Influence of neighbouring base sequence on N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the lacI gene of Escherichia coli

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced. In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations. This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine. The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis. This analysis revealed a strong influence by the 5' flanking base. On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.     

Immobilization of microorganisms by adhesion: interplay of electrostatic and nonelectrostatic interactions

The adhesion of three microorganisms (Saccharomyces cerevisiae, Acetobacter aceti, and Moniliella pollinis) to different materials has been studied using various supports (glass, metals, plastics), some of which were treated by an Fe(III) solution. The surface properties of the cells were characterized by the zeta potential and an index of hydrophobicity; characterization of the supports involved surface chemical analysis (XPS) and contact angle measurements. Cell suspensions in pure water at a given pH were left to settle on plates; the latter were then rinsed and examined microscopically, Saccharomyces cerevisiae and A. aceti adhere to metals under certain pH conditions but do not adhere to any of the other materials tested unless it is previously treated by ferric ions; adhesion of these hydrophilic cells is essentially controlled by electrostatic interactions. Moniliella pollinis adhere spontaneously to glass and to polymeric materials, but its attachment is also influenced by cell-cell or cell-support electrostatic repulsions; near the cell isoelectric point, cell flocculation is competing with adhesion to a support.     

Proposal for a scoring of the quality of the banding of chromosomes

Summary We propose an objective scoring of the quality of the banding of mitoses based on the number of bands (B), the length (L), and the width (W) of chromosome 7 in metaphase as used in the formula . When no figure shows the quality of mitoses from which a breakpoint is described, this scoring could give information about it. It could be applied in cytogenetic cancer studies as well as for preparations with high resolution banding.     

Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10^-4 to 10^-3 M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged ( 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10^-5 M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.     

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell-Descemet's membrane (DM) interface. Twenty-four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell-DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post-injury, a line of reaction product could still be demonstrated at the cell-DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10(-8) M colchicine or 2.5 X 10(-3) M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell-DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell-DM interface. However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell-DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.     

Glycosaminoglycan variants in the C2 muscle cell line

Using a replica technique, we have isolated and characterized five genetic variants of the C2 mouse muscle cell line that are defective in incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs). The variants incorporate free sulfate into GAGs at 5-20% of wild-type levels. None of the variants is defective in sulfate transport across the cell membrane, and in no case could the deficit in incorporation of sulfate be reversed by addition of an artificial initiator of GAG biosynthesis, p-nitrophenyl beta-D-xyloside. Analysis of the incorporation of [3H]glucosamine into GAGs by the variants revealed three different patterns: one variant incorporated [3H]glucosamine at the wild-type level; one, S27, at a severely reduced level; and three at intermediate levels. Four of the five variants showed marked deficits in their ability to differentiate and fuse. The remaining variant, S27, formed multinucleated myotubes and expressed acetylcholine receptor with a normal time course. Differentiation of the first four variants could not be restored by addition of exogenous GAGs or extracellular matrix. Because of the important roles that GAGs and proteoglycans are thought to play in the differentiation of muscle, these genetic variants should serve as useful tools in functional analyses of these molecules.     

Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships

Much progress has been made in recent years regarding the mechanisms of targeting of secretory proteins to, and across, the endoplasmic reticulum (ER) membrane. Many of the cellular components involved in mediating translocation across this bilayer have been identified and characterized. Polypeptide domains of secretory proteins, termed signal peptides, have been shown to be necessary, and in most cases sufficient, for entry of preproteins into the lumen of the ER. These NH2-terminal segments appear to serve multiple roles in targeting and translocation. The structural features which mediate their multiple functions are currently the subject of intense study.