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.     

Isolation of the MurineNbr1Gene Adjacent to the MurineBrca1Gene

The humanNBR1cDNA has previously been identified using polyclonal sera to CA125, an ovarian tumor antigen used in monitoring ovarian cancer. The gene was mapped to theBRCA1region on chromosome 17q21 and subsequently found to lie in close proximity to the recently identifiedBRCA1gene. The NBR1 protein has a B-box motif but the function of the protein is as yet unknown. To investigate the function and importance of this gene, we have studied the conservation of this gene in other species and in particular in the mouse. We have isolated murineNbr1cDNA and genomic clones. Translation of the cDNA sequence indicates that the protein is highly conserved, being 89% similar and 84% identical to the human. Analysis of the murineNbr1genomic clones indicates that it maps less than 1 kb from theBrca1gene and that, unlike that in human, this region is not duplicated.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.