Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit. The molecular mass of the complex is 163,700 daltons. Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration.     

Direct observation of cell wall structure in living plant tissues by solid-state C NMR spectroscopy

Solid-state ¹³C nuclear magnetic resonance (NMR) spectra of the following intact plant tissues were recorded by the crosspolarization magic-angle spinning technique: celery (Apium graveolens L.) collenchyma; carob bean (Ceratonia siliqua L.), fenugreek (Trigonella foenum-graecum L.), and nasturtium (Tropaeolum majus L.) endosperm; and lupin (Lupinus polyphyllus Lindl.) seed cotyledons. All these tissues had thickened cell walls which allowed them to withstand the centrifugal forces of magic angle spinning and which, except in the case of lupin seeds, dominated the NMR spectra. The celery collenchyma cell walls gave spectra typical of dicot primary cell walls. The carob bean and fenugreek seed spectra were dominated by resonances from galactomannans, which showed little sign of crystalline order. Resonances from β(1,4′)-d galactan were visible in the lupin seed spectrum, but there was much interference from protein. The nasturtium seed spectrum was largely derived from a xyloglucan, in which the conformation of the glucan core chain appeared to be intermediate between the solution form and solid forms of cellulose.     

Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.     

Stopped-flow mechanistic study of bromide substitution by an organonitrile at an iron(II) phosphinic centre; a π-electron driven process

The kinetics of the displacement reactions of the bromide ligands of trans-[FeBr₂(depe)₂] (depe = Et₂PCH₂CH₂PEt₂) by the organonitrile NCCH₂C₆H₄OMe-4, in tetrahydrofuran (either in the absence or in the presence of added Br⁻), to give the corresponding mono- and dinitrile complexes trans-[FeBr(NCCH₂C₆H₄OMe-4)(depe)₂]⁺ and trans-[Fe(NCCH₂C₆H₄OMe-4)₂(depe)₂]²⁺, have been investigated by stopped-flow spectrophotometry. The substitution reaction occurs by a mechanism involving rate-limiting dissociation of bromo ligands to form the unsaturated intermediates [FeBr(depe)₂]⁺ (k₁ = 1.52 ± 0.02 s⁻¹) and [Fe(NCR)(depe)₂]²⁺ (k₃ = 0.063 ± 0.008 s⁻¹) which add the nitrile ligand to form those nitrile complexes. The competition between the nitrile and Br⁻ for such metal centres has also been investigated and a stronger inhibiting effect of added Br⁻ is observed for the substitution of the second bromo ligand relative to the first one. The kinetic data are rationalized in terms of π-electronic effects of these unsaturated metal centres and of the bromide and nitrile ligands.     

Predation on artificial,solitary and aggregated wader nests on farmland

Predation rates on artificial wader nests, solitary curlew (Numenius arquata) and lapwing (Vanellus vanellus) nests and lapwing nests in colonies were studied on a farmland site in central Sweden. Predation rates were highest on artificial wader nests, intermediate on solitary curlew and lapwing nests and lowest on lapwing nests in colonies, probably because of active defence of adults at real nests and/or because of selection of nest sites with lower predation risk by breeding birds. A comparison of nests close to (50 m) and far away from (200 m) forest edges revealed no increased predation risk close to edges for any of the studied nest types. Predation risk changed during the season for artificial nests (highest in the middle of May), while predation rates on lapwing and curlew nests were more stable. Artificial nests seem to be inappropriate for measuring actual predation rates and temporal differences in predation rates on real nests, but they might be suitable for use as an index of spatial differences.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Comparison of techniques for measuring plasmid stability in yeasts

Summary Three techniques for measuring plasmid stability in yeasts are described and evaluated. The yeast used was aKluyveromyces lactis strain which was transformed with a plasmid, pCR1, to enable production of heterologous α-amylase. The techniques were based on plate counts on a selective antibiotic-containing medium and a non-selective medium, and on clearing zones on starch-supplemented plates for α-amylase detection. The plate ratio and clearing zones methods gave comparable results while the transfer colony method estimated much lower plasmid stabilities.