Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database

Abstract The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (Ptsl), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.     

An early Holocene reef in the western Atlantic: submersible investigations of a deep relict reef off the west coast of Barbados,W.I.

Submersible observations and collections reveal that a probable relict reef off the west coast of Barbados has a rich cover of sponges, along with algae and scattered corals, on a substrate of algal nodules in a muddy-sand matrix. The collections provide new data on the distributions of these fauna. This relict reef is about 20 km long, has a relief of up to 10 m, and is established at a depth of 80 m. Relict shallow-water features in other areas at similar depths along with data from core holes drilled off the south coast of Barbados suggest that this reef was probably established about 12,000 years ago and existed for no more than 2,000 years, during the Holocene sea-level transgression.     

The interaction of auramine O with calmodulin: location of the binding site on the connecting strand.

The cationic dye auramine O forms a fluorescent complex with Ca(2+)-liganded calmodulin. One moderately strong binding site is present, as well as one or more weaker sites. The binding site for auramine O is different from those for toluidinyl-naphthalene sulfonate. The dependence of binding upon electrolyte concentration suggests a substantial electrostatic component of the free energy of binding. The splitting of the bond between residues 77 and 78 by trypsin digestion abolishes auramine O binding; the N- and C-terminal half-molecules have virtually no binding capacity. This suggests that the primary binding site is located near the midpoint of the connecting strand and includes elements of both half-molecules. Thrombin digestion, which splits calmodulin between residues 106 and 107, also substantially reduces auramine O binding; this may be interpreted in terms of the stabilization of the structure of the connecting strand by interaction with residues within binding domain IV. The binding affinity at pH 5.0, where the helical organization of the connecting strand may be intact, is greater than at neutral pH.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detailed Analysis of Sequence Changes Occurring during vlsE Antigenic Variation in the Mouse Model of Borrelia burgdorferi Infection

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained “template-independent” sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.