Homing Endonucleases and Inteins PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Homing Endonucleases and Inteins PDF full book. Access full book title Homing Endonucleases and Inteins by Marlene Belfort. Download full books in PDF and EPUB format.
Author: Marlene Belfort
Publisher: Springer Science & Business Media
ISBN: 9783540852353
Category : Science
Languages : en
Pages : 404
Get Book Here
Book Description
This book provides the first and only comprehensive description and detailed summary of the genetics, structure, function, mechanisms of action, evolution and engineering of homing endonucleases and inteins. These two unique protein superfamilies, which are tied together through their frequent fusion and coevolution, have generated considerable excitement for their fundamental, structural, and functional properties, their evolution as parasitic elements, and their widespread applications as gene targeting agents and as instruments for the generation of modified proteins and novel protein combinations.
Author: Marlene Belfort
Publisher: Springer Science & Business Media
ISBN: 9783540852353
Category : Science
Languages : en
Pages : 404
Get Book Here
Book Description
This book provides the first and only comprehensive description and detailed summary of the genetics, structure, function, mechanisms of action, evolution and engineering of homing endonucleases and inteins. These two unique protein superfamilies, which are tied together through their frequent fusion and coevolution, have generated considerable excitement for their fundamental, structural, and functional properties, their evolution as parasitic elements, and their widespread applications as gene targeting agents and as instruments for the generation of modified proteins and novel protein combinations.
Author: David R. Edgell
Publisher:
ISBN: 9781627039680
Category : Endonucleases
Languages : en
Pages : 282
Get Book Here
Book Description
Homing Endonucleases: Methods and Protocols aims at providing molecular biologists with a comprehensive resource to identify and characterize homing endonucleases from genomic sequence, to deduce the biological basis of binding and cleavage specificity, as well as to provide protocols to redesign endonuclease target specificity for genome-editing applications. Engineering of designer homing endonucleases has set the stage for genome editing of complex eukaryotic genomes with a broad range of potential applications including targeted gene knockouts in model organisms and gene therapy in humans, making this book a valuable resource for future research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Homing Endonucleases: Methods and Protocols serves as a key reference for all labs studying site-specific DNA endonucleases.
Author: Alfred Pingoud
Publisher: Springer Science & Business Media
ISBN: 3642188516
Category : Science
Languages : en
Pages : 462
Get Book Here
Book Description
Restriction enzymes are highly specific nucleases which occur ubiquitously among prokaryotic organisms, where they serve to protect bacterial cells against foreign DNA. Many different types of restriction enzymes are known, among them multi-subunit enzymes which depend on ATP or GTP hydrolysis for target site location. The best known representatives, the orthodox type II restriction endonucleases, are homodimers which recognize palindromic sequences, 4 to 8 base pairs in length, and cleave the DNA within or immediately adjacent to the recognition site. In addition to their important biological role (up to 10 % of the genomes of prokaryotic organisms code for restriction/modification systems!), they are among the most important enzymes used for the analysis and recombination of DNA. In addition, they are model systems for the study of protein-nucleic acids interactions and, because of their ubiquitous occurence, also for the understanding of the mechanisms of evolution.
Author: Gregory K. Taylor
Publisher:
ISBN:
Category :
Languages : en
Pages : 97
Get Book Here
Book Description
Homing endonucleases, found in all forms of microbial life, facilitate the invasion of host genes often in concert with introns or inteins by generating double stranded breaks in conserved coding sequences. There are five homing endonuclease families distinct in their structural characteristics and each family appears to share a common ancestor with diverse host proteins of unrelated function. Such related proteins include restriction endonucleases, DNA mismatch repair proteins, transcription factors, four-way junction resolving enzymes, and colicins. Homing endonculeases are currently being computationally redesigned for applications in genome engineering and structures of three redesigned homing endonuclease variants are described. In these experiments, crystal structures uncovered unexpected shifts in the DNA backbone relative to the wild type endonucleases and have thus been informative in the redesign process. Recently, a sixth homing endonuclease family homologous to E. Coli DNA repair protein VSR was discovered. A series of biochemical and x-ray crystallographic experiments investigating binding specificity and catalytic mechanism of a representative family member I-Bth0305I are described. Finally, a database archiving experimentally characterized homing endonucleases and a web-base program supporting homing endonuclease target site search are discussed.
Author: Alireza Ghodsi Senejani
Publisher:
ISBN:
Category :
Languages : en
Pages : 234
Get Book Here
Book Description
Author: Kyle M. Jacoby
Publisher:
ISBN:
Category :
Languages : en
Pages : 98
Get Book Here
Book Description
LAGLIDADG Homing Endonucleases (LHEs) are a family of highly specific DNA-cutting enzymes capable of recognizing target sequences of ~20 bp. In many eukaryotes, including humans and yeast, double-strand breaks induced by LHEs stimulate repair by Homologous Recombination, which can be used to alter or repair a gene if the template is supplied in trans, and Non-Homologous End Joining, which can be used to knock out a gene. The potential for such precise genome editing would reduce worry about insertional mutagenesis or misregulation, as only the specific gene under its native promoter would be targeted. Thus, LHEs have drawn intense interest for their research, biotech and clinical applications. Methods for rational engineering of LHEs have been limited by a small number of high quality starting enzymes, and an extremely restricted understanding of how to modify them to create novel enzymes that efficiently cleave hybrid target sequences. Here I describe my attempts to address these limitations by using a homology-directed search method to acquire, characterize, and engineer a robust set of I-OnuI-related LHEs which recognize a diverse set of target sequences. A system of iterative binding selection using yeast surface display was also developed to identify target sites for, and perform non-directed analysis of, previously uncharacterized enzymes. This diverse family of LHEs will serve both as a platform from which to launch short-distance designs, and a dataset to improve our understanding of protein-DNA interactions.
Author: Austin BURT
Publisher: Harvard University Press
ISBN: 0674029119
Category : Science
Languages : en
Pages : 613
Get Book Here
Book Description
Covering all species from yeast to humans, this is the first book to tell the story of selfish genetic elements that act narrowly to advance their own replication at the expense of the larger organism.
Author: Tuhin Guha
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential 'on/off switch' for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies.
Author: Weihua Qiu (Ph. D.)
Publisher:
ISBN:
Category :
Languages : en
Pages : 226
Get Book Here
Book Description
Homing endonucleases (HEs) typically have one of four types of catalytic domains (LAGLIDADG, GIY-YIG, H-N-H, His-Cys), and a DNA-binding region(s) that provides specificity. I-CreII, which is encoded by the psbA4 intron from Chlamydomonas reinhardtii, is unusual in containing two catalytic motifs: H-N-H and GIY-YIG. A previous study showed that I-CreII cleavage leaves 2-nt 3' OH overhangs similar to GIYYIG endonucleases, but that it also has a flexible metal requirement like H-N-H enzymes. Also, alanine substitution of several conserved residues in the GIY-YIG motif and two in the H-N-H motif did not produce a clear catalytic mutant, although some variants had strongly reduced DNA binding. Thus, in order to identify the catalytic motif, I substituted additional amino acids in both domains with alanine, and identified three histidines in the H-N-H motif that are likely to be involved in catalysis. To gain insight into how I-CreII interacts with its ~30-bp homing-site DNA, three types of DNA protection analysis were performed. Hydroxyl-radical footprinting, which reveals regions of tight DNA binding, indicated that I-CreII binds strongly to a region downstream of the cleavage and intron-insertion sites, corresponding to bp 2-10 of exon 5. There was also partial protection around the cleavage site, but only on the top strand, which is consistent with the enzyme's tendency to cleave this strand first. DNase I protection, which can reveal less closely-bound regions of target DNA, gave a larger footprint than hydroxyl-radical protection, with the additional region lying upstream of the cleavage site. These results also suggest that DNA backbone-binding downstream of the cleavage site involves sugars and phosphates, whereas upstream it is mainly with phosphates. DMS protection, which probes guanines on the N-7 position in the major groove, did not provide any evidence of major groove binding (at least not through guanines). DNase I protection could also be performed on the I-CreII variants that had reduced DNA affinity. The N161A variant was instructive in that it showed reduced protection of a T-A bp very close to the cleavage site, providing support for a catalytic role for the H-N-H motif and a possible constraint for modeling. Of the GIY-YIG motif variants, the footprint of the G231E/K245A variant was distinctly useful in that it was preferentially effected downstream of the cleavage site. This result suggested the H-N-H and GIY-YIG motifs are co-linear with their targets in the homing site. Structural modeling of the GIY-YIG domain of I-CreII using the solved I-TevI domain as template provided evidence for a unique insertion in the I-CreII structure that disrupted a catalytic [alpha]-helix; the insertion is predicted to be a positively charged, hairpinlike loop anchored by two antiparallel [beta]-strands. I propose that this insertion can explain the evolutionary conversion of this catalytic endonuclease domain into a DNA-binding domain. These findings should also help to understand other dual-motif H-N-H/GIY-YIG endonucleases, such as I-CmoeI.
Author: Jue Lin
Publisher:
ISBN:
Category :
Languages : en
Pages : 402
Get Book Here
Book Description
