25 research outputs found
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
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 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
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>
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>
Exploring the Functional Residues in a Flavin-Binding Fluorescent Protein Using Deep Mutational Scanning
<div><p>Flavin mononucleotide (FMN)-based fluorescent proteins are versatile reporters that can monitor various cellular processes in both aerobic and anaerobic conditions. However, the understanding of the role of individual amino acid residues on the protein function has been limited and has restricted the development of better functional variants. Here we examine the functional amino acid residues of <i>Escherichia coli</i> flavin mononucleotide binding fluorescent protein (EcFbFP) using the application of high-throughput sequencing of functional variants, termed deep mutational scanning. The variants were classified into 329 function-retained (FR) and 259 function-loss (FL) mutations, and further the mutational enrichment in each amino acid residues was weighed to find the functionally important residues of EcFbFP. We show that the crucial amino acid residues of EcFbFP lie among the FMN-binding pocket, turns and loops of the protein where conformation changes occur, and spatially clustered residues near the E56-K97 salt bridges. In addition, the mutational sensitivity of the critical residues was confirmed by site-directed mutagenesis. The deep mutational scanning of EcFbFP has demonstrated important implications for constructing better functioning protein variants.</p></div
Critical residues determined by mutation frequency.
<p>(a) Common and unique mutations in the FL library and FR library. (b) Heatmap of the positions of unique mutations of the FR and FL library with mutation frequency drawn on a scale of 0 to 1. Positions with enriched mutations are shown in yellow. The positional effect bar shows the residues with enriched FL mutations in yellow and residues with enriched FR mutations in black. The asterisks indicate known FMN-binding sites.</p