Project description:We used ChIP-seq to map binding of the CRISPR surveillance complex, Cascade, in an E. coli strain lacking the endonuclease Cas3. These data enabled us to determine the precise sequence requirements for Cascade binding.
Project description:CRISPR/Cas loci commonly encode both proteins and small guide RNAs (crRNAs) to assemble ribonucleoproteins particles (RNPs) that confer a sequence-based, adaptive immunity against viruses and plasmids in prokaryotes. However, it has not been established whether this conserved synteny of RNA and protein genes is needed for the efficient RNP production in vivo. We show that the pathogenic bacterium Salmonella Typhimurium harbours two physically unlinked loci, CRISPR01 and CRISPR02, both of which produce mature crRNAs, although CRISPR02 lacks the protein genes for the type I-specific Cascade complex. Utilizing these coexisting complete and orphan CRISPR loci, we provide the first in vivo evidence for a crRNA-dependent assembly of Cascade. Strikingly, the full RNP assembles identically on crRNAs from either CRISPR cassette, i.e. regardless of their expression in cis or trans. Deep sequencing of Cas-bound transcripts identifies crRNAs derived from both loci as the sole bona fide targets of Cascade, although individual Cas proteins may interact with other cellular RNAs outside of the Cascade context. Altogether, our data demonstrate that CRISPR RNAs, independently of their origin, serve as the organizing centre of Cascade in vivo and are the main reason-to-be for its protein components in Salmonella.
Project description:We used Bio-Rad laboratories, lnc. PCR assay panel to analyze expression levels of MAPK genes in various tissues or organs. Cucumber plants of the ‘Jinyan No.4’ cultivar were reared in growth chambers at 28 ± 1 °C with a photoperiod of 16 h light/8 h dark and light intensity of 400 μmol m−2 s−1. The roots, leaves, stems, female flower buds (approximately 3 d before anthesis), male flower buds (approximately 1.0 cm in length), and fruits (10 d after pollination) were collected from flowering plants for tissue expression analysis. The experiment was designed to gain insight into the temporal and spatial transcription patterns of 58 MAPK cascade genes in cucumber.
Project description:IKK/beta-TrCP2 phosphorylation-ubiquitination cascade as a major mechanism that governs TFEB stability independently of other TFEB regulators.