scholarly journals In Vivo Site-Directed Mutagenesis of Yeast Plasmids Using a Three-Fragment Homologous Recombination System

BioTechniques ◽  
2002 ◽  
Vol 33 (2) ◽  
pp. 288-294 ◽  
Author(s):  
Katsumi Kitagawa ◽  
Rashid Abdulle
Keyword(s):  
Homologous Recombination ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Recombination System ◽  
Yeast Plasmids

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

scholarly journals Site-Directed Mutagenesis and Generation of Chimeric Viruses by Homologous Recombination in Yeast to Facilitate Analysis of Plant-Virus Interactions

2004 ◽  
Vol 17 (6) ◽  
pp. 571-576 ◽  
Author(s):  
Delin Liang ◽  
Stewart M. Gray ◽  
Igor Kaplan ◽  
Peter Palukaitis
Keyword(s):  
Homologous Recombination ◽  
Capsid Protein ◽  
Plant Virus ◽  
Shuttle Vector ◽  
Plant Viruses ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Recombination System ◽  
Virus Interactions

A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.

scholarly journals In vivo elimination of parental clones in general and site-directed mutagenesis

2015 ◽  
Vol 417 ◽  
pp. 67-75 ◽  
Author(s):  
Erika G. Holland ◽  
Felicity E. Acca ◽  
Kristina M. Belanger ◽  
Mary E. Bylo ◽  
Brian K. Kay ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis

FEBS Letters ◽  
1992 ◽  
Vol 299 (3) ◽  
pp. 267-272 ◽  
Author(s):  
Viktor Magdolen ◽  
Roland Schricker ◽  
Gertrud Strobel ◽  
Herbert Germaier ◽  
Wolfhard Bandlow
Keyword(s):  
Adenylate Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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