Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

Comparative study of different variants of Actinomyces roseoflavus producing the polyene antibiotic roseofungin

The respiration chain in the membranes of whole Actinomyces roseoflavus (var. roseofungini) cells from the parent and secondary cultures is sensitive to KCN, non-sensitive to Triton X-100 treatment removing the antibiotic roseofungin from the cells, and has a very high for the bacteria respiration control. When the cells are in contact with atomic tritium at the temperature of liquid nitrogen, roseofungin is tritiated and binds to A. roseoflavus isolated membranes and whole cells, mostly to those of the parent culture as compared to the secondary culture. A fraction of membranes which lost NADH dehydrogenase in the course++ of purification was isolated from the cells disintegrated in the frozen state.     

Effect of ascorbic acid prophylaxis on the frequency of chromosome aberrations, urine mutagenicity and nucleolus test in workers occupationally exposed to cytostatic drugs

The genetic risk of workers occupationally exposed to a series of newly developed cytostatic drugs and the presumed antimutagenic potential of ascorbic acid (AA) were studied in a group of 38 chemical laboratory personnel examined for chromosome aberrations in lymphocytes, urine mutagenicity and nucleolar RNA activity before and after a 6-month prophylactic administration of AA at daily doses of 1 g for 5 days a week. Chromosome aberration tests revealed elevated aberrant cell (AB.C) rates both prior to and after AA supplementation (3.9% and 3.65% of AB.C., respectively). These values were significantly higher than those found in 18 non-exposed matching controls (1.05% of AB.C.). Tests for mutagenic activity in the urine of drug-exposed workers revealed 64% positive urine samples prior to vitaminization and 60% positive urine specimens after it; positive urine samples in the group of controls accounted for 21% of samples. In the nucleolus test, numbers of inactivated micronuclei in the exposed were initially higher than those of controls (33.4% versus 24.3%), but dropped to 20.5% after AA supplementation. These findings show that AA prophylaxis alone cannot substantially reduce the hazards associated with exposure to anti-cancer drugs.     

ent-Kaurene Synthesis and Endogenous Levels of Gibberellins in a Shoot Forming Tobacco Crown Gall Tissue

The in vitro ent-Mcaurene synthesizing capacity, as well asthe endogenous GA content of shoot-forming tobacco crown gallsinduced by a nopaline-type Ti plasmid, was studied. For determinationof the ent-kaurene synthesizing capacity, an HPLC procedurepreceded by sample clean-up was used and the GA content wasexamined by GC-SIM. Kaurene synthesis reached a maximum at thebeginning of the logarithmic phase of growth. There was a clearcorrelation between the ent-kaurene synthesizing capacity andthe content of C₂₀-GAs. It seems that gibberellin synthesisis related to growth and development of the tissue. The natureof the GAs identified suggests, that the GA metabolism mightbe an unusual one. (Received October 12, 1987; Accepted April 11, 1988)     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

The composition of the murein of Escherichia coli

Escherichia coli murein, the polymer from which the shape-maintaining structure of the cell envelope is made, shows unexpected complexity. The separation of murein building blocks with high performance liquid chromatography reveals about 80 different types of muropeptides. Their behavior in high performance liquid chromatography and their chemical structure are described. The complexity of E. coli murein is due to the free combination of seven different types of side chains (L-Ala-D-Glu-R with R = -OH, -m-A2pm, -m-A2pm-D-Ala, -m-A2 pm-Gly, -m-A2pm-D-Ala-D-Ala, -m-A2pm-D-Ala-Gly, -m-A2pm-Lys-Arg) with two types of cross-bridges (D-Ala-m-A2pm, -m-A2pm-m-A2pm). The novel type of cross-bridge, A2pm-A2pm, contains an L,D-peptide bond, as shown by Edman degradation and chemical analysis of the reaction products. The A2pm-A2pm cross-bridge is assumed to play a role in the adaptation of the cross-linkage of murein to different growth conditions of the cell. The structural data of E. coli murein agree best with a model of a thin, however multilayered, murein sacculus.     

In vitro fusion of newt macrophages

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange-stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.     

Quantitation of the guanine nucleotide binding regulatory protein Gs in S49 cell membranes using antipeptide antibodies to alpha s

Polyclonal antibodies reactive against the guanine nucleotide binding stimulatory protein, Gs, were affinity-purified from two rabbits immunized with a synthetic peptide corresponding to amino acids 28-42 in the alpha-subunit, alpha s. On immunoblots, these antibodies recognized alpha s, but not alpha-subunits from two other guanine nucleotide binding regulatory proteins, Gi and Go. A competitive enzyme-linked immunosorbent assay was developed in which inhibition of antibody binding to peptide-coated microtiter plates was used to quantitate purified Gs or Gs in cholate extracts of cell membranes. Plasma membranes derived from wild type S49 lymphoma cells contained 18.9 +/- 2.3 pmol/mg of membrane protein of alpha s. The same membranes bound 169 +/- 12 fmol/mg of protein of [125I]iodocyanopindolol to beta-adrenergic receptors, indicating that the amount of Gs is far in excess of the amount of beta-adrenergic receptors. Thus, even if every beta-adrenergic receptor molecule were to activate 10 Gs molecules, in order for Gs to be limiting for the receptors to reach their high affinity state, it is likely that compartmentation exists for target cell membrane receptors and Gs. Moreover, a comparison of beta-adrenergic receptor number and Gs levels in several different S49 lymphoma cell mutants having lesions in receptors or Gs argues against a coordinate regulation of beta-adrenergic receptors and Gs.