scholarly journals Single-Primer PCR Procedure for Rapid Identification of Transposon Insertion Sites

BioTechniques ◽  
2000 ◽  
Vol 28 (6) ◽  
pp. 1078-1082 ◽  
Author(s):  
Andrey V. Karlyshev ◽  
Mark J. Pallen ◽  
Brendan W. Wren
Keyword(s):  
Rapid Identification ◽  
Transposon Insertion ◽  
Insertion Sites ◽  
Single Primer

scholarly journals Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

2012 ◽  
Vol 93 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Lopamudra Giri ◽  
Michael G. Feiss ◽  
Bryony C. Bonning ◽  
David W. Murhammer
Keyword(s):  
Large Scale ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Systematic Method ◽  
Defective Interfering Particles ◽  
Extracellular Virus ◽  
Transposon Insertion ◽  
Target Sites ◽  
Insertion Sites

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes

2006 ◽  
Vol 66 (2) ◽  
pp. 276-285 ◽  
Author(s):  
Johan H.J. Leveau ◽  
Saskia Gerards ◽  
Kathrin Fritsche ◽  
Gerben Zondag ◽  
Johannes A. van Veen
Keyword(s):  
Rapid Identification ◽  
Functional Genes ◽  
Insertion Sites

YES1 amplification as a mechanism of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs) identified by a transposon mutagenesis screen and clinical genomic testing.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 9043-9043 ◽  
Author(s):  
Pang-Dian Fan ◽  
Giuseppe Narzisi ◽  
Anitha Jayaprakash ◽  
Elisa Venturini ◽  
Nicolas Robine ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Transposon Mutagenesis ◽  
Src Family Kinase ◽  
Mechanisms Of Resistance ◽  
Transposon Insertion ◽  
Insertion Sites ◽  
Egfr Tki ◽  
Egfr Tkis

9043 Background: Overcoming AR to EGFR TKIs remains challenging, and in many cases the mechanisms are still unclear. To identify novel mechanisms of resistance to EGFR TKIs, we performed a forward genetic screen using transposon mutagenesis in EGFR-mutant lung adenocarcinoma cells. Methods: EGFR TKI-sensitive PC9 cells were co-transfected with plasmids encoding a mutagenic piggyBactransposon and hyperactive piggyBac transposase. Transposon-tagged, afatinib-resistant clones were generated by sequential selection of transfected cells with puromycin and 1µM afatinib. Transposon insertion sites were mapped using a modified TraDIS-type method and next-generation sequencing (NGS). Selected clones were characterized using Western blots, receptor tyrosine kinase (RTK) arrays, and viability assays following treatment with TKIs or siRNA-mediated gene knockdowns. We reviewed MSK-IMPACT™ NGS data on 100 patient tumors with EGFR TKI AR. Available tumor samples were analyzed by fluorescence in situ hybridization (FISH). Results: In 187/188 afatinib-resistant clones, transposon insertion sites consistent predominantly with gene upregulation were found in MET, the Src family kinase (SFK) member YES1, or both. Clones with activating YES1 insertions exhibited resistance to all three generations of EGFR TKIs; high levels of expression of tyrosine-phosphorylated YES1; sensitivity to the SFK TKI dasatinib and to siRNA-mediated knockdown of YES1; and tyrosine phosphorylation of YAP1 and ERBB3. A query of the MSK-IMPACT™ data on EGFR TKI AR patients revealed amplification of YES1 and no alteration of MET, ERBB2 or BRAF in 3/54 T790M-negative (95% CI 1 to 16%) and 1/46 (95% CI 1 to 12%) T790M-positive cases. Amplification of YES1was confirmed by FISH in 2/2 cases, and was absent in matched pre-TKI samples in 2/2 cases. Conclusions: YES1 amplification is found in 4% of patients with acquired resistance to EGFR TKIs and is potentially targetable by Src family kinase inhibitors. Forward genetic screens using transposon mutagenesis and routine clinical NGS of patient samples can identify novel mechanisms of resistance to targeted therapies.

Genome-wide sequence transposon insertion sites and analyze the essential genes of Brucella melitensis

2017 ◽  
Vol 112 ◽  
pp. 97-102 ◽  
Author(s):  
Yanyan De ◽  
Congyue Dong ◽  
Yanyang Cao ◽  
Xiaolei Wang ◽  
Xiaowen Yang ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Essential Genes ◽  
Transposon Insertion ◽  
Genome Wide ◽  
Insertion Sites

scholarly journals Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer

2017 ◽  
Vol 114 (5) ◽  
pp. E733-E740 ◽  
Author(s):  
Zuojian Tang ◽  
Jared P. Steranka ◽  
Sisi Ma ◽  
Mark Grivainis ◽  
Nemanja Rodić ◽  
...  
Keyword(s):  
Serous Carcinoma ◽  
Transposon Insertion ◽  
Human Genomes ◽  
Mammalian Genomes ◽  
Insertion Sites ◽  
Interspersed Repeats ◽  
Long Interspersed Element ◽  
Profiling Analysis ◽  
L1 Retrotransposon ◽  
Generation Sequencing

Mammalian genomes are replete with interspersed repeats reflecting the activity of transposable elements. These mobile DNAs are self-propagating, and their continued transposition is a source of both heritable structural variation as well as somatic mutation in human genomes. Tailored approaches to map these sequences are useful to identify insertion alleles. Here, we describe in detail a strategy to amplify and sequence long interspersed element-1 (LINE-1, L1) retrotransposon insertions selectively in the human genome, transposon insertion profiling by next-generation sequencing (TIPseq). We also report the development of a machine-learning–based computational pipeline, TIPseqHunter, to identify insertion sites with high precision and reliability. We demonstrate the utility of this approach to detect somatic retrotransposition events in high-grade ovarian serous carcinoma.

scholarly journals Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

PLoS Genetics ◽  
2009 ◽  
Vol 5 (11) ◽  
pp. e1000733 ◽  
Author(s):  
Sanzhen Liu ◽  
Cheng-Ting Yeh ◽  
Tieming Ji ◽  
Kai Ying ◽  
Haiyan Wu ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Open Chromatin ◽  
Maize Genome ◽  
Epigenetic Marks ◽  
Transposon Insertion ◽  
Insertion Sites

scholarly journals Sequence Analysis of TnphoA Insertion Sites in Vibrio cholerae Mutants Defective in Rugose Polysaccharide Production

2000 ◽  
Vol 68 (12) ◽  
pp. 6857-6864 ◽  
Author(s):  
Afsar Ali ◽  
Zahid Hayat Mahmud ◽  
J. Glenn Morris ◽  
Shanmuga Sozhamannan ◽  
Judith A. Johnson
Keyword(s):  
Vibrio Cholerae ◽  
Pyruvate Dehydrogenase Complex ◽  
Gene Encoding ◽  
Polysaccharide Biosynthesis ◽  
E Coli ◽  
Polysaccharide Production ◽  
General Utility ◽  
Transposon Insertion ◽  
Insertion Sites ◽  
El Tor

ABSTRACT Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated TnphoA-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the TnphoA insertions was mapped to the first gene of thevps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA-like gene, also in thevps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsDgene (encodes the “secretin” of the extracellular protein secretion apparatus), (ii) in a hydG-like gene (encodes a ς54-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli(encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy-d-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.

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