RPL12A/B double deletion strains: Control vs. Wt RPL12B or Mutant RPL12B-R66K Transformed
ABSTRACT: Transcriptional and translational profiling of RPL12A/B double deletion comparing control untransformed RPL12B strains with transformed with a pESC-RPL12B wild type or muated RPL12B-R66K gene. Keywords: Genetic modification Two-condition experiment, Transcriptional experiments from total mRNA and Translational experiments from polysomal mRNA from three strains including deleted RPL12A/B, retransformed WT RPL12B and mutated RPL12B-R66K strain. Biological replicates: independently grown and harvested. double, triple or more replication per array.
Project description:To understand the extent that Heat shock protein 90 (Hsp90) regulated its target proteins at the transcription level, transcriptomic change was profiled in yeast cells upon Hsp90 compromising. We genetically modified the R1158 strain (resulting genotype of mutant strain: TETp-HSC82 hsp82Δ arg4Δ lys5Δ car2Δ::URA3) and then reduced the Hsp90 amount with doxycycline treatment. Fold change of mRNA from untreated to treated cells indicated the transcriptomic change. Totally, we identified 1104 genes mis-regulated with a fold change of no less than 1.5 (P <0.05) upon Hsp90 compromising. Two-condition experiment, treated vs. untreated cells. Biological duplicates, independently grown and harvested. Technical triplicates for RNA isolation.
Project description:Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between B. abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real time RT-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression was evaluated in placentomes from experimentally infected cows. Expression of pro-inflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant up-regulation of CXC chemokines, namely CXCL6 (GCP-2) and CXCL8 (IL-8), was observed at 12, but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing expression of pro-inflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of pro-inflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis. Keywords: trophoblast response to Brucella Total RNA used for microarray analysis was obtained from 6 placentas with 12 infected and 12 control explants from each placenta. At 4 h after inoculation, total RNA was isolated from the trophoblastic surface of the explants. Three RNA pools from two placentas each were generated prior to cDNA synthesis.
Project description:Transcriptional profiling of SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) and control SAS cells (transfected with pLKO.1 vector, designated as CTL). Goal was to determine the effects of LYRIC knockdown on global SAS cells gene expression. Two-condition experiment, SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) v.s. control SAS cells (transfected with pLKO.1 vector, designated as CTL). Biological replicates: 4 control replicates, 4 transfected replicates.
Project description:The two-component system (TCS) is a specific regulatory system in bacteria and plays an important role in sensing and adapting to the environment. In this study, we evaluated the roles of TCSs in the major cariogenic pathogen Streptococcus mutans in resistance to several types of bacteriocin. In a comprehensive analysis using individual TCS mutants, we found that two novel TCSs were associated with resistance against distinct lantibiotics nisin A (class I type A[I]) and nukacin ISK-1(class I type A[II]). One TCS, SMU.659-660 (designated as NsrRS), was related to resistance against nisin A produced by Lactococcus lactis ATCC 11454. The other TCS, SMU.1146-1145 (designated as LcrRS), was related to resistance against nukacin ISK-1 produced by Staphylococcus warneri ISK-1. NsrRS induced the expression of SMU.658 (designated as NsrX), which constitutes an operon with nsrRS, in response to nisin A. Inactivation of nsrX increased susceptibility to nisin A. Additionally, NsrX expression in S. mutans increased the binding affinity to nisin A compared to a no-expression strain. LcrRS induced the expression of SMU.1148-50 (lctFEG), which encodes an ABC transporter and is located upstream of lcrRS, in response to nukacin ISK-1. Inactivation of lctFEG significantly increased susceptibility to nukacin ISK-1. Electrophoretic mobility shift assay analysis revealed that NsrR and LcrR bound directly to regions upstream of nsrX and lctFEG, respectively. This is the first report that two distinct TCSs, NsrRS and LcrRS, are independently involved in resistance to nisin A and nukacin ISK-1 in S. mutans. Total of 14 samples were analyzed. Total RNA from each test strain and control described below were labeled with Alexa Fluor® 555 and Alexa Fluor® 647, respectively, and were cohybridized on a single array. Labeling and hybridization were performed once or twice independently. UA159 wild type as control vs. UA159 with nisinA or nukacin ISK-1, UA159 wild type with nisinA or nukacin ISK-1 vs. nsrRS or lcrRS deletion mutant in UA159 with nisinA or nukacin ISK-1.
Project description:Identification of a stable gene expression signature with high classifying potential to discriminate post-radiotherapy-induced thyroid tumors (follicular adenomas and papillary carcinomas) from their sporadic counterparts.
Project description:Identification of a stable gene expression signature with high classifying potential to discriminate benign (Follicular adenomas) and malignant (papillary carcinomas) thyroid tumors
Project description:DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus of heifers fed to gain body weight at high (HG, n = 4) and low (LG, n = 4) rates from 4.5 to 8.5 mo of age. A methyl-CpG binding domain-based (MBD) protein assay was performed to capture fragments of methylated DNA (methylated-enriched DNA). Input (total) and methylated-enriched DNA were labeled with two different dyes and co-hybridized to a custom-designed oligonucleotide array targeted to genes associated with nutritional inputs and the control of puberty. The ratio of the log2 (enriched/input) of the normalized intensities, were determined. Data was analyzed comparing values of HG versus LG heifers. Two nutritional schedules: HG (n=4 heifers) and LG (n=4 heifers); one array per heifer; methylated enriched DNA (enriched) and total DNA (input) co-hybridyzed into each array Methylated-enriched DNA obtained from a methyl-CpG binding domain-based (MBD) protein assay
Project description:Transcript abundance in Escherichia coli O157:H7 was determined in the presence or absence of pulsed expression of the small RNA, AsxR. AsxR was cloned under the control the arabinose inducible promoter Para. Escherichia coli O157:H7 str. TUV93-0 with pAsxR or empty vector was cultured in MEM-HEPES media to an OD600 of 0.8 and 0.2% arabinose added. 10min after addition of arabinose 10ml of cells were harvested and and pellets resuspended in 1ml of Trizol and total RNA isolated. RNAs were labelled using the SuperScript Plus indirect cDNA labelling System. Triplicate control RNAs were pooled and hybridised to seperate AsxR test RNAs on three microarays. Arrays were hybridised using the Maui hybridisation platform and Scann using and Axon Autoloader Scanner. GenePix software was used to analyse images and GPR files were analysed using Genespring 7.3.1.
Project description:Transcription profiling of skeletal muscle (tibialis anterior) of 5-week-old male wild type and myostatin null mice given ad libitum access to 20ppm clenbuterol hydrochloride in drinking water or plain tap water for 2 weeks. The objective of the study was to determine overlap in the mechanisms of muscle hypertrophy of the two models. 2x2 factorial arrangement of genotype (wild type and myostatin null) and clenbuterol treatment (control and clenbuterol at 20 ppm) with 4 replicates.