Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.     

Physiological and social constraints on growth of Arctic charr, Salvelinus alpinus L.: an investigation of factors leading to stunting

When stunted (runt) Arctic charr, Salvelinus alpinus , were reared together in small groups growth rates were low. Contrary to expectation, there did not arise a rapidly growing despot within each group. It is suggested that the narrow size range offish within groups prevented the rapid formation of clear-cut dominance hierarchies. Fish reared in isolation had significantly higher and more variable rates of growth than those held in groups, demonstrating that social interactions between individuals were responsible for growth suppression of grouped-reared fish. There were no significant differences in growth rates between fish allowed visual contact with conspecifics and those held in visual isolation, indicating that visual contact is insufficient to cause growth depensation in Arctic charr. Despite the fact that growth rates of stunted (runt) Arctic charr improved when they were held in isolation, the growth rates recorded were substantially lower than those of normal individuals reared under hatchery conditions. It is concluded that physiological (genetic) factors are important in the determination of the slow growth rates of stunted (runt) Arctic charr and that this trend is exacerbated by social interactions with more rapidly growing siblings.     

Effect of feeding frequency on food intake and growth of Arctic charr, Salvelinus alpinus L.

Social interactions and dominance hierarchy effects are important factors governing rates of growth of Arctic charr, Salvelinus alpinus L. The effects of hierarchy were increased as access to food became more restricted, i.e. feeding frequency was reduced, but these effects could not be attributed to direct competition for food since fish were fed to satiation at each feeding period. The results suggest that, whilst some fish on the restricted feeding regime were able to maintain good rates of growth, feeding by the majority of the fish was inhibited by the presence of larger individuals. Due to the importance of these hierarchy effects it was not possible to demonstrate physiological adaptations in fish allowed infrequent access to food.     

Measurement of vitellogenin,a biomarker for exposure to oestrogenic chemicals,in a wide variety of cyprinid fish

There is increasing concern about man-made chemicals in the aquatic environment that mimic oestrogens because they may disrupt reproductive function. Vitellogenin, a precursor of egg-yolk in fish and other oviparous animals, may be used as a biomarker for “oestrogen” exposure. This study investigated the use of a radioimmunoassay developed to carp (Cyprinus carpio) vitellogenin to measure vitellogenin in other species of fish, especially cyprinids that would be of value for field and laboratory studies on oestrogenic xenobiotics. Of the nine families of fish studied, only vitellogenin from cyprinids (to which the carp belongs) showed good cross-reactivity in the carp vitellogenin radioimmunoassay. Vitellogenin from cyprinids native to Europe that cross reacted in the carp vitellogenin radioimmunoassay included: bream (Abramis brama), roach (Rutilus rutilus), rudd (Scardinius erythropthalmus), gudgeon (Gobio gobio) and minnow (Phoxinus phoxinus). Vitellogenin from cyprinids used widely in ecotoxicology that cross reacted in the carp vitellogenin radioimmunoassay included: fathead minnow (Pimephales promelas), zebrafish (Brachydanio rerio) and goldfish (Carassius auratus). In the cyprinids studies, the concentrations of vitellogenin in mature females were between a few hundred and a thousand microgram per millilitre. Concentrations of plasma vitellogenin in immature females were always greater than 200 ng·m^-1, whereas in males (with the exception of the fathead minnow) plasma vitellogenin concentrations were less than 20 ng·ml^-1 (and generally, much lower). The results suggest that the structure of vitellogenin is highly conserved within the cyprinid family and that the carp vitellogenin radioimmunoassay may be used to measure the concentrations of vitellogenin in plasma from a wide variety of cyprinids.     

The influence of environmental manipulations on inter– and intra–individual variation in food acquisition and growth performance of Arctic charr, Salvelinus alpinus

Repeated measurements of food intake made on juvenile Arctic charr, Salvelinus alpinus , held under different rearing conditions enabled examination of the effects of environmental manipulations on both intra– and inter–individual variations in food intake to be made. This permitted the assessment of the influences of differential food acquisition on individual growth rates and biomass gain. When charr were held in isolation individual fish showed relatively little day–to–day variability in food intake and inter–individual differences in intake were small ('base–fine' values). All fish exhibited positive rates of growth and the overall range was narrow. Nevertheless, there was a highly significant positive correlation between food intake and growth, indicating that those individuals that consumed the greatest quantities of food were also those that had the highest rates of weight gain. The rearing of charr in groups led to increases in both intra– and inter–individual variations in food intake to levels considerably above 'base–line'. This increased variability in food intake was reflected in rates of weight gain being more variable amongst the charr reared in groups, with fish that lost weight often being recorded. Manipulation of the rearing environment had marked influences upon intra–individual variability in food intake, inter–individual differences in food acquisition and rates of weight gain. High stocking densities and exposure of the fish to moderate water currents were most effective in reducing levels of variability to approach those observed under 'base–line' conditions.     

Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii

We describe the characterization of tandemly repeated DNA sequences, which resemble the satellite DNA sequences of multicellular eucaryotes, in the unicellular green alga Chlamydomonas reinhardtii. Restriction enzymes that cleave C. reinhardtii DNA relatively frequently produce a number of high molecular weight DNA fragments in addition to the bulk of low molecular weight DNA fragments. pTANC 1.5 contains a 1.5 kb Sau3A fragment cloned from one of these large bands. pTANC 1.5 hybridized to at least three large arrays (200 to 700 kb) of tandemly repeated DNA sequences in the cell-wall-deficient strain cw1.5. These arrays are composed of repeat units that are each cleaved once by BamHl into bands of 1.5, 1.9, 2.0 and 2.5 kb in size. The copy numbers of the 1.5, 1.9, 2.0 and 2.5 kb Bamhl bands vary between different C. reinhardtii strains. Chlamydomonas smithii and a number of C. reinhardtii strains are deficient in all four BamHl bands. Genetic analysis of wild-type strain 137c, which is deficient in the 2.0 kb BamHl band, indicates that the 1.5, 1.9 and 2.5 kb BamHl bands derive from at least five loci. The 1.5, 1.9 and 2.5 kb repeat units are not extensively interspersed with each other in strain 137c. Pulsed-field gel electrophoresis of intact C. reinhardtii chromosomes indicates that TANC arrays are present on more than one chromosome.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Exercise,agonistic behaviour and food acquisition in Arctic charr,Salvelinus alpinus

Synopsis Although swimming is energetically costly, a number of studies on salmonid species have demonstrated increased growth rates in fishes forced to swim for prolonged periods at moderate speeds (typically 1–2 body lengths per sec). This suggests that additional energetic costs of swimming are more than met by alternative compensatory gains. The mechanisms underlying such effects are not fully understood. In this paper, we describe an experiment designed to examine one possible mechanism, namely a swimming-induced inhibition of aggression, with consequent beneficial effects on growth. The study used Arctic charr,Salvelinus alpinus, a species for which a positive relationship between exercise and growth has been clearly established. Using direct behavioural observations on small groups, we demonstrate that individuals displaying high levels of aggressive behaviour are able to monopolise access to food and that enforced swimming at a moderate speed (1 body length per sec) reduces the incidence of aggression although not the degree of monopolisation of food shown by aggressive individuals. These results suggest that the enhanced growth rates accompanying enforced swimming may reflect lower energetic costs of reduced aggressive activity rather than improved access to food by subordinates.