Isolation and characterization of isoprene mutants of Escherichia coli.

Isoprenoid compounds are found in all organisms. In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis. Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis. This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E. coli. Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells. The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content. In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC 5.3.3.2), farnesyldiphosphate synthetase (EC 2.5.1.1), and higher prenyl transferases.     

Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate-ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts

The effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate-ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50-500 microM H2O2 for 20 min or 100 microM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250-500 microM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50-500 microM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant-related DNA damage is a function of the type of oxidative stress used and may be cell-specific.     

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.     

Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of Mr of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in the short 5''-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.     

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study.

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.     

Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies.     

Effects of nitrate and acetate availability on chloroform production during carbon tetrachloride destruction

Fed batch experiments were performed to test the effects of electron donor and electron acceptor availability on the production of chloroform (CF) during carbon tetrachloride (CT) destruction by a denitrifying bacterial consortium. In one series of tests, acetate (electron donor) was present in excess while nitrate and nitrite (electron acceptor) were limiting. In the other series of tests, acetate was the limiting nutrient, and nitrate and nitrite were in excess. Under nitrate limiting conditions, 50% (+/-17%) of the CT transformed by the microorganisms was converted to CF. However, under acetate limiting conditions, only 4% (+/-4%) of the CT that was degraded appeared as CF. Previous research had suggested that denitrifying bacteria can degrade CT via two competing pathways. One of these pathways produces CF as the predominant end product. The second pathway produces CO(2) as the primary end product. The results shown here suggest that the first pathway is dominant when nitrate and nitrite are depleted while the second pathway, which produces little CF, dominates when nitrate or nitrite are available.