Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Cold sensitive mutant of Escherichia coli with altered RNA polymerase

Summary Cold sensitive (cs) mutants of E. coli unable to grow at 26° C were isolated. Three of about 200 mutants tested have RNA polymerase which is about 5 times less active at low temperatures than the wild type RNA polymerase. One of such mutants cs 1 was studied in more detail. The mutation affects core enzyme and is closely linked to rif-r mutations. By transduction analysis the following order of markers was established: argH — rif-r — cs 1.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

RNA polymerase mutant with altered sigma factor in Escherichia coli.

Summary E. gracilis chloroplast DNA Bam fragments E and D, coding for rRNA were cloned separately using the plasmid pBR 322 as vector and E. coli as host. The newly constructed recombinant plasmids EgcKS 8 and EgcKS 11 (containing the Bam HI fragments E and D respectively) were analysed and characterized by gel electrophoresis, electronmicroscopy and analytical ultracentrifugation.Abbreviations Ap Ampicillin - Tc Tetracycline-hydrochloride - Bam HI endonuclease isolated from Bacillus amyloliquefaciens - Eco RI endonuclease isolated from E. coli RY13 - Bgl II endonuclease isolated from Bacillus globiggi - EDTA Ethylene-diamine-tetra-acetic-acid - ctDNA chloroplast DNA An abstract of this work was presented at the 10th annual meeting of the Union Schweizerischer Gesellschaften für Experimentelle Biologie, Davos 19th and 20th Mai, 1978. The recommendations of the Schweizerische Akademie für medizinische Wissenschaften for work with recombinant DNA-molecules were respected throughout this work.     

A temperature-sensitive mutant ofEscherichia coli with an alteration in ribosomal protein L22

A temperature-sensitive, protein synthesis-defective mutant ofEscherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to therplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.     

Assay of lysine by means of a mutant ofEscherichia coli

Описывается простой и доступный турбидиметрический метод определения L-лизина с помощью мутанта Escherichia coli ATCC 12408, чувствительного к лизину. Это метод надежного определения количества лизина в пределах от 20 до 100 μг. Присутствие высоких концентраций аргинина или орнитина не оказывает влияния на точность метода.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Human mutant cell lines with altered RNA polymerase II

A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to α-amanitin at concentrations up to 10 μg/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled α-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50-to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to α-amanitin is due to an alteration of RNA polymerase II.