Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.

The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.     

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.     

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.     

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

The eccentric cell of theLimulus lateral eye: encoder of circadian changes in visual responses

Efferent fibers from a central circadian clock innervate both photoreceptor cells and second-order neurons (eccentric cells) in the lateral compound eye ofLimulus, and release octopamine when activated. We have used intracellular microelectrodes to study the modulation of eccentric cell function by efferent optic-nerve activity, octopamine agonists, and a K⁺-channel blocker, TEA.

Chromosomal aberrations and sister-chromatid exchanges in lymphocytes from coke oven workers

To test whether coke oven workers, an occupational group known to be at increased cancer risk, manifest increased peripheral blood chromosomal aberration frequencies, we obtained samples from a group of 30 steelworker volunteers, who had worked several years at coke oven jobs. Exposure estimates were made using measurements of work place atmospheric coal tar pitch volatiles and work histories. No statistically significant positive regression of chromosomal aberrations on exposure estimates was found. The data from the coke oven workers were also compared with the obtained concurrently and employing precisely the same laboratory protocol from a group of male Brookhaven National Laboratory employees. The coke oven workers as a group were found to have statistically significantly elevated frequencies of chromatid aberrations and of sister-chromatid exchanges.     

Effects of simulated fire on vesicular-arbuscular mycorrhizae in pinyon-juniper woodland soil

Effects of fire on vesicular-arbuscular mycorrhizal fungi were tested using microcosms constructed from soil, litter, and duff collected beneath canopies of pinyon pine, Utah juniper, and in the open space (interspace). Burning was conducted over wet and dry soils. Soil temperatures were monitored continuously throughout the microcosms during burning. Plants grown in soils burned when dry had a lower VAM colonization than soils burned when wet. Juniper soils demonstrated the greatest reduction, over 95%, compared to their respective controls. Plants grown in interspace soils burned when wet were least affected. There was a positive correlation (r²=0.90) between the decrease in VAM colonization and the soil temperature as a result of the fire. Temperature effects, and associated reductions in VAM, were related to amount of litter burned in each microcosm and the moisture content of the soils.