Escherichiacoli mutants deficient in exoribonucleases

Strain S296, isolated by screening 2000 colonies after nitrosoguanidine mutagenesis, yields extracts with less than 1% of wild-type RNase activity against (³H) poly(U). Unlike other $\text{E.}\text{coli}$ strains, S296 grows with a doubling time of about 2 hr., both in nutrient broth and in minimal medium, and at 30°, 37° and 42°. The strain retains 10 to 20% of wild-type exonuclease activity against (³H) rRNA or T4 phage-specific mRNA; but two further mutants, made by screening mutagenized colonies of strain S296, are reduced to 3% of wild-type activity against those substrates as well.     

Molybdenum and chlorate resistant mutants in Escherichiacoli K12

Radioactive molybdenum is used to detect the existence of molybdo compounds in $\text{E.}\text{coli}$ K12. Three membrane bound Mo-proteins are found, using sodium dodecyl sulfate. One of them is the nitrate reductase. The nature of the other two is discussed. The soluble fraction of the cellular extract contains a small Mo binding molecule which could be peptidic in nature (MW is about 1,500). Different chlorate resistant mutants are analyzed on the basis of these molybdo-compounds. None of the mutants is found to contain radioactivity bound to nitrate reductase protein. Defects in the biosynthesis of a molybdenum coenzyme is deduced for chlorate resistant pleiotropic mutants.     

Associative properties of the Escherichiacoli galactose binding protein and maltose binding protein

The periplasmic galactose binding protein and maltose binding protein of $\text{Escherichia}\text{coli}$ are recovered mostly in dimeric form when purified, from osmotically-shocked bacteria, in the presence of protease inhibitors and 2-mercaptoethanol without dialysis and concentration of the shock fluid. The specific ligands, galactose (but not glucose) for galactose binding protein, and maltose for maltose binding protein, provoque the monomerisation of the dimeric native forms. These results are discussed in relation to the function of both binding proteins in transport and chemotaxis.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

Parental RF formation of phages ⊘X174 and M13 requires the dnaZ gene product of Escherichiacoli

A functional $\text{dnaZ}$ product, known to be essential for host DNA polymerization and for the growth of coliphages ?X174 and M13, is required for the $\text{in}\text{vivo}$ single strand to replicative form conversions of both of these phages.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

DNA postreplication repair gap filling in temperature-sensitive dnaG, dnaC, dnaA mutants of Escherichiacoli

Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type $\text{Escherichia}\text{coli}$ to ultraviolet light are filled during reincubation. In this study the $\text{dnaG}\text{,}\text{dnaC}$, and $\text{dnaA}$ gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.     

Chromosomal incompatibility in Escherichia,coli K-12: A new explanation for the observed instability of minichromosome inheritance

The minichromosome pWS6 was unstable in $\text{Escherichia}$,$\text{coli}$ K-12 but became stable upon transfer to $\text{Salmonella}$,$\text{typhimurium}$. The instability of pWS6 was restored when pWS6 was brought back to $\text{E.}$,$\text{coli}$, an observation consistent with the proposed phenomena of chromosomal incompatibility.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Metabolites of mating pheromone,rhodotorucine A, by a cells of Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ which induces mating tube formation of $\text{a}$ cells in $\text{Rhodosporidium}\text{toruloides}$ is metabolized rapidly by $\text{a}$ cells. By use of labeled rhodotorucine $\text{A}$, the degradation was found to be proteolytic. Two peptide fragments Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg and Asn-Gly-Cys(S-farnesyl) were identified as the metabolites. Proteolysis of the pheromone mainly occurred on the cell surface. Culture filtrate of $\text{a}$ cells at log phase did not metabolize rhodotorucine $\text{A}$.     

Importance of membrane fluidity in the induction of alkaline phosphatase,a periplasmic enzyme,in Escherichiacoli

An unsaturated fatty acid auxotroph was supplemented with either elaidate or oleate. After derepression of alkaline phosphatase by phosphate limitation at 38°C, the cells were shifted to incubation at various temperatures. Arrhenius plots of the rate of enzyme induction gave a steeper negative slope in the temperature range from 30°C to 35°C with elaidate-supplemented cells than with oleate-supplemented cells. At 25°C the induction was arrested in the former cells, while it was continued at a considerable rate in the latter. The arrest was released upon shift-back to 38°C, and precursors convertible to the active enzyme were not accumulated during incubation at 25°C. There was no marked difference in slope of Arrhenius plots of the rate of bulk protein synthesis between both types of cells, and the slope was almost equal to that of the rate of enzyme induction in the oleate-supplemented cells. The rate of β-galactosidase induction in the elaidate cells showed a similar temperature dependence to that of bulk protein synthesis.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.