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.     

INVITED SPECIAL PAPER: History of the botanical teaching laboratory in the United States

The establishment of teaching laboratories for botany in the United States was strongly influenced in the early part of the 19th century by the founding of a laboratory of natural history at the Rensselaer School by Amos Eaton who inspired numerous educators, particularly women. By midcentury and later, botany programs were established at land-grant colleges and the so-called “new Botany” movement spread from them. In the latter part of the century additional changes were brought about by the influence of German laboratory activity and botanists’ reactions to the introduction of the Huxley-Martin biology programs to America. During these times, Americans were improving their own manufactured microscopes, laboratory supplies, and equipment capabilities. By the beginning of the 20th century, laboratory teaching of botanical subjects was widely accepted as normal in universities and colleges, as well as in some high schools.     

The N-acylation-phosphodiesterase pathway and cell signalling

Long-chain N-acylethanolamines (NAEs) elicit a variety of biological and pharmacological effects, Anandamide (20:4n-6 NAE) and other polyunsaturated NAEs bind to the cannabinoid receptor and may thus serve as highly specific lipid mediators of cell signalling. NAEs can be formed by phospholipase D-catalyzed hydrolysis of N-acylethanolamine phospholipids or by direct condensation of ethanolamine and fatty acid, So far, most of the latter biosynthetic activity has been shown to be the reverse reaction of the NAE amidohydrolase that catalyzes NAE degradation. Thus, increasing evidence supports the hypothesis that the N-acylation-phosphodiesterase pathway yields not only saturated-monounsaturated NAEs, but polyunsaturated ones, including anandamide, as well.     

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.     

Molecular characterization of the marker chromosome associated with cat eye syndrome.

Cat eye syndrome (CES) is associated with a supernumerary bisatellited marker chromosome which is derived from duplicated regions of 22pter-22q11.2. In this study we have used dosage and RFLP analyses on 10 CES patients with marker chromosomes, by using probes to five loci mapped to 22q11.2. The sequences recognized by the probes D22S9, D22S43, and D22S57 are in four copies in all patients, but the sequences at the more distal loci, D22S36 and D22S75, are duplicated only in some individuals. D22S36 is present in three copies in some individuals, and D22S75 is present in two copies in the majority of cases. Only three individuals have a duplication of the most distal locus examined (D22S75), and these individuals have the largest marker chromosomes identified in this study. From the dosage analysis it was found that the marker chromosomes are variable in size and can be asymmetric in nature. There is no obvious correlation between the severity of the phenotype and the size of the duplication. The distal boundary of the CES critical region (D22S36) is proximal to that of DiGeorge syndrome, a contiguous-gene-deletion syndrome of 22q11.2.     

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.