7 expression in Burkitt lymphomas might contribute to the tumor phenotype.

  2. ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

    DEFF Research Database (Denmark)

    Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel;

    2012-01-01

    signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling......Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function...... as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified...

  3. A Comprehensive Catalog of Human KRAB-associated Zinc Finger Genes: Insights into the Evolutionary History of a Large Family of Transcriptional Repressors

    Energy Technology Data Exchange (ETDEWEB)

    Huntley, S; Baggott, D M; Hamilton, A T; Tran-Gyamfi, M; Yang, S; Kim, J; Gordon, L; Branscomb, E; Stubbs, L

    2005-09-30

    Krueppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotic species. In mammals, most ZNF proteins comprise a single class of transcriptional repressors in which a chromatin interaction domain, called the Krueppel-associated box (KRAB) is attached to a tandem array of DNA-binding zinc-finger motifs. KRAB-ZNF loci are specific to tetrapod vertebrates, but have expanded dramatically in numbers through repeated rounds of segmental duplication to create a gene family with hundreds of members in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the human genome for key motifs and used them to construct and manually curate gene models. The resulting KRAB-ZNF gene catalog includes 326 known genes, 243 of which were structurally corrected by manual annotation, and 97 novel KRAB-ZNF genes; this single family therefore comprises 20% of all predicted human transcription factor genes. Many of the genes are alternatively spliced, yielding a total of 743 distinct predicted proteins. Although many human KRAB-ZNF genes are conserved in mammals, at least 136 and potentially more than 200 genes of this type are primate-specific including many recent segmental duplicates. KRAB-ZNF genes are active in a wide variety of human tissues suggesting roles in many key biological processes, but most member genes remain completely uncharacterized. Because of their sheer numbers, wide-ranging tissue-specific expression patterns, and remarkable evolutionary divergence we predict that KRAB-ZNF transcription factors have played critical roles in crafting many aspects of human biology, including both deeply conserved and primate-specific traits.

  4. Gain, loss and divergence in primate zinc-finger genes: a rich resource for evolution of gene regulatory differences between species.

    Directory of Open Access Journals (Sweden)

    Katja Nowick

    Full Text Available The molecular changes underlying major phenotypic differences between humans and other primates are not well understood, but alterations in gene regulation are likely to play a major role. Here we performed a thorough evolutionary analysis of the largest family of primate transcription factors, the Krüppel-type zinc finger (KZNF gene family. We identified and curated gene and pseudogene models for KZNFs in three primate species, chimpanzee, orangutan and rhesus macaque, to allow for a comparison with the curated set of human KZNFs. We show that the recent evolutionary history of primate KZNFs has been complex, including many lineage-specific duplications and deletions. We found 213 species-specific KZNFs, among them 7 human-specific and 23 chimpanzee-specific genes. Two human-specific genes were validated experimentally. Ten genes have been lost in humans and 13 in chimpanzees, either through deletion or pseudogenization. We also identified 30 KZNF orthologs with human-specific and 42 with chimpanzee-specific sequence changes that are predicted to affect DNA binding properties of the proteins. Eleven of these genes show signatures of accelerated evolution, suggesting positive selection between humans and chimpanzees. During primate evolution the most extensive re-shaping of the KZNF repertoire, including most gene additions, pseudogenizations, and structural changes occurred within the subfamily homininae. Using zinc finger (ZNF binding predictions, we suggest potential impact these changes have had on human gene regulatory networks. The large species differences in this family of TFs stands in stark contrast to the overall high conservation of primate genomes and potentially represents a potent driver of primate evolution.

  5. Embryonic neural inducing factor churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed beta-sheet and zinc binuclear cluster.

    Science.gov (United States)

    Lee, Brian M; Buck-Koehntop, Bethany A; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2007-08-31

    Churchill is a zinc-containing protein that is involved in neural induction during embryogenesis. At the time of its discovery, it was thought on the basis of sequence alignment to contain two zinc fingers of the C4 type. Further, binding of an N-terminal GST-Churchill fusion protein to a particular DNA sequence was demonstrated by immunoprecipitation selection assay, suggesting that Churchill may function as a transcriptional regulator by sequence-specific DNA binding. We show by NMR solution structure determination that, far from containing canonical C4 zinc fingers, the protein contains three bound zinc ions in novel coordination sites, including an unusual binuclear zinc cluster. The secondary structure of Churchill is also unusual, consisting of a highly solvent-exposed single-layer beta-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveal that Churchill is unusually dynamic on a number of time scales, with the exception of regions surrounding the zinc coordinating sites, which serve to stabilize the otherwise unstructured N terminus and the single-layer beta-sheet. No binding of Churchill to the previously identified DNA sequence could be detected, and extensive searches using DNA sequence selection techniques could find no other DNA sequence that was bound by Churchill. Since the N-terminal amino acids of Churchill form part of the zinc-binding motif, the addition of a fusion protein at the N terminus causes loss of zinc and unfolding of Churchill. This observation most likely explains the published DNA-binding results, which would arise due to non-specific interaction of the unfolded protein in the immunoprecipitation selection assay. Since Churchill does not appear to bind DNA, we suggest that it may function in embryogenesis as a protein-interaction factor.

  6. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.; Khan, Amir R.

    2016-05-23

    Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

  7. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    Science.gov (United States)

    Oliveira, Tânia; Sharkey, Michael A; Engel, Paul C; Khan, Amir R

    2016-06-01

    Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)(+) as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP(+) cofactor from the parent E. coli domain II, although there are subtle differences in catalytic activity. PMID:27303899

  8. Mycobacterial and HIV infections up-regulated human zinc finger protein 134, a novel positive regulator of HIV-1 LTR activity and viral propagation.

    Directory of Open Access Journals (Sweden)

    Ronald Benjamin

    Full Text Available BACKGROUND: Concurrent occurrence of HIV and Tuberculosis (TB infections influence the cellular environment of the host for synergistic existence. An elementary approach to understand such coalition at the molecular level is to understand the interactions of the host and the viral factors that subsequently effect viral replication. Long terminal repeats (LTR of HIV genome serve as a template for binding trans-acting viral and cellular factors that regulate its transcriptional activity, thereby, deciding the fate of HIV pathogenesis, making it an ideal system to explore the interplay between HIV and the host. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using biotinylated full length HIV-1 LTR sequence as bait followed by MALDI analyses, we identified and further characterized human-Zinc-finger-protein-134 (hZNF-134 as a novel positive regulator of HIV-1 that promoted LTR-driven transcription and viral production. Over-expression of hZNF-134 promoted LTR driven luciferase activity and viral transcripts, resulting in increased virus production while siRNA mediated knockdown reduced both the viral transcripts and the viral titers, establishing hZNF-134 as a positive effector of HIV-1. HIV, Mycobacteria and HIV-TB co-infections increased hZNF-134 expressions in PBMCs, the impact being highest by mycobacteria. Corroborating these observations, primary TB patients (n = 22 recorded extraordinarily high transcript levels of hZNF-134 as compared to healthy controls (n = 16. CONCLUSIONS/SIGNIFICANCE: With these observations, it was concluded that hZNF-134, which promoted HIV-1 LTR activity acted as a positive regulator of HIV propagation in human host. High titers of hZNF-134 transcripts in TB patients suggest that up-regulation of such positive effectors of HIV-1 upon mycobacterial infection can be yet another mechanism by which mycobacteria assists HIV-1 propagation during HIV-TB co-infections. hZNF-134, an uncharacterized host protein, thus

  9. Transcriptional activation is a conserved feature of the early embryonic factor Zelda that requires a cluster of four zinc fingers for DNA binding and a low-complexity activation domain.

    Science.gov (United States)

    Hamm, Danielle C; Bondra, Eliana R; Harrison, Melissa M

    2015-02-01

    Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation. We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity, and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome.

  10. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    Science.gov (United States)

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  11. Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

    Energy Technology Data Exchange (ETDEWEB)

    Foster, Mark P.; Wuttke, Deborah S.; Clemens, Karen R.; Jahnke, Wolfgang; Radhakrishnan, Ishwar; Tennant, Linda; Reymond, Martine; Chung, John; Wright, Peter E. [Scripps Research Institute, Department of Molecular Biology and Skaggs Institute for Chemical Biology (United States)

    1998-07-15

    We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone{sup 1} H{sup N}, {sup 15}N,{sup 13} C{sup {alpha}} and{sup 13} C{sup {beta}} and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

  12. Engineered zinc-finger proteins can compensate genetic haploinsufficiency by transcriptional activation of the wild-type allele: application to Willams-Beuren syndrome and supravalvular aortic stenosis.

    Science.gov (United States)

    Zhang, Pei; Huang, Angela; Morales-Ruiz, Manuel; Starcher, Barry C; Huang, Yan; Sessa, William C; Niklason, Laura E; Giordano, Frank J

    2012-11-01

    Williams-Beuren syndrome (WBS) and supravalvular aortic stenosis (SVAS) are genetic syndromes marked by the propensity to develop severe vascular stenoses. Vascular lesions in both syndromes are caused by haploinsufficiency of the elastin gene. We used these distinct genetic syndromes as models to evaluate the feasibility of using engineered zinc-finger protein transcription factors (ZFPs) to achieve compensatory expression of haploinsufficient genes by inducing augmented expression from the remaining wild-type allele. For complex genes with multiple splice variants, this approach could have distinct advantages over cDNA-based gene replacement strategies. Targeting the elastin gene, we show that transcriptional activation by engineered ZFPs can induce compensatory expression from the wild-type allele in the setting of classic WBS and SVAS genetic mutations, increase elastin expression in wild-type cells, induce expression of the major elastin splice variants, and recapitulate their natural stoichiometry. Further, we establish that transcriptional activation of the mutant allele in SVAS does not overcome nonsense-mediated decay, and thus ZFP-mediated transcriptional activation is not likely to induce production of a mutant protein, a crucial consideration. Finally, we show in bioengineered blood vessels that ZFP-mediated induction of elastin expression is capable of stimulating functional elastogenesis. Haploinsufficiency is a common mechanism of genetic disease. These findings have significant implications for WBS and SVAS, and establish that haploinsufficiency can be overcome by targeted transcriptional activation without inducing protein expression from the mutant allele.

  13. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

    Science.gov (United States)

    Wang, Jianbin; DeClercq, Joshua J; Hayward, Samuel B; Li, Patrick Wai-Lun; Shivak, David A; Gregory, Philip D; Lee, Gary; Holmes, Michael C

    2016-02-18

    The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy. PMID:26527725

  14. Sda1, a Cys2-His2 zinc finger transcription factor, is involved in polyol metabolism and fumonisin B1 production in Fusarium verticillioides.

    Directory of Open Access Journals (Sweden)

    Martha Malapi-Wight

    Full Text Available The ubiquitous ascomycete Fusarium verticillioides causes ear rot and stalk rot of maize, both of which reduce grain quality and yield. Additionally, F. verticillioides produces the mycotoxin fumonisin B1 (FB1 during infection of maize kernels, and thus potentially compromises human and animal health. The current knowledge is fragmentary regarding the regulation of FB1 biosynthesis, particularly when considering interplay with environmental factors such as nutrient availability. In this study, SDA1 of F. verticillioides, predicted to encode a Cys-2 His-2 zinc finger transcription factor, was shown to play a key role in catabolizing select carbon sources. Growth of the SDA1 knock-out mutant (Δsda1 was completely inhibited when sorbitol was the sole carbon source and was severely impaired when exclusively provided mannitol or glycerol. Deletion of SDA1 unexpectedly increased FB1 biosynthesis, but reduced arabitol and mannitol biosynthesis, as compared to the wild-type progenitor. Trichoderma reesei ACE1, a regulator of cellulase and xylanase expression, complemented the F. verticillioides Δsda1 mutant, which indicates that Ace1 and Sda1 are functional orthologs. Taken together, the data indicate that Sda1 is a transcriptional regulator of carbon metabolism and toxin production in F. verticillioides.

  15. A Novel Role for the Zinc-Finger Transcription Factor EGL-46 in the Differentiation of Gas-Sensing Neurons in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Rojo Romanos, Teresa; Gramstrup Petersen, Jakob; Redo Riveiro, Alba;

    2014-01-01

    Oxygen (O2) and carbon dioxide (CO2) provoke distinct olfactory behaviors via specialized sensory neurons across metazoa. In the nematode Caenorhabditis elegans, the BAG sensory neurons are specialized to sense changes in both O2 and CO2 levels in the environment. The precise functionality of the......-13. Thereby, three conserved transcription factors collaborate to ensure neuron type-specific identity features of the BAG gas-sensing neurons.......Oxygen (O2) and carbon dioxide (CO2) provoke distinct olfactory behaviors via specialized sensory neurons across metazoa. In the nematode Caenorhabditis elegans, the BAG sensory neurons are specialized to sense changes in both O2 and CO2 levels in the environment. The precise functionality...... is partially, though not completely, controlled by ETS-5, an ETS-domain-containing transcription factor, and EGL-13, a Sox transcription factor. We report here, the identification of EGL-46, a zinc-finger transcription factor, which regulates BAG gas-sensing fate in partially parallel pathways to ETS-5 and EGL...

  16. A Missense Mutation in the Zinc Finger Domain of OsCESA7 Deleteriously Affects Cellulose Biosynthesis and Plant Growth in Rice.

    Science.gov (United States)

    Wang, Daofeng; Qin, Yanling; Fang, Jingjing; Yuan, Shoujiang; Peng, Lixiang; Zhao, Jinfeng; Li, Xueyong

    2016-01-01

    Rice is a model plant species for the study of cellulose biosynthesis. We isolated a mutant, S1-24, from ethyl methanesulfonate (EMS)-treated plants of the japonica rice cultivar, Nipponbare. The mutant exhibited brittle culms and other pleiotropic phenotypes such as dwarfism and partial sterility. The brittle culms resulted from reduced mechanical strength due to a defect in thickening of the sclerenchyma cell wall and reduced cellulose content in the culms of the S1-24 mutant. Map-based gene cloning and a complementation assay showed that phenotypes of the S1-24 mutant were caused by a recessive point mutation in the OsCESA7 gene, which encodes cellulose synthase A subunit 7. The missense mutation changed the highly conserved C40 to Y in the zinc finger domain. The OsCESA7 gene is expressed predominantly in the culm at the mature stage, particularly in mechanical tissues such as vascular bundles and sclerenchyma cells, consistent with the brittle phenotype in the culm. These results indicate that OsCESA7 plays an important role in cellulose biosynthesis and plant growth. PMID:27092937

  17. The Arabidopsis a zinc finger domain protein ARS1 is essential for seed germination and ROS homeostasis in response to ABA and oxidative stress

    Directory of Open Access Journals (Sweden)

    Dongwon eBaek

    2015-11-01

    Full Text Available The phytohormone abscisic acid (ABA induces accumulation of reactive oxygen species (ROS, which can disrupt seed dormancy and plant development. Here, we report the isolation and characterization of an Arabidopsis thaliana mutant called ars1 (aba and ros sensitive 1 that showed hypersensitivity to ABA during seed germination and to methyl viologen (MV at the seedling stage. ARS1 encodes a nuclear protein with one zinc finger domain, two nuclear localization signal (NLS domains, and one nuclear export signal (NES. The ars1 mutants showed reduced expression of a gene for superoxide dismutase (CSD3 and enhanced accumulation of ROS after ABA treatment. Transient expression of ARS1 in Arabidopsis protoplasts strongly suppressed ABA-mediated ROS production. Interestingly, nuclear-localized ARS1 translocated to the cytoplasm in response to treatment with ABA, H2O2, or MV. Taken together, these results suggest that ARS1 modulates seed germination and ROS homeostasis in response to ABA and oxidative stress in plants.

  18. Activation of the ustilagic acid biosynthesis gene cluster in Ustilago maydis by the C2H2 zinc finger transcription factor Rua1.

    Science.gov (United States)

    Teichmann, Beate; Liu, Lidan; Schink, Kay Oliver; Bölker, Michael

    2010-04-01

    The phytopathogenic basidiomycetous fungus Ustilago maydis secretes, under conditions of nitrogen starvation, large amounts of the biosurfactant ustilagic acid (UA). This secreted cellobiose glycolipid is toxic for many microorganisms and confers biocontrol activity to U. maydis. Recently, a large gene cluster that is responsible for UA biosynthesis was identified. Here, we show that expression of all cluster genes depends on Rua1, a nuclear protein of the C(2)H(2) zinc finger family, whose gene is located within the gene cluster. While deletion of rua1 results in complete loss of UA production, overexpression of rua1 promotes increased UA synthesis even in the presence of a good nitrogen source. Bioinformatic analysis allowed us to identify a conserved sequence element that is present in the promoters of all structural genes involved in UA biosynthesis. Deletion analysis of several promoters within the cluster revealed that this DNA element serves as an upstream activating sequence (UAS) and mediates Rua1-dependent expression. We used the yeast one-hybrid system to demonstrate specific recognition of this DNA element by Rua1. Introduction of nucleotide exchanges into the consensus sequence interfered with Rua1-dependent activation, suggesting that this sequence element acts as a direct binding site for Rua1. PMID:20173069

  19. Identification of a putative DEAD-box RNA helicase and a zinc-finger protein in Candida albicans by functional complementation of the S. cerevisiae rok1 mutation.

    Science.gov (United States)

    Kim, W I; Lee, W B; Song, K; Kim, J

    2000-03-30

    We identified two novel genes, CHR1 and CSR1, of the fungal pathogen Candida albicans, by functional complementation of the Saccharomyces cerevisiae rok1 mutation. The Rok1 protein is a member of the DEAD protein family of ATP-dependent RNA helicases. ROK1 is required for cell cycle progression and also for rRNA processing. The CHR1 gene product of 578 amino acids is highly homologous to the Rok1 protein (54% identity) and is considered to be a putative DEAD-box RNA helicase. We predict that the CSR1 gene encodes a 73 kDa protein of 612 amino acids with five zinc-finger motifs at the C-terminal region. CHR1 or CSR1 on a high-copy number plasmid showed a slow-growth phenotype in a condition where the ROK1 expression is turned on from the GAL1 promoter. This result is consistent with the lethality caused by the ROK1 overexpression. We conclude that CHR1 encodes a functional homologue of Rok1 protein and CSR1 is a heterologous suppressor of the rok1 mutation. PMID:10705369

  20. Characterization of 47 Cys2 -His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Cao, Huijuan; Huang, Pengyun; Zhang, Lilin; Shi, Yongkai; Sun, Dandan; Yan, Yuxin; Liu, Xiaohong; Dong, Bo; Chen, Guoqing; Snyder, John Hugh; Lin, Fucheng; Lu, Jianping

    2016-08-01

    The Cys2 -His2 (C2H2) zinc finger protein family is the second-largest family of transcription factors (TFs) in Magnaporthe oryzae, the causal fungus responsible for the destructive rice blast disease. However, little is known about the roles of most C2H2 TFs in the development and pathogenicity of M. oryzae. The roles of 47 C2H2 genes in development and pathogenicity were investigated by gene deletion in M. oryzae. The TF-dependent genes in mycelia or appressoria were analyzed with RNA sequencing and quantitative PCR (qPCR). Forty-four C2H2 genes are involved in growth (20 genes), conidiation (28 genes), appressorium formation (four genes) and pathogenicity (22 genes) in M. oryzae. Of these, MGG_14931, named as VRF1, is required for pathogenicity, specifically controlling appressorium maturation by affecting the expression of genes related to appressorial structure and function, including melanin biosynthesis, chitin catabolism, lipid metabolism, proteolysis, transmembrane transport, and response to oxidative stress; MGG_01776, named as VRF2, is required for plant penetration and invasive growth; conidiation-related gene CON7 is required for conidial differentiation; and MoCREA, encoding a carbon catabolite repression protein, is a novel repressor of lipid catabolism when glucose obtainable in M. oryzae. This study provides many insights into the regulation of growth, asexual development, appressorium formation, and pathogenicity by C2H2 TFs in M. oryzae. PMID:27041000

  1. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    Energy Technology Data Exchange (ETDEWEB)

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.; (Sydney)

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  2. Sda1, a Cys2-His2 zinc finger transcription factor, is involved in polyol metabolism and fumonisin B1 production in Fusarium verticillioides.

    Science.gov (United States)

    Malapi-Wight, Martha; Smith, Jonathon; Campbell, Jacquelyn; Bluhm, Burton H; Shim, Won-Bo

    2013-01-01

    The ubiquitous ascomycete Fusarium verticillioides causes ear rot and stalk rot of maize, both of which reduce grain quality and yield. Additionally, F. verticillioides produces the mycotoxin fumonisin B1 (FB1) during infection of maize kernels, and thus potentially compromises human and animal health. The current knowledge is fragmentary regarding the regulation of FB1 biosynthesis, particularly when considering interplay with environmental factors such as nutrient availability. In this study, SDA1 of F. verticillioides, predicted to encode a Cys-2 His-2 zinc finger transcription factor, was shown to play a key role in catabolizing select carbon sources. Growth of the SDA1 knock-out mutant (Δsda1) was completely inhibited when sorbitol was the sole carbon source and was severely impaired when exclusively provided mannitol or glycerol. Deletion of SDA1 unexpectedly increased FB1 biosynthesis, but reduced arabitol and mannitol biosynthesis, as compared to the wild-type progenitor. Trichoderma reesei ACE1, a regulator of cellulase and xylanase expression, complemented the F. verticillioides Δsda1 mutant, which indicates that Ace1 and Sda1 are functional orthologs. Taken together, the data indicate that Sda1 is a transcriptional regulator of carbon metabolism and toxin production in F. verticillioides.

  3. A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Akhil [Univ. of Hawaii, Manoa, HI (United States); Ohm, Robin A. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Oxiles, Lindsay [Univ. of Hawaii, Manoa, HI (United States); Brooks, Fred [Univ. of Hawaii, Manoa, HI (United States); Lawrence, Christopher B. [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Grigoriev, Igor V. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Cho, Yangrae [Univ. of Hawaii, Manoa, HI (United States)

    2011-10-26

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19- reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

  4. Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

    Science.gov (United States)

    Li, Haiyan S; Jin, Jin; Liang, Xiaoxuan; Matatall, Katie A; Ma, Ying; Zhang, Huiyuan; Ullrich, Stephen E; King, Katherine Y; Sun, Shao-Cong; Watowich, Stephanie S

    2016-09-01

    Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

  5. Expression Profiling and Functional Implications of a Set of Zinc Finger Proteins, ZNF423, ZNF470, ZNF521, and ZNF780B, in Primary Osteoarthritic Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Maria Mesuraca

    2014-01-01

    Full Text Available Articular chondrocytes are responsible for the maintenance of healthy articulations; indeed, dysregulation of their functions, including the production of matrix proteins and matrix-remodeling proteases, may result in fraying of the tissue and development of osteoarthritis (OA. To explore transcriptional mechanisms that contribute to the regulation of chondrocyte homeostasis and may be implicated in OA development, we compared the gene expression profile of a set of zinc finger proteins potentially linked to the control of chondrocyte differentiation and/or functions (ZNF423, ZNF470, ZNF521, and ZNF780B in chondrocytes from patients affected by OA and from subjects not affected by OA. This analysis highlighted a significantly lower expression of the transcript encoding ZNF423 in chondrocytes from OA, particularly in elderly patients. Interestingly, this decrease was mirrored by the similarly reduced expression of PPARγ, a known target of ZNF423 with anti-inflammatory and chondroprotective properties. The ZNF521 mRNA instead was abundant in all primary chondrocytes studied; the RNAi-mediated silencing of this gene significantly altered the COL2A/COL1 expression ratio, associated with the maintenance of the differentiated phenotype, in chondrocytes cultivated in alginate beads. These results suggest a role for ZNF423 and ZNF521 in the regulation of chondrocyte homeostasis and warrant further investigations to elucidate their mechanism of action.

  6. Identification of ‘safe harbor’ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair.

    Directory of Open Access Journals (Sweden)

    Christian eCantos

    2014-06-01

    Full Text Available Zinc-finger nucleases (ZFNs have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs at specific sites, which are further repaired through homologous recombination (HR or non-homologous end joining (NHEJ. However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3, harboring β-glucuronidase (GUS as a reporter gene, were used to identify safe harbor loci regions on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR. Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and it also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner.

  7. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

    Science.gov (United States)

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-01-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  8. Subtraction by addition: domesticated transposases in programmed DNA elimination

    OpenAIRE

    Motl, Jason A.; Chalker, Douglas L.

    2009-01-01

    The ciliate Paramecium tetraurelia must eliminate ∼60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478–2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed ...

  9. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    International Nuclear Information System (INIS)

    The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2-DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23-SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63-THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63-THEAC and Q2LQ23-SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2-DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63-THEAC and Q2LQ23-SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63-THEAC and Q2LQ23-SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63-THEAC, but is absent from the NTD of Q2LQ23-SYNAS. Second, whereas the structure of the CTD of Q2LQ23-SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure

  10. Screening and validation of specific zinc finger nucleases targeted pig CFTR gene%猪CFTR基因特异性锌指核酸酶的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    王瑞; 王令; 林娟; 张存芳; 张智英

    2012-01-01

    【目的】获得靶向猪囊性纤维化跨膜传导调节因子(Cystic fibrosis transmembrane conductance regulator,CFTR)基因的特异锌指核酸酶(Zinc finger nuclease,ZFN),为建立CFTR基因敲除猪细胞系提供技术支持。【方法】通过开源式(Oligomerized pool engineering,OPEN)方法筛选,首先以已知三锌指蛋白为框架,随机突变单个锌指关键位点氨基酸编码序列,建立人工三锌指蛋白随机库;然后应用细菌双杂交技术,从库中筛选出能够结合CFTR基因靶位点的三锌指蛋白;最后将获得的锌指蛋白与非限制性核酸内切酶FokⅠ组装成特异ZFN,通过酵母验证体系,检测ZFN靶向切割其识别序列的效率。【结果】获得3个人工三锌指蛋白随机库,每个库约含有2×106个单克隆,通过2轮细菌双杂交筛选,分别获得48个针对CFTR基因左右两侧靶位点的三锌指蛋白。ZFN活性验证结果表明,约90%的ZFN能够在酵母细胞内实现靶向切割,获得了高效特异的ZFN。【结论】获得了猪CFTR基因高效特异的ZFN。%【Objective】Specific zinc finger nucleases(ZFN)were screened to target pig cystic fibrosis transmembrane conductance regulator(CFTR)gene for the preparation of generating CFTR gene knockout pig cell line.【Method】Upon the oligomerized pool engineering(OPEN)strategy,firstly,three libraries of 3-zinc finger proteins were constructed.The DNA sequences of recognition amino acid residues of a finger motif were randomized by cassette mutagenesis.Secondly,bacterial twohybrid(B2H)system was used to screen for ZFPs from the three zinc finger protein libraries.Finally,selected ZFPs were fused to FokⅠdomain to generate specific ZFN for subsequent validation of ZFN cleavage activity in yeast.【Result】Three artificial randomly libraries of 3-zinc finger proteins were obtained,and each contained about 2×106 colonies.The three zinc finger random libraries were used

  11. Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-06-01

    Full Text Available Abstract Background Herpes simplex type II (HSV-2 is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M system, we previously identified the Escherichia coli restriction enzyme (REase EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome. Methods and results Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,, and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class, 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00 at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the

  12. Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation.

    Science.gov (United States)

    Qin, Zhenkui; Li, Yun; Su, Baofeng; Cheng, Qi; Ye, Zhi; Perera, Dayan A; Fobes, Michael; Shang, Mei; Dunham, Rex A

    2016-04-01

    Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes. PMID:26846523

  13. ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

    Science.gov (United States)

    Le, Cham Thi Tuyet; Brumbarova, Tzvetina; Ivanov, Rumen; Stoof, Claudia; Weber, Eva; Mohrbacher, Julia; Fink-Straube, Claudia; Bauer, Petra

    2016-01-01

    Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.

  14. Positional cloning of zinc finger domain transcription factor Zfp69, a candidate gene for obesity-associated diabetes contributed by mouse locus Nidd/SJL.

    Directory of Open Access Journals (Sweden)

    Stephan Scherneck

    2009-07-01

    Full Text Available Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1 contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO mice. A critical interval of distal chromosome 4 (2.1 Mbp conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT-PCR, and RACE-PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lep(ob background, the diabetogenic Zfp69(SJL allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes.

  15. The brain-specific neural zinc finger transcription factor 2b (NZF-2b/7ZFMyt1 suppresses cocaine self-administration in rats

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    Vijay Chandrasekar

    2010-04-01

    Full Text Available Brain-specific neural-zinc-finger transcription factor-2b (NZF2b/7ZFMyt1 is induced in the mesolimbic dopaminergic region after chronic cocaine exposure and lentiviral-mediated expression of NZF2b/7ZFMyt1 in the nucleus accumbens results in decreased locomotor activity (Chandrasekar and Dreyer, 2009. In this study the role of NZF2b/7ZFMyt1 in active cocaine seeking and of its interaction with histone deacetylase on the altered behavior has been observed. Localized expression of NZF2b/7ZFMyt1 in the nucleus accumbens resulted in attenuated cocaine self-administration, whereas silencing this transcription factor with lentiviruses expressing siRNAs increased the animal′s motivation to self-infuse cocaine. Low doses of sodium butyrate, a potent inhibitor of histone deacetylase, were sufficient to reverse the NZF2b/7ZFMyt1-mediated decrease in cocaine self-administration. NZF2b/7ZFMyt1 expression resulted in strong induction of transcription factors REST1 and NAC1 and of the dopamine D2 receptor, with concomitant inhibition of BDNF and its receptor TrkB. We show that NZF2b/7ZFMyt1 colocalizes with histone deacetylase-2 (HDAC2, probably overcoming the suppression of transcriptional activity caused by Lingo1. These findings show that molecular adaptations mediated by NZF2b/7ZFMyt1 expression possibly lead to decreased responsiveness to the reinforcing properties of cocaine and play a prominent role in affecting the behavioral changes induced by the drug.

  16. ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

    Science.gov (United States)

    Le, Cham Thi Tuyet; Brumbarova, Tzvetina; Ivanov, Rumen; Stoof, Claudia; Weber, Eva; Mohrbacher, Julia; Fink-Straube, Claudia; Bauer, Petra

    2016-01-01

    Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions. PMID:26556796

  17. Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis1[OPEN

    Science.gov (United States)

    Chen, Jian; Yan, Xingxing; Liu, Yunlei; Wang, Ren; Fan, Tingting; Ren, Yongbing; Tang, Xiaofeng; Xiao, Fangming

    2016-01-01

    Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis. PMID:26983992

  18. Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN.

    Directory of Open Access Journals (Sweden)

    Marwan Semaan

    Full Text Available Xenotransplantation has been proposed as a solution to the shortage of suitable human donors for transplantation and pigs are currently favoured as donor animals. However, xenotransplantation may be associated with the transmission of zoonotic microorganisms. Whereas most porcine microorganisms representing a risk for the human recipient may be eliminated by designated pathogen free breeding, multiple copies of porcine endogenous retroviruses (PERVs are integrated in the genome of all pigs and cannot be eliminated this way. PERVs are released as infectious particles and infect human cells. The zinc finger nuclease (ZFN technology allows knocking out specifically cellular genes, however it was not yet used to eliminate multiple integrated proviral sequences with a strong conservation in the target sequence. To reduce the risk of horizontal PERV transmission and to knock out as many as possible proviruses, for the first time the powerful tool of the ZFN technology was used. ZFN were designed to bind specifically to sequences conserved in all known replication-competent proviruses. Expression and transport of the ZFN into the nucleus was shown by Western blot analysis, co-localisation analysis, PLA and FRET. Survival of transfected cells was analysed using fluorescent ZFN and cell counting. After transfection a strong expression of the ZFN proteins and a co-localisation of the expressed ZFN proteins were shown. However, expression of the ZFN was found to be extremely toxic for the transfected cells. The induced cytotoxicity was likely due to the specific cutting of the high copy number of the PERV proviruses, which is also commonly observed when ZFN with low specificity cleave numerous off-target sites in a genome. This is the first attempt to knock out multiple, nearly identical, genes in a cellular genome using ZFN. The attempt failed, and other strategies should be used to prevent PERV transmission.

  19. Wheat zinc finger protein TaLSD1, a negative regulator of programmed cell death, is involved in wheat resistance against stripe rust fungus.

    Science.gov (United States)

    Guo, Jun; Bai, Pengfei; Yang, Qian; Liu, Furong; Wang, Xiaodong; Huang, Lili; Kang, Zhensheng

    2013-10-01

    Genetic characterization of the Arabidopsis lesion simulating disease 1 (lsd1) mutant, a lesion mimic mutant (LMM), has revealed the essential role of AtLSD1 in the negative regulation of cell death and disease resistance. The three zinc-finger motifs found in AtLSD1 revealed a novel plant-specific gene family, whose members are significantly related to programmed cell death (PCD). In this study, we characterized a functional homologue to AtLSD1, TaLSD1, in the wheat-stripe rust fungus pathosystem. The expression of TaLSD1 was differentially induced during incompatible and compatible interactions between wheat and Puccinia striiformis f. sp. tritici (Pst) and was up-regulated by oxidative stress generated by methyl viologen (MV). TaLSD1 was found to be predominately localized in the nucleus of onion epidermal cell. Transient overexpression assays in Nicotiana benthamiana demonstrated that TaLSD1 partially inhibited programmed cell death triggered by a mouse Bax protein, whereas expression of TaLSD1 alone had no influence on the phenotype of tobacco. Knocking down the expression of TaLSD1 through virus-induced gene silencing (VIGS) increased wheat resistance against Pst accompanied by an enhanced hypersensitive response (HR), an increase in PR1 gene expression and a reduction in Pst hyphal growth. Our results suggest that TaLSD1 functions negatively in regulating the plant hypersensitive cell death and is involved in disease resistance of wheat against the stripe rust pathogen.

  20. A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers

    Institute of Scientific and Technical Information of China (English)

    Tina S Mehta; Heng Lu; Xianhui Wang; Alison M Urvalek; Kim-Hang H Nguyen; Farah Monzur; Jojo D Hammond; Jameson Q Ma; Jihe Zhao

    2009-01-01

    Kruppel-like factor 8 (KLF8) transcription factor plays a critical role in cell cycle progression, oncogenic trans-formation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal(s) (NLS) has not been identified. KLF8 shares with other KLFs monopartite NLSs (mNLS) and C2H2 zinc fingers (ZFs), both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn2+-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of>1.5 ZFs from C-terminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid (aa) 151-200 re-gion almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear lo-calization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.

  1. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Genetically Modified Organisms (GMO

    2012-10-01

    Full Text Available

    The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3 which allows the integration of gene(s in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3 is used to describe the technique rather than ZFN-3 specifically. The EFSA GMO Panel considers that its guidance documents are applicable for the evaluation of food and feed products derived from plants developed using the SDN-3 technique and for performing an environmental risk assessment. However, on a case-by-case basis lesser amounts of event specific data may be needed for the risk assessment of plants developed using the SDN-3 technique. The EFSA GMO Panel compared the hazards associated with plants produced by the SDN-3 technique with those obtained by conventional plant breeding techniques and by currently used transgenesis. With respect to the genes introduced, the SDN-3 technique does not differ from transgenesis or from the other genetic modification techniques currently used, and can be used to introduce transgenes, intragenes or cisgenes. The main difference between the SDN-3 technique and transgenesis is that the insertion of DNA is targeted to a predefined region of the genome. Therefore, the SDN-3 technique can minimise hazards associated with the disruption of genes and/or regulatory elements in the recipient genome. Whilst the SDN-3 technique can induce off-target changes in the genome of the recipient plant these would be fewer than those occurring with most mutagenesis techniques. Furthermore, where such changes occur they would be of the same types as those produced by conventional breeding techniques.

  2. Diversity of Prdm9 zinc finger array in wild mice unravels new facets of the evolutionary turnover of this coding minisatellite.

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    Jérôme Buard

    Full Text Available In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs. High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons -1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.

  3. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    Science.gov (United States)

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae. PMID:25698512

  4. The BTB-zinc finger transcription factor abrupt acts as an epithelial oncogene in Drosophila melanogaster through maintaining a progenitor-like cell state.

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    Nezaket Turkel

    Full Text Available The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib, and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1, is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.

  5. The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in Drosophila melanogaster through Maintaining a Progenitor-like Cell State

    Science.gov (United States)

    Turkel, Nezaket; Sahota, Virender K.; Bolden, Jessica E.; Goulding, Karen R.; Doggett, Karen; Willoughby, Lee F.; Blanco, Enrique; Martin-Blanco, Enrique; Corominas, Montserrat; Ellul, Jason; Aigaki, Toshiro; Richardson, Helena E.; Brumby, Anthony M.

    2013-01-01

    The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state. PMID:23874226

  6. Perforator-based chimaeric thoracodorsal flap for foot reconstruction.

    Science.gov (United States)

    Rausky, Jonathan; Binder, Jean-Philippe; Mazouz-Dorval, Sarra; Hamou, Cynthia; Revol, Marc

    2013-12-01

    The reconstruction of severe defects of the ankle and foot is a challenge. The ideal solution should combine a thin skin flap on the dorsum to allow shoe fitting and a muscle flap with a split-thickness skin graft on the weight-bearing area. Perforator-based thoracodorsal chimaeric flaps allow us to achieve these two goals with minimal donor-site morbidity. We present a reconstruction of an extended circumferential defect of the ankle with an exposed heel using a chimaeric thoracodorsal perforator flap with a serratus muscle flap. The skin flap was transferred on the dorsal foot, whereas the serratus anterior muscle was transferred on the exposed heel. Postoperative recovery was uneventful and the patient began full weight bearing after 3 months. Twelve months after reconstruction, natural shape and walking function were successfully achieved.

  7. Expression, Purification and Characterization of the Zinc Finger Domain of PLZF Protein%重组PLZF蛋白锌指结构域的表达、提纯和活性分析

    Institute of Scientific and Technical Information of China (English)

    李伟

    2011-01-01

    PLZF is an important transcription repressor. It consists of an N-terminal BTB domain and a C-terminal zinc finger domain. To date, the three dimensional structure of the zinc finger domain is still not clear. Hence, expression and purification studies were performed. To produce the zinc finger domain of PLZF protein, the sequence encoding zinc finger domain fragment with a start codon added to the 5' was inserted into PET-lla expression vector. The expression plasmid was transformed into E. Coli BL21 ( DE3) strain and protein expressionwas induced by IPTG. The recombinant protein was found to be expressed as inclusion bodies. The inclusion body was solubilized using buffers containing SDS detergent and proteins were purified to more than 96 % homogeneity through gel filtration. The purified proteins were denatured and then refolded by reverse dialysis. The refolded proteins were characterized by DNA electrophoretic mobility shift assay ( EMS A) and found to be active. An important foundation has been laid for further three-dimensional structural study on the zinc finger domain of PLZF.%PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成.鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯.为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点.构建好的表达质粒转化到BI21( DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达.用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上.对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础.

  8. Subtraction by addition: domesticated transposases in programmed DNA elimination

    Science.gov (United States)

    Motl, Jason A.; Chalker, Douglas L.

    2009-01-01

    The ciliate Paramecium tetraurelia must eliminate ∼60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478–2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed silencing machinery can guide major genome reorganization. PMID:19884252

  9. A Zinc Finger Transcription Factor, αA-Crystallin Binding Protein 1, Is a Negative Regulator of the Chondrocyte-Specific Enhancer of the α1(II) Collagen Gene

    OpenAIRE

    Tanaka, Kazuhiro; Matsumoto, Yoshihiro; Nakatani, Fumihiko; Iwamoto, Yukihide; Yamada, Yoshihiko

    2000-01-01

    Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, αA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the ...

  10. Isolation of a Novel Family of C2H2 Zinc Finger Proteins Implicated in Transcriptional Repression Mediated by Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) Orphan Nuclear Receptors*

    OpenAIRE

    Avram, Dorina; Fields, Andrew; Top, Karen Pretty On; Nevrivy, Daniel J.; Ishmael, Jane E.; Leid, Mark

    2000-01-01

    Two novel and related C2H2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of A...

  11. U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3′ end processing

    OpenAIRE

    Azzouz, Teldja N.; Gruber, Andreas; Schümperli, Daniel

    2005-01-01

    The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100....

  12. Study on a cDNA sequence of cold inducible zinc finger protein in albinism tea cultivar "Xiaoxueya"%茶树品种“小雪芽”冷诱导锌指蛋白基因cDNA研究

    Institute of Scientific and Technical Information of China (English)

    王开荣; 李娜娜; 陆建良; 郑新强; 梁月荣; 吴颖; 李明

    2012-01-01

    A cDNA sequence of cold inducible zinc finger protein in leaf of albinism tea cultivar " Xiaoxueya" was investigated. The results shows that the cDNA sequence had 698 bp in length, with 83% and 82% identity to zinc finger protein mRNA of Glycine max and Ricinus communis respectively. It had an opening reading frame encoding 230 amino acids. Compared to that of common tea cuhivar " Fudingdabai" , there were three loci of nucleotide deletion and one locus of nucleotide substitution. Its deduced amino acid sequence had 99% identity to that of cuhivar " Fudingdabai" , among which there were 3 loci of amino acid substitution. The expression of cold inducible zinc finger protein in " Xiaoxueya" ,sas significantly lower than in " Fudingdabai". It is considered that the mutation in gene sequence resulted in the sensitivity of cultivar "Xiaoxueya" to low temperature through low expression and mutation of the cold inducible zinc finger protein.%分析了低温诱导型新梢白化茶树品种“小雪芽”叶片低温诱导锌指蛋白基因cDNA序列。该序列长度为698bp,与大豆锌指蛋白mRNA同源性为83%,与蓖麻锌指蛋白mRNA同源性为82%;具有可编码230个氨基酸的开放阅读框。与“福鼎大白茶”冷诱导锌指蛋白eDNA序列相比,该eDNA序列在50—51位上核苷酸AT缺失,第143位的A被置换为G,第654位T缺失。其翻译的蛋白质氨基酸序列与“福鼎大白茶”同源性达到99%,但有3个位点的氨基酸变异。该基因表达丰度明显低于“福鼎大白茶”。研究认为,基因结构差异,引起表达强度和蛋白质氨基酸序列的差异,可能是引起“小雪芽”品种对低温敏感的重要因素。

  13. Genome-Wide Analysis of C2H2 Zinc-Finger Family Transcription Factors and Their Responses to Abiotic Stresses in Poplar (Populus trichocarpa.

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    Quangang Liu

    Full Text Available C2H2 zinc-finger (C2H2-ZF proteins are a large gene family in plants that participate in various aspects of normal plant growth and development, as well as in biotic and abiotic stress responses. To date, no overall analysis incorporating evolutionary history and expression profiling of the C2H2-ZF gene family in model tree species poplar (Populus trichocarpa has been reported.Here, we identified 109 full-length C2H2-ZF genes in P. trichocarpa, and classified them into four groups, based on phylogenetic analysis. The 109 C2H2-ZF genes were distributed unequally on 19 P. trichocarpa linkage groups (LGs, with 39 segmental duplication events, indicating that segmental duplication has been important in the expansion of the C2H2-ZF gene family. Promoter cis-element analysis indicated that most of the C2H2-ZF genes contain phytohormone or abiotic stress-related cis-elements. The expression patterns of C2H2-ZF genes, based on heatmap analysis, suggested that C2H2-ZF genes are involved in tissue and organ development, especially root and floral development. Expression analysis based on quantitative real-time reverse transcription polymerase chain reaction indicated that C2H2-ZF genes are significantly involved in drought, heat and salt response, possibly via different mechanisms.This study provides a thorough overview of the P. trichocarpa C2H2-ZF gene family and presents a new perspective on the evolution of this gene family. In particular, some C2H2-ZF genes may be involved in environmental stress tolerance regulation. PtrZFP2, 19 and 95 showed high expression levels in leaves and/or roots under environmental stresses. Additionally, this study provided a solid foundation for studying the biological roles of C2H2-ZF genes in Populus growth and development. These results form the basis for further investigation of the roles of these candidate genes and for future genetic engineering and gene functional studies in Populus.

  14. C2H2 zinc finger proteins of the SP/KLF, Wilms tumor, EGR, Huckebein, and Klumpfuss families in metazoans and beyond.

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V

    2015-11-15

    Specificity proteins (SPs) and Krüppel-Like Factors (KLFs) are C2H2-type zinc finger transcription factors that play essential roles in differentiation, development, proliferation and cell death. SP/KLF proteins, similarly to Wilms tumor protein 1 (WT1), Early Growth Response (EGR), Huckebein, and Klumpfuss, prefer to bind GC-rich sequences such as GC-box and CACCC-box (GT-box). We searched various genomes and transcriptomes of metazoans and single-cell holozoans for members of these families. Seven groups of KLFs (KLFA-G) and three groups of SPs (SPA-C) were identified in the three lineages of Bilateria (Deuterostomia, Ecdysozoa, and Lophotrochozoa). The last ancestor of jawed vertebrates was inferred to have at least 18 KLFs (group A: KLF1/2/4/17, group B: KLF3/8/12; group C: KLF5/5l; group D: KLF6/7; group E: KLF9/13/16; group F: KLF10/KLF11; group G: KLF15/15l) and 10 SPs (group A: SP1/2/3/4; group B: SP5/5l; group C: SP6/7/8/9), since they were found in both cartilaginous and boned fishes. Placental mammals have added KLF14 (group E) and KLF18 (group A), and lost KLF5l (KLF5-like) and KLF15l (KLF15-like). Multiple KLF members were found in basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). Ctenophora has the least number of KLFs and no SPs, which could be attributed to its proposed sister group relationship to other metazoans or gene loss. While SP, EGR and Klumpfuss were only detected in metazoans, KLF, WT1, and Huckebein are present in nonmetazoan holozoans. Of the seven metazoan KLF groups, only KLFG, represented by KLF15 in human, was found in nonmetazoans. In addition, two nonmetazoan groups of KLFs are present in Choanoflagellatea and Filasterea. WT1 could be evolutionarily the earliest among these GC/GT-box-binding families due to its sole presence in Ichthyosporea. PMID:26187067

  15. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

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    Miguel G. Toscano

    2013-03-01

    Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS, which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

  16. Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats

    Science.gov (United States)

    Fan, Fan; Geurts, Aron M.; Pabbidi, Mallikarjuna R.; Smith, Stanley V.; Harder, David R.; Jacob, Howard; Roman, Richard J.

    2014-01-01

    We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats. PMID:25397684

  17. Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

    Directory of Open Access Journals (Sweden)

    Fan Fan

    Full Text Available We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art and middle cerebral artery (MCA and autoregulation of renal and cerebral blood flow (RBF and CBF were impaired in Fawn Hooded hypertensive (FHH rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5 were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2, were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

  18. Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

    Directory of Open Access Journals (Sweden)

    Susan Richter

    Full Text Available Zinc finger nucleases (ZFN are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK, in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116. All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002 and 4.3±0.8% (p = 0.001 for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

  19. Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

    Science.gov (United States)

    Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

    2013-01-01

    Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

  20. MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

    Directory of Open Access Journals (Sweden)

    Yamashita Shunichi

    2011-03-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC is often diagnosed at later stages until they are incurable. MicroRNA (miR is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC. Methods Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR. Results Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2

  1. Purification, crystallization and preliminary crystallographic analysis of the Hermes transposase

    Energy Technology Data Exchange (ETDEWEB)

    Perez, Zhanita N.; Musingarimi, Primrose [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States); Craig, Nancy L. [Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD (United States); Dyda, Fred; Hickman, Alison Burgess, E-mail: ahickman@helix.nih.gov [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States)

    2005-06-01

    Upon purification, an N-terminally deleted version of the Hermes transposase exists in solution as a mixture of two species that are approximately hexameric and dimeric. Crystals have been obtained of the smaller species that diffract to 2.1 Å resolution. DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations. One of these active transposons is Hermes, a 2749-base-pair element from Musca domestica that encodes its own transposase. An N-terminally deleted version of the Hermes transposase (residues 79–612) has been overexpressed and purified, and crystals that diffract to 2.1 Å resolution have been obtained at 277 K by the hanging-drop method.

  2. A hyperactive sleeping beauty transposase enhances transgenesis in zebrafish embryos

    Directory of Open Access Journals (Sweden)

    Lardelli Michael

    2010-11-01

    Full Text Available Abstract Background Transposons are useful molecular tools for transgenesis. The 'sleeping beauty' transposon is a synthetic member of the Tc1/mariner transposon family. Davidson et al. (2003 previously described a vector for zebrafish transgenesis consisting of the inverted repeats of 'sleeping beauty' flanking the gene to be transposed. Subsequently, there have been attempts to enhance the transpositional activity of 'sleeping beauty' by increasing the activity of its transposase. Recently, Mates et al. (2009 generated a hyperactive transposase giving a 100-fold increased transposition rate in mouse embryos. Findings The aim of this experiment was to determine whether this novel hyperactive transposase enhances transgenesis in zebrafish embryos. Using our previously characterised mitfa-amyloidβ-GFP transgene, we observed an eight-fold enhancement in transient transgenesis following detection of transgene expression in melanophores by whole mount in-situ hybridisation. However, high rates of defective embryogenesis were also observed. Conclusion The novel hyperactive 'sleeping beauty' transposase enhances the rate of transgenesis in zebrafish embryos.

  3. Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.

    Science.gov (United States)

    Naito, Mitsuru; Harumi, Takashi; Kuwana, Takashi

    2015-02-01

    Production of germline chimaeric chickens by the transfer of cultured primordial germ cells (PGC) is a useful system for germline manipulation. A novel culture system was developed for chicken PGC isolated from embryonic blood. The isolated PGC were cultured on feeder cells derived from chicken embryonic fibroblast. The cultured PGC formed colonies and they proliferated about 300-times during the first 30 days. The cultured PGC retained the ability to migrate to recipient gonads and were also chicken VASA homologue (CVH)-positive. Female PGC were present in the mixed-sex PGC populations cultured for more than 90 days and gave rise to viable offspring efficiently via germline chimaeric chickens. Male cultured PGC were transferred to recipient embryos and produced putative chimaeric chickens. The DNA derived from the cultured PGC was detected in the sperm samples of male putative chimaeric chickens, but no donor derived offspring were obtained. Donor-derived offspring were also obtained from germline chimaeric chickens by the transfer of frozen-thawed cultured PGC. The culture method for PGC developed in the present study is useful for manipulation of the germline in chickens, such as preservation of genetic resources and gene transfer.

  4. A hyperactive sleeping beauty transposase enhances transgenesis in zebrafish embryos

    OpenAIRE

    Lardelli Michael; Newman Morgan

    2010-01-01

    Abstract Background Transposons are useful molecular tools for transgenesis. The 'sleeping beauty' transposon is a synthetic member of the Tc1/mariner transposon family. Davidson et al. (2003) previously described a vector for zebrafish transgenesis consisting of the inverted repeats of 'sleeping beauty' flanking the gene to be transposed. Subsequently, there have been attempts to enhance the transpositional activity of 'sleeping beauty' by increasing the activity of its transposase. Recently...

  5. Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering.

    Science.gov (United States)

    Voigt, Franka; Wiedemann, Lisa; Zuliani, Cecilia; Querques, Irma; Sebe, Attila; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Barabas, Orsolya

    2016-03-30

    Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.

  6. Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering.

    Science.gov (United States)

    Voigt, Franka; Wiedemann, Lisa; Zuliani, Cecilia; Querques, Irma; Sebe, Attila; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Barabas, Orsolya

    2016-01-01

    Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases. PMID:27025571

  7. Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering

    Science.gov (United States)

    Voigt, Franka; Wiedemann, Lisa; Zuliani, Cecilia; Querques, Irma; Sebe, Attila; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Barabas, Orsolya

    2016-01-01

    Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases. PMID:27025571

  8. Nucleus Accumbens 1, a Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad protein binds to TAR DNA-binding protein 43 and has a potential role in Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Scofield, M D; Korutla, L; Jackson, T G; Kalivas, P W; Mackler, S A

    2012-12-27

    Protein degradation is a critical component of cellular maintenance. The intracellular translocation and targeting of the Ubiquitin Proteasome System (UPS) differentially coordinates a protein's half-life and thereby its function. Nucleus Accumbens 1 (NAC1), a member of the Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex (POZ/BTB) family of proteins, participates in the coordinated proteolysis of synaptic proteins by mediating recruitment of the UPS to dendritic spines. Here we report a novel interaction between NAC1 and TAR DNA-binding protein 43 (TDP-43), a protein identified as the primary component of ubiquitinated protein aggregates found in patients with Amyotrophic Lateral Sclerosis (ALS). In vitro translated full-length TDP-43 associated with both the POZ/BTB domain and the non-POZ/BTB domain of NAC1 in GST pulldown assays. Other POZ/BTB proteins (including zinc finger POZ/BTB proteins and atypical POZ/BTB proteins) showed weak interactions with TDP-43. In addition, NAC1 and TDP-43 were present in the same immunocomplexes in different regions of mouse brain and spinal cord. In primary spinal cord cultures, TDP-43 expression was mainly nuclear, whereas NAC1 was both nuclear and cytoplasmic. In order to mimic ALS-like toxicity in the spinal cord culture system, we elevated extracellular glutamate levels resulting in the selective loss of motor neurons. Using this model, it was found that glutamate toxicity elicited a dose-dependent translocation of TDP-43 out of the nucleus of cholinergic neurons and increased the co-localization of NAC1 and TDP-43. These findings suggest that NAC1 may function to link TDP-43 to the proteasome; thereby, facilitating the post-translational modifications of TDP-43 that lead to the development of ALS.

  9. Ultraviolet radiation dose to be applied in recipient zebrafish embryos for germ-line chimaerism is strain dependent.

    Science.gov (United States)

    Francisco-Simão, M; Cardona-Costa, J; Perez Camps, M; García-Ximénez, F

    2010-12-01

    Germ-line chimaerism is a powerful technique that has proved to be useful to produce viable gametes when transplanted blastomeres colonize the germinal ridges in recipient embryos and obtaining offspring from such transplanted cells. In fish, ionizing radiations were commonly used for embryo penalization to cancelling the cell participation of recipient embryos in development and in gamete production. The ultraviolet (UV) radiation when compared with other radiation types is cheaper, easier and no special installations are required for its use. So, the aim of this work was to establish the optimal UV radiation dose to be applied in zebrafish embryos at mid-blastula transition stage of development, in order to use them as penalized recipient embryos in futures chimaerism assays. A UV germicide lamp was used as radiation source (0.529 mW/cm(2)). Four exposure levels and three exposure times of UV radiation were tested. The survival rates obtained with the non-dechorionated embryos without lid group suggested that it could be the optimal exposure level to achieve the objective proposed. With the obtained results, we concluded that this UV radiation dose for 60 and 30 s are optimal parameters to penalize recipient wild and gold strain zebrafish embryos, respectively in chimaerism assays, but without involving their survival and apparently normal development.

  10. The C3H-type zinc finger protein GDS1/C3H42 is a nuclear-speckle-localized protein that is essential for normal growth and development in Arabidopsis.

    Science.gov (United States)

    Kim, Dae Won; Jeon, Su Jeong; Hwang, Sung Min; Hong, Jong Chan; Bahk, Jeong Dong

    2016-09-01

    Eukaryotic C3H-type zinc finger proteins (Znfs) comprise a large family of regulatory proteins involved in many aspects of plant stress response, growth and development. However, compared to mammalian, only a few plant Znfs have been functionally characterized. Here, T-DNA inserted gds1 (growth, development and splicing 1) mutant, displayed abnormal growth throughout the lifecycle owing to the reduction of cell size and number. Inverse PCR analysis revealed that the abnormal growth was caused by the disruption of At3g47120, which encodes a C3H42 protein belonging to the C-X7-C-X5-C-X3-H class of the Znf family. GDS1 was ubiquitously transcribed, but shows high levels of expression in young seedling and unexpanded new leaves. In gds1, the transcripts of many growth- and development-related genes were down-regulated, and the auxin response was dramatically reduced. A fluorescence-based assay revealed that the GDS1 protein was localized to the nucleus, prominently in the speckle compartments. Its arginine/serine dipeptide-rich-like (RS-like) domain was essential for nuclear localization. In addition, the SR1, SRm102 and U1-70K components of the U1 spliceosome interacted with GDS1 in the nuclear speckle compartments. Taken together, these suggest that GDS1, a nuclear-speckle-associated Znf, might play a significant role in splicing during plant growth and development. PMID:27457991

  11. Research Advance on Zinc-finger Nucleases Technology in Mammalian Transgenesis%锌指核酸酶技术在动物转基因中的研究进展

    Institute of Scientific and Technical Information of China (English)

    袁玉国; 彭秋玲

    2014-01-01

    锌指核酸酶(zinc finger nucleases,ZFNs)技术是近年来发展起来的一种基因修饰技术,利用ZFNs可特异识别靶位点DNA序列并使其断裂.与传统基于同源重组的基因打靶相比,ZFNs能使打靶效率提高100000倍,并使基因断裂与突变修复达到同样效果.ZFNs已经应用于多种生物,包括昆虫、两栖类生物、线虫、植物、多种动物和人类细胞.这些应用提高了人们对复杂生理系统的理解,使ZFNs成为一种生产转基因动物、细胞系和植物的有力工具,并在治疗人类疾病中发挥重要作用.文章对ZFNs技术原理及其应用于哺乳类动物转基因的研究进行了回顾和展望.

  12. Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

    Directory of Open Access Journals (Sweden)

    Kimura Hiroshi

    2011-05-01

    Full Text Available Abstract Background Herpes simplex viruses (HSVs rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1, a host cell protein markedly up-regulated by HSV infection. Results INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA. Conclusions The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.

  13. The mouse nac1 gene, encoding a cocaine-regulated Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger protein, is regulated by AP1.

    Science.gov (United States)

    Mackler, S A; Homan, Y X; Korutla, L; Conti, A C; Blendy, J A

    2003-01-01

    NAC1 cDNA was identified as a novel transcript induced in the nucleus accumbens from rats chronically treated with cocaine. NAC1 is a member of the Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger family of transcription factors and has been shown by overexpression studies to prevent the development of behavioral sensitization resulting from repeated cocaine treatment. This paper reports the cloning and characterization of the corresponding gene. The mouse Nac1 gene consist of six exons, with exon 2 containing an alternative splice donor, providing a molecular explanation of the splice variants observed in mouse and rat. Transcripts of Nac1 were ubiquitously detected in different mouse tissues with prominent expression in the brain. The mouse Nac1 gene was localized to chromosome 8, suggesting a highly plausible candidate gene to explain differences in cocaine-induced behaviors between C57BL6/J and DBA/2J mice that had previously been mapped to the area. In addition, a functional AP1 binding site has been identified in an intron 1 enhancer of the Nac1 gene that plays an essential role in the activation of the gene in differentiation of neuroblastoma cells. Co-transfection with c-jun and c-fos expression plasmids, which encode the two subunits of AP1, activated the wild type Nac1 intron 1 enhancer two-fold over basal, nearly at the level of NAC1 enhancer activity seen in differentiated N2A cells. Mutation of the AP1 site completely abrogated all activation of the NAC1 enhancer in differentiated N2A cells. Activation of immediate early genes such as c-fos and c-jun following chronic drug treatments has been well characterized. The present data describe one potential regulatory cascade involving these transcription factors and activation of NAC1. Identification of drug induced alterations in gene expression is key to understanding the types of molecular adaptations underlying addiction.

  14. An A20/AN1-zinc-finger domain containing protein gene in tea is differentially expressed during winter dormancy and in response to abiotic stress and plant growth regulators

    Directory of Open Access Journals (Sweden)

    Asosii Paul

    2015-03-01

    Full Text Available The present manuscript describes cloning and expression characterization of A20/AN1-zinc-finger domain containing protein (CsZfp gene in an evergreen tree tea [Camellia sinensis (L. O. Kuntze] in response to winter dormancy (WD, abiotic stresses (polyethylene glycol, hydrogen peroxide, and sodium chloride and plant growth regulators [abscisic acid (ABA, and gibberellic acid (GA3]. CsZfp encoded a putative protein of 173 amino acids with a calculated molecular weight of 18.44 kDa, an isoelectric point (pI of 6.50 and grand average of hydropathicity (GRAVY value of −0.334. The gene did not have an intron, and belonged to a multi-gene family. During the period of active growth (PAG, CsZfp showed maximum expression in root and fruit as compared to leaf, floral bud and stem. Interaction studies between temperature and plant growth regulators on the expression of CsZfp showed that ABA upregulated CsZfp expression at growth temperature (GT; 25 °C but had no effect at low temperature (LT; 4 °C. In response to GA3, upregulation was observed at LT but not at GT. Further, the expression was not modulated by LT either in the tissue harvested during PAG or during WD. It was interesting to record that the expression of CsZfp was upregulated by hydrogen peroxide and sodium chloride, whereas it was non-responsive to polyethylene glycol. The possible role of CsZfp in playing key but differential roles in tea to various abiotic stresses is discussed.

  15. Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase.

    Science.gov (United States)

    Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra

    2016-01-01

    The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer. PMID:27574698

  16. Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase.

    Science.gov (United States)

    Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra

    2016-01-01

    The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

  17. Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

    Energy Technology Data Exchange (ETDEWEB)

    Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao; Perez, Zhanita N.; Ghirlando, Rodolfo; Chandler, Michael; Dyda, Fred (Centre Nat); (NIH)

    2010-07-20

    Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.

  18. 锌指蛋白185对胶质瘤细胞增殖影响的研究%The effect of zinc finger protein 185 on the proliferation of human glioma cells

    Institute of Scientific and Technical Information of China (English)

    陆斌; 郑全辉

    2015-01-01

    目的:探讨锌指蛋白ZNF185在人脑胶质瘤细胞增殖中的作用。方法标本取自2011年1月—2013年12月于唐山市工人医院就诊,经病理确诊为胶质瘤患者的肿瘤组织,以瘤旁组织作对照。提取不同组织总蛋白, Western-blot检测ZNF185的表达。提取瘤旁组织总RNA,反转录扩增ZNF185编码序列并克隆至pEGFPC2质粒,构建ZNF185表达载体。采用Lipofactamine2000将ZNF185表达载体转染人胶质瘤细胞系SF767,以转染pEGFPC2空载体细胞作为对照。采用流式细胞仪检测细胞周期变化;MTT法检测细胞增殖活性。结果与瘤旁组织相比, ZNF185在人脑胶质瘤组织中表达显著降低(P<0.01);转染ZNF185表达载体的胶质瘤细胞与对照细胞相比ZNF185的表达显著增加(P<0.01);过表达ZNF185导致SF767细胞G0~G1期细胞比例显著增加(P<0.05),而S期细胞比例减少(P<0.05)。同时,过表达ZNF185也导致SF767细胞的增殖速度显著降低(P<0.05)。结论ZNF185在人脑胶质瘤细胞中发挥抑制细胞增殖的作用。%Objective To explore the role of zinc finger protein (ZNF)185 in the proliferation of human glioma cells. Methods Human glioma tissues and tumor adjacent tissues were obtained from glioma patients diagnosed pathologically in Tangshan Gongren Hospital from January 2011 to December 2013. Total protein was extracted from different tissues. The ZNF185 expression was detected by Western-blot assay. Total RNA was extracted from tumor adjacent tissues. ZNF 185 cod⁃ing sequence was obtained by RT-PCR and inserted into pEGFPC2 plasmid to construct the ZNF185 expression vector. Li⁃pofactamine2000 was used to transfect the ZNF185 expression vector to human glioma cell SF767. pEGFPC2 blank vector transfected SF767 cells were used as control. Changes of cell cycle were analyzed by flow cytometry, and cell proliferation was analyzed by MTT assay. Results The expression of ZNF185 decreased

  19. Cloning and Molecular Characterization of a RING Zinc-Finger Gene of Hevea brasiliensis%巴西橡胶树环锌指蛋白基因克隆及其分子特性

    Institute of Scientific and Technical Information of China (English)

    朱家红; 李辉亮; 屠发志; 田维敏; 彭世清

    2006-01-01

    Using the information about the sequence from a differentially expressed clone (designated as HbSSH10)encodes a protein specifying a cysteine-rich sequence containing a putative "RING finger" or "C3HC4" consensus motif that was cloned recently by the subtractive hybridization between latex and leaves from rubber tree (Hevea brasiliensis). A full-length cDNA encoding C3HC4 type zinc-finger protein was isolated and characterized from rubber tree. Sequence analysis revealed that the ORFs of HbRZF encode 156 amino acid residues with a total predicted molecular mass of 17.2 kD,HbRZF protein having a putative "RING finger" segment (amino acid residues 100-144). The deduced amino acid sequences of HbRZF showed high identities of 48%,52% and 50% to those of the ring zinc protein from Poncirus trifoliata, Arabidopsis thaliana, Thellungiella halophila. The result of Northern blot analysis indicated that the transcripts of the HbRZF were expressed more in the latex than in the leaves, whereas little expression was detected in roots and flowers. The transcription of HbRZF was induced by jasmonic acid, whereas ethylene had little effect.%在先前的研究中通过抑制缩减杂交获得了一个在巴西橡胶树胶乳中特异表达的片段(HbSSH10),该片段含有"RING finger"或"C3HC4"保守序列.根据HbSSH10的序列信息设计引物并通过3'-RACE和5'-RACE的方法,获得了一个全长的cDNA(HbRZF).该cDNA含有589个核苷酸,含有完整的阅读框架,编码156个氨基酸.从它推导出的氨基酸序列中含有"RING finger"或"C3HC4"保守区(氨基酸100~144).该氨基酸序列与Poncirus trifoliata、Arabidopsis thaliana和Thellungiella halophila的环锌指蛋白的同源性分别为48%、52%和50%.Northern杂交分析表明HbRZF在胶乳中大量表达,在叶片中微量表达,而在根和花中几乎没有表达.茉莉酸处理可以诱导胶乳中HbRZF的表达,而乙烯对胶乳中HbRZF的表达基本上没有影响.

  20. The Potential of the Combination of CRISPR/Cas9 and Pluripotent Stem Cells to Provide Human Organs from Chimaeric Pigs

    Directory of Open Access Journals (Sweden)

    Wanyou Feng

    2015-03-01

    Full Text Available Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9, in combination with induced pluripotent stem cells (iPSC, may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient’s immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.

  1. Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive Sleeping Beauty transposase for somatic integration

    OpenAIRE

    Zhang, W; Muck-Hausl, M.; Wang, J.; C. Sun; Gebbing, M.; Miskey, C.; Ivics, Z.; Izsvak, Z; Ehrhardt, A.

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We foun...

  2. Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

    OpenAIRE

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We foun...

  3. A Domesticated PiggyBac Transposase Interacts with Heterochromatin and Catalyzes Reproducible DNA Elimination in Tetrahymena

    OpenAIRE

    Alexander Vogt; Kazufumi Mochizuki

    2013-01-01

    The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activ...

  4. Microbial co-habitation and lateral gene transfer: what transposases can tell us

    Energy Technology Data Exchange (ETDEWEB)

    Hooper, Sean D.; Mavromatis, Konstantinos; Kyrpides, Nikos C.

    2009-03-01

    Determining the habitat range for various microbes is not a simple, straightforward matter, as habitats interlace, microbes move between habitats, and microbial communities change over time. In this study, we explore an approach using the history of lateral gene transfer recorded in microbial genomes to begin to answer two key questions: where have you been and who have you been with? All currently sequenced microbial genomes were surveyed to identify pairs of taxa that share a transposase that is likely to have been acquired through lateral gene transfer. A microbial interaction network including almost 800 organisms was then derived from these connections. Although the majority of the connections are between closely related organisms with the same or overlapping habitat assignments, numerous examples were found of cross-habitat and cross-phylum connections. We present a large-scale study of the distributions of transposases across phylogeny and habitat, and find a significant correlation between habitat and transposase connections. We observed cases where phylogenetic boundaries are traversed, especially when organisms share habitats; this suggests that the potential exists for genetic material to move laterally between diverse groups via bridging connections. The results presented here also suggest that the complex dynamics of microbial ecology may be traceable in the microbial genomes.

  5. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  6. 青花菜C3H型锌指蛋白基因 BoCCCH2的克隆与表达%Cloning and expression of a C3H-type zinc finger protein gene BoCCCH2 from Brassica oleracea var . italica

    Institute of Scientific and Technical Information of China (English)

    蒋明; 刘青娥; 章燕如; 祝琦; 龚秀; 俞可可; 周秀倩

    2016-01-01

    以青花菜为材料,在克隆C3 H型锌指蛋白基因 BoCCC H2的基础上,研究该基因在不同器官及霜霉菌和灰葡萄孢菌侵染叶片中的表达模式。测序结果表明,BoCCC H2没有内含子,编码区全长为1740 bp ,编码579个氨基酸,推导的蛋白质具2个 ANK结构域和2种CCCH 锌指结构,锌指结构的类型分别为C—X8—C—X5—C—X3—H和C—X5—C—X4—C—X3—H 。反转录聚合酶链反应表明:BoCCC H2在根、叶、花茎、嫩角果、花蕾和花中均有表达,其中在根中的表达量最高;经霜霉菌和灰葡萄孢菌侵染后,该基因表达量均有不同程度的增加,其中在霜霉菌侵染下,表达量在24 h后开始增加,72 h时下降,而在灰葡萄孢菌侵染下,6 h时的表达量最大,12 h时开始缓慢下降。聚类结果表明,BoCCCH2与其他十字花科植物的同源序列聚为一类,支持率达100%,而与豆科、大戟科和蔷薇科等植物的序列处于不同分支。对 BoCCC H2基因的克隆和表达分析为该基因功能研究奠定了基础。%Summary Brassicaoleraceavar.italicaisanimportantvegetablecropworldwide,andinChina,TaizhouCity of Zhejiang Province is one of the major broccoli production areas . Downy mildew and grey mold rot are two common fungal diseases caused by Hyaloperonospora parasitica and Botrytis cinerea , respectively . In recent years , broccoli production in Taizhou was frequently affected by these two fungal diseases , resulting in yield and quality loss . Broccoli germplasm resources resistance to disease is scarce; therefore , molecular breeding is regarded as an effective solution to solve the problem . This is critically important to isolate genes associated with disease resistance , which will act as potential target genes for broccoli breeding . Zinc finger proteins are kinds of important transcription factors in eukaryotic organisms , which involve in various biological activities , such as replication , transcription

  7. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    Science.gov (United States)

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-07-19

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

  8. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    Science.gov (United States)

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose. PMID:27434682

  9. RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.

    Directory of Open Access Journals (Sweden)

    Christina Rauschhuber

    Full Text Available BACKGROUND: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. PRINCIPAL FINDINGS: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.

  10. Birth of a chimeric primate gene by capture of the transposase gene from a mobile element.

    Science.gov (United States)

    Cordaux, Richard; Udit, Swalpa; Batzer, Mark A; Feschotte, Cédric

    2006-05-23

    The emergence of new genes and functions is of central importance to the evolution of species. The contribution of various types of duplications to genetic innovation has been extensively investigated. Less understood is the creation of new genes by recycling of coding material from selfish mobile genetic elements. To investigate this process, we reconstructed the evolutionary history of SETMAR, a new primate chimeric gene resulting from fusion of a SET histone methyltransferase gene to the transposase gene of a mobile element. We show that the transposase gene was recruited as part of SETMAR 40-58 million years ago, after the insertion of an Hsmar1 transposon downstream of a preexisting SET gene, followed by the de novo exonization of previously noncoding sequence and the creation of a new intron. The original structure of the fusion gene is conserved in all anthropoid lineages, but only the N-terminal half of the transposase is evolving under strong purifying selection. In vitro assays show that this region contains a DNA-binding domain that has preserved its ancestral binding specificity for a 19-bp motif located within the terminal-inverted repeats of Hsmar1 transposons and their derivatives. The presence of these transposons in the human genome constitutes a potential reservoir of approximately 1,500 perfect or nearly perfect SETMAR-binding sites. Our results not only provide insight into the conditions required for a successful gene fusion, but they also suggest a mechanism by which the circuitry underlying complex regulatory networks may be rapidly established. PMID:16672366

  11. Full-length dysferlin transfer by the hyperactive Sleeping Beauty transposase restores dysferlin-deficient muscle

    OpenAIRE

    Escobar, H.; Schoewel, V.; Spuler, S.; Marg, A.; Izsvak, Z.

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target geno...

  12. A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

    Institute of Scientific and Technical Information of China (English)

    Qingshu Meng; Kaifu Chen; Lina Ma; Songnian Hu; Jun Yu

    2011-01-01

    Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ~ 160 Mb) has been sequenced and is composed of ~65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.

  13. Chimeric piggyBac transposases for genomic targeting in human cells.

    Science.gov (United States)

    Owens, Jesse B; Urschitz, Johann; Stoytchev, Ilko; Dang, Nong C; Stoytcheva, Zoia; Belcaid, Mahdi; Maragathavally, Kommineni J; Coates, Craig J; Segal, David J; Moisyadi, Stefan

    2012-08-01

    Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy. PMID:22492708

  14. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    Science.gov (United States)

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.

  15. 锌指核酸酶技术制备肌肉生长抑制素基因敲除的五指山小型猪成纤维细胞%Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

    Institute of Scientific and Technical Information of China (English)

    曹随忠; 岳成鹤; 李西睿; 冯冲; 龙川; 潘登科

    2013-01-01

    Disruption of myostatin (MSTN) gene in pigs may improve porcine lean meat percentage (LMP), and create an animal model for certain human diseases. Using zinc-finger nucleases (ZFNs) technology, MSTN gene was deleted in Wuzhishan miniature pig fibroblasts by transfection of either ZFNs plasmids or ZFNs mRNA in high efficiency. Strikingly, ZFNs encoding mRNA could produce MSTN+/-and MSTN-/- cell colonies with single or double allele deletion by single transfection. Sequencing results demonstrated that 92.18% of the mutations were short fragment deletions or insertions (≤10 bp). Prediction of amino acids sequences indicated that more than half of the mutations cause premature transla-tional-termination codon. MSTN+/+, MSTN+/-, and MSTK-/- cell colonies were used as nuclear donor for somatic cell nuclear transfer (SCNT), and developmental potential of SCNT embryos were measured by the blastocyst rate. The results revealed no significant difference in development competence among the three kinds of reconstructed embryos (14.29% vs. 19.64% vs. 16.13%), which provides the possibility of making myostatin knock out pigs in the future.%敲除猪肌肉生长抑制素(Myostatin,MSTN)基因可能提高猪瘦肉率,MSTN 基因敲除猪也可作为相关疾病的动物模型.文章利用锌指核酸酶(Zinc-finger nucleases,ZFNs)技术敲除五指山小型猪胎儿成纤维细胞MSTN基因,为制备MSTN 基因敲除猪奠定基础.ZFNs 质粒或编码ZFNs 的mRNA 均能高效敲除MSTN 基因,使用ZFNs mRNA 能直接得到MSTN+/-和MSTN-/-两种基因型的细胞克隆.DNA 序列测定与分析发现,细胞克隆的突变类型多为ZFNs 作用靶位点处不大于10 bp 的碱基插入或缺失(92.18 %); 氨基酸预测发现,突变型MSTN 基因的终止密码子常常提前出现.将MSTN 基因敲除的细胞进行体细胞核移植(Somatic cell nuclear transfer,SCNT)发现,胚胎体外早期发育潜力与野生型无显著差异,表明这些细胞可用于后续MSTN 基因敲除猪的制备.

  16. Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

    Science.gov (United States)

    Hickman, Alison B; Ewis, Hosam E; Li, Xianghong; Knapp, Joshua A; Laver, Thomas; Doss, Anna-Louise; Tolun, Gökhan; Steven, Alasdair C; Grishaev, Alexander; Bax, Ad; Atkinson, Peter W; Craig, Nancy L; Dyda, Fred

    2014-07-17

    Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA. PMID:25036632

  17. Structural Basis for Transposon End Recognition by Hermes, an Octameric hAT DNA Transposase from Musca domestica

    Science.gov (United States)

    Hickman, Alison B.; Ewis, Hosam E.; Li, Xianghong; Knapp, Joshua A.; Laver, Thomas; Doss, Anna-Louise; Tolun, Gökhan; Steven, Alasdair C.; Grishaev, Alexander; Bax, Ad; Atkinson, Peter W.; Craig, Nancy L.; Dyda, Fred

    2014-01-01

    SUMMARY Hermes is a member of the hAT transposon superfamily which has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. While isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple non-specific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA. PMID:25036632

  18. Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.

    Directory of Open Access Journals (Sweden)

    Wenli Zhang

    Full Text Available We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR and linear amplification-mediated PCR (LAM-PCR. Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.

  19. The Response of a Putative Maize Zinc-Finger Protein Gene ZmAN14 in Transgenic Tabacco to Abiotic Stress%玉米锌指蛋白基因ZmAN14过表达转基因烟草对非生物胁迫的响应

    Institute of Scientific and Technical Information of China (English)

    宣宁; 柳絮; 张华; 陈高; 刘国霞; 边斐; 姚方印

    2015-01-01

    [Objective]ZmAN14is a member of the maize A20/AN1 zinc finger protein gene family. This gene family of rice is involved in the response to abiotic stress. The expression ofZmAN14inthe maize inbred line H21 and transgenic ZmAN14tobacco under abiotic stress was analyzed. The results will provide novel information for the comprehensive analysis of the functional and molecular mechanisms of maizeZmAN14and the entire gene family.[Method] Sequence analysis ofZmAN14was performed to confirm that this gene is a member of the maize A20/AN1 zinc finger protein gene family. The experiment was conducted by using maize inbred line H2. At the three-leaf stage, the seedlings were treated with multiple abiotic stress or induced by ABA. After treatment for 0, 1, 3, 6, 12 and 24 h, the whole plants were harvested. In the meantime, the roots, stems, leaves, coleptiles, pistil, stamen, silks and bract leaves were harvested from different growing periods of maize. Real-time quantitative fluorescence PCR was used to analyze the expression profile in different tissues and abiotic stress response ofZmAN14and its ABA-induced expression profile. Thecis-acting element of the promoter area was cloned to perform the comparative analysis. TheZmAN14coding sequence was cloned onto the GFP expressing vector pMDC85. A subcellular localization method was used to verify the localization of the ZmAN14 protein in the cell. TheZmAN14coding sequence was cloned onto the GAL4 DNA-binding domain vector. This vector was used to transform yeast. The transformed yeast was spotted onto the defect medium, and the resulted transcriptional activation activity was analyzed. TheZmAN14coding region was ligated to the plant expression vector p1300-221 to create an overexpression vector. The overexpression vector was transformed into tobacco. T2homozygous transgenic lines with highZmAN14 mRNA expression level were used to conduct salt-, drought- and ABA induced experiments, and its response to abiotic stress was

  20. Functional characterization of sugarcane mustang domesticated transposases and comparative diversity in sugarcane, rice, maize and sorghum

    Directory of Open Access Journals (Sweden)

    Daniela Kajihara

    2012-01-01

    Full Text Available Transposable elements (TEs account for a large portion of plant genomes, particularly in grasses, in which they correspond to 50%-80% of the genomic content. TEs have recently been shown to be a source of new genes and new regulatory networks. The most striking contribution of TEs is referred as “molecular domestication”, by which the element coding sequence loses its movement capacity and acquires cellular function. Recently, domesticated transposases known as mustang and derived from the Mutator element have been described in sugarcane. In order to improve our understanding of the function of these proteins, we identified mustang genes from Sorghum bicolor and Zea mays and performed a phenetic analysis to assess the diversity and evolutionary history of this gene family. This analysis identified orthologous groups and showed that mustang genes are highly conserved in grass genomes. We also explored the transcriptional activity of sugarcane mustang genes in heterologous and homologous systems. These genes were found to be ubiquitously transcribed, with shoot apical meristem having the highest expression levels, and were downregulated by phytohormones. Together, these findings suggest the possible involvement of mustang proteins in the maintenance of hormonal homeostasis.

  1. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    Science.gov (United States)

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  2. Sequence diversity and copy number variation of Mutator-like transposases in wheat

    Directory of Open Access Journals (Sweden)

    Nobuaki Asakura

    2008-01-01

    Full Text Available Partial transposase-coding sequences of Mutator-like elements (MULEs were isolated from a wild einkorn wheat, Triticum urartu, by degenerate PCR. The isolated sequences were classified into a MuDR or Class I clade and divided into two distinct subclasses (subclass I and subclass II. The average pair-wise identity between members of both subclasses was 58.8% at the nucleotide sequence level. Sequence diversity of subclass I was larger than that of subclass II. DNA gel blot analysis showed that subclass I was present as low copy number elements in the genomes of all Triticum and Aegilops accessions surveyed, while subclass II was present as high copy number elements. These two subclasses seemed uncapable of recognizing each other for transposition. The number of copies of subclass II elements was much higher in Aegilops with the S, Sl and D genomes and polyploid Triticum species than in diploid Triticum with the A genome, indicating that active transposition occurred in S, Sl and D genomes before polyploidization. DNA gel blot analysis of six species selected from three subfamilies of Poaceae demonstrated that only the tribe Triticeae possessed both subclasses. These results suggest that the differentiation of these two subclasses occurred before or immediately after the establishment of the tribe Triticeae.

  3. Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome

    Indian Academy of Sciences (India)

    Susana Mariconda; Soon-Young Namgoong; Ki-Hoon Yoon; Hong Jiang; Rasika M Harshey

    2000-12-01

    Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (III and III, respectively). Although the central domain II (the ‘DDE’ domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within III also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, III or III domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain III or III function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.

  4. Association analysis of zinc finger protein 804A gene with bipolar disorders and its subtypes in Han Chinese%锌指蛋白804A基因与中国汉族双相障碍及其亚型的关联分析

    Institute of Scientific and Technical Information of China (English)

    张晨; 汪作为; 吴志国; 洪武; 陈俊; 彭代辉; 方贻儒

    2015-01-01

    Objective To investigate the potential association between zinc finger protein 804A (ZNF804A) rs1344706 polymorphism and bipolar disorder as well as its subtypes.Methods A total of 304 bipolar Ⅰ disorder,442 bipolar Ⅱ disorder and 762 healthy controls subjects were recruited from Division of Mood Disorders,Shanghai Mental Health Center,Shanghai Jiao Tong University School of Medicine,from November 2006 to August 2014.Rs1344706 was genotyped by SNaPshot genotyping assay.Results In comparison between the patients with bipolar disorder and controls,there were significant differences in the genotypic (TT:210(28.2%) vs.175(23.0%),TG:378(50.7%) vs.399(52.4%),GG:158 (21.2%) vs.188(24.7%)) and allelic (T:798(53.5%) vs.749(49.1%),G:694(46.5%) vs.775(50.9%)) distributions (x2=6.18,P=0.04,x2=5.68,P=0.017,respectively).The frequency of T allele in patients with bipolar disorder was higher than that in controls (OR=1.19,95%CI 1.03-1.37).After stratified by diagnosed subtypes,there were significant differences in the genotypic and allelic distribution between patients with bipolar Ⅰ disorder and controls (x2=7.57,P=0.02,x2=7.22,P=0.007).The frequency of T allele in patients with bipolar Ⅰ disorder was higher than those in controls (OR=1.30,95% CI 1.07-1.56).In comparison between patients with bipolar Ⅱ disorder and controls,there was no significant difference in the distributions of genotype and allele.Conclusion There is positive association between rs1344706 polymorphism and bipolar Ⅰ disorder in a Chinese Han population.ZNF804A gene may confer susceptibility to bipolar Ⅰ disorder in Han Chinese.%目的 探讨位于锌指蛋白804A(zinc finger protein 804A,ZNF804A)内含子2区的多态性位点rs 1344706与双相障碍及其亚型的关系.方法 收集2006年11月至2014年8月在上海交通大学医学院附属精神卫生中心心境障碍科就诊的双相障碍患者746例(患者组),根据DSM-Ⅳ中双相障碍诊断亚型

  5. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

    Directory of Open Access Journals (Sweden)

    Emeline Dubois

    2012-01-01

    Full Text Available Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus, ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes.

  6. Electrochemically modified carbon and chromium surfaces for AFM imaging of double-strand DNA interaction with transposase protein.

    Science.gov (United States)

    Esnault, Charles; Chénais, Benoît; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Pilard, Jean-François

    2013-02-01

    Carbon and chromium surfaces were modified by electrochemical reduction of a diazonium salt formed in situ from the sulfanilic acid. The organic layer formed was activated by phosphorus pentachloride (PCl(5)) to form a benzene sulfonil chloride (Ar-SO(2)Cl). An electrochemical study of the blocking effect and the activity of this surface was carried out on a carbon electrode. The chromium surface study was completed by X-ray photoelectron spectroscopy and atomic force microscopy to characterize the formation of a compact monolayer (0.8 nm height and roughness 0.2-0.3 nm). The compactness and the activity of this organic monolayer allowed us to affix a length dsDNA with the aim of analyzing the formation of a complex between dsDNA and a protein. The interaction of a transposase protein with its target dsDNA was investigated. The direct imaging of the nucleoproteic complex considered herein gives new insights in the comprehension of transposase-DNA interaction in agreement with biochemical data.

  7. A Domesticated piggyBac Transposase Plays Key Roles in Heterochromatin Dynamics and DNA Cleavage during Programmed DNA Deletion in Tetrahymena thermophila

    OpenAIRE

    Cheng CY; Vogt A; Mochizuki K.; Yao MC

    2010-01-01

    Transposons comprise large fractions of eukaryotic genomes and provide genetic reservoirs for the evolution of new cellular functions. We identified TPB2, a homolog of the piggyBac transposase gene that is required for programmed DNA deletion in Tetrahymena. TPB2 was expressed exclusively during the time of DNA excision, and its encoded protein Tpb2p was localized in DNA elimination heterochromatin structures. Notably, silencing of TPB2 by RNAi disrupts the final assembly of these heterochrom...

  8. Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

    Directory of Open Access Journals (Sweden)

    Fraser Tresa S

    2008-08-01

    Full Text Available Abstract Background The piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS near the c-terminus, just upstream of a putative ZnF (ZnF. Results We fused the piggyBac TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2 cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulphate. Conclusion Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.

  9. Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.

    Science.gov (United States)

    Li, Zicong; Zeng, Fang; Meng, Fanming; Xu, Zhiqian; Zhang, Xianwei; Huang, Xiaoling; Tang, Fei; Gao, Wenchao; Shi, Junsong; He, Xiaoyan; Liu, Dewu; Wang, Chong; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2014-05-01

    The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

  10. The periodontal pathogen Porphyromonas gingivalis induces expression of transposases and cell death of Streptococcus mitis in a biofilm model.

    Science.gov (United States)

    Duran-Pinedo, Ana E; Baker, Vinesha D; Frias-Lopez, Jorge

    2014-08-01

    Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage. PMID:24866802

  11. Tethering of the conserved piggyBac transposase fusion protein CSB-PGBD3 to chromosomal AP-1 proteins regulates expression of nearby genes in humans.

    Directory of Open Access Journals (Sweden)

    Lucas T Gray

    2012-09-01

    Full Text Available The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3 transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program.

  12. Expression and purification of a novel ZNF191 zinc finger protein——ZNF191 protein and its truncated zinc finger region ZNF191 (243—368)

    Institute of Scientific and Technical Information of China (English)

    刘玉奇; 余龙; 余文浩; 施少林; 孙炳耘; 吴国俊; 黄仲贤

    1999-01-01

    ZNF191 is a new zine finger gene whieh has a 1107 bp open reading frame (ORF) and eneodes a 368 amino avid protein including a putative DNA-binding domain of four Cys2 His2 zine finger motifs at its C-terminal region. The ZNF191 cDNA is loeated in the chromosome 18q12.1 region where it is known that a variety of hereditary diseases are related to. Probably, this protein has potential function of stimulating the gene franscription. In order to examine the function and structure of ZNF191 protein, the ORF of ZNF191 and its zine finger region ZNF191(243--368) genes were inserted into PTSA-18 expression vector by PCR amplification, then the constructed genes were expressed in the PTSA-18/B121 (DE3) system. The two proteins were purified by DEAE-52, CM-23 and Heparin-Sepharose 4B columns. The pooled proteins showed a single band as assayed by Coomasie Brillian Blue Staining of an SDS/polyacryamide gel.

  13. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

    Science.gov (United States)

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence Jn; Rosenberg, Steven A

    2016-06-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

  14. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

    Science.gov (United States)

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg) conferring optimal levels of long-term expression (>1011 photons/second/cm2). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2) was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  15. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

    Science.gov (United States)

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence Jn; Rosenberg, Steven A

    2016-06-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006

  16. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    Science.gov (United States)

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  17. Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

    Directory of Open Access Journals (Sweden)

    Dong Chan Moon

    Full Text Available Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs, (225RKRKRK(230. Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1 gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

  18. AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

    OpenAIRE

    Weber, Nicholas D.; Daniel Stone; Ruth Hall Sedlak; De Silva Feelixge, Harshana S.; Pavitra Roychoudhury; Schiffer, Joshua T.; Martine Aubert; Jerome, Keith R.

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatme...

  19. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B;

    2011-01-01

    targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAca or NeuAca2-6GalNAca O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O......-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O...

  20. Brittle Cornea Syndrome Associated with a Missense Mutation in the Zinc-Finger 469 Gene

    DEFF Research Database (Denmark)

    Christensen, Anne Elisabeth; Knappskog, Per Morten; Midtbø, Marit;

    2010-01-01

    Purpose: To investigate the diverse clinical manifestations, identify the causative mutation and explain the association with red hair in a family with brittle cornea syndrome (BCS). Methods: Eight family members in three generations underwent ophthalmic, dental, and general medical examination...... mapping with SNP markers, DNA sequencing, and MC1R genotyping. Results: At 42 and 48 years of age, respectively, both affected individuals were blind due to retinal detachment and secondary glaucoma. They had extremely thin and bulging corneas, velvety skin, chestnut colored hair, scoliosis, reduced BMD......, dental anomalies, hearing loss and minor cardiac defects. The morphologies of the skin biopsies were normal except that in some areas slightly thinner collagen fibrils were seen in one of the affected individuals. Molecular genetic analysis revealed a novel missense mutation of ZNF469, c.10016G...

  1. TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

    DEFF Research Database (Denmark)

    Le Douarin, B; You, J; Nielsen, Anders Lade;

    1998-01-01

    Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new...... family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

  2. Expression of Zinc Finger Protein 804A (ZNF804A) in the brain

    DEFF Research Database (Denmark)

    Benedikz, Eirikur

    Schizophrenia is a severe psychiatric disorder with lifetime prevalence between 0.5 and 1%. The disease is characterized by delusions, hallucinations, altered cognition, emotional reactivity and disorganized behavior. Research increasingly suggests that schizophrenia is a subtle disorder of brain...

  3. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

    2013-01-01

    Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome ...

  4. The zinc finger protein ZAT11 modulates paraquat-induced programmed cell death in Arabidopsis thaliana

    NARCIS (Netherlands)

    Qureshi, Muhammad Kamran; Sujeeth, Neerakkal; Gechev, Tsanko S.; Hille, Jacques; Liu, J.-H.

    2013-01-01

    Plants use programmed cell death (PCD) as a tool in their growth and development. PCD is also involved in defense against different kinds of stresses including pathogen attack. In both types of PCD, reactive oxygen species (ROS) play an important role. ROS is not only a toxic by-product but also act

  5. E(var)3-9 of Drosophila melanogaster Encodes a Zinc Finger Protein

    OpenAIRE

    Weiler, Karen S.

    2007-01-01

    The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (w) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)...

  6. Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice.

    Science.gov (United States)

    Davies, Benjamin; Hatton, Edouard; Altemose, Nicolas; Hussin, Julie G; Pratto, Florencia; Zhang, Gang; Hinch, Anjali Gupta; Moralli, Daniela; Biggs, Daniel; Diaz, Rebeca; Preece, Chris; Li, Ran; Bitoun, Emmanuelle; Brick, Kevin; Green, Catherine M; Camerini-Otero, R Daniel; Myers, Simon R; Donnelly, Peter

    2016-02-11

    The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

  7. The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action

    Science.gov (United States)

    Abbondanza, Ciro; Medici, Nicola; Nigro, Vincenzo; Rossi, Valentina; Gallo, Luigi; Piluso, Giulio; Belsito, Angela; Roscigno, Annarita; Bontempo, Paola; Puca, Annibale A.; Molinari, Anna Maria; Moncharmont, Bruno; Puca, Giovanni A.

    2000-01-01

    Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control. PMID:10706618

  8. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle

    Institute of Scientific and Technical Information of China (English)

    Shengli Yu; Junjie Luo; Zhiyuan Song; Fangrong Ding; Yunping Dai; Ning Li

    2011-01-01

    Dear Editor,Gene targeting is in widespread use as a gold standard for determining the function of genes in mice and human embryonic stem cells [1].However,the poor efficiency of this technology has hindered its application to domestic animals,for which embryonic stem cells are not available.Although gene-targeted large domestic animals have been produced successfully by combination of homologous recombination-based targeting strategy and cloning [2-4],the efficiency is very low and,more importantly,the disruption of the targeted gene is usually mono-allelic.It thus takes a long time to obtain a null mutant.

  9. Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

    Directory of Open Access Journals (Sweden)

    Nezaket Turkel

    2015-08-01

    Full Text Available The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib and overexpression of the BTB-ZF protein Abrupt (Ab. Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems.

  10. gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

    Science.gov (United States)

    Chevrier, Sandy; Boidot, Romain

    2014-01-01

    The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA. PMID:25350069

  11. Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

    Science.gov (United States)

    Krois, Alexander S.; Ferreon, Josephine C.; Martinez-Yamout, Maria A.; Wright, Peter E.

    2016-01-01

    An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2–p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1–p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification. PMID:26976603

  12. The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival

    Science.gov (United States)

    Wallis, Deeann; Hamblen, Melanie; Zhou, Yi; Venken, Koen J T.; Schumacher, Armin; Grimes, H. Leighton; Zoghbi, Huda Y.; Orkin, Stuart H.; Bellen, Hugo J.

    2003-01-01

    Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.

  13. Reactivity of Cys4 Zinc Finger Domains with Gold(III) Complexes : Insights into the Formation of "Gold Fingers"

    NARCIS (Netherlands)

    Jacques, Aurélie; Lebrun, Colette; Casini, Angela; Kieffer, Isabelle; Proux, Olivier; Latour, Jean-Marc; Sénèque, Olivier

    2015-01-01

    Gold(I) complexes such as auranofin or aurothiomalate have been used as therapeutic agents for the treatment of rheumatoid arthritis for several decades. Several gold(I) and gold(III) complexes have also shown in vitro anticancer properties against human cancer cell lines, including cell lines resis

  14. A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator

    OpenAIRE

    Bird, Amanda J.; Zhao, Hui; Luo, Huan; Jensen, Laran T.; Srinivasan, Chandra; Evans-Galea, Marguerite; Winge, Dennis R.; Eide, David J.

    2000-01-01

    The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change...

  15. Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Vakhrushev, Sergey Y; Kong, Yun;

    2012-01-01

    Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc...... to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific O-glycosylation inhibits proprotein...... activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme...

  16. The Zinc Finger Protein Mig1 Regulates Mitochondrial Function and Azole Drug Susceptibility in the Pathogenic Fungus Cryptococcus neoformans.

    Science.gov (United States)

    Caza, Mélissa; Hu, Guanggan; Price, Michael; Perfect, John R; Kronstad, James W

    2016-01-01

    The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions, and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 under low-iron conditions and Mig1 regulation of mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Consistently with these regulatory functions, a mig1Δ mutant had impaired growth on inhibitors of mitochondrial respiration and inducers of ROS. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole, thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. IMPORTANCE Fungal pathogens of humans are difficult to treat, and there is a pressing need to identify new targets for antifungal drugs and to obtain a detailed understanding of fungal proliferation in vertebrate hosts. In this study, we examined the roles of the regulatory proteins Mig1 and HapX in mitochondrial function and antifungal drug susceptibility in the fungus Cryptococcus neoformans. This pathogen is a particular threat to the large population of individuals infected with human immunodeficiency virus (HIV). Our analysis revealed regulatory interactions between Mig1 and HapX, and a role for Mig1 in mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Importantly, loss of Mig1 increased susceptibility to the antifungal drug fluconazole, which is commonly used to treat cryptococcal disease. These studies highlight an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. PMID:27303693

  17. Epigenetic reprogramming of endogenous genes for permanent modulation of gene expression : Targeted interventions by self-engineered zinc finger proteins

    NARCIS (Netherlands)

    Huisman, Christian

    2015-01-01

    De epigenetische componenten van een gen, waarvan DNA methylatie en histonmodificaties het belangrijkste zijn, hebben een belangrijke rol bij de regulatie van genexpressie. Deregulatie van de epigenetische informatie kan er toe leiden dat genen afwijkend tot expressie komen, wat vervolgens tot ziekt

  18. Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Daria A Gaykalova

    Full Text Available Head and Neck Squamous Cell Carcinoma (HNSCC is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas. Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

  19. ZIP4 Regulates Pancreatic Cancer Cell Growth by Activating IL-6/STAT3 Pathway via Zinc Finger Transcription Factor CREB

    Science.gov (United States)

    Zhang, Yuqing; Bharadwaj, Uddalak; Logsdon, Craig D.; Chen, Changyi; Yao, Qizhi; Li, Min

    2010-01-01

    Purpose Recent studies indicate a strong correlation of zinc transporter ZIP4 and pancreatic cancer progression; however, the underlying mechanisms are unclear. We have recently found that ZIP4 is overexpressed in pancreatic cancer. In this study, we investigated the signaling pathway through which ZIP4 regulates pancreatic cancer growth. Experimental Design The expression of cyclin D1, IL-6, and STAT3 in pancreatic cancer xenografts and cells were examined by real time PCR, Bio-Plex cytokine assay, and Western blot, respectively. The activity of CREB is examined by a promoter activity assay. Results Cyclin D1 was significantly increased in the ZIP4 overexpressing MIA PaCa-2 cells (MIA-ZIP4)-injected orthotopic xenografts and was downregulated in the ZIP4 silenced ASPC-1 (ASPC-shZIP4) group. The phosphorylation of signal transducer and activator of transcription 3 (STAT3), an upstream activator of cyclin D1, was increased in MIA-ZIP4 cells, and decreased in ASPC-shZIP4 cells. IL-6, a known upstream activator for STAT3, was also found to be significantly increased in the MIA-ZIP4 cells and xenografts, and decreased in the ASPC-shZIP4 group. Overexpression of ZIP4 led to a 75% increase of IL-6 promoter activity, and caused increased phosphorylation of cAMP response element binding protein (CREB). Conclusions Our study suggest that ZIP4 overexpression causes increased IL-6 transcription via CREB, which in turn activates STAT3, and leads to increased cyclin D1 expression, resulting in increased cell proliferation and tumor progression in pancreatic cancer. These results elucidated a novel pathway in ZIP4-mediated pancreatic cancer growth, and suggest new therapeutic targets including ZIP4, IL-6, and STAT3 in pancreatic cancer treatment. PMID:20160059

  20. DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

    DEFF Research Database (Denmark)

    Danielsen, Jannie Michaela Rendtlew; Povlsen, Lou Klitgaard; Villumsen, Bine Hare;

    2012-01-01

    Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMO...

  1. Antagonistic control of oxidative stress-induced cell death in Arabidopsis by two related, plant-specific zinc finger proteins

    OpenAIRE

    Epple, Petra; Mack, Amanda A.; Morris, Veronica R. F.; Dangl, Jeffery L.

    2003-01-01

    The most familiar form of plant programmed cell death is the hypersensitive response (HR) associated with successful plant immune responses. HR is preceded by an oxidative burst and the generation of both reactive oxygen intermediates (ROI) and NO. The Arabidopsis LSD1 gene encodes a negative regulator of plant programmed cell death that meets several criteria for a regulator of processes relevant to ROI management during pathogen responses. Here we demonstrate that a highly conserved L...

  2. Convergent evidence from mouse and human studies suggests the involvement of zinc finger protein 326 gene in antidepressant treatment response.

    Directory of Open Access Journals (Sweden)

    Ying-Jay Liou

    Full Text Available OBJECTIVES: The forced swim test (FST is a commonly used model to predict antidepressant efficacy. Uncovering the genetic basis of the model may unravel the mechanism of antidepressant treatment. METHODS: FVB/NJ (FVB and C57BL/6J (B6 were first identified as the response and non-response strains to fluoxetine (a serotonin-specific reuptake inhibitor antidepressant treatment in the mouse FST. Simple-interval (SIM and composite-interval (CIM mappings were applied to map the quantitative trait loci (QTLs of the anti-immobility effect of fluoxetine in FST (FST(FLX in 865 male B6×FVB-F2 mice. The brain mRNA expressions of the gene with the maximum QTL-linkage signal for FST(FLX after the FST were compared between B6 and FVB mice and also compared between fluoxetine and saline treatment. The association of the variants in the human homologue of the mouse FST(FLX-QTL gene with major depressive disorder (MDD and antidepressant response were investigated in 1080 human subjects (MDD/control = 582/498. RESULTS: One linkage signal for FST(FLX-QTL was detected at an intronic SNP (rs6215396 of the mouse Zfp326 gene (maximal CIM-LOD = 9.36. The Zfp326 mRNA expression in the FVB thalamus was significantly down-regulated by fluoxetine in the FST, and the higher FVB-to-B6 Zfp326 mRNA expressions in the frontal cortex, striatum and hypothalamus diminished after fluoxetine treatment. Two coding-synonymous SNPs (rs2816881 and rs10922744 in the human homologue of Zfp326, ZNF326, were significantly associated with the 8-week antidepressant treatment response in the MDD patients (Bonferroni-corrected p = 0.004-0.028. CONCLUSIONS: The findings suggest the involvement of the Zfp326 and ZNF326 genes in antidepressant treatment response.

  3. The Zinc Finger Protein Mig1 Regulates Mitochondrial Function and Azole Drug Susceptibility in the Pathogenic Fungus Cryptococcus neoformans

    OpenAIRE

    Caza, Mélissa; Hu, Guanggan; Price, Michael; Perfect, John R.; Kronstad, James W.

    2016-01-01

    ABSTRACT The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions, and Hap4 is subject to glucose repression via ...

  4. The zinc finger SET domain gene Prdm14 is overexpressed in lymphoblastic lymphomas with retroviral insertions at Evi32.

    Directory of Open Access Journals (Sweden)

    E J Dettman

    Full Text Available BACKGROUND: AKXD recombinant inbred strains of mice have proven to be very useful in the identification of potential oncogenes and tumor suppressors involved in the development of lymphoid and myeloid malignancies. In these tumors, the hematopoietic insertion of an active AKV murine leukemia virus (MuLV is associated with the onset of disease. Common sites of retroviral insertion (CIS identify genes causally associated with the development or initiation of lymphoma. METHODOLOGY: In the present study, we analyzed a previously uncharacterized CIS, Ecotropic Viral Integration Site 32 (Evi32, which is located on mouse chromosome 1. We analyzed candidate genes in the region to identify those involved in Evi32 mediated oncogenesis. RESULTS: Here we show that proviral insertion at Evi32 correlates with significant overexpression of a putative transcription factor, PR-domain containing 14 (Prdm14. Tumors with insertions at Evi32 are consistently lymphoid in nature. Prdm14 is normally expressed early in embryonic development with the highest expression in undifferentiated embryonic stem (ES cells. This study implicates Prdm14 as a proto-oncogene involved in lymphoblastic lymphoma formation.

  5. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

    Science.gov (United States)

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-07-15

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  6. Biological functions of zinc finger protein ZBTB2: recent advance%锌指蛋白ZBTB20生物学功能的研究进展

    Institute of Scientific and Technical Information of China (English)

    刘淦; 陈李斌佶; 谢志芳; 章卫平

    2015-01-01

    锌指蛋白ZBTB20是ZBTB锌指蛋白亚家族的新成员,与已知的BCL6和PLZF同源.通过基因打靶小鼠和疾病人群研究,学者们发现ZBTB20在肝脏、胰岛、神经系统和免疫系统等多个器官与系统中具有重要生理学功能,是个体发育、学习记忆、痛觉感受、糖脂代谢等正常生理过程所必需的调节分子.ZBTB20表达或功能异常可导致个体发育障碍,与智障、肿瘤和代谢性疾病等多种重大疾病的病理生理过程密切相关.作为甲胎蛋白基因的主要转录抑制因子,ZBTB20在出生后肝脏甲胎蛋白基因的表达失活中发挥了关键性作用,且与肝癌预后密切相关.人群中ZBTB20基因的缺失与多种肿瘤有关,其点突变可导致Primrose综合征.本文就ZBTB20的生物学功能研究进展作一综述.

  7. Aspergillus parasiticus CrzA, Which Encodes a Calcineurin Response Zinc-Finger Protein, is Required for Aflatoxin Production Under Calcium Stress

    Science.gov (United States)

    Calcium has been reported to be required for aflatoxin production. Calcium, like cAMP, is a second messenger. Cacineurin, a calmodulin-dependent serine/threonine protein phosphatase, is an important component of the calcium signaling pathway. The control of calcineurin-dependent gene expression is v...

  8. A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1

    DEFF Research Database (Denmark)

    Negishi, Masamitsu; Saraya, Atsunori; Mochizuki, Shinobu;

    2010-01-01

    BACKGROUND: Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood. METHODOL...... is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways....... direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated...

  9. The Coronary Artery Disease-associated Coding Variant in Zinc Finger C3HC-type Containing 1 (ZC3HC1) Affects Cell Cycle Regulation.

    Science.gov (United States)

    Jones, Peter D; Kaiser, Michael A; Ghaderi Najafabadi, Maryam; McVey, David G; Beveridge, Allan J; Schofield, Christine L; Samani, Nilesh J; Webb, Tom R

    2016-07-29

    Genome-wide association studies have to date identified multiple coronary artery disease (CAD)-associated loci; however, for most of these loci the mechanism by which they affect CAD risk is unclear. The CAD-associated locus 7q32.2 is unusual in that the lead variant, rs11556924, is not in strong linkage disequilibrium with any other variant and introduces a coding change in ZC3HC1, which encodes NIPA. In this study, we show that rs11556924 polymorphism is associated with lower regulatory phosphorylation of NIPA in the risk variant, resulting in NIPA with higher activity. Using a genome-editing approach we show that this causes an effective decrease in cyclin-B1 stability in the nucleus, thereby slowing its nuclear accumulation. By perturbing the rate of nuclear cyclin-B1 accumulation, rs11556924 alters the regulation of mitotic progression resulting in an extended mitosis. This study shows that the CAD-associated coding polymorphism in ZC3HC1 alters the dynamics of cell-cycle regulation by NIPA. PMID:27226629

  10. Transcriptional Regulation of the AP-2α Promoter by BTEB-1 and AP-2rep, a Novel wt-1/egr-Related Zinc Finger Repressor

    OpenAIRE

    Imhof, Axel; Schuierer, Marion; Werner, Oliver; Moser, Markus; Roth, Christina; Bauer, Reinhard; BUETTNER, REINHARD

    1999-01-01

    AP-2 transcription factors have been suggested to exert key regulatory functions in vertebrate embryonic development, in tumorigenicity of various cancer cell types, and in controlling cell cycle and apoptotic effector genes. In this study, we investigated transcriptional regulation of the AP-2α gene promoter mediated by an autoregulatory element (referred to as A32) with a core consensus AP-2 binding site at position −336 relative to the mRNA initiation site. AP-2 and multiple different nucl...

  11. Zinc-finger transcription factors are associated with guanine quadruplex motifs in human, chimpanzee, mouse and rat promoters genome-wide

    OpenAIRE

    Yadav, V. K.; Baral, A.; Kumar, P.; D. Saha; Chowdhury, S.

    2011-01-01

    Function of non-B DNA structures are poorly understood though several bioinformatics studies predict role of the G-quadruplex DNA structure in transcription. Earlier, using transcriptome profiling we found evidence of widespread G-quadruplex-mediated gene regulation. Herein, we asked whether potential G-quadruplex (PG4) motifs associate with transcription factors (TF). This was analyzed using 220 position weight matrices [designated as transcription factor binding sites (TFBS)], representing ...

  12. The zinc finger protein PtaZFP2 negatively controls stem growth and gene expression responsiveness to external mechanical loads in poplar.

    Science.gov (United States)

    Martin, Ludovic; Decourteix, Mélanie; Badel, Eric; Huguet, Stéphanie; Moulia, Bruno; Julien, Jean-Louis; Leblanc-Fournier, Nathalie

    2014-07-01

    Mechanical cues are essential signals regulating plant growth and development. In response to wind, trees develop a thigmomorphogenetic response characterized by a reduction in longitudinal growth, an increase in diameter growth, and changes in mechanical properties. The molecular mechanisms behind these processes are poorly understood. In poplar, PtaZFP2, a C2H2 transcription factor, is rapidly up-regulated after stem bending. To investigate the function of PtaZFP2, we analyzed PtaZFP2-overexpressing poplars (Populus tremula × Populus alba). To unravel the genes downstream PtaZFP2, a transcriptomic analysis was performed. PtaZFP2-overexpressing poplars showed longitudinal and cambial growth reductions together with an increase in the tangent and hardening plastic moduli. The regulation level of mechanoresponsive genes was much weaker after stem bending in PtaZFP2-overexpressing poplars than in wild-type plants, showing that PtaZFP2 negatively modulates plant responsiveness to mechanical stimulation. Microarray analysis revealed a high proportion of down-regulated genes in PtaZFP2-overexpressing poplars. Among these genes, several were also shown to be regulated by mechanical stimulation. Our results confirmed the important role of PtaZFP2 during plant acclimation to mechanical load, in particular through a negative control of plant molecular responsiveness. This desensitization process could modulate the amplitude and duration of the plant response during recurrent stimuli.

  13. A stage-specific open reading frame from three-day old adult worms of Trichinella spiralis encodes zinc-finger motifs

    Directory of Open Access Journals (Sweden)

    Zhu X.P.

    2005-06-01

    Full Text Available The aim of the study was to isolate genes coding for stage-specific antigens of T. spiralis. Such antigens may then be associated with local and systemic immune responses against adult T. spiralis. Recombinant clones were obtained with an adult stage specific probe from a cDNA library of three-day old adult T. spiralis. Several cDNA clones encoding the same peptide were identified and their stage specificity was confirmed by northern blot analysis. Three independent clones were fully sequenced, and the resulting sequence found to code for a 487 amino acid peptide with a deduced molecular weight of ≈ 55 kDa. Sequence analysis showed that the 55 kDa peptide contained putative DNA binding motifs, suggesting that this protein may be involved in transcriptional regulation during the early development of the parasite.

  14. Synthesis and characterization of mixed ligand complexes of Zn(II) and Co(II) with amino acids: Relevance to zinc binding sites in zinc fingers

    Indian Academy of Sciences (India)

    P Rabindra Reddy; M Radhika; P Manjula

    2005-05-01

    Mixed ligand complexes of Zn(II) and Co(II) with cysteine, histidine, cysteinemethylester, and histidinemethylester have been synthesized and characterized by elemental analysis, conductivity, magnetic susceptibility measurements, and infrared, 1H NMR, TGA and FAB mass spectra. In these complexes, histidine, and histidinemethylester act as bidentate ligands involving amino and imidazole nitrogens in metal coordination. Similarly, cysteine, and cysteinemethylester also act as bidentate ligands coordinating through thiol sulphur and amino nitrogen. Tetrahedral geometry has been proposed for Zn(II) and Co(II) complexes based on experimental evidence.

  15. DNA methylation patterns in human tissues of uniparental origin using a zinc-Finger gene (ZNF127) from the Angelman/Prader-Willi region

    Energy Technology Data Exchange (ETDEWEB)

    Mowery-Rushton, P.A.; Surti, U.; Locker, J. [Univ. of Pittsburgh, PA (United States)] [and others

    1996-01-11

    In order to further our understanding of the epigenetic modification of DNA and its role in imprinting, we examined DNA methylation patterns of human tissues of uniparental origin. We used complete hydatidiform moles (CHM), which are totally androgenetic conceptions, to examine the paternal methylation pattern in the absence of a maternal contribution and we used ovarian teratomas to represent the maternal counterpart. We carried out an analysis of DNA methylation of a gene which has been shown to contain sites which are differentially methylated in a parent-specific fashion. The gene, ZNF127, is located on chromosome 15q11-q13 in the region associated with Prader-Willi and Angelman syndromes. The parent-of-origin DNA methylation has been postulated to reflect the presence of an imprint and recent studies have confirmed that ZNF127 is differentially expressed only from the paternal chromosome. We identified a unique pattern of hyper- and hypomethylated sites in androgenetic conceptions which was nearly identical to the paternal pattern found in sperm. This may represent the paternal germ-line methylation imprint. We also studied partial hydatidiform moles, non-molar triploid conceptions, normal chorionic villi, and somatic tissue. These all demonstrated a modified DNA methylation pattern characteristic of normal chorionic villi with only limited findings of the imprint. Our results suggest that human androgenetic conceptions may provide an excellent model to analyze epigenetic DNA modifications, such as methylation, in imprinted genes. The paternal allele-specific methylation imprint will also be useful clinically to confirm the androgenetic nature of suspected molar conceptions in which parental blood samples may not be available. 55 refs., 3 figs.

  16. Advances in molecular genetic systems in malaria.

    Science.gov (United States)

    de Koning-Ward, Tania F; Gilson, Paul R; Crabb, Brendan S

    2015-06-01

    Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

  17. Evaluation of the protection aganst Pelargonium containing a chimaeric cecropin gene

    NARCIS (Netherlands)

    Renou, J.P.; Mary, I.; Hanteville, S.; Narcy, J.P.; Florack, D.; Cadic, A.

    2000-01-01

    The phytopathogenic bacterium Xanthomonas campestris pv. pelargonii causes severe losses in vegetatively propagated Pelargonium cultivars. Symptoms are characterized by wilting of the plant, localized water-soaked lesions and stem rot (1) To overcome this problem horticulturists have to produce Xant

  18. The zinc-finger transcription factor SALL4 is frequently expressed in human cancers: association with clinical outcome in squamous cell carcinoma but not in adenocarcinoma of the esophagus.

    Science.gov (United States)

    Kilic, Ergin; Tennstedt, Pierre; Högner, Anica; Lebok, Patrick; Sauter, Guido; Bokemeyer, Carsten; Izbicki, Jakob R; Wilczak, Waldemar

    2016-04-01

    SALL4 is a transcription factor originally identified as a homeotic gene essential for organ development. Early studies suggested that SALL4 is a useful marker to identify testicular and ovarian germ cell tumors. The aim of the study was to evaluate the diagnostic potential of SALL4 immunohistochemistry. Immunohistochemical staining was performed on a tissue microarray (TMA) with 3966 samples from 94 different tumor types and on a further TMA with 492 esophagus carcinomas. SALL4 immunostaining was by far most prevalent and most intensive in testicular tumors with a positivity rate of 93.1 % in seminomas, 80 % in mixed germ cell tumors (embryonic carcinomas/yolk sac tumors), and 18.5 % in teratomas, respectively. However, SALL4 expression is not specific to germ cell tumors. We observed SALL4 positivity in non-germ cell tumors as carcinomas of the kidney (28.9 % of chromophobe, 34.4 % of clear cell carcinoma), in intestinal type adenocarcinoma of the stomach (10.9 %), in adenocarcinoma (10.5 %) and squamous cell carcinoma (7.2 %) of the esophagus, and in malignant melanoma (8.1 %) and invasive urothelial bladder carcinoma (20 %). SALL4 expression was not found in lymphomas, in soft tissue tumors or breast tumors. At analysis of esophagus carcinoma TMA, no significant association was seen between SALL4 expression and overall survival in adenocarcinoma. However, SALL4 expression was strongly associated with worse overall survival in squamous cell carcinoma. SALL4 expression can be found at relevant frequencies in various tumors of different primary sites. SALL4 expression in squamous cell carcinoma of the esophagus may constitute a sign of dedifferentiation leading to poor patient prognosis. PMID:26818834

  19. The κB transcriptional enhancer motif and signal sequences of V(DJ recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study

    Directory of Open Access Journals (Sweden)

    Wu Lai-Chu

    2002-08-01

    Full Text Available Abstract Background The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N and the carboxyl-DNA binding domain (ZAS-C of a representative family member, named κB DNA binding and recognition component (KRC, were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains. Results Both fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(DJ recombination signal sequences (RSS from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS. Conclusions The RSS are cis-acting DNA motifs which are essential for V(DJ recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(DJ recombination.

  20. Angiotensinogen gene-inducible enhancer-binding protein 1, a member of a new family of large nuclear proteins that recognize nuclear factor kappa B-binding sites through a zinc finger motif.

    OpenAIRE

    Ron, D; Brasier, A R; Habener, J F

    1991-01-01

    Transcriptional activation of the rat angiotensinogen gene during the acute-phase response is dependent on a previously characterized acute-phase response element (APRE) that binds at least two types of nuclear proteins: a cytokine-inducible activity indistinguishable from nuclear factor kappa-B (NF kappa B) and a family of C/EBP-like proteins. We screened a rat liver cDNA expression library with a labeled APRE DNA probe and isolated a single clone that encodes a sequence-specific APRE-bindin...

  1. Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: Possible reaction of cadmium with the zinc finger domain of Sp1

    OpenAIRE

    Kothinti, Rajendra K.; Blodgett, Amy B.; Petering, David H.; Tabatabai, Niloofar M.

    2010-01-01

    Cadmium (Cd) exposure causes glucosuria (glucose in the urine). Previously, it was shown that Cd exposure of primary cultures of mouse kidney cells (PMKC) decreased mRNA levels of the glucose transporters, SGLT1 and SGLT2 and that Sp1 from Cd-exposed cells displayed reduced binding to the GC boxes of the mouse SGLT1 promoter in vitro. Here, we identified a GC box upstream of mouse SGLT2 gene. ChIP assays on PMKC revealed that exposure to 5 μM Cd abolished Sp1 binding to SGLT1 GC box while it ...

  2. Protein (Viridiplantae): 255079374 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available steine-rich protein with zinc finger Micromonas sp. RCC299 MMDARPVNVGLGSAPAQRGVKRQRDWSDKPTPKSGFCEHGRRRSRCKECGGSAICEHGRQRAQCKECG...GSAICEHGRQRSHCKECGGASICEHGRRRSQCKECGGSQICEHGRHRSQCKECGGSQICEHGRRRSVCKECGGSEICEHGRQRAQCKECGGASICEHGRVRSRCKECG...GASVCEHGRQRRYCKECGGASICEHGRQRAQCKQCGGSAICEHGRQRSHCKECGGASVCEHGRRRSQCKECG

  3. Protein (Viridiplantae): 255074333 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available steine-rich protein with zinc finger Micromonas sp. RCC299 MEGSESEEEEKHATKKGVKRKRAPPTKGPCEHGVKYRSNCKVCSACPHGKRRCHCKECG...GASICEHGRIRSTCKECGGATICEHGRRRSQCKECGGSQICEHGRHRPQCKECGGSQICEHGRRRTECKECGGGSICEHDRVRSQCKECGGSQICEHGRQRSKCKECGGSQICEHGRRRTECKECGGGSICEHDRVRSQCKECGGGSICEHDRVRSKCKECRAAKARTG ...

  4. Protein (Viridiplantae): 255086954 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available steine-rich protein with zinc finger Micromonas sp. RCC299 MPAIWRPNVRIELSDVDVEVKATEEEVTKKGTKRKRAPPTKGPCQHGVKYRSNCKVCSACPHGRQRHRCKECG...GSQICEHGRERSYCKECGGSQICEHGRQRSRCKDCSGASICEHGRQRRYCKECGGSAFCEHGRIRSTCKECGGASICEHGRHRIQCKECGGSQICEHGRRRSQCKECG...GSQICEHGRIRSTCKECGGSQICEHGRHRQYCKECGGSAFCEHGRIRSTCKECDGSQICEHGRIHSLCKECGGSGICEHGRQRNQCKECGGSGICEHGRQRYSCKECGGSGICEHGRRRYRCKECRAAKHI ...

  5. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: Signaling proteins AZF1 OZAKGYO, ZF1 At5g67450, Cys2/His2-type zinc finger protein... 1, Zinc finger protein OZAKGYO, Zinc-finger protein 1 3702 Arabidopsis thaliana 836881 Q9SSW1 21852415 ...

  6. Whole genome characterisation and engineering of chimaeric rotavirus-like particles using African rotavirus field strains / Khuzwayo Chidiwa Jere

    OpenAIRE

    Jere, Khuzwayo Chidiwa

    2012-01-01

    Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. N...

  7. Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

    Institute of Scientific and Technical Information of China (English)

    LI Dong-dong; SHI Wei; DENG Xiu-xin

    2003-01-01

    Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cultivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg L-1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

  8. Conformational toggling controls target site choice for the heteromeric transposase element Tn7.

    Science.gov (United States)

    Shi, Qiaojuan; Straus, Marco R; Caron, Jeremy J; Wang, Huasheng; Chung, Yu Seon; Guarné, Alba; Peters, Joseph E

    2015-12-15

    The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified. PMID:26384427

  9. A genome-wide transcriptional analysis of producer and non-producer NS0 myeloma cell lines.

    Science.gov (United States)

    Khoo, Soo Hean Gary; Falciani, Francesco; Al-Rubeai, Mohamed

    2007-06-01

    'Genome-wide' or 'global' gene expression profiling provides a powerful approach to the characterization of a cell's transcriptional state. Such technology has been used in animal cell culture to create genome-wide snapshots of transcriptional activity in response to environmental factors or cellular triggers under bioprocessing conditions. Furthermore, it allows us to have a fundamental understanding of genetic mechanisms involved in recombinant protein production. One such mechanism adversely affecting the growth of recombinant bacteria is the increased metabolic burden resulting from the maintenance of plasmid copy number and heterologous protein expression. There have also been some reports on the effect of metabolic burden in mammalian cell systems. In the present study, we have used a mouse array representing 6400 genes to assess the expression profile of a WT (wild-type) mouse plasmacytoma cell line, NS0 WT, and a GS (glutamine synthetase)-NS0 6A1-100 cell line expressing chimaeric monoclonal antibody. The producer cells did not exhibit a slower growth as the result of any metabolic burden, but showed differences in metabolic activity. Gene expression profiling revealed that the producer cell line was selected for a higher expression of chromosomal genes, genes for zinc-finger proteins as well as cell-cycle-related events. On the other hand, protein synthesis is greater and ribosomal genes were more expressed in the WT cells. A possible shift from expressing antigen presenting proteins to recombinant protein could also be seen. Hence, gene expression profiling suggests that the effect of the metabolic burden in slowing growth can be mostly negated in producer cell lines by careful clonal selection, where stable transfected cells are selected for both high productivity as well as high growth rates. PMID:17223793

  10. AcEST: DK952752 [AcEST

    Lifescience Database Archive (English)

    Full Text Available HLLAVYLSQVMRTCQQLSVSDKQLTDILL 133 >sp|Q9CWQ2|ZSWM7_MOUSE Zinc finger SWIM domain-containing protein 7 OS=Mus musculus GN=Zswim...DSLLCKHLLAIYLSQLLRNCQQLHVSDKQLTDLLME 134 >sp|A4FVI0|ZSWM7_DANRE Zinc finger SWIM domain-containing protein 7 OS=Danio rerio GN=zswim...AMGLCQQEQVSDQQM 128 >sp|Q55FI7|ZSWM7_DICDI Zinc finger SWIM domain-containing protein 7 homolog OS=Dictyostelium discoideum GN=zswim

  11. Clinical response, pharmacokinetics, development of human anti-chimaeric antibodies, and synovial tissue response to rituximab treatment in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    R.M. Thurlings; O. Teng; K. Vos; D.M. Gerlag; L. Aarden; S.O. Stapel; J.M. van Laar; P.P. Tak; G.J. Wolbink

    2010-01-01

    Objectives: To analyse whether persistence of synovial B lineage cells and lack of clinical response to rituximab treatment in patients with rheumatoid arthritis (RA) are associated with low rituximab serum levels and anti-rituximab antibody (ARA) formation. Methods: Fifty-eight patients with RA wer

  12. Chimaeric anti-CD20 monoclonal antibody (rituximab) in post-transplant B-lymphoproliferative disorder following stem cell transplantation in children.

    Science.gov (United States)

    Faye, A; Quartier, P; Reguerre, Y; Lutz, P; Carret, A S; Dehée, A; Rohrlich, P; Peuchmaur, M; Matthieu-Boué, A; Fischer, A; Vilmer, E

    2001-10-01

    Post-transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8-22% of patients with high-risk factors. We retrospectively investigated tolerance and efficacy of humanized anti-CD20 monoclonal antibody (rituximab) as first-line treatment in 12 children with B-cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein-Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/m2 once a week by intravenous infusion (1-9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non-responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in non-responders. Rituximab was an effective and well-tolerated treatment of B-cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre-emptive treatment should be considered and evaluated in further longitudinal multicentre studies.

  13. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At1g80790.1 68414.m09479 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-122 ...

  14. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At3g48670.2 68416.m05314 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-118 ...

  15. Arabidopsis CDS blastp result: AK059669 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059669 001-031-F06 At4g00380.1 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 7e-49 ...

  16. Arabidopsis CDS blastp result: AK069077 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069077 J023007G13 At3g48670.2 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-139 ...

  17. Arabidopsis CDS blastp result: AK063522 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063522 001-116-H09 At1g15910.1 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-118 ...

  18. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At3g48670.1 68416.m05313 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-118 ...

  19. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At1g15910.1 68414.m01908 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-128 ...

  20. Arabidopsis CDS blastp result: AK064511 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064511 002-111-E08 At1g15910.1 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 4e-31 ...

  1. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At1g13790.1 68414.m01619 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-95 ...

  2. Arabidopsis CDS blastp result: AK065750 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065750 J013039A03 At1g15910.1 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 7e-84 ...

  3. Arabidopsis CDS blastp result: AK242745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242745 J090050D05 At3g12550.1 68416.m01562 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 1e-100 ...

  4. Arabidopsis CDS blastp result: AK107535 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107535 002-129-H03 At1g80790.1 XH/XS domain-containing protein / XS zinc finger domain...-containing protein contains Pfam domains PF03469: XH domain, PF03468: XS domain and PF03470: XS zinc finger domain 2e-32 ...

  5. Arabidopsis CDS blastp result: AK241364 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241364 J065152E11 At3g61850.1 68416.m06945 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  6. Arabidopsis CDS blastp result: AK287447 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287447 J043016O04 At3g61850.2 68416.m06946 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  7. Arabidopsis CDS blastp result: AK288349 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288349 J090023P19 At3g61850.2 68416.m06946 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  8. Arabidopsis CDS blastp result: AK241364 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241364 J065152E11 At3g61850.2 68416.m06946 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  9. Arabidopsis CDS blastp result: AK288349 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288349 J090023P19 At3g61850.1 68416.m06945 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  10. Arabidopsis CDS blastp result: AK287447 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287447 J043016O04 At3g61850.1 68416.m06945 Dof zinc finger protein DAG1 / Dof affecting... germination 1 (DAG1) / transcription factor BBFa (BBFA) identical to SP|Q43385 DOF zinc finger protein DAG1 (Dof affecting

  11. Arabidopsis CDS blastp result: AK288349 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288349 J090023P19 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affecting... germination 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 1e-23 ...

  12. Gclust Server: 5714 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available TED: similar to zinc finger and BTB domain containing 8 opposite strand ; no annotation 19 1.00e-14 42.86 22...resentative annotation XP_001133395.1 PREDICTED: similar to zinc finger and BTB domain containing 8 opposite

  13. Arabidopsis CDS blastp result: AK287566 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287566 J065027L04 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 2e-77 ...

  14. Arabidopsis CDS blastp result: AK289209 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK289209 J100058I16 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 1e-12 ...

  15. Arabidopsis CDS blastp result: AK243285 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243285 J100051N01 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 1e-24 ...

  16. NCBI nr-aa BLAST: CBRC-BTAU-01-1535 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1535 ref|NP_001074390.1| IKAROS family zinc finger 2 (Helios) [Bos tau...rus] gb|AAI26591.1| IKAROS family zinc finger 2 (Helios) [Bos taurus] NP_001074390.1 2e-06 72% ...

  17. Arabidopsis CDS blastp result: AK241364 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241364 J065152E11 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affecting germination... 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 2e-20 ...

  18. Arabidopsis CDS blastp result: AK287447 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287447 J043016O04 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affecting germination... 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 2e-30 ...

  19. SwissProt search result: AK104521 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104521 006-303-E08 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-11 ...

  20. SwissProt search result: AK067216 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067216 J013098P16 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-23 ...

  1. SwissProt search result: AK103046 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103046 J033117G06 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-24 ...

  2. SwissProt search result: AK105286 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105286 001-116-E08 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-11 ...

  3. SwissProt search result: AK069995 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069995 J023037B09 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 6e-20 ...

  4. SwissProt search result: AK072007 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072007 J013098F24 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 6e-11 ...

  5. SwissProt search result: AK068989 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068989 J023002F22 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-27 ...

  6. SwissProt search result: AK072236 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072236 J023003N03 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-20 ...

  7. SwissProt search result: AK119840 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119840 002-178-C07 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-20 ...

  8. SwissProt search result: AK067295 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067295 J013106J21 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-29 ...

  9. SwissProt search result: AK069264 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069264 J023011B03 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-13 ...

  10. SwissProt search result: AK104353 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104353 001-034-G12 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-18 ...

  11. SwissProt search result: AK066313 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066313 J013065E23 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-14 ...

  12. SwissProt search result: AK073653 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073653 J033048F17 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 1e-20 ...

  13. SwissProt search result: AK066352 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066352 J013063O03 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-14 ...

  14. SwissProt search result: AK101919 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101919 J033071J10 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 9e-11 ...

  15. SwissProt search result: AK121806 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121806 J033097L10 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-23 ...

  16. SwissProt search result: AK072584 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072584 J023132A01 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-12 ...

  17. SwissProt search result: AK071527 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071527 J023099O15 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-25 ...

  18. SwissProt search result: AK060894 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060894 001-035-C07 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-11 ...

  19. SwissProt search result: AK119444 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119444 001-133-C11 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-17 ...

  20. SwissProt search result: AK072630 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072630 J023130P03 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-11 ...

  1. SwissProt search result: AK101292 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101292 J033033G18 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 9e-11 ...

  2. SwissProt search result: AK060188 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060188 001-001-D10 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 5e-27 ...

  3. SwissProt search result: AK100156 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100156 J023019I03 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 8e-23 ...

  4. SwissProt search result: AK106099 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106099 001-207-C04 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 4e-18 ...

  5. SwissProt search result: AK121573 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121573 J033036A12 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 8e-12 ...

  6. SwissProt search result: AK119393 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119393 001-132-D02 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-15 ...

  7. SwissProt search result: AK064076 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064076 002-100-A06 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduce...d expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 2e-19 ...

  8. SwissProt search result: AK071686 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071686 J023109M13 (Q9UIJ5) Zinc finger DHHC domain containing protein 2 (Zinc finger protein 372) (Reduced... expression associated with metastasis protein) (Ream) (Reduced expression in cancer protein) (Rec) ZDHC2_HUMAN 3e-22 ...

  9. InterProScan Result: FS919613 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS919613 FS919613_2_ORF2 C3A4180337AD2E2D PIR PIRSF037956 Uncharacterised protein w...ith zinc finger Ran-binding domain, ZRANB2 type 4.6e-92 T IPR017337 Uncharacterised conserved protein UCP037956, zinc finger Ran-binding ...

  10. The Periodontal Pathogen Porphyromonas gingivalis Induces Expression of Transposases and Cell Death of Streptococcus mitis in a Biofilm Model

    OpenAIRE

    Duran-Pinedo, Ana E.; Baker, Vinesha D.; Frias-Lopez, Jorge

    2014-01-01

    Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most ...

  11. Sequence Classification: 776671 [

    Lifescience Database Archive (English)

    Full Text Available ar zinc finger protein, Mog interacting, Ectopic P granules MEP-1, GEX (Gut on EXterior) Interacting protein GEI-2 (97.4 kD) (mep-1) || http://www.ncbi.nlm.nih.gov/protein/25152712 ...

  12. Experiment list: SRX100559 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ortant regulator of T-cell development and plays an important role in endothelial c...o the GATA family of transcription factors. The protein contains two GATA-type zinc fingers and is a n imp

  13. Experiment list: SRX186638 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available targetdescription=Transcriptional regulator with 11 highly conserved zinc finger domains. Depending on con...text, can bind a histone acetyltransferase (HAT)-containing complex and function as

  14. Experiment list: SRX186611 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available y Target: CTCF || antibody targetdescription=Transcriptional regulator with 11 highly conserved zinc finger domains. Depending on con...text, can bind a histone acetyltransferase (HAT)-containing complex and function as

  15. Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs

    DEFF Research Database (Denmark)

    Rowe, R.G.; Li, X.Y.; Hu, Y.;

    2009-01-01

    Epithelial-mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whet...

  16. Overexpression of VOZ2 confers biotic stress tolerance but decreases abiotic stress resistance in Arabidopsis

    OpenAIRE

    Nakai, Yusuke; Fujiwara, Sumire; Kubo, Yasuyuki; Sato, Masa H.

    2013-01-01

    VOZ (vascular plant one zinc-finger protein) is a plant specific one-zinc finger type transcriptional activator, which is highly conserved through land plant evolution. We have previously shown that loss-of-function mutations in VOZ1 and VOZ2 showed increased cold and drought stress tolerances whereas decreased biotic stress resistance in Arabidopsis. Here, we demonstrate that transgenic plants overexpressing VOZ2 impairs freezing and drought stress tolerances but increases resistance to a fu...

  17. Role of LRF/Pokemon in lineage fate decisions

    OpenAIRE

    Lunardi, Andrea; Guarnerio, Jlenia; Wang, Guocan; Maeda, Takahiro; Pandolfi, Pier Paolo

    2013-01-01

    In the human genome, 43 different genes are found that encode proteins belonging to the family of the POK (poxvirus and zinc finger and Krüppel)/ZBTB (zinc finger and broad complex, tramtrack, and bric à brac) factors. Generally considered transcriptional repressors, several of these genes play fundamental roles in cell lineage fate decision in various tissues, programming specific tasks throughout the life of the organism. Here, we focus on functions of leukemia/lymphoma-related factor/POK e...

  18. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  19. Methods for Identification and Characterization of Protein Unexpectedly Expressed in Escherichia Coli:A Case Study Involvingβ-Lactamase Observed during the Expression of Zinc Finger 2-8 of NRSF/REST%原核表达过程中非目标蛋白质识别与确认的方法:NRSF/REST蛋白功能结构域ZnF2-8原核表达过程中β-内酰胺酶的确认

    Institute of Scientific and Technical Information of China (English)

    张岩; 赵玢; 杨中正; 申杰; 胡伟; 蓝文贤; 吴厚铭; 曹春阳

    2015-01-01

    Escherichia coli (E. coli) is often used to produce recombinant proteins rapidly with high yield. However, the bacteria expresses non-target proteins unexpectedly in many cases, some of which may later be proven useful. Full characterization of these unpredicted proteins are usually expensive and time-consuming. In this study, we used E. coli to express neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) functional motif ZnF2-8, which is involved in the interaction of NRSF/REST with neuron-restrictive silencer element (NRSE/RE1) dsDNA or small non-coding dsRNA for neuron gene transcriptional repression or activation. Overexpression of a non-target protein was observed. Two-dimensional 1H-15N HSQC NMR spectroscopy, X-ray crystallography and other biochemical assays were used in combination to characterize the non-target protein to beb-lactamase.%大肠杆菌常被用来大量快速制备重组蛋白质。但是,在原核表达目标蛋白质时非目标蛋白质经常会意外表达。有时这些非目标蛋白质也非常有使用价值,但是最终确认这些非目标蛋白质的过程昂贵又及其耗时。基于此,该文发展了一个新的基于二维核磁共振波谱技术、X-单晶衍射技术、结合其他生物化学方法,确认在原核表达神经限制性沉默因子 NRSF/REST 蛋白(该蛋白能够特异性识别神经限制性沉默因子 RE1 dsDNA及神经限制性激活因子dsRNA,以调节神经元干细胞的发育)功能结构域ZnF2-8时非目标蛋白b-内酰胺酶(b-lactamase)。

  20. Accelerated evolution of the Prdm9 speciation gene across diverse metazoan taxa.

    Directory of Open Access Journals (Sweden)

    Peter L Oliver

    2009-12-01

    Full Text Available The onset of prezygotic and postzygotic barriers to gene flow between populations is a hallmark of speciation. One of the earliest postzygotic isolating barriers to arise between incipient species is the sterility of the heterogametic sex in interspecies' hybrids. Four genes that underlie hybrid sterility have been identified in animals: Odysseus, JYalpha, and Overdrive in Drosophila and Prdm9 (Meisetz in mice. Mouse Prdm9 encodes a protein with a KRAB motif, a histone methyltransferase domain and several zinc fingers. The difference of a single zinc finger distinguishes Prdm9 alleles that cause hybrid sterility from those that do not. We find that concerted evolution and positive selection have rapidly altered the number and sequence of Prdm9 zinc fingers across 13 rodent genomes. The patterns of positive selection in Prdm9 zinc fingers imply that rapid evolution has acted on the interface between the Prdm9 protein and the DNA sequences to which it binds. Similar patterns are apparent for Prdm9 zinc fingers for diverse metazoans, including primates. Indeed, allelic variation at the DNA-binding positions of human PRDM9 zinc fingers show significant association with decreased risk of infertility. Prdm9 thus plays a role in determining male sterility both between species (mouse and within species (human. The recurrent episodes of positive selection acting on Prdm9 suggest that the DNA sequences to which it binds must also be evolving rapidly. Our findings do not identify the nature of the underlying DNA sequences, but argue against the proposed role of Prdm9 as an essential transcription factor in mouse meiosis. We propose a hypothetical model in which incompatibilities between Prdm9-binding specificity and satellite DNAs provide the molecular basis for Prdm9-mediated hybrid sterility. We suggest that Prdm9 should be investigated as a candidate gene in other instances of hybrid sterility in metazoans.

  1. Role of LRF/Pokemon in lineage fate decisions.

    Science.gov (United States)

    Lunardi, Andrea; Guarnerio, Jlenia; Wang, Guocan; Maeda, Takahiro; Pandolfi, Pier Paolo

    2013-04-11

    In the human genome, 43 different genes are found that encode proteins belonging to the family of the POK (poxvirus and zinc finger and Krüppel)/ZBTB (zinc finger and broad complex, tramtrack, and bric à brac) factors. Generally considered transcriptional repressors, several of these genes play fundamental roles in cell lineage fate decision in various tissues, programming specific tasks throughout the life of the organism. Here, we focus on functions of leukemia/lymphoma-related factor/POK erythroid myeloid ontogenic factor, which is probably one of the most exciting and yet enigmatic members of the POK/ZBTB family. PMID:23396304

  2. AcEST: BP912567 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Sbjct: 272 ERGPVVPGAGLRRPTYAPPSIMPQK----HWVYVK 302 >sp|A7GS60|MTNW_BACCN 2,3-diketo-5-methylthiopentyl-1-pho...2,3-diketo-5-methylthiopentyl-1-phosphate e... 30 4.6 sp|Q5KSV0|GTAK_DICDI GATA zinc finger domain-containin...m... 32 1.6 sp|Q5TJE2|ZBT22_CANFA Zinc finger and BTB domain-containing prot... 30 4.6 sp|A7GS60|MTNW_BACCN

  3. AcEST: BP916265 [AcEST

    Lifescience Database Archive (English)

    Full Text Available -type protein 2 OS=Mus musc... 29 6.3 sp|Q9UBW7|ZMYM2_HUMAN Zinc finger MYM-type protein 2 OS=Homo sap... 29...R C NM TQ Sbjct: 782 WRGEMKHFCDQHCLLRFYCQQNEPNMTTQ 810 >sp|Q9UBW7|ZMYM2_HUMAN Zin

  4. Experiment list: SRX104409 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available two GATA-type zinc fingers and is a n important regulator of T-cell development and plays an important role in endothelial cell biol...ogy. Defects in this gene are the cause of hypoparathyroidism with sensorineural de

  5. Znf202 Affects High Density Lipoprotein Cholesterol Levels and Promotes Hepatosteatosis in Hyperlipidemic Mice

    NARCIS (Netherlands)

    Vrins, C.L.J.; Out, R.; Santbrink, P. van; Zee, A. van der; Mahmoudi, T.; Groenendijk, M.; Havekes, L.M.; Berkel, T.J.C. van; Dijk, K.W. van; Biessen, E.A.L.

    2013-01-01

    Background: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. Methodology and Principal Findings: We generated mouse Znf202 ex

  6. Znf202 Affects High Density Lipoprotein Cholesterol Levels and Promotes Hepatosteatosis in Hyperlipidemic Mice

    NARCIS (Netherlands)

    C.L.J. Vrins (Carlos L.J.); R. Out (Ruud); P. van Santbrink (Peter); A. van der Zee (Anneke); T. Mahmoudi (Tokameh); M. Groenendijk (Martine); L.M. Havekes (Louis); Th.J.C. van Berkel (Theo); J.A.P. Willems van Dijk (Ko); E.A.L. Biessen (Erik)

    2013-01-01

    textabstractBackground: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. Methodology and Principal Findings: We generated mou

  7. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site

    NARCIS (Netherlands)

    S.R. Eberhardy; J. Goncalves; S. Coelho; D.J. Segal; B. Berkhout; C.F. Barbas

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjac

  8. GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

    NARCIS (Netherlands)

    G.T. Ma (Grace); M.E. Roth (Matthew); J.C. Groskopf (John); F.G. Grosveld (Frank); J.D. Engel (Douglas); D.I.H. Linzer (Daniel); F.Y. Tsai (Fong-Ying); S.H. Orkin (Stuart)

    1997-01-01

    textabstractWe previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that bo

  9. Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators

    Science.gov (United States)

    Belaguli, N. S.; Sepulveda, J. L.; Nigam, V.; Charron, F.; Nemer, M.; Schwartz, R. J.

    2000-01-01

    Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

  10. Role of the domains of human gene ZNF569 in MAPK pathway

    Institute of Scientific and Technical Information of China (English)

    YUAN Wuzhou; HUANG Xinqiong; ZHU Chuanbing; WANG Yuequn; LI Yongqing; WU Xiushan

    2006-01-01

    Mitogen-activated protein kinase (MAPK) signal pathways are important components in signal transduction connecting cell-surface receptors to critical regulatory targets within cells,mediating multiple intracetlular signal cascades and phosphorylating their target proteins,such as transcriptional factors ELK-l,SRE and AP-1,and finally activating the expression of intracellular functional genes.Zinc finger genes are some of the largest gene families in humans,and Zinc finger proteins play an essential role in altering gene expression by acting as transcription factors.Zinc finger proteins are also involved in multiple cell processes,including proliferation,differentiation and development,by interacting with DNA.Here,we reported the transcriptional activities of the domains of zinc finger gene ZNF569 taking advantage of MAPK pathway.Overexpression of ZNF569 in COS-7 cells dramatically inhibited the transcriptional repressor activities of SRE and AP-1,which was also confirmed by subcellular localization analysis.Report assays indicated that the potent repression domains of ZNF569 were the KRAB and ZNF motifs.The results suggested that ZNF569 protein might act as a transcriptional repressor in MAPK signaling pathway to regulate cellular processes.

  11. Amino acid substitutions of cysteine residues near the amino terminus of Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    Science.gov (United States)

    The amino-terminal half of HC-Pro of Wheat streak mosaic virus (WSMV) is required for semi-persistent transmission by the wheat curl mite (Aceria tosichella Keifer). The amino-proximal region of WSMV HC-Pro is cysteine-rich with a zinc finger-like motif. Amino acid substitutions were made in this re...

  12. A Ttach lmportance to Vascular lmplications of Kruppel-like Factors

    Institute of Scientific and Technical Information of China (English)

    范自力; 贺石林

    2009-01-01

    @@ Kruppel-like factors(KLFs) are a subclass of the zinc finger family of DNA-binding transcription factors. The nomenclature is based on the homology to the DNA-binding domain of the drosophila protein kruppel. To date,17 members of KLF family have been identified in mammalian cells.

  13. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation

    DEFF Research Database (Denmark)

    Adhikary, Sovana; Marinoni, Federica; Hock, Andreas;

    2005-01-01

    The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linke...

  14. AcEST: BP918343 [AcEST

    Lifescience Database Archive (English)

    Full Text Available |Q9NDJ2|DOM_DROME Helicase domino OS=Drosophila melanogaster G... 48 4e-05 sp|O60315|ZEB2_HUMAN Zinc finger ...EF+ N E+ ED+ T+ A+E L Sbjct: 393 PWHPDEDDEEFT-ANEEEAEDEEDTIAAEEQL 423 >sp|Q9NDJ2|DOM_DROME Helicase domino O

  15. Experiment list: SRX103011 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available =wgEncodeEH000528 || labversion=baseAlignCounts.pl v 1,fseq v 1.84, iff_GM12878,p-value cutoff: 0.05,1% ENCODE array platform validat...ion tests || replicate=1,3,2 || antibody targetdescription=CTCF zinc finger transcr

  16. Domain Modeling: NP_009062.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_009062.2 chrX CRYSTAL STRUCTURE OF A DESIGNED ZINC FINGER PROTEIN BOUND TO DNA c1meyf_ chrX/NP_009062....2/NP_009062.2_holo_145-216.pdb psi-blast 146E,150L,151G,152G,153T,154A,155V,156A,159F,

  17. Domain Modeling: NP_001071092.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_001071092.1 chr19 ZINC FINGER-DNA RECOGNITION: CRYSTAL STRUCTURE OF A ZIF268-DNA COMPLEX AT 2....1 ANGSTROMS c1zaac_ chr19/NP_001071092.1/NP_001071092.1_holo_446-524.pdb blast 450C,452C,455C,

  18. Domain Modeling: NP_005072.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_005072.2 chr2 STRUCTURE OF A CYS2HIS2 ZINC FINGER/TATA BOX COMPLEX (TATAZF;CLONE... #6) c1g2ff_ chr2/NP_005072.2/NP_005072.2_holo_1429-1508.pdb psi-blast 1432C,1435C,1437R,1438A,1439F,1441W,1

  19. Domain Modeling: NP_005332.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_005332.1 chr1 Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain... Bound to DNA p2prta_ chr1/NP_005332.1/NP_005332.1_holo_463-600.pdb blast 467C,468E,469F,470C,472H,483H,487H

  20. Genetics of Estrogen-Related Traits; From Candidate Genes to GWAS

    NARCIS (Netherlands)

    L. Stolk (Lisette)

    2009-01-01

    textabstractIn the first part of this thesis, the association of polymorphisms in three candidate genes (estrogen receptor alpha (ESR1), retinoblastoma interacting zinc finger domain (RIZ1) and catechol-O-methyltransferase (COMT)) with estradiol levels, age at natural menopause, BMD and fracture ris

  1. AcEST: DK948004 [AcEST

    Lifescience Database Archive (English)

    Full Text Available HUMAN Zinc finger FYVE domain-containing protein... 32 3.3 sp|P27197|AURB_CHLAA Auracyanin-B OS=Chloroflexus...R Sbjct: 74 CKGLSGHLPGVRQR 87 >sp|P27197|AURB_CHLAA Auracyanin-B OS=Chloroflexus aurantiacus (strain ATCC 29

  2. Protein: FEB6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEB6 Photoresponse regulatory proteins HD1 SE1 Zinc finger protein HD1 Protein CONSTANS-like, Protein... HEADING DATE 1, Protein PHOTOPERIOD SENSITIVITY 1 39947 Oryza sativa subsp. japonica 4340746 Q9FDX8 21952207, 19246394 #shimamoto ...

  3. EST Table: CA946165 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946165 KI000253R 10/09/28 42 %/154 aa ref|XP_001952754.1| PREDICTED: similar to Z...el KRAB box and zinc finger, C2H2 type domain containing protein [Tribolium castaneum] CK539334 L9 ...

  4. A ribosomal protein L23-nucleophosmin circuit coordinates Miz1 function with cell growth

    DEFF Research Database (Denmark)

    Wanzel, Michael; Russ, Annika C; Kleine-Kohlbrecher, Daniela;

    2008-01-01

    The Myc-associated zinc-finger protein, Miz1, is a negative regulator of cell proliferation and induces expression of the cell-cycle inhibitors p15(Ink4b) and p21(Cip1). Here we identify the ribosomal protein L23 as a negative regulator of Miz1-dependent transactivation. L23 exerts this function ...

  5. Domain Modeling: NP_660281.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_660281.2 chr1 Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain... Bound to DNA p2prta_ chr1/NP_660281.2/NP_660281.2_holo_261-368.pdb psi-blast 265T,266S,267E,268K,270Q,271G,

  6. Domain Modeling: NP_001005851.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_001005851.1 chr19 ZINC FINGER-DNA RECOGNITION: CRYSTAL STRUCTURE OF A ZIF268-DNA... COMPLEX AT 2.1 ANGSTROMS c1zaac_ chr19/NP_001005851.1/NP_001005851.1_holo_275-356.pdb blast 275K,279C,282C,

  7. NCBI nr-aa BLAST: CBRC-GGAL-35-0420 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GGAL-35-0420 ref|NP_001001588.1| MYST histone acetyltransferase (monocytic leukemia...) 3 [Danio rerio] gb|AAT11171.1| monocytic leukemia zinc finger protein; MYST3; ZNF220 [Danio rerio] NP_001001588.1 0.23 36% ...

  8. Metal Ion Controlled Polymorphism of a Peptide

    DEFF Research Database (Denmark)

    Hemmingsen, Lars Bo Stegeager; Jancso, Attila; Szunyogh, Daniel;

    2011-01-01

    , …) in the peptide, and the ligand and structural preferences of the metal ion (in our studies Zn2+, Cd2+, Hg2+, Cu+/2+). Simultaneously, new species such as metal ion bridged ternary complexes or even oligomers may be formed. In recent previous studies we have observed similar polymorphism of zinc finger model...

  9. Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry

    Science.gov (United States)

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and ex...

  10. Gold finger formation studied by high-resolution mass spectrometry and in silico methods

    NARCIS (Netherlands)

    Laskay, Ü.A.; Garino, C.; Tsybin, Y.O.; Salassa, L.; Casini, A.

    2015-01-01

    High-resolution mass spectrometry and quantum mechanics/molecular mechanics studies were employed for characterizing the formation of two gold finger (GF) domains from the reaction of zinc fingers (ZF) with gold complexes. The influence of both the gold oxidation state and the ZF coordination sphere

  11. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

  12. Experiment list: SRX150646 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ich belongs to the GATA family of transcription factors. The protein contains two GATA-type zinc fingers and is a n important... regulator of T-cell development and plays an important role in endothelial cell biology. D

  13. Domain Modeling: NP_066574.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_066574.2 chr2 CRYSTAL STRUCTURE OF A DESIGNED ZINC FINGER PROTEIN BOUND TO DNA c1meyf_ chr2/NP_066574.2.../NP_066574.2_holo_254-335.pdb blast 255K,259C,261E,262C,263G,264K,265A,266F,267F,268D

  14. Recurrent deletion of ZNF630 at Xp11.23 is not associated with mental retardation

    DEFF Research Database (Denmark)

    Lugtenberg, Dorien; Zangrande-Vieira, Luiz; Kirchhoff, Maria;

    2010-01-01

    ZNF630 is a member of the primate-specific Xp11 zinc finger gene cluster that consists of six closely related genes, of which ZNF41, ZNF81, and ZNF674 have been shown to be involved in mental retardation. This suggests that mutations of ZNF630 might influence cognitive function. Here, we detected...

  15. Experiment list: SRX150655 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available to the cap'n'collar type of basic region leucine zipper factor family (CNC-bZip). The encoded protein conta...ins broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domains, which is atypical of C

  16. A20 Inhibits β-Cell Apoptosis by Multiple Mechanisms and Predicts Residual β-Cell Function in Type 1 Diabetes

    DEFF Research Database (Denmark)

    Fukaya, Makiko; Brorsson, Caroline A; Meyerovich, Kira;

    2016-01-01

    Activation of the transcription factor nuclear factor kappa B (NFkB) contributes to β-cell death in type 1 diabetes (T1D). Genome-wide association studies have identified the gene TNF-induced protein 3 (TNFAIP3), encoding for the zinc finger protein A20, as a susceptibility locus for T1D. A20 res...

  17. Fine mapping of ZNF804A and genome-wide significant evidence for its involvement in schizophrenia and bipolar disorder

    NARCIS (Netherlands)

    Williams, H.J.; Norton, N.; Dwyer, S.; Moskvina, V.; Nikolov, I.; Carroll, L.; Georgieva, L.; Williams, N.M.; Morris, D.W.; Quinn, E.M.; Giegling, I.; Ikeda, M.; Wood, J.; Lencz, T.; Hultman, C.; Lichtenstein, P.; Thiselton, D.; Maher, B.S.; Malhotra, A.K.; Riley, B.; Kendler, K.S.; Gill, M.; Sullivan, P.; Sklar, P.; Purcell, S.; Nimgaonkar, V.L.; Kirov, G.; Holmans, P.; Corvin, A.; Rujescu, D.; Craddock, N.; Owen, M.J.; O'Donovan, M.C.; GROUP investigators, [No Value

    2011-01-01

    A recent genome-wide association study (GWAS) reported evidence for association between rs1344706 within ZNF804A (encoding zinc-finger protein 804A) and schizophrenia (P = 1.61 x 10(-7)), and stronger evidence when the phenotype was broadened to include bipolar disorder (P = 9.96 x 10(-9)). In this

  18. AcEST: DK953266 [AcEST

    Lifescience Database Archive (English)

    Full Text Available CLPP1_PSEAE ATP-dependent Clp protease proteolytic sub... 32 3.2 sp|P31264|HMPB_D...ROME Homeotic protein proboscipedia OS=Drosophil... 31 4.1 sp|Q9UEG4|ZN629_HUMAN Zinc finger protein 629 OS=

  19. Screening of 99 Danish patients with congenital heart disease for GATA4 mutations

    DEFF Research Database (Denmark)

    Zhang, Litu; Tümer, Zeynep; Jacobsen, Joes Ramsøe;

    2006-01-01

    Congenital heart disease (CHD) affects nearly 1% of the population, but only few genes involved in human CHD are presently known. Germ-line mutations in the zinc finger transcription factor GATA4 have been associated with familial cases of atrial and ventricular septal defects and pulmonary steno...

  20. Domain Modeling: NP_064611.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_064611.3 chr4 Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain... Bound to DNA p2prta_ chr4/NP_064611.3/NP_064611.3_holo_471-589.pdb psi-blast 475G,476G,478T,479G,496E,497R,

  1. Domain Modeling: NP_003411.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_003411.3 chr3 CRYSTAL STRUCTURE OF A DESIGNED ZINC FINGER PROTEIN BOUND TO DNA c1meyf_ chr3/NP_003411.3.../NP_003411.3_holo_219-301.pdb blast 224C,226V,227C,228G,229K,230G,231F,232S,233Q,234S

  2. Mapping functional regions of transcription factor TFIIIA.

    Science.gov (United States)

    Vrana, K E; Churchill, M E; Tullius, T D; Brown, D D

    1988-04-01

    Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

  3. Functional analysis of genes involved in the regulation of development of reproductive organs in rice (Oryza sativa)

    NARCIS (Netherlands)

    Chen, Yi

    2011-01-01

    Quality of the rice grain is determined mainly by starch and protein contents of the endosperm. In this thesis, the analyses of four genes involved in the regulation of development of rice grain and floret are presented. Two CCCH type zinc finger proteins, OsGZF1 and OsGZF2, were identified as novel

  4. Sequence Classification: 889124 [

    Lifescience Database Archive (English)

    Full Text Available in the processing of pre-rRNA to mature rRNA; contains a C2/C2 zinc finger motif; srd1 mutation suppresses d...efects caused by the rrp1-1 mutation; Srd1p || http://www.ncbi.nlm.nih.gov/protein/10383786 ...

  5. ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

    NARCIS (Netherlands)

    S. Goossens (Steven); E. Radaelli (Enrico); O. Blanchet (Odile); K. Durinck (Kaat); J. van der Meulen (Joni); S. Peirs (Sofie); T. Taghon (Tom); C.S. Tremblay (Cedric S.); M. Costa (Magdaline); M.F. Ghahremani; J. De Medts (Jelle); S. Bartunkova (Sonia); K. Haigh (Katharina); C. Schwab (Claire); N. Farla (Natalie); T. Pieters (Tim); F. Matthijssens (Filip); N. van Roy (Nadine); J.A. Best (J. Adam); K. Deswarte (Kim); P. Bogaert (Pieter); C. Carmichael (Catherine); A. Rickard (Adam); S. Suryani (Santi); L.S. Bracken (Lauryn S.); R. Alserihi (Raed); K. Canté-Barrett (Kirsten); L. Haenebalcke (Lieven); E. Clappier; P. Rondou (Pieter); K. Slowicka (Karolina); D. Huylebroeck (Danny); A.W. Goldrath (Ananda W.); V. Janzen (Viktor); M.P. McCormack (Matthew P.); R.B. Lock (Richard B.); D.J. Curtis (David J.); C.J. Harrison (Christine); G. Berx (Geert); F. Speleman (Franki); J.P.P. Meijerink (Jules); J. Soulier (Jean); P. van Vlierberghe (Pieter); K. Haigh (Katharina)

    2015-01-01

    textabstractEarly T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using

  6. AcEST: BP916073 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 4 sp|Q5TJE2|ZBT22_CANFA Zinc finger and BTB domain-containing prot... 30 8.3 sp|A7GS60|MTNW_BACCN 2,3-diketo-5-methylthiopentyl...IMPQK----HWVYVK 302 >sp|A7GS60|MTNW_BACCN 2,3-diketo-5-methylthiopentyl-1-phospha

  7. Unigene BLAST: CBRC-MMUS-04-0075 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUS-04-0075 gnl|UG|Mm#S10845368 Mus musculus 12 days embryo embryonic body between diaphragm...o ZINC FINGER PROTEIN 13 (FRAGMENT) [Rattus norvegicus], full insert sequence /cds=p(137,1846) /gb=AK034880 /gi=26330267 /ug=Mm.358911 /len=3228 5e-43 48% ...

  8. Unigene BLAST: CBRC-MMUS-07-0621 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUS-07-0621 gnl|UG|Mm#S10845368 Mus musculus 12 days embryo embryonic body between diaphragm...o ZINC FINGER PROTEIN 13 (FRAGMENT) [Rattus norvegicus], full insert sequence /cds=p(137,1846) /gb=AK034880 /gi=26330267 /ug=Mm.358911 /len=3228 2e-38 45% ...

  9. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

    NARCIS (Netherlands)

    M.M. Jaegle (Martine); M. Ghazvini (Mehrnaz); W.J. Mandemakers (Wim); M. Piirsoo (Marko); S. Driegen (Siska); F. Levavasseur (Francoise); S. Raghoenath; F.G. Grosveld (Frank); D. Meijer (Daniëlle)

    2003-01-01

    textabstractThe genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during devel

  10. A short Gfi-1B isoform controls erythroid differentiation by recruiting the LSD1-corest complex through the dimethylation of its SNAG domain

    NARCIS (Netherlands)

    B. Laurent (Benoît); V. Randrianarison-Huetz (Voahangy); E. Frisan (Emilie); C. Andrieu-Soler (Charlotte); E. Soler (Eric); M. Fontenay (Michaela); I. Dusanter-Fourt (Isabelle); D. Dumenil (Dominique)

    2012-01-01

    textabstractGfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domain

  11. AcEST: BP915293 [AcEST

    Lifescience Database Archive (English)

    Full Text Available response protein 1 OS=Bos taur... 30 4.2 sp|Q499R0|ZF518_RAT Zinc finger protein 518 OS=Rattus norvegicus... 30 5.5 sp|Q4K9T4|NUOCD...PVHISERSETRVSRSKANCT----IERNFNKRKTCKNKFAKIKTRI 1258 >sp|Q4K9T4|NUOCD_PSEF5 NADH-q

  12. Hypermethylated in cancer 1 (HIC1) recruits polycomb repressive complex 2 (PRC2) to a subset of its target genes through interaction with human polycomb-like (hPCL) proteins

    DEFF Research Database (Denmark)

    Boulay, Gaylor; Dubuissez, Marion; Van Rechem, Capucine;

    2012-01-01

    Hic1-deficient mice die perinatally and exhibit gross developmental defects throughout the second half of development. HIC1 encodes a transcriptional repressor with five C(2)H(2) zinc fingers mediating sequence-specific DNA binding and two repression domains: an N-terminal BTB/POZ domain and a central...

  13. SwissProt search result: AK063555 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063555 001-117-E08 (Q9UIG0) Bromodomain adjacent to zinc finger domain protein 1B (William...s-Beuren syndrome chromosome region 9 protein) (WBRS9) (Williams syndrome transcription factor) (hWALP2) BAZ1B_HUMAN 2e-11 ...

  14. SwissProt search result: AK101745 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101745 J033061O13 (Q9UIG0) Bromodomain adjacent to zinc finger domain protein 1B (William...s-Beuren syndrome chromosome region 9 protein) (WBRS9) (Williams syndrome transcription factor) (hWALP2) BAZ1B_HUMAN 9e-11 ...

  15. UniProt search blastx result: AK288287 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288287 J090018F21 Q9UIG0|BAZ1B_HUMAN Bromodomain adjacent to zinc finger domain protein 1B (William...s-Beuren syndrome chromosome region 9 protein) (Williams syndrome transcription factor) (hWALP2) - Homo sapiens (Human) 0 ...

  16. SwissProt search result: AK067393 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067393 J013106B07 (Q9UIG0) Bromodomain adjacent to zinc finger domain protein 1B (William...s-Beuren syndrome chromosome region 9 protein) (WBRS9) (Williams syndrome transcription factor) (hWALP2) BAZ1B_HUMAN 4e-11 ...

  17. Dysregulation of crypt cell proliferation and lineage determination, and villus cell migration in the small intestines of mice lacking the intestinal smooth muscle-expressed transcription factor, Krüppel-like factor 9 (Klf9)

    Science.gov (United States)

    Krüppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation and cell fate. Mice with targeted inactivation of Klf9 gene exhibit postnatal growth retardation and increased mortality after birth. Using these mice, we have...

  18. AcEST: DK947950 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 10071|GLI3_HUMAN Zinc finger protein GLI3 OS=Homo sapiens GN... 31 4.9 sp|Q8WXX7|AUTS2_HUMAN Autism suscepti...FSPPHPYINPYMDYIRSLHSSPSLSMI 216 >sp|Q8WXX7|AUTS2_HUMAN Autism susceptibility gene 2 protein OS=Homo sapiens

  19. Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities

    NARCIS (Netherlands)

    Uil, T.G.; Haisma, H.J.; Rots, Marianne

    2003-01-01

    Designer molecules that can specifically target pre-determined DNA sequences provide a means to modulate endogenous gene function. Different classes of sequence-specific DNA-binding agents have been developed, including triplex-forming molecules, synthetic polyamides and designer zinc finger protein

  20. Arabidopsis CDS blastp result: AK065522 [KOME

    Lifescience Database Archive (English)

    Full Text Available g protein low similarity to SP|P46718 Programmed cell death protein 2 (Zinc finger protein Rp-8) {Mus muscul...us}; contains Pfam profile PF04194: Programmed cell death protein 2, C-terminal domain 3e-62 ...

  1. EST Table: CK534064 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available Mariner transposase delivery vector pFA545] gb|ABB59013.1| transposase [Himar1-delivery and mutagenesis vector... pFNLTP16 H3] gb|ACK37846.1| transposase [Himar1-delivery and mutagenesis vector pHBurk3] gb|ACK37849.1| t...ransposase [Himar1-delivery and mutagenesis vector pHBurk5] 10/08/31 38 %/106 aa

  2. Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xi; Zhou, Xixi [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Du, Libo [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Liu, Wenlan [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Yang [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hudson, Laurie G. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Ke Jian, E-mail: kliu@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

    2014-01-15

    Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

  3. Insights into Strand Exchange in BTB Domain Dimers from the Crystal Structures of FAZF and Miz1

    Energy Technology Data Exchange (ETDEWEB)

    Stogios, Peter J.; Cuesta-Seijo, Jose Antonio; Chen, Lu; Pomroy, Neil C.; Privé, Gilbert G. (Toronto); (OCI)

    2010-09-22

    The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel {beta}-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 {angstrom} and 2.6 {angstrom}, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminal {beta}-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric a brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain.

  4. Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

    International Nuclear Information System (INIS)

    Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

  5. Caspases in plants: metacaspase gene family in plant stress responses.

    Science.gov (United States)

    Fagundes, David; Bohn, Bianca; Cabreira, Caroline; Leipelt, Fábio; Dias, Nathalia; Bodanese-Zanettini, Maria H; Cagliari, Alexandro

    2015-11-01

    Programmed cell death (PCD) is an ordered cell suicide that removes unwanted or damaged cells, playing a role in defense to environmental stresses and pathogen invasion. PCD is component of the life cycle of plants, occurring throughout development from embryogenesis to the death. Metacaspases are cysteine proteases present in plants, fungi, and protists. In certain plant-pathogen interactions, the PCD seems to be mediated by metacaspases. We adopted a comparative genomic approach to identify genes coding for the metacaspases in Viridiplantae. We observed that the metacaspase was divided into types I and II, based on their protein structure. The type I has a metacaspase domain at the C-terminus region, presenting or not a zinc finger motif in the N-terminus region and a prodomain rich in proline. Metacaspase type II does not feature the prodomain and the zinc finger, but has a linker between caspase-like catalytic domains of 20 kDa (p20) and 10 kDa (p10). A high conservation was observed in the zinc finger domain (type I proteins) and in p20 and p10 subunits (types I and II proteins). The phylogeny showed that the metacaspases are divided into three principal groups: type I with and without zinc finger domain and type II metacaspases. The algae and moss are presented as outgroup, suggesting that these three classes of metacaspases originated in the early stages of Viridiplantae, being the absence of the zinc finger domain the ancient condition. The study of metacaspase can clarify their assignment and involvement in plant PCD mechanisms.

  6. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  7. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  8. Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.

    Science.gov (United States)

    Carballar-Lejarazú, R; Brennock, P; James, A A

    2016-08-01

    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.

  9. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  10. Nac1 interacts with the POZ-domain transcription factor, Miz1.

    Science.gov (United States)

    Stead, Mark A; Wright, Stephanie C

    2014-06-05

    Nac1 (nucleus accumbens 1) is a POZ (poxvirus and zinc finger)-domain transcriptional repressor that is expressed at high levels in ovarian serous carcinoma. Here we identify Nac1 as a novel interacting partner of the POZ-domain transcriptional activator, Miz1 (Myc-interacting zinc-finger protein 1), and using chemical crosslinking we show that this association is mediated by a heterodimeric interaction of the Nac1 and Miz1 POZ domains. Nac1 is found in discrete bodies within the nucleus of mammalian cells, and we demonstrate the relocalization of Miz1 to these structures in transfected HeLa cells. We show that siRNA (small interfering RNA)-mediated knockdown of Nac1 in ovarian cancer cells results in increased levels of the Miz1 target gene product, p21Cip1. The interaction of Nac1 with Miz1 may thus be relevant to its mechanism of tumourigenesis in ovarian cancer.

  11. AcEST: BP911717 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000008_D01 438 Adiantum capillus-veneris mRNA. clone: YMU001_000008_D01. BP911717... CL1298Contig1 Show BP911717 Clone id YMU001_000008_D01 Library YMU01 Length 438 Definition Adiantum ca...pillus-veneris mRNA. clone: YMU001_000008_D01. Accession BP911717 Tissue type prothallium Developmental stag...cleic Acids Res. 25:3389-3402. Query= BP911717|Adiantum capillus-veneris mRNA, cl...|ZSC29_HUMAN Zinc finger and SCAN domain-containing pro... 35 0.14 sp|P17040|ZSC20_HUMAN Zinc finger and SCA

  12. Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.

    Science.gov (United States)

    Carballar-Lejarazú, R; Brennock, P; James, A A

    2016-08-01

    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes. PMID:27110891

  13. Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

    OpenAIRE

    Hongge Jia; Nian Wang

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been succes...

  14. Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the Early Stage of Seed Germination.

    Science.gov (United States)

    Li, Ming; Yin, Xiaojian; Sakata, Katsumi; Yang, Pingfang; Komatsu, Setsuko

    2015-07-01

    The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, we performed gel-and label-free nuclear phosphoproteomics. A total of 3467 phosphopeptides belonging to 102 nuclear phosphoproteins from rice embryos were identified. Protein-synthesis-related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified, and significant changes in the phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide-binding proteins and zinc finger CCCH- and BED-type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that the Sas10/Utp3 protein, which functions in snoRNA binding and gene silencing, was the center of the phosphoprotein network in nuclei. The germination rate of seeds was significantly slowed with phosphatase inhibitor treatment. The mRNA expression of the zinc finger CCCH-type protein did not change, and the zinc finger BED-type protein was upregulated in rice embryos during the early stage of germination with phosphatase inhibitor treatment. These results suggest that the phosphorylation and dephosphorylation of nuclear proteins are involved in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH- and BED-type proteins might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might interact with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination. PMID:26035336

  15. Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

    OpenAIRE

    Maheswaran, Shyamala; Englert, Christoph; Zheng, Gang; Lee, Sean Bong; Wong, Jenise; Harkin, D Paul; Bean, James; Ezzell, Robert; Garvin, A. Julian; McCluskey, Robert T.; DeCaprio, James A.; Haber, Daniel A.

    1998-01-01

    The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Co...

  16. Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells

    OpenAIRE

    Bandyopadhyay, Sanmay; Duré, Myrianne; Paroder, Monika; Soto-Nieves, Noemí; Puga, Irene; Macián, Fernando

    2007-01-01

    In T cells anergy may be evoked by an unbalanced stimulation of the T-cell receptor in the absence of costimulation. Anergic T cells are unresponsive to new antigen receptor engagement and do not produce interleukin 2. We present evidence that anergizing stimuli induce changes in histone acetylation, which mediates transcriptional repression of interleukin 2 expression. In response to calcium signaling, anergic T cells up-regulate the expression of Ikaros, a zinc finger transcription factor e...

  17. Induction of Extracellular Matrix-Remodeling Genes by the Senescence-Associated Protein APA-1

    OpenAIRE

    Benanti, Jennifer A.; Williams, Dawnnica K.; Robinson, Kristin L; Ozer, Harvey L.; Galloway, Denise A.

    2002-01-01

    Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-imm...

  18. A novel genetic mechanism regulates dorsolateral hinge-point formation during zebrafish cranial neurulation

    OpenAIRE

    Nyholm, Molly K.; Abdelilah-Seyfried, Salim; Grinblat, Yevgenya

    2009-01-01

    During neurulation, vertebrate embryos form a neural tube (NT), the rudiment of the central nervous system. In mammals and birds, a key step in cranial NT morphogenesis is dorsolateral hinge-point (DLHP) bending, which requires an apical actomyosin network. The mechanism of DLHP formation is poorly understood, although several essential genes have been identified, among them Zic2, which encodes a zinc-finger transcription factor. We found that DLHP formation in the zeb...

  19. Funktionsanalyse ausgewählter DOF-Transkriptionsfaktoren bei der Modellpflanze Arabidopsis thaliana

    OpenAIRE

    Skirycz, Aleksandra

    2008-01-01

    Transcription factors (TFs) are global regulators of gene expression playing essential roles in almost all biological processes, and are therefore of great scientific and biotechnological interest. This project focused on functional characterisation of three DNA-binding-with-one-zinc-finger (DOF) TFs from the genetic model plant Arabidopsis thaliana, namely OBP1, OBP2 and AtDOF4;2. These genes were selected due to severe growth phenotypes conferred upon their constitutive over-expression. To ...

  20. Domain Modeling: NP_005072.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_005072.2 chr2 Solution structure of the N-terminal C2H2 type zinc-binding domain... of the Zinc finger protein 64, isoforms 1 and 2 c2dmda_ chr2/NP_005072.2/NP_005072.2_holo_380-476.pdb psi-blast 383C,386C,388Y,399H,404H,412C,415C,428H,429K,447A,449Q,450E _ZN 0 ...

  1. Domain Modeling: NP_055612.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_055612.1 chr6 STRUCTURE OF A CYS2HIS2 ZINC FINGER/TATA BOX COMPLEX (TATAZF;CLONE... #6) c1g2ff_ chr6/NP_055612.1/NP_055612.1_holo_135-214.pdb psi-blast 136K,140L,143A,145A,146P,148V,149V,150I

  2. Domain Modeling: NP_055532.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_055532.1 chr19 STRUCTURE OF A CYS2HIS2 ZINC FINGER/TATA BOX COMPLEX (TATAZF;CLON...E #6) c1g2ff_ chr19/NP_055532.1/NP_055532.1_holo_2-80.pdb psi-blast 298K,302C,304L,305C,307F,308A,310S,311Q,

  3. Recurrent deletions of IKZF1 in pediatric acute myeloid leukemia

    OpenAIRE

    de Rooij, Jasmijn D.E.; Beuling, Eva; Marry M van den Heuvel-Eibrink; Obulkasim, Askar; Baruchel, André; Trka, Jan; Reinhardt, Dirk; Sonneveld, Edwin; Gibson, Brenda E.S.; Pieters, Rob; Zimmermann, Martin; Zwaan, C. Michel; Fornerod, Maarten

    2015-01-01

    IKAROS family zinc finger 1/IKZF1 is a transcription factor important in lymphoid differentiation, and a known tumor suppressor in acute lymphoid leukemia. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. To investigate whether IKZF1 deletions also play a role in pediatric acute myeloid leukemia, we screened a panel of pediatric acute myeloid leukemia samples for deletions of the IKZF1 locus using multiplex ligation-dependent probe amplification and for mutations...

  4. Progress and prospects: techniques for site-directed mutagenesis in animal models

    OpenAIRE

    Yan, Z.; X. Sun; Engelhardt, JF

    2009-01-01

    In the past 2 years, new gene-targeting approaches using adeno-associated virus and designer zinc-finger nucleases have been successfully applied to the production of genetically modified ferrets, pigs, mice and zebrafish. Gene targeting using these tools has been combined with somatic cell nuclear transfer and germ cell transplantation to generate gene-targeted animal models. These new technical advances, which do not require the generation of embryonic stem cell-derived chimeras, will great...

  5. Krüpple-like factors in the central nervous system: novel mediators in Stroke

    OpenAIRE

    Yin, Ke-jie; Hamblin, Milton; Fan, Yanbo; Zhang, Jifeng; Chen, Y. Eugene

    2013-01-01

    Transcription factors play an important role in the pathophysiology of many neurological disorders, including stroke. In the past three decades, an increasing number of transcription factors and their related gene signaling networks have been identified, and have become a research focus in the stroke field. Krüppel-like factors (KLFs) are members of the zinc finger family of transcription factors with diverse regulatory functions in cell growth, differentiation, proliferation, migration, apop...

  6. Efficient genome editing in zebrafish using a CRISPR-Cas system

    OpenAIRE

    Hwang, Woong Y.; Fu, Yanfang; Reyon, Deepak; Maeder, Morgan L.; Tsai, Shengdar Q.; Sander, Jeffry D.; Peterson, Randall T.; Yeh, J.-R. Joanna; Joung, J Keith

    2013-01-01

    In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nuclea...

  7. ZEB1 Expression in Endometrial Biopsy Predicts Lymph Node Metastases in Patient with Endometrial Cancer

    OpenAIRE

    Gang Feng; Xiangming Wang; Xiaozhi Cao; Lijuan Shen; Jiansheng Zhu

    2014-01-01

    Purpose. The purpose of this study was to analyze the expression of zinc-finger E-box-binding homeobox 1 (ZEB1) in endometrial biopsy and its correlation with preoperative characteristics, including lymph node metastases in patient with endometrial cancer. Methods. Using quantitative RT-PCR, ZEB1 expressions in endometrial biopsy from 452 patients were measured. The relationship between ZEB1 expression and preoperative characteristics was analyzed. Results. ZEB1 expressions were significantly...

  8. Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Material

    OpenAIRE

    Dayton, Melody; Koskinen, Mikko T; Tom, Bradley K.; Mattila, Anna-Maria; Johnston, Eric; Halverson, Joy; Fantin, Dennis; DeNise, Sue; Budowle, Bruce; Smith, David Glenn; Kanthaswamy, Sree

    2009-01-01

    Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio det...

  9. Tristetraprolin and Its Family Members Can Promote the Cell-Free Deadenylation of AU-Rich Element-Containing mRNAs by Poly(A) Ribonuclease

    OpenAIRE

    Lai, Wi S.; Kennington, Elizabeth A; Blackshear, Perry J.

    2003-01-01

    Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TT...

  10. Phosphorylation site analysis of the anti-inflammatory and mRNA destabilizing protein tristetraprolin

    OpenAIRE

    Cao, Heping; Deterding, Leesa J.; Blackshear, Perry J.

    2007-01-01

    Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been use...

  11. RNA-destabilizing Factor Tristetraprolin Negatively Regulates NF-κB Signaling*

    OpenAIRE

    Liang, Jian; Lei, Tianhua; Song, Yuting; Yanes, Natalie; Qi, Yongfen; Fu, Mingui

    2009-01-01

    Tristetraprolin (TTP) is a CCCH zinc finger-containing protein that destabilizes mRNA by binding to an AU-rich element. Mice deficient in TTP develop a severe inflammatory syndrome mainly because of overproduction of tumor necrosis factor α. We report here that TTP also negatively regulates NF-κB signaling at the transcriptional corepressor level, by which it may repress inflammatory gene transcription. TTP expression inhibited NF-κB-dependent transcription. However, overexpression of TTP did...

  12. Regulation and localization of endogenous human tristetraprolin

    OpenAIRE

    Fairhurst, Anna-Marie; Connolly, John E.; Hintz, Katharine A; Goulding, Nicolas J; Rassias, Athos J.; Yeager, Mark P.; Rigby, William; Wallace, Paul K

    2003-01-01

    Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expr...

  13. Phosphorylation of Recombinant Tristetraprolin in Vitro

    OpenAIRE

    Cao, Heping; Lin, Rui

    2008-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potent...

  14. Glucocorticoids Regulate Tristetraprolin Synthesis and Posttranscriptionally Regulate Tumor Necrosis Factor Alpha Inflammatory Signaling▿

    OpenAIRE

    Smoak, Kathleen; Cidlowski, John A.

    2006-01-01

    Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3′ untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment lead...

  15. Quantitative Evaluation of His-Tag Purification and Immunoprecipitation of Tristetraprolin and Its Mutant Proteins from Transfected Human Cells

    OpenAIRE

    Cao, Heping; Lin, Rui

    2009-01-01

    Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of current study was to evaluate His-tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti-inflammatory property. Human embryonic kidney 293 cells were tran...

  16. Creation of a split-cas9 system for effective plant genome editing

    OpenAIRE

    Costa Carrió, Pedro

    2015-01-01

    Precise and efficient genome-targeting technologies are needed to enable both systematic genome editing and interrogation of gene function. Although technologies such as designer zinc fingers (ZFs), transcription activator-like effectors (TALENs), and homing meganucleases have begun to enable targeted genome modifications, there is a need for new technologies that are scalable, affordable, and easy to engineer. Easy programmability of the RNA-guided DNA-endonuclease Cas9 of the type II CRISPR...

  17. CRISPR-Cas: Development and applications for mammalian genome editing

    OpenAIRE

    Ran, Fei Ann

    2014-01-01

    The ability to introduce targeted modifications into genomes and engineer model organisms holds enormous promise for biomedical and technological applications, and has driven the development of tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). To facilitate genome engineering in mammalian cells, we have engineered the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 programmable nuclease systems from Streptococcus p...

  18. Combined clinical and genetic testing algorithm for cervical cancer diagnosis

    OpenAIRE

    Liou, Yu-Ligh; Zhang, Tao-Lan; Yan, Tian; Yeh, Ching-Tung; Kang, Ya-Nan; Cao, Lanqin; Wu, Nayiyuan; Chang, Chi-Feng; Wang, Huei-Jen; Yen, Carolyn; Chu, Tang-Yuan; Zhang, Yi; Zhang, Yu; Zhou, Honghao

    2016-01-01

    Background Opportunistic screening in hospitals is widely used to effectively reduce the incidence rate of cervical cancer in China and other developing countries. This study aimed to identify clinical risk factor algorithms that combine gynecologic examination and molecular testing (paired box gene 1 (PAX1) or zinc finger protein 582 (ZNF582) methylation or HPV16/18) results to improve diagnostic accuracy. Methods The delta Cp of methylated PAX1 and ZNF582 was obtained via quantitative methy...

  19. Crystal structure of the BTB domain from the LRF/ZBTB7 transcriptional regulator

    OpenAIRE

    Peter J. Stogios; Chen, Lu; Privé, Gilbert G.

    2007-01-01

    BTB-zinc finger (BTB-ZF) proteins are transcription regulators with roles in development, differentiation, and oncogenesis. In these proteins, the BTB domain (also known as the POZ domain) is a protein–protein interaction motif that contains a dimerization interface, a possible oligomerization surface, and surfaces for interactions with other factors, including nuclear co-repressors and histone deacetylases. The BTB-ZF protein LRF (also known as ZBTB7, FBI-1, OCZF, and Pokemon) is a master re...

  20. The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B

    OpenAIRE

    Schmidlin, Martin; Lu, Min; Leuenberger, Sabrina A; Stoecklin, Georg; Mallaun, Michel; Gross, Brigitte; Gherzi, Roberto; Hess, Daniel; Brian A Hemmings; Moroni, Christoph

    2004-01-01

    Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3′ untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylatin...