Project description:Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by Dehalococcoides organisms. A Dehalococcoides-containing microbial consortium (ANAS) with the ability to degrade TCE to ethene, an innocuous end-product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of Dehalococcoides ethenogenes 195 (strain 195). This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray revealed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria which is essential in developing effective strategies for bioremediation of PCE and TCE in the environment. Keywords: comparative genomic hybridization
Project description:Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by Dehalococcoides organisms. A Dehalococcoides-containing microbial consortium (ANAS) with the ability to degrade TCE to ethene, an innocuous end-product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of Dehalococcoides ethenogenes 195 (strain 195). This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray revealed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria which is essential in developing effective strategies for bioremediation of PCE and TCE in the environment. Keywords: comparative genomic hybridization Genomic DNA from each culture was divided into replicate samples which were independently fragmented, labeled, and hybridized to arrays. Two microarrays were processed for the positive control (strain 195), two for the negative control (D. restrictus), and five for the ANAS enrichment culture(two analyses from one biological sample followed one year later by three analyses of a second biological sample).
Project description:Our aim was to classify and quantify transcripts identified in 24-h-cultured primary duck hepatocytes and construct a protein–protein interaction network to serve as a reference for host factors associated with hepadnavirus infection. Methods: The transcriptome of 24h-cultured PDHs was analyzed by the pair-end sequencing on the Illumina Solexa platform. High-quality reads were mapped to the Anas platyrhynchos genome with TopHat v2.0.12 software. TopHat allows multiple alignments per read and default parameters were used. Cufflinks v2.2.1 software was later used for analyses that included transcript assembly and FPKM value calculations to quantify gene expression; this program was also run with default parameters. Results: A total of 87.8 million high-quality reads were obtained from three primary duck hepatocyte samples isolated from three separate 1-day-old Anas domesticus ducklings. The reads (mean length 92.21 bases) were mapped to the Anas platyrhynchos genome. A total of 13,541 genes with > 1 fragments per kilobase of transcript per million mapped reads values were expressed in the 24-h-cultured primary duck hepatocyte samples.Using gene ontology analysis, expressed genes were assigned to functional categories. A total of 182 genes expressed in all three separate primary duck hepatocyte samples were identified as liver-specific genes. Conclusions: Transcriptome and gene ontology analyses of 24-h-cultured primary duck hepatocytes indicate that these cells retain hepatocyte-specific biological characteristics and can be used as a model system for hepadnavirus infection. A novel protein–protein interaction network suggests that host factors regulating or inhibiting innate immunity are directly associated with hepadnavirus. The transcriptome of 24h-cultured PDHs was analyzed by the paired-end sequencing on the Illumina Solexa platform.
Project description:Our aim was to classify and quantify transcripts in primary duck hepatocytes cultured in medium with 5% FBS or 1.5% DMSO for 8 days. Methods: The transcriptome of PDHs under different conditions was analyzed by the pair-end sequencing on the Illumina Solexa platform. High-quality reads were mapped to the Anas platyrhynchos genome with TopHat v2.0.12 software. TopHat allows multiple alignments per read and default parameters were used. Cufflinks v2.2.1 software was later used for analyses that included transcript assembly and FPKM value calculations to quantify gene expression; this program was also run with default parameters.
Project description:Our aim was to classify and quantify transcripts identified in 24-h-cultured primary duck hepatocytes and construct a protein–protein interaction network to serve as a reference for host factors associated with hepadnavirus infection. Methods: The transcriptome of 24h-cultured PDHs was analyzed by the pair-end sequencing on the Illumina Solexa platform. High-quality reads were mapped to the Anas platyrhynchos genome with TopHat v2.0.12 software. TopHat allows multiple alignments per read and default parameters were used. Cufflinks v2.2.1 software was later used for analyses that included transcript assembly and FPKM value calculations to quantify gene expression; this program was also run with default parameters. Results: A total of 87.8 million high-quality reads were obtained from three primary duck hepatocyte samples isolated from three separate 1-day-old Anas domesticus ducklings. The reads (mean length 92.21 bases) were mapped to the Anas platyrhynchos genome. A total of 13,541 genes with > 1 fragments per kilobase of transcript per million mapped reads values were expressed in the 24-h-cultured primary duck hepatocyte samples.Using gene ontology analysis, expressed genes were assigned to functional categories. A total of 182 genes expressed in all three separate primary duck hepatocyte samples were identified as liver-specific genes. Conclusions: Transcriptome and gene ontology analyses of 24-h-cultured primary duck hepatocytes indicate that these cells retain hepatocyte-specific biological characteristics and can be used as a model system for hepadnavirus infection. A novel protein–protein interaction network suggests that host factors regulating or inhibiting innate immunity are directly associated with hepadnavirus.