Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

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.     

The potential of production of sulfated polysaccharides from Porphyridium

Summary The environmental conditions prevailing in Israel make marine algae an attractive crop for the production of valuable chemicals. A marine species of Porphyridium seems to fit this purpose.The unicellular red alga Porphyridium is encapsulated by a polysaccharide envelope that is present in the gel state. This polysaccharide is an acidic heteropolymer composed of sulfated sugars. It forms ionic bridges through divalent cations, thus reaching a very high molecular weight. The thickness of the polysaccharide capsule varies according to the phase of growth and the growth conditions. Its outer part dissolves in the growth medium, which becomes progressively more viscous. Sulfated polysaccharides form theramlly reversible gels similar to agar and carrageenan, which are usually extracted from marine macroalgae. These gels have been finding increasing use in commercial applications as gelling agents, thickeners, stabilizers, and emulsifiers.We have done experiments on the cultivation of a marine species of Porphyridium for the production of polysaccharides. This unicellular alga has an advantage over the macroalgae due to its relatively faster growth rate and the possibility to regulate its growth. The potential for production of the polysaccharide, both that dissolved in the external medium and that attached to the cell (including an intracellular fraction), and the effects of growth conditions on productivity were suudied in the laboratory. Porphyridium was also cultivated outdoors in seawater in 1-m² ponds and its growth potential investigated.     

Purification and partial characterization of the extracellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602

The gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase, or endopeptidase I, from Bacillus sphaericus 9602 was purified to apparent protein homogeneity. The purification was achieved by a six-step procedure: ammonium sulfate fractionation, phenyl-Sepharose chromatography, two consecutive DEAE-Trisacryl chromatographies, chromatofocusing and Sephacryl S-200 permeation chromatography. The enzyme was purified 5000-fold with a 38% recovery of lytic activity. It is an acidic protein (pI 5.4) of hydrophobic nature. Kinetic studies have shown a Km value of 0.57 mM and an apparent Vmax of 8.3 mumol min-1 (mg enzyme)-1 with N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)meso-diaminopimelyl (L)-D-[14C]alanine as substrate. The enzyme was inhibited by o-phenanthroline and EDTA and was reactivated by zinc, cobalt and manganese ions; thus endopeptidase I is a metallo enzyme, probably a zinc enzyme. Moreover it is a heat-stable protein with an apparent inactivation temperature of 80 degrees C.     

The ;heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis.