We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple rapid can be applied to most genes, even those that are essential. What makes this unique particularly effective the use of temperature-sensitive pSC101 replicon facilitate replacement. proceeds by homologous recombination between on chromosome sequences carried plasmid temperature sensitive DNA replication. Thus, after transformation into an appropriate host, it possible select...

The isolation of a temperature-sensitive allele RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that and polynucleotide phosphorylase (PNPase) are required for cell viability mRNA turnover Escherichia coli. Double-mutant strains carrying pnp-7 rnb-500 alleles (PNPase deficient thermolabile) ceased growing Luria broth within 30 min after shift to nonpermissive temperature. Cessation growth was accompanied an accumulation fragments 100-1500 nucleotides long. In contrast,...

The indirect suppression of recB(-) and recB(-)recC(-) mutations by the sbcB(-) allele is caused loss a nuclease active on denatured DNA. Results from enzyme purifications studies with specific antiserum demonstrate that activity present in sbcB(+) strains, lost exonuclease I. It likely sbcB structural gene for I restores recombination proficiency Escherichia coli cells has been recB and/or recC genes. This indicates absence recB-recC-determined enzyme, prevents recombination. Hypothetical...

An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. is a recombinant Escherichia coli carrying the N. qa-2 + gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) part of qa cluster. The recipient strain carries stable - mutation an arom-9 mutation, thus lacking both biosynthetic activities. Transformants were selected as colonies able to grow in absence aromatic amino acid supplement....

The Escherichia coli peptide methionine sulfoxide reductase gene (msrA) encodes a single-subunit polypeptide of 212 amino acid residues (M. A. Rahman, H. Nelson, Weissbach, and N. Brot, J. Biol. Chem. 267:15549-15551, 1992). RNA blot analysis showed that the is transcribed into an mRNA about 850 nucleotides. promoter region was characterized, transcription initiation site identified by primer extension. synthesis MsrA protein increased threefold in growth-phase-dependent fashion. In attempt...

In Escherichia coli , the global regulatory protein CsrA (carbon store regulator A) binds to leader segments of target mRNAs, affecting their translation and stability. activity is regulated by two noncoding RNAs, CsrB CsrC, which act sequestering multiple dimers. Here, we describe a (CsrD) that controls degradation CsrB/C RNAs. The dramatic stabilization RNAs in csrD mutant altered expression CsrA-controlled genes manner predicted from previously described Csr circuitry. A deficiency RNase...

As part of our genetic analysis mRNA decay in Escherichia coli K-12, we examined the effect pcnB gene [encoding poly(A) polymerase I] on message stability. Eliminating I (delta pcnB) dramatically stabilized lpp, ompA, and trxA transcripts. The half-lives individual mRNAs were increased both a delta single mutant pnp-7 rnb-500 rne-1 multiple mutant. We also found intermediates mutants that not detected control strains. By end-labeling total E. RNA with [32P]pCp T4 ligase then digesting RNase...

In vitro , polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade in the presence inorganic phosphate. However, because high vivo concentration phosphate exponentially growing cells, it has been assumed that enzyme works exclusively an exonuclease. Here we demonstrate that, contrary to this prediction, not only synthesizes long, highly heteropolymeric tails but also accounts for all observed...

The bimodal-incision nature of the reaction UV-irradiated DNA catalyzed by Escherichia coli uvrABC protein complex potentially leads to excision a 12- 13-nucleotide-long damaged fragment. However, oligonucleotide fragment containing UV-induced pyrimidine dimer is not released under nondenaturing in vitro conditions. Also, proteins are stably bound incised and do turn over after incision event. In this communication it shown that release from parental uvrABC-incised dependent upon either...

The in vitro and vivo analysis of the ribonuclease E-deficient (rne-) altered mRNA stability protein-deficient (ams-) strains Escherichia coli has demonstrated that they carry mutations same structural gene. Strains encoding either thermolabile RNase E (rne-3071) or Ams protein (ams-1) are defective both rRNA processing turnover. Immediately after a shift to nonpermissive temperature, chemical decay rate bulk is slowed 2- 3-fold, within 70 min, precursors 5S begin accumulate. In addition,...

The degradation of mRNA in Escherichia coli is thought to occur through a series endonucleolytic and exonucleolytic steps. By constructing multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), ams-1 (altered message stability) alleles, it was possible study general turnover as well specific mRNAs. Of most interest triple mutant which half-life total pulse-labeled RNA increased three- fourfold at nonpermissive temperature. RNA-DNA hybridization analysis...

Summary In Escherichia coli , the post‐transcriptional addition of poly(A) tails by polymerase I (PAP I, pcnB ) plays a significant role in cellular RNA metabolism. However, many important features this system, including its regulation and selection polyadenylation sites, are still poorly understood. Here we show that inactivation Hfq ( hfq ), an abundant RNA‐binding protein, leads to reduction ability PAP add at 3′ termini mRNAs containing Rho‐independent transcription terminators even...

RNase E, an essential endoribonuclease in Escherichia coli, is involved 9S rRNA processing, the degradation of many mRNAs, and processing M1 RNA subunit P. However, reason that E required for cell viability still not fully understood. In fact, recent experiments have suggested defects mRNA decay are responsible lack growth mutants. By using several new rne alleles, we confirmed these observations also ruled out by viability. Rather, our data suggest critical vivo role initiation tRNA...

RNA snap ™ is a simple and novel method that recovers all intracellular quantitatively (>99%), faster (<15 min) less expensively (∼3 cents/sample) than any of the currently available isolation methods. In fact, none bacterial methods, including commercial kits, are effective in recovering species RNAs (76–5700 nt) with equal efficiency, which can lead to biased results genome-wide studies involving microarray or RNAseq analysis. The procedure yields ∼60 µg from 10 8Escherichia coli cells be...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEnzymic repair of DNA. III. Properties the uv-endonuclease and uv-exonucleaseJoan C. Kaplan, Sidney R. Kushner, Lawrence GrossmanCite this: Biochemistry 1971, 10, 18, 3315–3324Publication Date (Print):August 1, 1971Publication History Published online1 May 2002Published inissue 1 August 1971https://doi.org/10.1021/bi00794a001RIGHTS & PERMISSIONSArticle Views45Altmetric-Citations134LEARN ABOUT THESE METRICSArticle Views are COUNTER-compliant sum...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTExperimental Chemotherapy of Tuberculosis. II. The Synthesis Pyrazinamides and Related Compounds1S. Kushner, H. Dalalian, J. L. Sanjurjo, F. Bach Jr., S. R. Safir, V. K. Smith WilliamsCite this: Am. Chem. Soc. 1952, 74, 14, 3617–3621Publication Date (Print):July 1, 1952Publication History Published online1 May 2002Published inissue 1 July 1952https://pubs.acs.org/doi/10.1021/ja01134a045https://doi.org/10.1021/ja01134a045research-articleACS...

recB - and recC strains of Escherichia coli K12 are recombination deficient sensitive to ultraviolet light the drug, mitomycin C. We have reported that sbcB mutations indirectly suppress all three phenotypes result in loss exonuclease I. In this publication we report occurrence other lead I UV sensitivity but not deficiency. These (called xonA ) cotransducible with his closely linked . Both mutant appear identical residual amounts nuclease activity on single-stranded DNA. It is hypothesized...

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased led a decrease half-life total pulse-labelled along with decreased half-lives rpsO, trxA, lpp and ompA transcripts. In contrast, transcripts for rne (RNase E) pnp (polynucleotide phosphorylase, PNPase), enzymes involved mRNA decay,...

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation transduction proficiencies ability to form Lac+ recombinants an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA rep mutants as deficient (20% wild type) recB recC their produce progeny. All the other exhibited increased frequencies recombinant formation, compared with type,...