Effect of de-phosphorylation of linearized pAN7-1 and of addition of restriction enzyme on plasmid integration in Penicillium paxilli

1997 ◽  
Vol 32 (2) ◽  
pp. 147-151 ◽  
Author(s):  
Y. Itoh ◽  
Barry Scott
Keyword(s):  
Restriction Enzyme ◽  
Plasmid Integration ◽  
Penicillium Paxilli

Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration

1998 ◽  
Vol 33 (5) ◽  
pp. 368-377 ◽  
Author(s):  
Carolyn Young ◽  
Yasuo Itoh ◽  
R. Johnson ◽  
I. Garthwaite ◽  
Christopher O. Miles ◽  
...  
Keyword(s):  
Plasmid Integration ◽  
Penicillium Paxilli

scholarly journals AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration

1997 ◽  
Vol 87 (9) ◽  
pp. 967-972 ◽  
Author(s):  
H. Akamatsu ◽  
Y. Itoh ◽  
M. Kodama ◽  
H. Otani ◽  
K. Kohmoto
Keyword(s):  
Restriction Enzyme ◽  
Alternaria Alternata ◽  
Restriction Enzymes ◽  
Toxin Production ◽  
Molecular Genetic Analysis ◽  
Transformation Frequency ◽  
Fungal Genome ◽  
Plasmid Integration ◽  
Tomato Pathotype ◽  
Aal Toxin

Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.

scholarly journals Restriction Enzyme-Mediated Integration Used to Produce Pathogenicity Mutants of Colletotrichum graminicola

2000 ◽  
Vol 13 (12) ◽  
pp. 1356-1365 ◽  
Author(s):  
M. R. Thon ◽  
E. M. Nuckles ◽  
L. J. Vaillancourt
Keyword(s):  
Restriction Enzyme ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Transformation Efficiency ◽  
Colletotrichum Graminicola ◽  
Wild Type ◽  
Plasmid Integration ◽  
Integration Sites ◽  
Enzyme Recognition

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Multiple, Tandem Plasmid Integration in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (4) ◽  
pp. 747-749
Author(s):  
Terry L. Orr-Weaver ◽  
Jack W. Szostak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Enzyme ◽  
Tandem Array ◽  
Plasmid Integration ◽  
Genomic Location ◽  
Homologous Site ◽  
Multiple Copies

Nonreplicating plasmids transform Saccharomyces cerevisiae by recombining with a homologous site in the genome. Frequently, multiple copies of the plasmid integrate in a tandem array. We show that, after transformation with restriction enzyme-cut plasmids, most, if not all, multimers arise by sequential integration of plasmid molecules into the same genomic location.

scholarly journals Multiple, Tandem Plasmid Integration inSaccharomyces cerevisiae

1983 ◽  
Vol 3 (4) ◽  
pp. 747-749 ◽  
Author(s):  
Terry L. Orr-Weaver ◽  
Jack W. Szostak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Enzyme ◽  
Tandem Array ◽  
Plasmid Integration ◽  
Genomic Location ◽  
Homologous Site ◽  
Multiple Copies

Nonreplicating plasmids transformSaccharomyces cerevisiaeby recombining with a homologous site in the genome. Frequently, multiple copies of the plasmid integrate in a tandem array. We show that, after transformation with restriction enzyme-cut plasmids, most, if not all, multimers arise by sequential integration of plasmid molecules into the same genomic location.

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