Project description:Gene expression profiling was performed to compare RNA abundances in mycelium of Puf knockout strains compared to that in mycelium of wild-type N. crassa
Project description:The molecular mechanisms of aging are unsolved and fascinating fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding to the 3’ untranslated regions (3’ UTR) in the mRNA of target genes. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF, a mitochondrial fission factor, and subsequently regulate longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. We generate these mutations in C. elegans, those mutations reduced the fertility and extended the lifespan. We deep sequenced total mRNAs from wild-type and puf-8 mutant worms and conducted in vitro RNA pull-down experiments. Six PUF-8 regulated genes were identified, in which their mRNA 3’ UTRs contain at least one PUF-binding element (PBE). One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8(-) animals. Therefore, PUF-8 may regulate the lifespan of C. elegans via modulating pqm-1-related pathways
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose.
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004.
Project description:Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose. N. crassa was pregrown in Sucrose and transferred to Avicel (cellulose), Cellobiose, Sucrose or media with no carbon added. Biological triplicates used to identify differentially expressed genes in WT on Avicel. Single libraries for mutant strains identify which genes show similair expression on cellobiose as in the WT on cellulose.