scholarly journals Shotgun Cloning of Transposon Insertions in the Genome ofCaenorhabditis elegans

2004 ◽  
Vol 5 (3) ◽  
pp. 225-229 ◽  
Author(s):  
Alexander M. van der Linden ◽  
Ronald H. A. Plasterk
Keyword(s):  
Transposable Elements ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Genomic Sequence ◽  
Mutagenesis Screen ◽  
Mutator Strain ◽  
C Elegans ◽  
Large Numbers ◽  
Transposon Insertions ◽  
Insertional Mutagenesis Screen

We present a strategy to identify and map large numbers of transposon insertions in the genome ofCaenorhabditis elegans. Our approach makes use of the mutator strainmut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence ofC. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predictedC. elegansgenes. The strategy presented provides an approach to isolate insertions of natural transposable elements in manyC. elegansgenes and to create a large-scale collection ofC. elegansmutants.

scholarly journals Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare

2019 ◽  
Author(s):  
Ken Harata ◽  
Tetsuro Okuno
Keyword(s):  
Host Plants ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Morphological Differentiation ◽  
Wild Type ◽  
Colletotrichum Orbiculare ◽  
Mutagenesis Screen ◽  
Threonine Synthase ◽  
Infection Processes ◽  
Insertional Mutagenesis Screen

AbstractUpon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.

scholarly journals A large-scale insertional mutagenesis screen in zebrafish

1999 ◽  
Vol 13 (20) ◽  
pp. 2713-2724 ◽  
Author(s):  
A. Amsterdam ◽  
S. Burgess ◽  
G. Golling ◽  
W. Chen ◽  
Z. Sun ◽  
...  
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Mutagenesis Screen ◽  
Insertional Mutagenesis Screen

scholarly journals A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma

2009 ◽  
Vol 27 (3) ◽  
pp. 264-274 ◽  
Author(s):  
Vincent W Keng ◽  
Augusto Villanueva ◽  
Derek Y Chiang ◽  
Adam J Dupuy ◽  
Barbara J Ryan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Insertional Mutagenesis ◽  
Mutagenesis Screen ◽  
Insertional Mutagenesis Screen

Retroviral Insertional Mutagenesis Screen in a C/EBPalpha Proliferative Genetic Background.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4342-4342
Author(s):  
Marie S. Hasemann ◽  
Annette B. Sørensen ◽  
Finn S. Pedersen ◽  
Claus Nerlov ◽  
Bo Porse
Keyword(s):  
Myeloid Leukemia ◽  
Insertional Mutagenesis ◽  
Southern Blotting ◽  
Insertion Mutagenesis ◽  
Neutrophil Granulocytes ◽  
Mutagenesis Screen ◽  
T Cell Lymphomas ◽  
Enhancer Binding Protein ◽  
Regulation Of Growth ◽  
Insertional Mutagenesis Screen

Abstract The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours after birth. In contrast, homozygous knockin mice in which the wild type Cebpa allele has been replaced with a mutant allele (BRM2) deficient in repressing the activity of E2F family members, are viable. At 8 weeks of age these animals display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older BRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia. These findings strongly suggest that secondary mutations in other loci must occur during the phenotypic progression. In order to identify genes that cooperate with C/EBPalpha in the development of leukemia in BRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn BRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus and when disease is evident the mice are euthanized and analyzed. As expected the BRM2/BRM2 mice have a shorter latency than wildtype mice (182 vs. 260 days). The mice have enlarged spleen, thymus, and lymph nodes and were further characterized by histology, flow cytometry and Southern blotting in order to determine the hematopoietic phenotypes. Most abundantly was the AML-like phenotype, but also T-cell lymphomas are developing. Finally, the loci carrying retroviral insertions loci are identified through a splinkerette-aided PCR strategy. This study provides a better understanding of the genes involved in the development of myeloid leukemia.

A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2990-2990 ◽  
Author(s):  
Zhe Li ◽  
Jan-Henning Klusmann ◽  
Frank J. Godinho ◽  
Hee-Won Lee ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Insertional Mutagenesis ◽  
Fetal Liver ◽  
Myeloproliferative Disorder ◽  
Mutagenesis Screen ◽  
Retroviral Integration ◽  
Genome Wide ◽  
A Genome ◽  
Integration Sites ◽  
Insertional Mutagenesis Screen

Abstract Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.

scholarly journals High-throughput imaging of Caenorhabditis elegans aging using collective activity monitoring

2021 ◽  
Author(s):  
Anthony D Fouad ◽  
Matthew A Churgin ◽  
Julia Hayden ◽  
Joyce Xu ◽  
Jeong-Inn Park ◽  
...  
Keyword(s):  
Large Scale ◽  
Imaging System ◽  
Activity Monitoring ◽  
Automated System ◽  
Aging Research ◽  
C Elegans ◽  
Collective Activity ◽  
Large Numbers ◽  
Natural Isolates ◽  
Rate Of Decline

The genetic manipulability and short lifespan of C. elegans make it an important model for aging research. Widely applied methods for measurements of worm aging based on manual observation are labor intensive and low-throughput. Here, we describe the Worm Collective Activity Monitoring Platform (WormCamp), a system for assaying aging in C. elegans by monitoring activity of populations of worms in standard 24-well plates. We show that metrics based on the rate of decline in collective activity can be used to estimate the average lifespan and locomotor healthspan in the population. Using the WormCamp, we assay a panel of highly divergent natural isolates of C. elegans and show that both lifespan and locomotor healthspan display substantial heritability. To facilitate analysis of large numbers of worms, we developed a robotic imaging system capable of simultaneous automated monitoring of activity, lifespan, and locomotor healthspan in up to 2,304 populations containing a total of ~90,000 animals. We applied the automated system to conduct a large-scale RNA interference screen for genes that affect lifespan and locomotor healthspan. The WormCamp system is complementary to other current automated methods for assessing C. elegans aging and is well suited for efficiently screening large numbers of conditions.

scholarly journals Genome ARTIST: a robust, high-accuracy aligner tool for mapping transposon insertions and self-insertions

2015 ◽  
Author(s):  
Alexandru Al. Ecovoiu ◽  
Iulian Constantin Ghionoiu ◽  
Andrei Mihai Ciuca ◽  
Attila Cristian Ratiu
Keyword(s):  
Insertional Mutagenesis ◽  
Genomic Sequence ◽  
Fruit Fly ◽  
High Accuracy ◽  
Model Organisms ◽  
Robust Solution ◽  
Small Indels ◽  
Mapping Tool ◽  
Transposon Insertions ◽  
User Friendly

A critical topic of insertional mutagenesis experiments performed on model organisms is mapping the hits of artificial transposons (ATs) at nucleotide level accuracy. Obviously, mapping errors may occur when sequencing artifacts or mutations as SNPs and small indels are present very close to the junction between a genomic sequence and a transposon inverted repeat (TIR). Another particular item of insertional mutagenesis is mapping of the transposon self-insertions and, to our best knowledge, there is no publicly available mapping tool designed to analyze such molecular events. We developed Genome ARTIST, a pairwise gapped aligner tool which works out both issues by means of an original, robust mapping strategy. Genome ARTIST is not designed to use NGS data but to analyze ATs insertions obtained in small to medium-scale mutagenesis experiments. Genome ARTIST employs a heuristic approach to find DNA sequence similarities and harnesses a multi-step implementation of a Smith-Waterman adapted algorithm to compute the mapping alignments. The experience is enhanced by easily customizable parameters and a user-friendly interface that describes the genomic landscape surrounding the insertion. Genome ARTIST deals with many genomes of bacteria and eukaryotes available in Ensembl and GenBank repositories. Our tool specifically harnesses/exploits the sequence annotation data provided by FlyBase for Drosophila melanogaster (the fruit fly), which enables mapping of insertions relative to various genomic features such as natural transposons. Genome ARTIST was tested against other alignment tools using relevant query sequences derived from the D. melanogaster and Mus musculus (mouse) genomes. Real and simulated query sequences were also comparatively inquired, revealing that Genome ARTIST is a very robust solution for mapping transposon insertions. Genome ARTIST is a stand-alone user-friendly application, designed for high-accuracy mapping of transposon insertions and self-insertions. The tool is also useful for routine aligning assessments like detection of SNPs or checking the specificity of primers and probes. Genome ARTIST is an open source software and is available for download at www.genomeartist.ro and at www.bioinformatics.org.

scholarly journals Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

2011 ◽  
Vol 55 (1) ◽  
pp. 73-84 ◽  
Author(s):  
Vladimir B. Brukhin ◽  
Miloslawa Jaciubek ◽  
Arturo Bolanos Carpio ◽  
Vera Kuzmina ◽  
Ueli Grossniklaus
Keyword(s):  
Arabidopsis Thaliana ◽  
Insertional Mutagenesis ◽  
Gene Trap ◽  
Mutagenesis Screen ◽  
Insertional Mutagenesis Screen
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