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.     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

18O studies of the peroxidase-catalyzed oxidation of N-methylcarbazole. Mechanisms of carbinolamine and carboxaldehyde formation

Chloroperoxidase, horseradish peroxidase, hemoglobin, myoglobin, lactoperoxidase, and microperoxidase catalyzed the ethyl hydroperoxide-dependent oxidation of N-methylcarbazole to N-(hydroxymethyl)carbazole and N-formylcarbazole as major products. Mass spectral analysis of the N-(hydroxymethyl)carbazole formed during the peroxidase-catalyzed N-demethylation of N-methylcarbazole in 18O-enriched medium indicated partial incorporation (7.5-25.9%) of solvent water oxygen into the carbinolamine intermediate in all systems investigated, suggesting that the peroxidase active site is partially accessible to solvent water during N-demethylation. In contrast, solvent water oxygen was not incorporated into the N-formylcarbazole formed during the peroxidase-catalyzed oxidation of N-methylcarbazole. N-(Hydroxymethyl)carbazole was not further metabolized by the peroxidases in the presence of ethyl hydroperoxide, indicating that it is not an intermediate in N-formylcarbazole formation. The horseradish peroxidase-catalyzed formation of N-formylcarbazole was decreased by 77% when the hydroperoxide-supported reactions were carried out in a nitrogen atmosphere, while the formation of N-(hydroxymethyl)carbazole was decreased by 46%. When the horseradish peroxidase-catalyzed reactions were carried out in a 18O2 atmosphere, 18O incorporation into N-(hydroxymethyl)carbazole was 64.4% of the total oxygen, while 81.8% of the oxygen incorporated into N-formylcarbazole came from 18O2. These results suggest that there are two different mechanisms for the formation of N-(hydroxymethyl)carbazole, both involving the initial oxidation of N-methylcarbazole to a neutral carbon-centered radical. The radical can be further oxidized in the enzyme active site to an iminium cation, which reacts with water derived from either the oxidant or the medium to form the carbinolamine. Alternatively, the substrate radical can react with molecular oxygen to form a hydroperoxy radical, which decomposes to form the carboxaldehyde and carbinolamine.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole

Inhibition studies were used to investigate the identity of the microsomal enzyme(s) responsible for the NADPH-dependent N-hydroxylation of 2-amino-6-nitrotoluene. The N-hydroxylation reaction was inhibited by several cytochrome P-450 inhibitors as well as by methimazole, a substrate for flavin-containing monooxygenase. Heat inactivation of flavin-containing monooxygenase had no effect on the rate of the reaction but abolished the inhibition by methimazole. These results indicate that the flavin-containing monooxygenase-mediated metabolism of methimazole produced an inhibitor of the cytochrome P-450-catalyzed N-hydroxylation reaction. When glutathione was included in the incubation the inhibition by methimazole was abolished, presumably due to the reduction of oxygenated metabolites of methimazole. These results show that methimazole inhibition does not necessarily implicate flavin-containing monooxygenase in microsomal N-hydroxylation reactions.     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.