pi pspi  (New England Biolabs)

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkr242

Figure Lengend 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 .

Article Snippet: Extra-cellular cleavage assays A quantity of 1 µg of a plasmid [pGEM derivative ( a and b) or pDELT derivative ( d)] carrying a native, mutated or predicted target of PI-SceI ( a and d) or PI-PspI ( b) were subjected to cleavage using 1 U of each enzyme as provided by New Englands Biolabs (at 29 pmol/U for PI-SceI and 80 fmol/U for PI-PspI), in a 50 µl reaction at 37°C (PI-SceI) or 65°C (PI-PspI).

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

Journal: Journal of Virology

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

doi:

Figure Lengend 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.

Article Snippet: Restriction enzymes, intron endonucleases I-CeuI and PI-PspI, and Moloney murine leukemia virus reverse transcriptase (RT) were purchased from New England Biolabs, Stratagene, or Promega.

Techniques: Plasmid Preparation, Sequencing, Infection, Expressing