The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.     

Seasonal change in photoperiodic response of the larvae of the lacewing Nineta pallida

Larvae of the lacewing Nineta pallida (Schneider), collected in the field during two seasons, from September to July, were reared in the laboratory under short- or long-day light conditions at 21°C. In autumn and winter, artificial short days delayed the first ecdysis. The influence on the duration of the first instar was maximal (3.4 times longer) when the short days began at hatching time, and later regularly diminished. In spring, the second and third instars showed a reversed response so that the long days now increased the duration of development, although development took no more than 1.4 time as long as in short days. A similar effect appeared in field-collected third instars on and after mid June, reaching its maximum (1.8 time until the cocoon spinning) in July. This sort of photoperiodic effect on the larval development is new to the seasonal adaptation of the life cycle in insects.

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Résumé Des formes préimaginales (oeufs, puis larves) de N. pallida sont récoltées sur des conifères de montagne (Pyrénées), chaque mois depuis septembre jusqu'en juillet en deux saisons (1983/84 et 1985/86). Elles sont ensuite élevées au laboratoire à 21°C, soit en jours longs (JL=L16:D8), soit en jours courts (JC=L8:D16).Le développement embryonnaire est légèrement plus long s'il se fait en JC. Pour les larves de premier stade récoltées en automne et en hiver, les JC retardent considérablement la première mue et prolongent aussi le deuxième stade qui en provient. L'influence retardatrice est maximale (3,4 fois) lorsque les JC agissent dès l'éclosion. Elle diminue ensuite progressivement et devient insignificante pour les larves récoltées à artir de février.Au printemps, les larves récoltées au deuxième stade ainsi que les troisièmes stades qui en découlent présentent une réaction inverse: ce sont alors les JL qui augmentent la durée du dévelopement, toutefois, pas plus de 1,4 fois par rapport aux JC. Un effet de même ordre se manifeste sur les larves de troisième stade récoltées à partir de juin, atteignant son maximum (1,8 fois) dans le lot de larves de juillet, c'est-à-dire peu avant la fin de la croissance pondérale larvaire et le coconnage.Un tel retardement du développement larvaire hivernal, prolongé au printemps et au début de l'été par une inversion de la réponse à la photopériode, est nouveau comme élément d'adaptation saisonnière du cycle naturel chez les insectes.

     

Regulation of carbon flow in Selenomonas ruminantium grown in glucose-limited continuous culture.

We have applied a model that permits the estimation of the sensitivity of flux through branch point enzymes (D. C. LaPorte, K. Walsh, and D. E. Koshland, J. Biol. Chem. 259:14068-14075, 1984) in order to analyze the control of flux through the lactate-acetate branch point of Selenomonas ruminantium grown in glucose-limited continuous culture. At this branch point, pyruvate is the substrate of both the NAD-dependent L-(+)-lactate dehydrogenase (LDH) and the pyruvate:ferredoxin oxidoreductase (PFOR). The LDH was purified, and it exhibited positive cooperativity for the binding of pyruvate. The LDH had an [S].5 for pyruvate of 0.43 mM, a Hill coefficient of 2.4, and a K' equal to 0.13 mM. The PFOR, assayed in cell extracts, exhibited Michaelis-Menten kinetics for pyruvate, with a Km of 0.49 mM. Carbon flux through the LDH and the PFOR increased 80-fold and 3-fold, respectively, as the dilution rate was increased from 0.07 to 0.52 h-1 in glucose-limited continuous culture. There was nearly a twofold increase, from 6.5 to 11.2 mumol min-1 mg of protein-1 in the specific activity (i.e., maximum velocity) of the LDH at dilution rates of 0.11 and 0.52 h-1, respectively. A flux equation was used to calculate the intracellular concentration of pyruvate; a fourfold increase in pyruvate, from 0.023 to 0.093 mM, was thereby predicted as the dilution rate was increased from 0.07 to 0.52 h-1. When these calculated values of intracellular pyruvate concentration were inserted into the flux equation, the predicted values of flux through the LDH and the PFOR were found to match closely the flux actually measured in the chemostat-grown cells. Thus, the 80-fold increase in flux through the LDH was due to a twofold increase in the maximum velocity of the LDH and a fourfold increase in the intracellular pyruvate concentration. In addition, the flux through the LDH exhibited ultrasensitivity to changes in both the maximum velocity of the LDH and the intracellular concentration of pyruvate. The flux through the PFOR exhibited ultrasensitivity to changes in the maximum velocity of the LDH and hyperbolic sensitivity to changes in the intracellular concentration of pyruvate.     

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.     

Mixtures of a series of homologous hydrophobic peptides with lipid bilayers: a simple model system for examining the protein-lipid interface

The interactions of several members of a homologous series of peptides with the phospholipid bilayer have been examined by using fluorescence and deuterium NMR spectroscopy, differential scanning calorimetry, and measurements of water-to-bilayer partition coefficients. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers and tripeptides of the form Ala-X-Ala-O-tert-butyl are used as a model system to probe the influence of amino acid side-chain substitution on the insertion of peptides into membranes and the behavior of peptide/bilayer mixtures. Tripeptides with X = Gly, Ala, Phe, and Trp have been examined. All of the tripeptides are water soluble, and all partition into DMPC bilayer vesicles to some extent. The Gly-containing peptide is the least soluble and the Trp-containing peptide the most soluble in the bilayer. The extent of perturbation of the bilayer structure induced by the peptides parallels their bilayer solubility: the Gly and Ala peptides act as simple impurities while peptides containing bulky aromatic rings cause a phase separation. Changes in the fluorescence properties of the Trp analogue upon incorporation into the bilayer indicate that the Trp side chain is probably immersed in the hydrocarbon region of the bilayer. Peptides of this form should serve as easily modifiable model systems with which to examine details of how the bilayer environment affects peptide conformation, as well as how hydrophobic peptides affect the bilayer structure.     

A comparison of fragile X expression in lymphocyte and lymphoblastoid cultures

This study compares fragile X expression in peripheral blood lymphocyte cultures with expression in lymphoblastoid cell lines established from 23 individuals from families in which the fragile X is segregating. Most patients expressed the fragile X in lymphoblastoid cell lines treated with FUdR under optimal conditions at approximately the same frequency as in peripheral blood cultures from the same individual. No fragile X cells were seen in the lymphoblastoid cell lines from three phenotypically normal males who had transmitted the fragile X gene to offspring or in the lines from three phenotypically normal obligate-carrier females, all of whom were also negative in peripheral blood cultures. Two individuals, however, who expressed at high levels in peripheral blood lymphocytes expressed in lymphoblastoid cells only at low levels or not at all. We describe the considerations needed for the consistent demonstration of the fragile X in lymphoblastoid cell lines.     

Effect of hypoxia on the initiation of secondary wool follicles in the fetus

The development of secondary wool follicles in single fetal sheep subjected to hypobaric hypoxaemia was studied. One group of pregnant ewes were exposed to 57.1 kPa from 30 to 135 days gestation. Fetal weights (mean +/- s.d.) for the hypoxaemic group (3.35 +/- 0.53 kg; n = 4) were significantly lower than for the controls (4.19 +/- 0.31 kg; n = 3, P less than 0.05). At 110 days gestation, a second group had arterial and venous catheters surgically implanted into the ewe and fetus and skin samples were taken from the fetus. At 120 days gestation (10 days after surgery) these animals were subjected to hypoxia for 20 days, at a level to maintain fetal carotid pO2 between 1.47 and 1.87 kPa (mean carotid pO2 for the control fetuses was 2.84 +/- 0.28 kPa). Fetal weight at 140 days was not significantly different in the hypoxaemic and control groups. Morphometric analysis revealed that the secondary to primary follicle ratio (S:P) was less in both groups of hypoxaemic fetuses than in their respective controls. Although hypoxia for 20 days did not significantly alter fetal weight, it produced a low S:P ratio similar to the longer-term hypoxaemic animals. It is concluded that hypoxia has a marked effect in reducing the initiation of secondary follicles in the last third of gestation.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.