Evidence of transcription in intergenic sequences in Rickettsia conorii
Ontology highlight
ABSTRACT:
ORGANISM(S): Rickettsia conorii str. Malish 7
PROVIDER: GSE24157 | GEO | 2011/09/27
SECONDARY ACCESSION(S): PRJNA130075
REPOSITORIES: GEO
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Project description:The model cyanobacterium Synechocystis sp. PCC 6803 was used for a systematic survey on the number and types of antisense (as)RNAs. A tiled microarray was constructed with probes for both strands of the genes or intergenic spacers with candidate transcripts predicted by an algorithm and an equally sized and distributed control set. The resulting 102,739 individual probes cover an accumulated length of 1,441,146 nt or about 40% of the total chromosome sequence of Synechocystis 6803. Hybridization of this array with total RNA isolated from cultures raised under different growth conditions identified a high number of transcripts from intergenic spacers and in antisense orientation to known genes (natural cis-asRNAs). Extrapolated to the whole genome, around 10% of all open reading frames in Synechocystis 6803 may have a corresponding asRNA, suggesting a much more important role of chromosomally encoded asRNAs in bacteria then anticipated so far. Keywords: stress response 4 RNA hybridizations, 2 DNA controls. The raw data from the DNA hybridizations are in the GSE14410_DNA_1.txt and GSE14410_DNA_2.txt files.
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Project description:By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.
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Project description:The model cyanobacterium Synechocystis sp. PCC 6803 was used for a systematic survey of differential expression with a focus on antisense (as)RNAs and non-coding (nc)RNAs. A microarray was constucted with on average 5 probes for each transcript known thus far, including ncRNAs and asRNAs. The resulting 20,431 individual probes are duplicated on the array (Agilent 4x44k custom array) representing a technical replicate. Hybridization of this array with total RNA isolated from cultures raised under different growth conditions identified transcripts from intergenic spacers and in antisense orientation to known genes (natural cis-asRNAs) with differential expression compared to control hybridizations. This shows the involvement of such transcripts in the regulation of adaptation to various stresses.
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Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent 4x44k custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used. 6 replicate samples
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Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used. 6 replicate samples
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Project description:The model cyanobacterium Synechocystis sp. PCC 6803 was used for a systematic survey of differential expression with a focus on antisense (as)RNAs and non-coding (nc)RNAs. A microarray was constucted with on average 5 probes for each transcript known thus far, including ncRNAs and asRNAs. The resulting 20,431 individual probes are duplicated on the array (Agilent 4x44k custom array) representing a technical replicate. Hybridization of this array with total RNA isolated from cultures raised under different growth conditions identified transcripts from intergenic spacers and in antisense orientation to known genes (natural cis-asRNAs) with differential expression compared to control hybridizations. This shows the involvement of such transcripts in the regulation of adaptation to various stresses. 12 RNA hybridizations (1 control & 3 stress conditions, 3 times each)
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