Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.     

Seasonal occurrence of Campylobacter spp. in surface waters and their correlation with standard indicator bacteria

Campylobacter jejuni, Campylobacter coli, and a Campylobacter-like organism were isolated from a number of natural water sources in central Washington, including ponds, lakes, and small mountain streams at elevations ranging from 1,460 to 5,400 feet (ca. 445 to 1,646 m) above sea level. At the two sites where extensive sampling was done, the bacteria were recovered throughout the year. Generally, the recovery rates were highest in the fall and winter months and lowest during the spring and summer months. Campylobacter density did not show significant correlation with microbiological (plate counts of fecal and total coliforms, fecal streptococci, and heterotrophic bacteria) or physical (water temperature, pH, and conductivity) parameters.     

The coupling of biogeochemical cycles of nutrients

For any element which is incorporated into biomass, the biogeochemical cycle of that element in a given ecosystem will be coupled to that of any other element similarly incorporated. The mutual interaction of two such cycles is examined using a simple model in which each cycle is constrained into four compartments. In each cycle the assimilation rate (primary productivity) is related in a non-linear fashion to the two nutrients and to biomass. The interactions are represented by combining a hyperbolic dependence for each nutrient (involving a "Michaelis constant") with a logistic equation governing the dependence of rate on biomass (involving a "carrying capacity"). The response of the model to perturbation (e.g. mobilization of an abiotic reserve) is strongly governed by the values assigned to these constants. The coupled cycles can exhibit positive feed-back with anomalous responses of the steady state and time-dependent solutions may exhibit complex oscillatory behaviour. Both the steady-state sensitivity and the kinetic behaviour of such coupled systems are simplified if the range of atomic ratios permitted by the assimilation process is restricted. It will therefore be of importance to determine under what conditions the assimilation rates for different elements are governed by mass-action effects (Liebig's Law) or by stoichiometric constraints (Redfield ratios).     

The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with O-linked glycosylation of one third of its extracellular amino acids.

Leukosialin is one of the major glycoproteins of thymocytes and T lymphocytes and is notable for a very high content of O-linked carbohydrate structures. The full protein sequence for rat leukosialin as translated from cDNA clones is now reported. The molecule contains 371 amino acids with 224 residues outside the cell, one transmembrane sequence and 124 cytoplasmic residues. Data from the peptide sequence and carbohydrate composition suggest that one in three of the extracellular amino acids may be O-glycosylated with no N-linked glycosylation sites. The cDNA sequence contained a CpG rich region in the 3' coding sequence and a large 3' non-coding region which included tandem repeats of the sequence GGAT.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.