Epigenetic mutant gene expression analysis_2
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE19269 | GEO | 2009/12/10
SECONDARY ACCESSION(S): PRJNA120943
REPOSITORIES: GEO
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Project description:The aim of this project is to identify endogenous targets of yet unidentified epigenetic mutants. Mutants were discovered in a forward genetic approach using T-DNA insertion mutagenesis. Pools of 25 seedlings, grown in-vitro, will be used to examine changes in gene expression. 9 samples were used in this experiment.
2009-12-09 | E-GEOD-19269 | biostudies-arrayexpress
Project description:The aim of this project is to identify endogenous targets of yet unidentified epigenetic mutants. Mutants were discovered in a forward genetic approach using T-DNA insertion mutagenesis.Pools of each 50 seedlings, grown in-vitro, will be used to examine changes in gene-expression. Keywords: Expression profilling by array
2009-11-18 | GSE18977 | GEO
Project description:The aim of this project is to identify endogenous targets of yet unidentified epigenetic mutants. Mutants were discovered in a forward genetic approach using T-DNA insertion mutagenesis.Pools of each 50 seedlings, grown in-vitro, will be used to examine changes in gene-expression. Keywords: Expression profilling by array 9 samples were used in this experiment
2009-11-17 | E-GEOD-18977 | biostudies-arrayexpress
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
2010-12-31 | E-GEOD-24511 | biostudies-arrayexpress
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Project description:ngs2018_05_tor-analysis of arabidopsis mutants tor/lst8/yak1/maktor-Analysis of Arabidopsis insertion and suppressor mutants linked to the TOR signaling pathway.-Comparison of in vitro grown plantlets for lst8, yak1 mutants and double mutants corresponding to suppressor lines. Comparison of in vitro grown plantlets for mutants affected in LST8 interacting proteins (MAKTOR) compared to wild type.
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Project description:The transposon site hybridization (TraSH) technique (Sassetti, CM et al. 2001. PNAS 98:12712-7) was utilized to identify genes important for the survival of Y. pestis within murine macrophages. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool) in order to identify Y. pestis genes important for survival in macrophages. Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs can be used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool). Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs was used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe.
2011-11-30 | E-GEOD-33982 | biostudies-arrayexpress
Project description:This study employed an unbiased forward genetic screen to identify suppressors of the maternal-effect lethal phenotype caused by tost-1 dysfunction in Caenorhabditis elegans. The temperature-sensitive tost-1(xf196 ts) mutant exhibits complete embryonic lethality at the restrictive temperature (25°C), providing a robust screening platform. Following EMS mutagenesis of L4 larvae, we screened F2 progeny for viable individuals at 25°C, indicating suppression of the lethal phenotype. After backcrossing to confirm heritability, we performed whole genome sequencing on 21 suppressor strains to identify causative mutations. Additionally we also sequenced the strain used for the mutagenesis as the reference genome.
2025-11-11 | E-MTAB-15751 | biostudies-arrayexpress
Project description:Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development, and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2Î/Î and arp2Î/Îarp3Î/Î mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery. By forward genetic screening, we have identified genes that are essential for hyphal formation. One of the hits is the Arp2/3 complex, which is essential for hyphal formation, but not for viability in Candida albicans. To gain insights into cellular processes affected by disrupting Arp2/3 complex functions, we performed transcriptional profiling under yeast growth conditions (YPD at 30°C for three hours) or hyphal induction (YPD + 10% FBS at 37°C for three hours) and compared transcriptional consequences of deleting ARP2 to MYO5 and SLA2 microarray data sets (Oberholzer et al., 2006).
2010-10-08 | E-GEOD-19565 | biostudies-arrayexpress