Journal: Genes & Development
Article Title: Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes
Figure Lengend Snippet: Overexpression of the Pol V largest subunit CTD induces a dominant-negative RdDM phenotype suppressed in EMS-induced mutants. ( A ) RNA blot analysis of small RNA of wild-type (Col-0), pol IV mutant ( nrpd1 ), pol V mutant ( nrpe1-11 ), or CTD-OX plants. The blot was sequentially probed for small RNAs matching the 45S rRNA gene promoter, 5S rRNA gene intergenic spacer ( siR1003 ), or microRNA miR160 . An image of the ethidium bromide (EtBr)-stained gel is shown at the bottom . ( B ) Analysis of AtSN1 and SoloLTR transposon DNA methylation levels using Chop-PCR. Genomic DNA of wild-type Col-0, Pol V mutant ( nrpe1 - 11 ), or CTD-OX plants was digested (chopped) with the indicated methylation-sensitive endonucleases (the sequence context of queried cytosines are shown in parentheses) or left uncut as a control and then amplified using PCR primers specific for AtSN1 or soloLTR retrotransposons. PCR products were resolved by agarose gel electrophoresis and visualized with EtBr staining. ( C ) RT–PCR analyses of SDC , CTD , AtSN1 , and soloLTR expression levels relative to a ubiquitin ( UBQ ) control. The genotypes of plants tested are indicated at the top of each lane. Reactions in which reverse transcriptase was omitted (no RT) control for DNA contamination. The drm1 drm2 cmt3 genotype is known to induce SDC overexpression, serving as a positive control for the cmt3 CTD-OX genotype. The CTD reactions control for transgene expression. ( D ) CHG and CHH methylation-deficient cmt3 CTD-OX plants display the SDC overexpression phenotype. ( E ) RT–PCR analysis of CTD and native NRPE1 expression. The genotypes of plants tested are indicated at the top of each lane. Reactions lacking reverse transcriptase (no RT) control for DNA contamination. UBQ reactions control for the amount of RNA tested. ( F ) RT–PCR analysis of SDC , AtSN1 , soloLTR , and CTD expression in the cmt3 CTD-OX parental line and in the suppressor mutants m17, m65, m71, and m73. The nrpe1 - 11 ( pol V ) mutant served as control for derepression of AtSN1 and SoloLTR elements silenced by RdDM in wild type (Col-0). UBQ served as a loading control. Reactions without reverse transcriptase (no RT) served as controls for DNA contamination. ( G ) Analysis of AtSN1 and SoloLTR DNA methylation levels using Chop-PCR. Assays were conducted as in B , comparing wild-type Col-0 with the indicated mutants.
Article Snippet: RNA (1.5 µg) was then treated using a Turbo DNA-free kit (Thermo Fisher Scientific) and used for random-primed cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).
Techniques: Over Expression, Dominant Negative Mutation, Northern blot, Mutagenesis, Staining, DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Sequencing, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control