Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

Preferential binding of an unfolded protein to DsbA.

The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

On sex determination in the Turkish desert woodlouse Hemilepistus elongatus (Crustacea, Isopoda, Oniscidea): searching for sex chromosomes and for sex-specific differences in simple DNA repeats.

The karyotype of male and female Hemilepistus elongatus was investigated by means of C-banding. The diploid chromosome number in both sexes is 2n = 50. By scrutinizing general morphology and localization of the constitutive heterochromatin, no heteromorphic sex chromosomes were found. All chromosome pairs in males are well paired during diakinesis. Hybridization of genomic DNA with (GACA)4 and (GATA)4 oligonucleotides revealed no sex-specific patterns. Key words : karyotype, C-banding, sex determination, simple DNA-repeats, Isopoda.     

Endpoint Bias in Large Tn10-Catalyzed Inversions in Salmonella Typhimurium

A genetic strategy identified Salmonella typhimurium strains carrying large (>40 kb) Tn10-catalyzed inversions; the inverted segments were characterized by XbaI digestion and pulsed field gel electrophoresis. Two size classes of large inversions were found. More than half of the inversions extended 40-80 kb either clockwise or counterclockwise of the original Tn10 site. The remaining inversions extended up to 1620 kb (33% of the genome), but the distal endpoints of these inversions were not randomly scattered throughout the chromosome. Rather, each Tn10 repeatedly yielded similar (though not identical) inversions. The biased endpoint selection may reflect the limited search for target DNA sequences by the Tn10 transposase, and the spatial proximity of the donor and target regions in the folded S. typhimurium nucleoid. Using this interpretation, the data suggest that DNA sequences 40-80 kb clockwise and counterclockwise of the insertion site are in spatial proximity with the insertion, perhaps reflecting the organization of DNA into ~120-kb nucleoid domains. In addition, the data predict the spatial proximity of several distant DNA regions, including DNA sequences equidistant from the origin of DNA replication.     

Aspects of morphogenesis and function of diatom cell walls with implications for taxonomy

Summary Aspects of morphogenesis and morphology of diatom cell walls are reviewed to highlight functional correlations between wall structures and three-dimensional cytoplasmic activities during the cell cycle. Morphogenesis of the siliceous valve within the silica deposition vesicle is discussed in the light of the dependency on a precisely orchestrated moulding machinery, involving the cytoskeleton, mitochondria, endoplasmic reticulum, spacer vesicles produced by the Golgi apparatus, and the plasmalemma, in combination with adhesion of the cells to parts of the parental wall and localized plasmolyses. Sensitivity of morphogenetic events to fluctuations of external factors has implications for taxonomy.Abbreviations CF cleavage furrows - cPL cleavage plasmalemma - GB girdle bands - LP labiate process - LPA labiate process apparatus - MC microtubule center - mLP macro labiate process - MT microtubule - MTOC microtubules organizing center - PL plasmalemma - SDV silica deposition vesicle - SL SDV membrane - SpV spacer vesicles Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.