Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi

1999 ◽  
Vol 380 (7-8) ◽  
pp. 855-864 ◽  
Author(s):  
F. J. Maier ◽  
W. Schäfer
Keyword(s):  
Restriction Enzyme ◽  
Molecular Basis ◽  
Insertional Mutagenesis ◽  
Transformation Efficiency ◽  
Pathogenic Fungi ◽  
Transformation Protocol ◽  
Vector Integration ◽  
Future Developments ◽  
Genetically Determined ◽  
Remi Mutagenesis

Abstract Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

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.

SOYBEAN AND FLAX SELECTION METHODS

1970 ◽  
pp. 19-23
Author(s):  
V. М. Lukomets ◽  
S. V. Zelentsov
Keyword(s):  
High Resistance ◽  
Fungal Pathogens ◽  
Selection Process ◽  
Pathogenic Fungi ◽  
Cultivated Plants ◽  
Practical Implementation ◽  
Physiological Basis ◽  
Oil Crops ◽  
Soybean Genotypes ◽  
Genetically Determined

To improve the effectiveness of the soybeans and oil flax breeding, research to improve existing and develop new breeding methods are conducting in all-Russia Research institute of Oil Crops (Krasnodar). One of the improved methods for the soybean breeding, based on the use of sources of complexes of compensatory genes, is the CCG technology, which allows to create varieties with an increased yield of a heterotic level transmitted along the progeny for the entire life cycle of the variety. For the purpose of non-transgenic production of new traits, a theory of polyploid recombination of the genome (TPR) was formulated, which models the mechanism of the natural formation of polymorphism in the centers of origin of cultivated plants. On the basis of this theory, a method of breeding (TPR-technology) has been developed, which makes it possible to obtain recombinant reploids of soybeans and oil flax with an extended spectrum of traits. Of these reploids, the soybean lines with increased sucking force of the roots, providing high drought resistance, were distinguished; cold-resistant soybean lines, which stand in the phase of shoots of freezing to minus 5 °С; lines of oil flax with complete resistance to flax sickness of soil and high resistance to Fusarium; winter-hardy flax lines that withstand winter frosts down to minus 20–23 °С and ripen one and a half months earlier than spring sowings. Another original developed method is the ODCS-technology for isolating and selecting soybean genotypes with high resistance to fungal pathogens. The physiological basis of ODCS-technology is the blocking of osmotic nutrition of pathogenic fungi due to genetically determined increased osmotic pressure in the tissues of host plants. The practical implementation of CCG-, TPR- and ODKS-technologies in the selection process, allowed to create a whole series of soybean and oil flax varieties with improved or new traits.

scholarly journals The diversity of fungi colonizing necrotic inflorescence buds of rhododendron (Rhododendron L.)

2013 ◽  
Vol 66 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Małgorzata Żołna ◽  
Barbara Kierpiec-Baran ◽  
Maria Kowalik
Keyword(s):  
Dominant Species ◽  
Trichoderma Viride ◽  
Ornamental Plants ◽  
Pathogenic Fungi ◽  
Lower Number ◽  
Botanical Species ◽  
Fusarium Poae ◽  
Identification Of Fungi ◽  
Improved Cultivars ◽  
Genetically Determined

<p>The infection of rhododendron (<em>Rhododendron </em>L.) inflorescence buds caused by pathogenic fungi induces its browning, withering, and dieback. The identification of fungi causing the infection of rhododendron inflorescence buds can be a reason for creating new improved cultivars with genetically determined resistance to pathogens. The investigations were carried out in 2010–2011 on the collection of ornamental plants of the Faculty of Horticulture, University of Agriculture in Kraków. The material comprised infected inflorescence buds collected from nine newly bred taxa and one botanical species of rhododendron. 596 colonies of fungi belonging to 31 species were isolated from infected rhododendron inflorescence buds. The dominant species were: <em>Pestalotiopsis sydowiana</em>, <em>Truncatella truncata</em>, <em>Alternaria alternata</em>, <em>Phialophora asteris,</em> and <em>Trichoderma viride</em>, which constituted almost 74% of the isolated fungi population.<em> Boeremia exigua </em>var<em>. exigua</em>,<em> Epicoccum nigrum</em>, <em>Fusarium poae</em>, <em>Mammaria echinobotryoides</em>, <em>Paraphoma chrysanthemicola</em>, <em>Phialophora cyclaminis</em>,<em> Phoma eupyrena</em>, <em>Talaromyces wortmannii</em>, <em>Umbelopsis isabellina</em>, and other fungi were isolated in a lower number.</p><p>The results of mycological analysis confirm the diversity of species colonizing necrotic inflorescence buds of rhododendron.</p>.

scholarly journals Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

2007 ◽  
Vol 189 (24) ◽  
pp. 8914-8921 ◽  
Author(s):  
Anna Skoglund ◽  
Britta Björkholm ◽  
Christina Nilsson ◽  
Anders F. Andersson ◽  
Cecilia Jernberg ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Restriction Enzyme ◽  
Dna Methyltransferase ◽  
Insertional Mutagenesis ◽  
Phase Variation ◽  
Restriction Enzyme Digestion ◽  
Wild Type ◽  
Gene Mutant ◽  
H Pylori

ABSTRACT A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

scholarly journals CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER), a novel method for selective transformation

2015 ◽  
Author(s):  
Lynn J. Rothschild ◽  
Daniel T. Greenberg ◽  
Jack R. Takahashi ◽  
Kirsten A. Thompson ◽  
Akshay J. Maheshwari ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Convenient Method ◽  
Transformation Efficiency ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Gene Insertion ◽  
Dna Targeting ◽  
Selective Transformation ◽  
Novel Method ◽  
Selection For

The CRISPR/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be applied to facilitate efficient plasmid selection for transformation as well as selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts after restriction enzyme digestion and ligation. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes unwanted ligation byproducts and increases transformation efficiency of desired inserts from 20% up to 97% ± 3%. This CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient method for obtaining specific cloning products.

scholarly journals Linear Amplification Mediated PCR for the retrieval of lentiviral vector integration sites from tumors induced by insertional mutagenesis.

2013 ◽  
Author(s):  
Marco Ranzani ◽  
Marco Ranzani ◽  
Stefano Annunziato ◽  
Fabrizio Benedicenti ◽  
Pierangela Gallina ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Lentiviral Vector ◽  
Linear Amplification ◽  
Vector Integration ◽  
Integration Sites

Fifty Years of Medical Genetics

PEDIATRICS ◽  
1959 ◽  
Vol 23 (3) ◽  
pp. 581-581
Keyword(s):  
Medical Literature ◽  
Medical Science ◽  
Medical Genetics ◽  
Future Developments ◽  
General Terms ◽  
Hereditary Factors ◽  
Metabolic Reactions ◽  
Genetically Determined ◽  
Made In

Many articles are appearing in the medical literature which aim to provide a discussion of the application of genetics to medical science in general terms for the benefit of the clinician without training in genetics. The present lecture is an especially lucid and stimulating account of the elements of genetics and the progress that is being made in the elucidation of diseases by the application of genetics. The manner in which metabolic reactions may be affected, directly or indirectly, by genetically determined enzymic defects is briefly and clearly presented. The interplay between environmental and hereditary factors is considered. Sufficient background is provided to enable one to observe future developments in the application of genetics to medicine with a better understanding and consequently keener interest.

scholarly journals Frequency and Spectrum of Genomic Integration of Recombinant Adeno-Associated Virus Serotype 8 Vector in Neonatal Mouse Liver

2008 ◽  
Vol 82 (19) ◽  
pp. 9513-9524 ◽  
Author(s):  
Katsuya Inagaki ◽  
Chuncheng Piao ◽  
Nicole M. Kotchey ◽  
Xiaolin Wu ◽  
Hiroyuki Nakai
Keyword(s):  
Insertional Mutagenesis ◽  
Neonatal Period ◽  
Integration Site ◽  
Adeno Associated Virus ◽  
Genomic Integration ◽  
Vector Integration ◽  
Multiple Organs ◽  
Vector Genome ◽  
Virus Serotype ◽  
Integration Sites

ABSTRACT Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 × 1011 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a β-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ∼0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was ∼0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 103 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.

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