RpoN sites predicted with PromScan. DNA array with w-t and overexpressed RpoN (rpoN mutant was not viable). Further validation of expression with primer extension.
Regulated genes for each binding site are displayed below. Gene regulation diagrams
show binding sites, positively-regulated genes,
negatively-regulated genes,
both positively and negatively regulated
genes, genes with unspecified type of regulation.
For each indvidual site, experimental techniques used to determine the site are also given.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.