Nitrous acid mutagenesis of duplex DNA as a three-component system.

Purified native Hemophilus influenzae DNA is relatively insusceptible to nitrous acid (NA) mutagenesis in vitro, but is readily mutated following denaturation. NA mutagenicity for duplex DNA is significantly increased in the presence of various alcohols, glycols, phenols or primary amines. Phenol-extracted DNA contains dissociable contaminants of low molecular weight that enhance NA mutagenesis. Enhancement of NA mutagenesis by phenol and by spermine is due to the formation of unstable molecular species. We propose that reactive organic nitroso compounds are formed which then serve as delivery vehicles to promote mutagenicity of native DNA, perhaps via transnitrosation reactions. Similar reactions probably occur in vivo to promote NA-induced base substitution (but not frameshift) mutations in Salmonella typhimurium and in Escherichia coli. The possible significance of these observations to carcinogenesis is discussed.     

Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.     

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.     

Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in absence of recombinational repair

Near-ultraviolet (NUV) radiation killing of Escherichia coli K-12 can be enhanced by a sub-lethal concentration of hydrogen peroxide. This can be divided into a “RecA-dependent” and “RecA-independent” synergistic killing action. Stationary phase wild-type and 8 closely related repair-deficient mutants were examined for their NUV sensitivities in the presence and absence of H₂O₂. All exhibited the “RecA-independent” synergism; i.e., H₂O₂ enhanced NUV lethality when RecA repair was not operating. The “RecA-independent” synergism did not result from destruction of repair enzymes. Very few DNA—protein crosslinks could be detected following NUV plus H₂O₂ treatment. However, double-strand (DS) DNA breaks were produced, apparently by conversion of closely spaced single-strand (SS) breaks on opposite strands. The correlation between DS-break formation and lethality in wild-type and a polA mutant indicates that the RecA-independent synergistic killing results from the conversion of SS into lethal DS breaks.     

Extracellular Maltase of Bacillus brevis

Bacillus brevis NRRL B-4389 produced extracellular maltase (alpha-glucosidase; EC 3.2.1.20) only in the presence of short alpha-1,4-glucosidic polymers, such as maltose and maltotriose. An optimum medium was developed; it contained 2.5% maltose, 0.5% nonfat dry milk, 0.4% yeast extract, and 0.01% CaCl(2). The enzyme was produced extracellularly during the logarithmic phase of growth; no cell-bound activity was detected at any time. Partial purification of the maltase was accomplished by using diethylaminoethyl cellulose batch adsorption, ammonium sulfate precipitation, and Sephadex G-200 gel filtration. Maltase, isomaltase (oligo-1,6-glucosidase), and glucosyltransferase activities were purified 20.0-, 19.1-, and 11.5-fold, respectively. Some properties of the partially purified maltase were determined: optimum pH, 6.5; optimum temperature, 48 to 50 degrees C; pH stability range, 5.0 to 7.0; temperature stability range, 0 to 50 degrees C; isoelectric point, pH 5.2; and molecular weight, 52,000. The relative rates of hydrolysis of maltose (G(2)), maltotriose (G(3)), G(4), methyl-alpha-d-maltoside, G(40), dextrin, and isomaltose were 100, 22, 12, 10, 10, 8, and 5%, respectively; the K(m) on maltose was 5.8 mM; d-glucose, p-nitrophenyl-alpha-d-glucoside, and tris (hydroxymethyl) aminomethane were competitive inhibitors; transglucosylase activity of the enzyme on maltose resulted in the synthesis of isomaltose, isomaltotroise, and larger oligosaccharides.     

Does high-mobility-group non-histone protein HMG 1 interact specifically with histone H1 subfractions?

The interaction of the non-histone chromosomal protein HMG (high-mobility group) 1 with histone H1 subfractions was investigated by equilibrium sedimentation and n.m.r. sectroscopy. In contrast with a previous report [Smerdon & Isenberg (1976) Biochemistry 15, 4242--4247], it was found, by using equilibrium-sedimentation analysis, that protein HMG 1 binds to all three histone H1 subfractions CTL1, CTL2, and CTL3, arguing against there being a specific interaction between protein HMG 1 and only two of the subfractions, CTL1 and CTL2. Raising the ionic strength of the solutions prevents binding of protein HMG 1 to total histone H1 and the three subfractions, suggesting that the binding in vitro is simply a non-specific ionic interaction between acidic regions of the non-histone protein and the basic regions of the histone. Protein HMG 1 binds to histone H5 also, supporting this view. The above conclusions are supported by n.m.r. studies of protein HMG 1/histone H1 subfraction mixtures. When the two proteins were mixed, there was little perturbation of the n.m.r. spectra and there was no evidence for specific interaction of protein HMG 1 with any of the subfractions. It therefore remains an open question as to whether protein HMG 1 and histone H1 are complexed together in chromatin.     

Infection of the heart cockle, Clinocardium nuttallii, from Yaquina Bay,Oregon, with an endosymbiotic alga

Clinocardium nuttallii from Yaquina Bay, Oregon, were found to harbor an endosymbiotic alga. Some aspects of this relationship are presented. The areas of infection include the siphon, mantle, and occasionally the foot. C. nuttallii under 2 years old are not infected; the incidence and intensity of the infection increases in the older age groups. For all ages the incidence averages 35%. Pigment composition, morphology, and growth characteristics of the alga are similar to those found in the genus Chlorella. In situ, the algae form dense, sometimes massive colonies but do not appear to cause any host reaction or enfeeblement. The thick layers surrounding the algal cells in situ, the dense colonies, and the in vitro reaction to host extract suggest the alga is parasitic. However, the presence of chloroplasts, the location of the algal cells only in illuminated tissue, the seemingly unhampered reproduction in situ, and the eventual adaptation to mineral medium suggest the alga is a facultative parasite. Experimental infection was achieved by feeding mature uninfected cockles a diet of only symbiont cells. In vitro observations found the symbiont cells readily engulfed by host blood amoebocytes. It is believed that the animal acquires the infection through phagocytosis of the symbiont cells and subsequent diapedesis across epithelial barriers by host amoebocytic cells.     

Deterioration of High-Moisture Corn

Two small, leaky silos were filled with normal high-moisture corn (HMC), and two with HMC severely infested by Helminthosporium maydis. Counts of mesophilic bacteria, lactobacilli, coliforms, yeasts, and molds were made on corn samples as received and periodically thereafter during 220 days of storage. Temperature and gas levels also were monitored. Sequential changes in the populations of lactobacilli, yeasts, and molds were determined during spoilage of HMC. These population changes were compared on the basis of the variables encountered in the present study as well as with the results of previous studies conducted on normal HMC stored under adequate conditions. Heavy infestation by H. maydis had no appreciable effect on HMC preservation.     

Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking in digests of the substrate-protected enzyme. Peptides representing two sites of modification have been obtained from the inactivated carboxylase. Both sites of reaction have been identified as lysyl residues based on the conversion of the derivatives to free lysine by oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel electrophoretic experiments show that both essential lysyl residues are contained within the large subunit of ribulosebisphosphate carboxylase. In addition to lysyl residues, sulfhydryl groups of the carboxylase are also modified, but their modification seems to play little role in the inactivation process. The carboxylase modified in the presence of substrate contains sulfhydryl derivatives but is essentially lacking in lysyl derivatives. By comparing the profiles from ion exchange chromatography of labeled peptides in digests of inactivated and substrate-protected enzyme, we conclude that the same sulfhydryl groups are modified in the absence and presence of substrate.