Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Interaction of allosteric effectors (ATP,CO2, H+) modulating oxygen affinity of the hemoglobin in the carp,Cyprinus carpio,in vitro

Summary The interaction of allosteric effectors (CO₂, ATP, H⁺) with respect to the oxygen affinity of carp hemoglobin was analyzed by determining oxygen binding curves spectrophotometrically in dilute solutions of stripped hemoglobin at 20°C. The pH range studied was 6.8–8.2.P _CO2 was 0, 10 and 70 mmHg (0, 1.33 and 9.3 kPa). ATP/Hb₄ was 0, 8 and 24. In the presence of either CO₂ or ATP, the effects of the cofactors onP ₅₀ were as expected over the whole pH range. In contrast to other published data, each cofactor also had a significant effect onP ₅₀ in the presence of the other cofactor. Evidence was obtained that oxylabile carbamate is formed by carp hemoglobin and that the formation of carbamate persists at a lower level in the presence of ATP. The results support the view that the binding of ATP to carp hemoglobin requires only one terminal amino group, leaving the other N-terminal of the -chain free to react with CO₂.     

Delimitation ofPelargonium sect.Glaucophyllum (Geraniaceae)

Pelargonium otaviense Knuth andP. spinosum Willd. are excluded from sect.Glaucophyllum, whileP. grandiflorum (Andr.)Willd.,P. patulum Jacq. andP. tabulare (Burm. f.)L'Hérit. of sect.Eumorpha are included. Sect.Glaucophyllum is characterized by green to glaucous vegetative organs and zygomorphic white to pink corolla with five narrow petals. All the species have an identical pollen and chromosome morphology, the same basic chromosome number (x = 11) and similar flavonoid patterns. A close relationship between sect.Glaucophyllum and sect.Pelargonium is indicated by the occurrence of natural hybrids and concordant characters. Isorhamnetin and luteolin have been detected in the genus for the first time.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Using jackknife methods for estimating the parameter in dilution series

Dilution assays are quantal dose-response assays that detect a positive or negative response in each individual culture within groups of replicate cultures that vary in the dose of cells/organisms tested. We propose three jackknife versions of the maximum likelihood estimator of the unknown parameter, i.e., the frequency of a well-defined cell within the context of limiting dilution assays or the density of organisms within the context of serial dilution assays. The methods have been evaluated with artificial data from extensive Monte Carlo experiments. As a result of these experiments and theoretical considerations, the jackknife version based on deleting one individual culture at a time is proposed as the statistical procedure of choice. The next best method is the jackknife version based on leaving out the same replicate from each of the culture groups at a time.     

Secretion of a lipid transfer protein by human monocyte-derived macrophages

Human monocyte-derived macrophages in culture were shown to synthesize and secrete a lipid transfer protein. The human monocyte-derived macrophage transfer protein showed the following characteristics: (i) linear secretion rate over a 24-h period, which was blocked completely by cycloheximide and stimulated by phorbol myristate acetate (67% increase over nonstimulated values); (ii) apparent Mr = approximately 62,000 off Sephacryl S-200; (iii) isoelectric point of 5.0; (iv) binding to phenyl-Sepharose, but not to heparin-Sepharose; (v) facilitation of the transfer of both neutral lipids (cholesteryl esters and triglycerides) and phosphatidylcholine between high density lipoproteins and d less than 1.063 g/ml lipoproteins; and (vi) thermal stability (stable for 1 h at 56 degrees C). The last five of these properties are similar to those of the plasma lipid transfer protein. Thus, macrophages secrete a lipid transfer protein that closely resembles the neutral lipid transfer protein found in human plasma and may be a source of this plasma protein in vivo.     

Effects of oligomycin on the partial reactions of the sodium plus potassium-stimulated adenosine triphosphatase

The effects on phosphoenzyme (E-P) formation of ligands which activate Electrophorus (Na,K)-ATPase were investigated in the presence of oligomycin. When the enzyme was allowed to bind oligomycin in the presence of NaCl and MgCl2, subsequent addition of ATP plus KCl produced a monoexponential time course of E-P formation with a rate of 56 s-1, similar to the rate obtained in the uninhibited enzyme phosphorylated by ATP in the absence of KCl. Pi liberation under these conditions was slow and showed no initial burst phase, consistent with the inhibitory effect oligomycin has on the E1-P to E2-P conformational transition. Addition to KCl to a preincubation medium containing oligomycin, NaCl, and MgCl2 had no further effect on E-P formation. However, equilibration with oligomycin, KCl, and MgCl2 prior to the addition of NaCl plus ATP gave a much slower rate of E-P formation (5 s-1) and resulted in an initial rapid release of Pi similar to that found in the uninhibited enzyme. The slow increase in E-P level observed after incubation with oligomycin, KCl, and MgCl2 may be due to secondary formation of an inhibition complex following rapid binding of oligomycin. In contrast to the monophasic behavior which resulted from pre-exposure to NaCl or KCl, preincubation with oligomycin in the presence of MgCl2 plus Tris or Tris alone gave a biphasic pattern of E-P formation in which about 50% of the intermediate accumulated at a rate of 56 s-1 and the remainder at a rate of 5 s-1. In addition, the Pi burst amplitude was reduced, indicating partial inhibition of the enzyme. These results suggest that in the absence of Na+ and K+ only half of the enzyme is inhibited by oligomycin while the remainder undergoes inhibition subsequent to initiation of phosphorylation. Since the oligomycin concentration was saturating, the partial inhibition reflected in the biphasic pattern of E-P formation may be due to half-of-the-sites reactivity in which only half of the subunits bind oligomycin in the absence of monovalent cations.