Project description:blanc-08-01_2012_01_rnapaths_03 - rnapaths--3_02/2012 - Identify the transcript overlap and specificity between the PTGS and decapping/exoribonuclease pathways b identifying transcripts that are significantly changed in double mutants versus single mutants, and transcripts that are commonly changed among the single and double mutants compared to WT. - Identify transcripts that are significantly changed in double mutants (L1 vcs sgs2) (xrn4-5/sgs3-11) versus their respective single mutants (L1 vcs and L1 sgs2) (xrn4-5 and sgs3-11) , and identify transcripts that are changed among the single and double mutants compared to WT (Col) reference or to mutant L1 reference. 20 dye-swap - genotype comparaison
Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.
Project description:The aim was to examine changes in gene expression of the endometrium exposed to long-term tamoxifen treatment in comparison to age matched controls. To achieve this, endometrial tissues were obtained from women receiving tamoxifen treatment who were undergoing a hysterectomy. Using cDNA microarrays, gene expression changes in the postmenopausal endometrium of these women was compared with that in endometrium of age matched women not receiving tamoxifen. Endometrial tissue from post-menopausal women was obtained following ethical approval from the Leicester NHS Trust. None of the women had received any hormonal treatment for two months prior to the procurement of the specimens. Tissues were taken from untreated women (n=6) or those treated for 4 to 5 years with tamoxifen (20mg/day) (n=4), aged 58-82 (65 ± 9.1, mean ± SD). Total RNA was extracted. Controls were pooled. RNA labelling, hybridisation and analysis of fluorescence was carried out as described by Turton et al (2001). Cy3/Cy5 Dye swap labelling was carried out on samples from each patient. Reference: Turton NJ et. al. (Oncogene (2001) 20, 1300-1306
Project description:Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA (rRNA) and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.
Project description:This SuperSeries is composed of the following subset Series: GSE24037: Salivary cytokine alterations in HIV infection part 1 GSE24064: Salivary cytokine alterations in HIV infection part 2 Refer to individual Series
Project description:+Gly 20, 40, 80: Cells were grown to early log phase (OD600 ~ 0.2) at 30oC in 1 liter of minimal B medium (Cherest, H., and Surdin-Kerjan, Y. (1992) Genetics 130, 51-58) and t=0 time point samples were harvested (250 ml). The remainder of the medium was supplemented with 10 mM glycine incubated at 30oC and samples (250 ml) harvested at 20, 40 and 80 minutes. All cells were harvested by rapid centrifugation at room temperature then flash frozen in liquid nitrogen. -Gly 20, 40, 80: Cells were grown to early log phase (OD600 ~ 0.2) at 30oC in 1 liter of minimal B medium (Cherest, H., and Surdin-Kerjan, Y. (1992) Genetics 130, 51-58) supplemented with 10 mM glycine and t=0 time point samples were harvested (250 ml). The remainder of the medium was filtered and the cells were rapidly resuspended in 750 ml of pre-warmed B minimal medium, incubated at 30oC and samples (250 ml) harvested at 20, 40 and 80 minutes. All cells were harvested by rapid centrifugation at room temperature then flash frozen in liquid nitrogen. Keywords: time-course
Project description:Shotgun protein sequencing with meta-contig assembly.
Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings.
Project description:Cells were grown to early log phase (OD600 ~ 0.2) at 30oC in 1 liter of minimal B medium (Cherest, H., and Surdin-Kerjan, Y. (1992) Genetics 130, 51-58) with or without supplementation with 10 mM glycine and harvested. All cells were harvested by rapid centrifugation at room temperature then flash frozen in liquid nitrogen. Arrays compared expression of Gly- cells to the Gly+ controls. Strain BY4741 was the wild type and several deletion strains were also used. Keywords: parallel sample
Project description:The endothelium is the frontline target of multiple metabolic stressors and pharmacological agents. As a consequence, endothelial cells (ECs) display highly dynamic and diverse proteome profiles. We describe here the culture of human aortic ECs from healthy and type 2 diabetic donors, the treatment with a small molecular conformation of trans-resveratrol and hesperetin (tRES+HESP), followed by proteomic analysis of whole-cell lysate. A number of 3666 proteins were presented in all the samples and thus further analyzed. We found that 179 proteins had a significant difference between diabetic ECs vs. healthy ECs, while 81 proteins had a significant change upon the treatment of tRES+HESP in diabetic ECs. Among them, 16 proteins showed a difference between diabetic ECs and healthy ECs and the difference was reversed by the tRES+HESP treatment, with the top 5 drastically altered proteins being ACVRL1, ADAM9, ITGAV, PCCB, and TGFBR2. Follow-up functional assays identified ACVRL1 and TGFBR2 as the most pronounced mediator for tRES+HESP-induced protection of angiogenesis in vitro. Our study has revealed the global changes in proteins and biological pathways in ECs from diabetic donors, which are potentially reversible by the tRES+HESP formula. Furthermore, we have identified the TGFβ signaling axis as a responding mechanism in ECs treated with this formula, shedding light for future studies for deeper molecular characterization
Project description:Hepatocellular carcinoma (HCC) remains a major health problem worldwide, and HCC patients have a poor prognostic outcome. In this study, we systematically disclose mechanisms of hepatocarcinogenesis, and also effectively identify and validate novel anticancer targets in HCCs. Keywords: disease state analysis total 58 cDNA microarrays, all experiment samples are hepatocellular carcinoma. RNAs from 33 corresponding noncancerous livers and 5 normal livers were used as the reference, respectively. The tumor samples were labeled with Cy5-dUTP.The nontumor samples were labeled with Cy3-dUTP.