Additional file 2: Table S2. of Functional elucidation of the non-coding RNAs of Kluyveromyces marxianus in the exponential growth phase

Annotation of Kluyveromyces genome. (XLSX 286 kb

Additional file 4: Figure S1. of Functional elucidation of the non-coding RNAs of Kluyveromyces marxianus in the exponential growth phase

Determination of optimal cutoff value to differentiate non-coding transfrags from coding ones. The optimal cutoff value was determined by R script within the CPAT package with appropriate modification to fit our circumstance. (DOC 37 kb

Additional file 8: Figure S4. of Functional elucidation of the non-coding RNAs of Kluyveromyces marxianus in the exponential growth phase

Significantly enriched KEGG pathways of genes with lancRNAs in S. cerevisiae. Significantly enriched KEGG pathways of genes with lancRNA which covers more than half of coding region at ME (mid exponential), ES (early stationary), and HS (heat shock) conditions. Red words indicate carbohydrate metabolism or energy metabolism pathways. In constrast to K. marxianus with no KEGG annotation, gene-pathway link information within KEGG annotation was used rather than inferred by homology search. (DOC 49 kb

High-Level dCas9 Expression Induces Abnormal Cell Morphology in <i>Escherichia coli</i>

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in <i>Escherichia coli</i> mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including <i>fimA</i>, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in <i>E. coli</i>

High-Level dCas9 Expression Induces Abnormal Cell Morphology in <i>Escherichia coli</i>

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in <i>Escherichia coli</i> mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including <i>fimA</i>, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in <i>E. coli</i>

High-Level dCas9 Expression Induces Abnormal Cell Morphology in <i>Escherichia coli</i>

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in <i>Escherichia coli</i> mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including <i>fimA</i>, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in <i>E. coli</i>

The ratio of reaction rate constants of () stem II, stem I and stem I/III modified aptazymes for HCV replicase and () stem II, stem III and stem I/III modified aptazymes at 10 mM Tris–HCl, 100 mM KCl and 10 mM MgCl (pH 7

<p><b>Copyright information:</b></p><p>Taken from "Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal"</p><p>Nucleic Acids Research 2005;33(20):e177-e177.</p><p>Published online 27 Nov 2005</p><p>PMCID:PMC1292994.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>6). The ratio of reaction rate constants means ‘the ratio of the rate constant in the presence of protein to that in the absence of protein’. The concentrations of the aptazymes and substrate in the reaction mixture are 1 µM. Reaction rate constants were obtained from {ln[( − )/]/Δ} ( and are the fluorescence intensities at 5 and at 0 min, respectively). The blank rate constant of stem II mono-aptazyme, stem I mono-aptazyme and stem I/III bis-aptazymes in the absence of HCV replicase were 0.660, 0.757 and 0.254 min, respectively. And the blank rate constants of the stem II mono-aptazyme, stem III mono-aptazyme and stem I/III bis-aptazymes in the absence of HCV helicase (b) were 0.715, 0.782 and 0.019 min, respectively

Mutation frequency of amino acid residues involved in hydrophobic interaction with FMN.

<p>Mutation frequency of amino acid residues involved in hydrophobic interaction with FMN.</p

High-Level dCas9 Expression Induces Abnormal Cell Morphology in <i>Escherichia coli</i>

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in <i>Escherichia coli</i> mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including <i>fimA</i>, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in <i>E. coli</i>

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p