In vitro activation of bacteriophage P2 late gene expression by extracts from phage P4-infected cells.

We have used a cell-free, DNA-dependent protein-synthesizing system to study the stimulation of phage P2 late gene expression by satellite phage P4. An activity is present in extracts prepared from P4-infected cells, which, when added to the in vitro system with P2 DNA template, stimulates the synthesis of a number of P2 proteins. These stimulated proteins include the major P2 capsid protein (N gene product) and a major component of the P2 phage tail (FII gene product). Extracts prepared from P4-infected cells are also able to stimulate the synthesis from P4 DNA of two low-molecular-weight proteins (18,500 and 17,000 Mr). The stimulating activity has no effect on the synthesis of proteins from lambda plac5 template. Extracts prepared from cells infected with P4 alpha amber mutants lack this stimulating activity.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Reduced osmotic potential inhibition of photosynthesis : site-specific effects of osmotically induced stromal acidification

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH₄Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.

Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH₄Cl, and the substrate for phosphoribulokinase to increase in the presence of NH₄Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH₄Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.

Additions of NH₄Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

     

Interactions of porphyrins with nucleic acids

The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

Formation of glycolate by a reconstituted spinach chloroplast preparation

A reconstituted preparation requiring fructose 6-phosphate, transketolase, triphosphopyridine nucleotide, ferredoxin, fragmented spinach chloroplasts, and light capable of forming glycolate at rates of about 10 micromoles per milligram of chlorophyll per hour has been characterized. The glycolaldehyde-transketolase addition product could be substituted for fructose 6-phosphate and transketolase. The stoichiometry of the reaction was: 1 mole of fructose 6-phosphate consumed for each mole of glycolate and of reduced triphosphopyridine nucleotide produced. Evidence was presented indicating that glycolate formation was coupled to the photosystems of the photosynthetic electron transport chain. Synthesis of glycolate is envisaged as the result of either (a) a reaction between the upper two carbon atoms derived from fructose 6-phosphate and an uncharacterized oxidant generated by photosystem 2 or (b) hydrogen peroxide produced by the reoxidation of reduced triphos-phopyridine nucleotide or reduced ferredoxin by molecular oxygen.     

Involvement of Photosynthetic Carbon Reduction Cycle Intermediates in CO(2) Fixation and O(2) Evolution by Isolated Chloroplasts

The photosynthetic carbon reduction cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO₂ evolution by whole spinach (Spinacia oleracea) chloroplasts and on their ability to affect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, antimycin A) of photosynthesis: class I (maximal): fructose 1, 6-diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate; class 2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class 3 (variable): glycerate 3-phosphate. While class 1 compounds influence the photosynthetic rate, they do not lower the Michaelis constant of the chloroplast for bicarbonate or affect strongly other photosynthetic properties such as the isotopic distribution pattern. It was concluded that the class 1 compounds influence the chloroplast by not only supplying components to the carbon cycle but also by activating or stabilizing a structural component of the chloroplast.     

ERYTHROID CELL DIFFERENTIATION AND THE INHIBITION OF CYTOKINESIS BY CYTOCHALASIN

Cytochalasin B produces multinucleated erythroid cells in tissue cultures of very young chick blastoderms. There is no apparent qualitative interference with differentiation and maturation of erythroid cells, but the amounts produced are reduced 4- and 10-fold. These effects of cytochalasin are readily reversible.     

Transduction and Plasmid Deoxyribonucleic Acid Analysis in a Multiply Antibiotic-Resistant Strain of Staphylococcus epidermidis

A genetic analysis of a multiply antibiotic-resistant strain of Staphylococcus epidermidis was performed. Experiments designed to show reversion of organisms to antibiotic susceptibility, as well as studies of the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol (cml), tetracycline (tet), and neomycin (neo) resistance are present on separate plasmids, but the streptomycin marker is chromosomal. In 2 to 6% of tetracycline-resistant transductants, co-transduction of cml was also observed. By using CsCl-dye density gradients followed by neutral sucrose gradients, the plasmids carrying cml, tet, and neo could be isolated and their molecular weights could be determined. The tetracycline plasmid is shown to be incompatible with one of the cryptic plasmids of a recipient strain.