Zinc Finger Nucleases

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

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

okeanokoites bacteria restriction endonuclease, FokI [34-36]. In-vivo, as noted above, the zinc finger nucleases function as dimers (or pairs) [54-57]. Therefore, considering our three ZF array based ZFNs, eighteen (9 × 2) or more (plus the 5-7 spacer- sequence) nucleotide base pairs will be recognized and cleaved. As a result, unlike the five base-pair specificity palindrome of EcoRII {5’-CCWGG-3’ (W = A or T)} [31], ZFNs possess longer (> 18 bp) sequence recognition abilities, a unique feature that endows ZFNs with more target-specificity to HSV-2 genomic DNA, without risking off-target damage to host genomic DNA. Although the ZFNs identified in Table 1 are specifically proposed for excision of over 60% of the HSV-2 genomic DNA from infected host cells through NHEJ, it is possible to alternatively target and disrupt HSV- 2 virulence genes using those ZFNs that cleave several times within the contextual position corresponding to the target-gene of interest (see additional file 3 for all ZFNs cleaving within the HSV-2 genome), although there are questions surrounding these propositions.

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Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

Ultimately, the bcr-abl gene is the underlying cause of the CML pathogenesis and TKI-resistance. Therefore, in theory, a method to destroy bcr-abl gene will fundamen- tally solve the problem of CML onset and drug resist- ance. The technique known as ‘genome editing’ such as zinc finger nucleases (ZFNs) [14–16], transcription acti- vatorlike effector nucleases (TALENs) [17–19] and clus- tered regulatory interspaced short palindromic repeats (CRISPR) / Cas9 [20, 21] is opening the possibility of disrupting bcr-abl oncogene. Analysis of the bcr-abl se- quence analysis shows that it is ideal for the construc- tion of ZFNs. ZFNs are generated by fusing the sequence-specific DNA-binding domain to endonuclease domain of FokI [22, 23]. DNA-binding domain is com- posed of C2H2 zinc-finger proteins (ZFPs). Each finger recognizes 3-base pairs of DNA [24, 25] and has the highest affinity to the 5’-GNN-3′ nucleotide triplet [26]. Three to four zinc-finger domains linked together in tandem to constitute each of ZFN dimers and bind to 18-bp to 24-bp targeting DNA, such a long site is rarer cleavage targets even in complex genomes [27]. In the coding sequence of bcr-abl, there are 15 sites containing the best binding 5 ’ -GNN-3 ′ sequence fit to generate ‘three fingers ZFNs’ and among these sites even have 3 sites suitable to construct ‘four fingers ZFNs’. The design of ZFNs aim at these sites can effectively and specifically modify bcr-abl and also reduce the off-target cleavage. Therefore, ZFNs are the preferred technology for bcr-abl gene editing. Fok I domain, a nonspecific restriction en- zyme, cuts the DNA sequence identified by the ZFPs and introduces a DNA double strand break (DSB) [22]. Off-target effects and cellular toxicity by ZFNs can be induced by the homodimers formation of wild-type Fok I [27–29]. To address this problem, FokI nuclease variants have been used to eliminate the unwanted homodimers and cleave DNA only as a heterodimer pair [28 – 30].

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

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

I report here SIV/HIV-pol gene and HIV-1 whole genome specific zinc finger nucleases, which are proposed for use towards targeted HIV gene therapy. Specifically, because of the notoriety and promiscuousness of HIV at evading previous therapeutic and vaccine attempts, we - building on the bacterial R-M enzymatic machinery as a primitive anti-viral model and prior work identifying bacterial REases against SIV/HIV genomes - postulated that ex-vivo or even in-vivo disruption of viral gene action or excision of over 80% of proviral HIV DNA from within infected cells can irreversibly inactivate both active and latent virus [4-7,12-14]. The SIV/HIV-specific bacterial REases previously identified towards this purpose also target short palindromic targets present within the human genome and thereby carry the risk for toxicity [15]. Now, however, in the wake of advances in zinc finger technology, I have assembled 114 ZFAs (Figure 1, Table 1, and Additional file 2) and constructed 2 ZFNs (Figure 2, Table 2, and Additional file 2) with unique specificity to >18 bp sequences present only within the SIV/HIV pol gene. In addition, another 15 ZFNs were constructed that target and cleave within the >18 bp sequences present only within the proviral DNA of the whole HIV-1 genome (see Figure 3 for graphic distribution of the cleavage sites and pattern. For details of the latter, see additional file 3). It is therefore speculated that lentiviral vectors carrying either genotype (LV-2xZif HIV-pol F N or LV- 2xZif HIV- 1 F N ) may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV repli-

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Targeted Mutagenesis of Beta Lactoglobulin Gene in Caprine Fetal Fibroblasts by Context Dependent Assembly Zinc Finger Nucleases

Targeted Mutagenesis of Beta Lactoglobulin Gene in Caprine Fetal Fibroblasts by Context Dependent Assembly Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are artificial, hybrid restriction enzymes created by covalently linking a DNA- binding zinc finger (Zif) domain (composed of three to six finger-arrays) to the non-specific DNA cleavage do- main (or simply FN) of the Flavobacterium okeanokoites bacterial restriction endonuclease-Fok I. ZFNs have recently become a powerful tool for genome modification since they are able to generate site- specific double- strand breaks (DSBs) [1]. Repair of DSBs provides the molecular basis for gene disruption, gene correction, or gene addition during ZFN-mediated genome editing [2]. Specifically, a given DSB may be repaired by non- homologous end joining (NHEJ) or homology-directed repair (HDR) pathways [1]. HDR utilizes a homologous donor sequence as template for the conservative repair of the DNA break. Provision of a suitably designed donor DNA molecule can, therefore, specify gene addition or gene correction at the ZFN-induced DSB [3]-[5]. NHEJ, on the other hand, catalyzes the rejoining of the two DNA ends, a process that can result in the deletion or inser- tion of nucleotides at the repair junction. DNA repair via NHEJ is, therefore, mutagenic [6]-[9]. To date, ZFN-induced DNA repair via HDR or NHEJ has been utilized to target modifications to the genomes of nu- merous species [10].

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Zinc Finger Nucleases as tools to understand and treat human diseases

Zinc Finger Nucleases as tools to understand and treat human diseases

A further breakthrough in the utility of designer zinc fin- ger proteins has been the ability to cleave specific sites in large genomes. The initial proof-of-concept experiment in this area was the demonstration that the FokI endonu- clease from the prokaryote Flavobacterium okeanokoites, can be separated into two functional domains - one that binds DNA in a sequence specific manner, and one that cleaves in a sequence independent manner. Moreover, the cleavage domain can be fused to unrelated DNA recogni- tion modules and retain DNA cleavage, which occurs at 9 and 13 bps from the recognition site [40]. Structural and experimental analysis have shown that the nuclease domain of FokI requires dimerization for endonuclease activity on the target DNA [41,42]. This fact conveniently increases the potential specificity for the use of ZFPs linked to the FokI endonuclease (hereafter termed zinc finger nucleases, or ZFNs). Therefore by fusing a FokI monomer to two ZFPs, which bind to adjacent sequences, will generate at least an 18 bp sequence specific DNA nuclease that in principle should allow selective targeting within mammalian genomes. This approach is depicted in Figure 3, in which two zinc finger trimers are bound to their cognate DNA sequences creating an active FokI dimer. The effectiveness of this strategy was initially shown with artificial DNA substrates in vitro [43] and integrated transgenes in Xenopus eggs [44]. Subsequently, advances in the design of ZFPs led to the successful appli- cations of this technique to endogenous genomic loci in drosophila [45], human cells [46], plants [47] and C. ele- gans [48]. The remainder of this review will focus on the consequences and applications of targeted single stranded breaks induced by such ZFNs.

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Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study

Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study

the Flavobacterium okeanokoites bacteria restriction endo- nuclease, FokI [33]; have recently become a powerful tool for primarily editing host genomes. Following induction of a double strand break (DSB) within the target DNA, repair occurs, either by homologous recombination (HR) or non- homologous end-joining (NHEJ) [26-32]. Perez et E al. [30]-using engineered ZFNs targeting human CCR5, previ- ously demonstrated establishment of HIV-1 resistance in CD4+ T cells through generation of a doublestrand break (DSB) at predetermined sites in the CCR5 coding region up- stream of the natural CCR5D32 mutation. Holmes N et al. [31] have demonstrated control of HIV-1 infection within NSG-mice transplanted with human hematopoietic stem/ progenitor cells modified by zinc-finger nucleases targeting CCR5. Most recently, Wilen CB, et al. [32] successfully engineered HIV-Resistant Human CD4+ T Cells using CXCR4-Specific Zinc-Finger Nucleases (ZFN). This evi- dence, along with on-going improvements in the design and engineering of lentiviral [33,34] and parvovirus [35] vectors (LV and PV, respectively) for ex-vivo or in-vivo gene-delivery and transduction of erythroid precursors, suggests that the sickle cell mutation may be abrogated in erythroid bone marrow precursor with appropriate ZFNs. Indeed, we are aware that Sangamo Biosciences (http://www.sangamo. com/index.html) – one of the leading industries in ZFN- technology, has already focused its ZFN-mediated gene- editing technology to providing a unique solution for the treatment of monogenic diseases like hemophilia and SCA. Their ZFAs or ZFNs are, however, not publically available.

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Evaluation and application of modularly assembled zinc finger nucleases in zebrafish

Evaluation and application of modularly assembled zinc finger nucleases in zebrafish

This is likely to be due to fraying effects at the edges of the protein- DNA complex that reduce the specificity of the determinants at these positions (Choo, 1998). Comparison of PWM values across all individual modules revealed that those targeting purine-rich triplets were among the most robust, regardless of their finger position. For example, the GAG module consistently displayed excellent specificity in multiple finger positions in most ZFPs (Fig. 2B), as did modules targeting GAT, GGG and GGT (see Tables S5 and S6 in the supplementary material). We also identified four modules within the archive that displayed poor specificity at a specific position or in a context-dependent manner. For example, the TTG module displays context-dependent alterations in specificity. We originally utilized the TTG module at finger 3 in ZFPs targeting the zebrafish kdrl locus, where it displays moderate preference (NtG) for its triplet (Meng et al., 2008; Gupta et al., 2011), similar to the efnb2a 5p ZFP in this study (Fig. 2C). However, when it is placed at the finger 2 position adjacent to a C- terminal module bearing an RSD motif at positions –1, 1 and 2, the specificity shifts to a strong preference for a GNG binding site [Fig. 2C, zgc:66439 (clec14a – Zebrafish Information Network), 3p ZFP]. This dramatic alteration in specificity is not observed when the neighboring C-terminal module lacks an RSD motif (Fig. 2C, kif1b, 5p ZFP). Overall, the majority of analyzed modules have favorable recognition properties in multiple contexts, although several might specify incorrect triplets in particular contexts.

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The therapeutic landscape of HIV-1 via genome editing

The therapeutic landscape of HIV-1 via genome editing

TALENs have not yet been applied for HIV-1 treat- ment in clinical studies. However, several experimen- tal studies have shown promising results with potential for optimization for large scale anti-HIV-1 treatment. Shi and colleagues recently generated 28 CCR5-TALEN screens to target several domains on the CCR5 of CD4+ T cells [82]. CCR5-ZFNs similar to the anti-HIV-1 ZFNs currently being applied for ongoing clinical trials were used as controls in this study for comparative studies. The results were remarkable and showed increased edit- ing efficiency and minimal cytotoxicity activity com- pared to the CCR5-ZFN currently undergoing clinical trials [83]. Furthermore, cleavage of HIV-1 matrix pro- tein P17 gene sequences with custom designed TALENs delivered by lentiviral vectors to Jurkat-HIV-1 cell lines has been reported with editing efficiency of ~43% [68]. HIV-1 P17 helps in the assembly and budding of HIV- 1, and shows relatively little sequence diversity, therefore serves as a suitable candidate for clinical applications. TALENS  have been used  to target a highly conserved sequence in the transcription response element of the HIV-1 proviral DNA [84]. The HIV-1 TALEN constructs Fig. 1 Zinc finger nucleases

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Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

Targeted genome engineering refers to technologies that are used for site- specific genome modifications such as knockout, knockin and transcriptional regulation of genes of interest in organisms. Site-specific recombination sys- tem, zinc finger nucleases (ZFNs), transcriptional activator-like effector nuc- leases (TALENs) and clustered regularly interspaced short palindromic re- peats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) technologies are the representatives of targeted genome engineering and have been widely used in crop basic and applied research. In this review, we intro- duce the basic information and action modes of these different genome engi- neering technologies, summarize the recent progresses of targeted genome engineering technologies and their applications in crop improvement, and propose perspectives for genome engineering-mediated modifications of crop plants in the future.

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Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect

Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect

A critical aspect of APO3G’s biological activity is its ability to strongly inhibit HIV-1 infectivity in the absence of Vif. We have asked a key question: Is the antiviral effect a consequence of APO3G’s deaminase activity or is another mechanism in- volved? One approach has been to study the effects of point mutations in the zinc finger domains. A number of investiga- tors have found that deamination is linked to antiviral activity (26, 30, 40, 54), although there has been disagreement over whether both zinc finger domains (26, 54) or primarily the second domain (30, 40) is involved. In contrast, in a recent study it was reported that point mutations in either the first or second domain had similar inhibitory effects on antiviral activ- ity. These findings led to the proposal that the antiviral func- tion of APO3G can be dissociated from deaminase activity and that some other mechanism, as yet undefined, is responsible for antiviral activity (31). These authors, who used untagged proteins for their work, suggested that conflicting data ob- tained by others might result from the use of epitope-tagged proteins, which could interfere with mutant APO3G expres- sion and function (31). However, one of us (K. Strebel) has found that epitope-tagged and untagged WT or mutant APO3G have equivalent activities in a cell-based system (32). It is possible that these disparate results concerning antiviral activity reflect differences in experimental protocols, which would affect APO3G expression and/or encapsidation into virions.

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Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes

Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes

The two fingered ZFPS of Arabidopsis fall into five groups based on their protein sequences (2i-A-D with outlier X) [3]. Aside from protein sequence similarity it is unclear whether these different phylogenetic groups play a different role in expression or interaction with dif- ferent proteins. Most ZFPs have been studied one gene at a time and one group does not seem to fall into any specific expression category. ZFPs are often induced by a stress (biotic and/or abiotic) and due to the EAR motif, act to negatively regulate genes that they bind. ZFPs may modulate the stress response by binding activator tran- scription factors via the zinc fingers while the EAR motif binds to bridging proteins such as TPL or TPL-like pro- teins in Arabidopsis that subsequently interacts with HDAC proteins such as HDA19. This interaction results

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KRAB zinc finger proteins

KRAB zinc finger proteins

Together, these findings have led to the ‘ arms race ’ model (Fig. 3A), which asserts that dynamic competition between TEs and KRAB-ZFPs drives their co-evolution, with TEs that are controlled by a KRAB-ZFP mutating away to escape repression while the pool of KRAB-ZFP genes evolves proteins with novel zinc finger arrays, which get fixed once they can recognize the renegade TE (Imbeault and Trono, 2014). This model is best exemplified by the primate- specific L1PA subfamily of LINE elements, as these TEs, devoid of an extracellular phase, display a linear evolutionary path, each new subfamily deriving from the one previously expanded in the genome of its host species and ancestors. Indeed, compelling evidence for the arms race model stems from the characterization of ZNF93 and its binding to L1PA elements – in particular the loss (via deletion) of the ZNF93 recognition site in newer L1PA subfamilies (Jacobs et al., 2014). Additional support comes from the recent identification (Imbeault et al., 2017) of TE targets in a large set of human KRAB-ZFPs, which reveals the sequential recruitment at the 5 ′ ends of primate-specific L1 elements of not only ZNF93 but also ZNF141, ZNF649 and ZNF765, with zinc finger mutations accumulating coincidentally with the appearance of new L1PA

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Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins

Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins

WSTF including the nucleosome assembly complex (WINAC) is another SWI/SNF-type complex that has ATP-dependent chromatin-remodeling activity. It interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF, also named bromodomain adjacent zinc finger, BAZ1B). WINAC is a key complex for repressing and activating transcription [76], and is required to enable DNA repli- cation through highly condensed regions of chromatin [77]. The BAZ1B protein contains a Brd, a PHD-type zinc finger motif, a WAKZ motif, and a leucine-rich hel- ical domain (Figure 1). BAZ1B is ubiquitously expressed in both adult and fetal tissues, such as limb buds, tail and brain from around E11.5 in the mouse embryo [7]. It is also expressed strongly in the cranial neural crest- derived mesenchyme, which drives facial morphogenesis. Phosphorylation of BAZ1B in a MAPK-dependent man- ner is important to maintain the WINAC complex as- sembly [76]. BAZ1B has been previously identified as contributing to WS [78] (Table 1). Total deletion was detected in 50 of 50 WS individuals using fluorescence in situ hybridization analysis. WS (OMIM ID: 194050) is a microdeletion or contiguous gene deletion syndrome characterized by hemizygous deletion of 1.5 to 1.8 Mb on chromosome 7q11.23 [78]. The frequency of WS is estimated to be 1 in 10,000. Subjects with WS show typ- ical craniofacial dysmorphology (a small upturned nose with a flat nasal bridge, mandibular hypoplasia, mal- occlusion, bi-temporal narrowing and prominent fore- head), supravalvular aortic stenosis, multiple peripheral pulmonary arterial stenosis, statural deficiency, infantile hypocalcemia and a distinct cognitive profile with mild mental retardation [79,80]. The patients suffer from spe- cific cognitive deficits, including poor visual-motor inte- gration and attention deficit. In terms of molecular

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Zinc finger proteins in cancer progression

Zinc finger proteins in cancer progression

The post-translational modifications (PTMs) of ZNFs, especially acetylation and phosphorylation, add another layer of regulation for ZNFs in which transcription may be activated or repressed. GATA1, a transcription factor that contains 2 highly conserved zinc finger motifs, is found acetylated at the lysine residues adjacent to the C terminal zinc finger by acetyltransferase CBP and p300. Acetylation of GATA1 shows stable association with chromatin probably by facilitating protein interactions, such as bromodomain-containing protein Brd3 [30–32]. Erythroid Krüppel-like factor, also known as EKLF, is acet- ylated at lysine residues 288 and 302 near its zinc finger motif mediated by CBP and p300 [33]. The acetylated EKLF at lysine residue 288 can transactivate β-globin expression through recruiting the large erythroid complex (ERC-1) that contains SWI/SNF chromatin-remodeling proteins and histone 3.3 [33, 34]. Another C2H2 zinc finger protein, YY1, is acetylated by p300/CBP associated factor (PCAF) at its zinc finger motif and inhibits its DNA

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Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response

Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response

FIG 1 Schematic of PbZfp and gene-targeting plasmids. PbZfp was identified by PSI-BLAST sequence profiling for C2H2 ZnF and BLAST analysis of PlasmoDB database entries using the protein sequence of mouse PRDM9 as a query. (A) Schematic of PbZfp protein (1,248 amino acids) and relative positions of 11 zinc fingers (boundaries are indicated in the text box), identified by PSI-BLAST analysis (panel I). A schematic of truncated PbZfp protein lacking Zfd expression is shown in panel II. (B) Schematic of targeting plasmids and parasite genomic locus, before and after transfection. Details of sequential steps used to construct plasmids are given in Materials and Methods. Key features of the plasmids used to generate PbZfp knockout and truncated parasites are shown in panels I and II, respectively. In the truncation plasmid shown in panel II, a stop codon (*) was inserted near the 5= end of the sequence coding for the Zfd. Various restriction sites are also identified. The wild-type Pbzfp genomic locus is represented in panel III. Panel IV shows the genomic locus in Pbzfp knockout (KO) parasites after homologous recombination events at the 5=utr and 3=utr sequences present in the targeting plasmid (panel I) and the wild-type genomic locus (panel III). Panel V shows the expected genomic locus after homologous recombination events at the PbZfp coding sequence and 3=utr sequences present in the plasmid (panel II) and wild-type genomic locus (panel III). Various oligonucleotide primers used for PCR amplification and characterization are shown by numbers (panels I and III). Forward primers are indicated in black numbers and reverse primers in red.

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The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs

The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3 ⴕ long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP’s activity, whereas disruption of the first and third fingers just slightly lowered its activity.

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Analysis of the interaction between Zinc finger protein 179 (Znf179) and promyelocytic leukemia zinc finger (Plzf)

Analysis of the interaction between Zinc finger protein 179 (Znf179) and promyelocytic leukemia zinc finger (Plzf)

Background: Zinc finger protein 179 (Znf179), also known as ring finger protein 112 (Rnf112), is a member of the RING finger protein family and plays an important role in neuronal differentiation. To investigate novel mechanisms of Znf179 regulation and function, we performed a yeast two-hybrid screen to identify Znf179-interacting proteins. Results: Using a yeast two-hybrid screen, we have identified promyelocytic leukemia zinc finger (Plzf) as a specific interacting protein of Znf179. Further analysis showed that the region containing the first two zinc fingers of Plzf is critical for its interaction with Znf179. Although the transcriptional regulatory activity of Plzf was not affected by Znf179 in the Gal4-dependent transcription assay system, the cellular localization of Znf179 was changed from cytoplasm to nucleus when Plzf was co-expressed. We also found that Znf179 interacted with Plzf and regulated Plzf protein expression.

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Role of Murine Leukemia Virus Nucleocapsid Protein in Virus Assembly

Role of Murine Leukemia Virus Nucleocapsid Protein in Virus Assembly

The retroviral nucleocapsid protein (NC) originates by cleavage of the Gag polyprotein. It is highly basic and contains one or two zinc fingers. Mutations in either the basic residues or the zinc fingers can affect several events of the virus life cycle. They frequently prevent the specific packaging of the viral RNA, affect reverse transcription, and impair virion assembly. In this work, we explore the role of NC in murine leukemia virus (MLV) particle assembly and release. A panel of NC mutants, including mutants of the zinc finger and of a basic region, as well as truncations of the NC domain of Gag, were studied. Several of these mutations dramatically reduce the release of virus particles. A mutant completely lacking the NC domain is apparently incapable of assembling into particles, although its Gag protein is still targeted to the plasma membrane. By electron microscopy on thin sections of virus-producing cells, we observed that some NC mutants exhibit various stages of budding defects at the plasma membrane and have aberrant particle morphology; electron micrographs of cells expressing some of these mutants are strikingly similar to those of cells expressing “late-domain” mutants. However, the defects of NC mutants with respect to virus release and infectivity could be complemented by an MLV lacking the p12 domain. Therefore, the functions of NC in virus budding and infectivity are completely distinct from viral late-domain function.

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Utility of I SceI and CCR5 ZFN nucleases in excising selectable marker genes from transgenic plants

Utility of I SceI and CCR5 ZFN nucleases in excising selectable marker genes from transgenic plants

Results: In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.

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Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases

Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases

Lethality of the yA nuclease: In earlier experiments with y and all of those with bw, the temperature used for induction of the ZFNs was limited to 35° because flies carrying the yA or bwB transgene survived poorly after heat shocks at higher temperatures (B ibikova et al. 2002, 2003). The yB, bwA, ryA, and ryB ZFNs conferred no lethality at any temperature. It seemed likely that the lethality was due to excessive cleavage when particular ZFNs were overexpressed. To test this, we made a single amino acid substitution in the FokI cleavage domain of yA. Replacement of aspartate with alanine at position 450 of natural FokI abolished cleavage without affecting DNA binding (W augh and S auer 1993). We made the corresponding mutation (D175A) in the coding se- quence for yA and introduced it into the genome behind a heat-shock promoter, exactly as was done for yA originally. Two independent transformants were isolated and tested for lethality after a 37° heat shock. Neither showed any reduction in viability relative to flies having no ZFN transgene. Western blot analysis con- firmed that the mutant protein was expressed at a level comparable to that of the original yA nuclease (data not shown). This demonstrates that the lethality of yA was likely due to excessive cleavage and rules out the alter- native hypothesis (B ibikova et al. 2002) that simple binding at one or more sites recognized by the mono- meric protein interferes with an essential chromosomal function, since the nuclease mutant, as well as the active yA protein, should still bind. Because the cleavage do- main must dimerize to cut DNA, we presume that, at high levels of expression, the yA zinc fingers bind to noncanonical sequences leading to cleavage at unantic- ipated sites.

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