We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

/pdf/construction-of-escherichia-coli-k-12-in-frame-single-gene-4c68fx6pb1.pdf

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

/pdf/role-for-a-bidentate-ribonuclease-in-the-initiation-step-of-4ptw8d0dcg.pdf

Role for a bidentate ribonuclease in the initiation step of RNA interference

Roles moleculaires des proteines de choc thermique dans le fonctionnement des organismes a des temperatures normales et suite a des chocs thermiques; differents genes impliques

The heat-shock proteins

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3 ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.

/pdf/rna-interference-is-mediated-by-21-and-22-nucleotide-rnas-1tlpza9thy.pdf

RNA interference is mediated by 21- and 22-nucleotide RNAs

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

/pdf/an-abundant-class-of-tiny-rnas-with-probable-regulatory-vtv94uvmvm.pdf

An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans

https://mmbr.asm.org/content/mmbr/53/1/1.full.pdf

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

https://www.cell.com/cell/pdf/0092-8674(84)90493-8.pdf

The htpR gene product of E. coli is a sigma factor for heat-shock promoters

The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

/pdf/identification-of-the-sigma-e-subunit-of-escherichia-coli-1x5wd57bbs.pdf

Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.

cr ~ and r ~2 are two heat- and ethanol-inducible g-factors in Escherichia coli. The cr 32 regulon is also induced by unfolded and misfolded proteins in the cytoplasm, and the function of many of the proteins in the cr 32 regulon is to bind to cytoplasmic proteins and assist them in folding or unfolding. To further understand the function of the cr F regulon, we searched for mutants that affected cr E activity. Our results indicate that a signal generated by expression of outer membrane proteins modulates cr E activity. Specifically, r ~ activity is induced by increased expression of OMPs and is reduced by decreased expression of OMPs. In addition, mutations that cause misfolded OMPs induce cr E activity. This signal is generated after the fate of OMPs and periplasmic proteins diverge in the secretory pathway and is not the result of an accumulation of OMP precursors in the cytoplasm. Our results indicate that this effect of OMPs is specific to the r E regnlon, because none of the above mutations affect r 32 activity. We propose that the ~r ~ regnlon is involved in processes that occur in extracytoplasmic compartments and that these two heat-inducible regulons may have distinct but complementary roles of monitoring the state of proteins in the cytoplasm (or 32) and outer membrane ((]rE).

/pdf/the-activity-of-sigma-e-an-escherichia-coli-heat-inducible-6zs1ugadh4.pdf

James W. Erickson

Papers

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

The htpR gene product of E. coli is a sigma factor for heat-shock promoters

Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.

The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins.

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos.