Construction and properties of a chimeric bacteriophage lysis gene

Abstract The genomes of DNA phage ΦX174 and of RNA phage MS2 each encode a single lysis protein, E protein and L protein, respectively. Based on the known DNA and protein sequences, and with the aid of structural predictions of the proteins, a chimeric gene was constructed. The resulting protein was composed of the N-terminal sequence of E protein and the C-terminal sequence of L protein. The truncated E and L polypeptides used in this construction were functionally inactive. However, the chimeric gene product had very high lysis-inducing activity. This construction is an example which could be extended to the engineering of other lysis proteins designed with specific biotechnological processes in mind.     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

Enclosure experiment on the control mechanism of planktonic bacterial standing stock

In order to understand the control mechanisms of a large, stable bacterial standing stock, enclosure experiments were conducted in a eutrophic lake, where both bacterial productivity and grazing pressure were very high. Total bacterial number in the different enclosures ranged from 1.2 to 2.7×10⁷ cells mL⁻¹ throughout the experiment. The average bacterial cell production rate estimated from a grazer eliminating experiment was 6.3×10⁵ cells mL⁻¹ h⁻¹. Difference in the bacterial cell production rate between shaded and unshaded enclosures was not apparent. Bacteria showed a reduction in standing stock of only about 25–30% even after the supply of light was cut to 1%. Bacteria in the shaded enclosures then recovered their production rate in the first 12 days of perturbation. Grazing pressure in the shaded enclosures was not less than that for the control. Thus, it was considered a control mechanism of bacterial stable standing stock that the bacteria shifted their organic substrate from extracellular dissolved organic carbon freshly released from phytoplankton to that already stocked in the water column, though it is not known whether the dominant bacteria were the same.     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.     

The emergence of transgenic potatoes as commercial products and tools for basic science

The potato has tremendous potential as a transgenic crop and is a good model system by which to analyse metabolic regulation and gene expression. The potato’s difficult genetics, but ease of genetic transformation and its clonal means of propagation make it ideal for applied agricultural molecular genetics. Thus, the next 4 years promise to put the potato (with a diversity of transgenic constructs expressed) in the limelight as many of the first transgenic agricultural products enter the marketplace.     

Effect of glucagon and some other alpha and beta adrenergic agonists and antagonists on alanine amino transferase of perfused rat liver

Summary Glucagon increased alanine amino transferase (AAT) activity in perfused rat liver by about 90% over control. Propranolol, the beta receptor antagonist, abolished the effect of glucagon on this enzyme. Well known beta receptor agonists like isoproterenol, norepinephrine and epinephrine also increased the enzyme activity under identical condition and the enhancement was similarly abolished by propranolol. These experiments suggest that the effect of glucagon on AAT was mediated through beta adrenergic receptor. However, the interesting observation was that phenylephrine, alpha receptor agonist and phenoxybenzamine and tolazoline, two alpha receptor antagonists, increased the AAT activity like glucagon in perfusion experiments and the effects of all these three agents were also abolished by propranolol. Glucagon, when perfused with phenoxybenzamine showed some additive effect. From all these results we are proposing that in our system phenoxybenzamine is acting as beta agonist although it is known to be an alpha antagonist.