The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl-CoA is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of a pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required the simultaneous expression of E. faecalis genes encoding its E1a, E1ß, E2 and E3 subunits, as well as genes involved in lipoylation of E2 and addition of lipoate to growth media. A strain lacking ACS, that expressed these E. faecalis genes, grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial micro-organisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Overall design: For both strains - mutant strain IMY104 and reference strain CEN.PK113-7D'' three independent chemostat cultures were performed. Each of the chemosta was sampled for transcriptome analysis. Samples were processed as described below.
Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.
Cell line, Subject
View SamplesAdjacent alternative 3’ splice sites, those separated by =18nt, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron''s 3'' end depends upon sequence elements that define the branchpoint, polypyrimidine tract and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched C. elegans samples, we identify hundreds of introns with adjacent alternative 3’ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is mono-directional, with somatic cells preferring to splice at the distal 3'' splice site and germline cells showing a distinct shift towards usage of the adjacent proximal 3'' splice site. Splicing patterns in somatic cells follow consensus rules of 3’ splice site definition, using sites with a short stretch of pyrimidines and an AG dinucleotide. Splicing in germline cells occurs at proximal 3'' splice sites that frequently lack a polypyrimidine tract or, occasionally, the AG dinucleotide. We provide evidence that use of germline-specific proximal 3'' splice sites is conserved across Caenorhabditis species. We propose that divergent mechanisms exist between germline and somatic cells in determining an intron terminus at adjacent alternative 3’ splice sites. Overall design: Examination of alternative splicing changes between germline- and somatic-cell enriched samples as well as nonsense-mediated decay mutants.
Coordinated tissue-specific regulation of adjacent alternative 3' splice sites in C. elegans.
Specimen part, Cell line, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.
Sex, Age, Specimen part, Disease stage
View SamplesComparison of DNA methylome, mRNA transcriptome, and copy number variation in tumors with global loss of DNA methylation to tumors with normal global methylation.
Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.
Sex, Age, Specimen part, Disease stage
View SamplesSkeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle.
Calprotectin is released from human skeletal muscle tissue during exercise.
Sex, Subject, Time
View SamplesTotal RNA microarray data from Fresh-Frozen Glioblastoma tumor samples.
Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.
Specimen part, Disease stage
View SamplesWe report the transcriptome changes that result of the genomic deletion of one or two alleles of an islet-specific long non-coding RNA (Blinc1) in isolated pancreas from e15.5 mouse embryos. Overall design: Pancreas from e15.5 embryos were dissected and total RNA extracted. Libraries were prepared from total RNA (RIN>8) with the TruSeq RNA prep kit (Illumina) and sequenced using the HiSeq2000 (Illumina) instrument. More than 20 million reads were mapped to the mouse genome (UCSC/mm9) using Tophat (version 2.0.4) with 4 mismatches and 10 maximum multiple hits. Significantly differentially expressed genes were calculated using DEseq.
βlinc1 encodes a long noncoding RNA that regulates islet β-cell formation and function.
Specimen part, Subject
View SamplesWe performed mRNA-seq of a PRKACA-mutant adrenal tumor and demonstrated that the mutation is expressed at the mRNA level. Overall design: Total RNA obtained from a cortisol-producing adrenal tumor with a PRKACA p.Leu206Arg mutation.
Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors.
No sample metadata fields
View SamplesThis study investigated the use of three different established cell sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines.
Isolation and genomic characterization of stem cells in head and neck cancer.
Cell line
View SamplesWe detected fusion genes in 274 fresh surgical samples of gliomas using whole transcriptome sequencing. Using this approach we screened a panel of glioma samples and identified a number of activating novel fusion transcripts. Overall design: Fusion detection in 274 glioma patients
RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas.
Subject
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