pi pspi  (New England Biolabs)


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    Structured Review

    New England Biolabs pi pspi
    Characterization of HEase plasticity in target recognition and its use for finding HEase targets in the human and other genomes. ( a ) and ( b ): HEase cleavage is tolerant of concomitant mutations at all wobble positions along the target site. The cleavage efficiency of the HEases <t>PI-SceI</t> (from S. cerevisiae) (a) and <t>PI-PspI</t> (from Pyrococcus species GB-D) (b) was assayed on different targets cloned on a bacterial plasmid. The cloned vectors were then fragmented by a restriction enzyme for the sake of visual clarity. Both HEases cleave targets mutated at all wobble positions (top DNA sequence) with higher efficiency than they cleave their native targets (bottom DNA sequence). Conversely, a single non-synonymous mutation (red arrow) is sometimes sufficient to abolish cleavage by PI-SceI and to reduce cleavage by PI-PspI. ( c ) and ( d ) : PI-SceI can cleave its predicted targets from the human ATP6V1A1 gene and its homologs in the genomes of animal models. (c) Alignment of the native target of the PI-SceI HEase from S. cerevisiae with the predicted targets in the human ATP6V1A1 gene and its homologs in the genomes of animal models. (d) Results of an in vitro cleavage assay demonstrating that PI-SceI can cleave its predicted targets from the genomes of diverse organisms. UC, uncut; RE, cut by a restriction enzyme (XbaI); HEase, cut by an HEase, RE + HEase, cut by XbaI AND by PI-SceI. S. cerevisiae, Saccharomyces cerevisiae; H. sapiens, Homo sapiens; C. familiaris, Canis familiaris; C. jacchus, Callithrix jacchus; M. musculus, Mus musculus; G. gallus, Gallus Gallus; R. norvegicus, Rattus norvegicus; D. rerio, Danio rerio .
    Pi Pspi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi pspi/product/New England Biolabs
    Average 94 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    pi pspi - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Native homing endonucleases can target conserved genes in humans and in animal models"

    Article Title: Native homing endonucleases can target conserved genes in humans and in animal models

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr242

    Characterization of HEase plasticity in target recognition and its use for finding HEase targets in the human and other genomes. ( a ) and ( b ): HEase cleavage is tolerant of concomitant mutations at all wobble positions along the target site. The cleavage efficiency of the HEases PI-SceI (from S. cerevisiae) (a) and PI-PspI (from Pyrococcus species GB-D) (b) was assayed on different targets cloned on a bacterial plasmid. The cloned vectors were then fragmented by a restriction enzyme for the sake of visual clarity. Both HEases cleave targets mutated at all wobble positions (top DNA sequence) with higher efficiency than they cleave their native targets (bottom DNA sequence). Conversely, a single non-synonymous mutation (red arrow) is sometimes sufficient to abolish cleavage by PI-SceI and to reduce cleavage by PI-PspI. ( c ) and ( d ) : PI-SceI can cleave its predicted targets from the human ATP6V1A1 gene and its homologs in the genomes of animal models. (c) Alignment of the native target of the PI-SceI HEase from S. cerevisiae with the predicted targets in the human ATP6V1A1 gene and its homologs in the genomes of animal models. (d) Results of an in vitro cleavage assay demonstrating that PI-SceI can cleave its predicted targets from the genomes of diverse organisms. UC, uncut; RE, cut by a restriction enzyme (XbaI); HEase, cut by an HEase, RE + HEase, cut by XbaI AND by PI-SceI. S. cerevisiae, Saccharomyces cerevisiae; H. sapiens, Homo sapiens; C. familiaris, Canis familiaris; C. jacchus, Callithrix jacchus; M. musculus, Mus musculus; G. gallus, Gallus Gallus; R. norvegicus, Rattus norvegicus; D. rerio, Danio rerio .
    Figure Legend Snippet: Characterization of HEase plasticity in target recognition and its use for finding HEase targets in the human and other genomes. ( a ) and ( b ): HEase cleavage is tolerant of concomitant mutations at all wobble positions along the target site. The cleavage efficiency of the HEases PI-SceI (from S. cerevisiae) (a) and PI-PspI (from Pyrococcus species GB-D) (b) was assayed on different targets cloned on a bacterial plasmid. The cloned vectors were then fragmented by a restriction enzyme for the sake of visual clarity. Both HEases cleave targets mutated at all wobble positions (top DNA sequence) with higher efficiency than they cleave their native targets (bottom DNA sequence). Conversely, a single non-synonymous mutation (red arrow) is sometimes sufficient to abolish cleavage by PI-SceI and to reduce cleavage by PI-PspI. ( c ) and ( d ) : PI-SceI can cleave its predicted targets from the human ATP6V1A1 gene and its homologs in the genomes of animal models. (c) Alignment of the native target of the PI-SceI HEase from S. cerevisiae with the predicted targets in the human ATP6V1A1 gene and its homologs in the genomes of animal models. (d) Results of an in vitro cleavage assay demonstrating that PI-SceI can cleave its predicted targets from the genomes of diverse organisms. UC, uncut; RE, cut by a restriction enzyme (XbaI); HEase, cut by an HEase, RE + HEase, cut by XbaI AND by PI-SceI. S. cerevisiae, Saccharomyces cerevisiae; H. sapiens, Homo sapiens; C. familiaris, Canis familiaris; C. jacchus, Callithrix jacchus; M. musculus, Mus musculus; G. gallus, Gallus Gallus; R. norvegicus, Rattus norvegicus; D. rerio, Danio rerio .

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Cleavage Assay

    2) Product Images from "Episomal Segregation of the Adenovirus Enhancer Sequence by Conditional Genome Rearrangement Abrogates Late Viral Gene Expression"

    Article Title: Episomal Segregation of the Adenovirus Enhancer Sequence by Conditional Genome Rearrangement Abrogates Late Viral Gene Expression

    Journal: Journal of Virology

    doi:

    Linear AdV that resolves into a circular episome. The elements involved in the self-directed rearrangement of the vector are shown schematically in pLEP1BHCRGFP/EBV and in the corresponding AdV. Starting from the left ITR (L.ITR), the elements are shown in the following sequence: L.ITR, 147 bp; first 34-bp loxP site; 185-bp enhancer/packaging signal; 64-bp splicing acceptor (SA) from EF1α gene first intron; 720-bp GFP cDNA; 230-bp simian virus 40 (SV40) poly(A) site; 1.7-kb TK-EBNA-1/OriP; 970-bp HCR12 promoter; 1-kb EF1α gene first intron containing splicing donor (SD) and acceptor (SA) sites with the second loxP site inserted at 64 bp upstream of the 3′ end; 1.2-kb Cre gene tagged with AU1 and a nuclear localization signal; ∼120-bp poly(A) signal and PI-PspI site. After infection of liver cells, the HCR12 promoter drives the expression of Cre, which results in cleavage of the two loxP sites. This results in circularization of the fragment containing the EBV replicon. The excision severs the connection between the enhancer/packaging signals and the remainder of the AdV genome. The Cre gene becomes promoterless and is left on the AdV genome fragment. After excision, the HCR12 promoter drives the expression of the GFP reporter gene. The EBV replicon maintains the excised circle as an episome in host cells.
    Figure Legend Snippet: Linear AdV that resolves into a circular episome. The elements involved in the self-directed rearrangement of the vector are shown schematically in pLEP1BHCRGFP/EBV and in the corresponding AdV. Starting from the left ITR (L.ITR), the elements are shown in the following sequence: L.ITR, 147 bp; first 34-bp loxP site; 185-bp enhancer/packaging signal; 64-bp splicing acceptor (SA) from EF1α gene first intron; 720-bp GFP cDNA; 230-bp simian virus 40 (SV40) poly(A) site; 1.7-kb TK-EBNA-1/OriP; 970-bp HCR12 promoter; 1-kb EF1α gene first intron containing splicing donor (SD) and acceptor (SA) sites with the second loxP site inserted at 64 bp upstream of the 3′ end; 1.2-kb Cre gene tagged with AU1 and a nuclear localization signal; ∼120-bp poly(A) signal and PI-PspI site. After infection of liver cells, the HCR12 promoter drives the expression of Cre, which results in cleavage of the two loxP sites. This results in circularization of the fragment containing the EBV replicon. The excision severs the connection between the enhancer/packaging signals and the remainder of the AdV genome. The Cre gene becomes promoterless and is left on the AdV genome fragment. After excision, the HCR12 promoter drives the expression of the GFP reporter gene. The EBV replicon maintains the excised circle as an episome in host cells.

    Techniques Used: Plasmid Preparation, Sequencing, Infection, Expressing

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    • pi pspi  (New England Biolabs)
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    New England Biolabs pi pspi
    Pi Pspi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi pspi/product/New England Biolabs
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pi pspi - by Bioz Stars, 2022-12
    94/100 stars
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