Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase.

As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, J. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to l3 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP.     

Rapid glutamate decarboxylase assay for detection of Escherichia coli.

A rapid test procedure for the enzyme glutamate decarboxylase was developed for detection of Escherichia coli. The assay procedure was able to confirm the presence of E. coli in enteric broth cultures with 95% specificity for both pure cultures and environmental samples. The procedure was capable of detecting survivors among chlorine-exposed cells.     

Improving efficiency of breeding for higher crop yield

Summary Exclusive selection for yield raises, the harvest index of self-pollinated crops with little or no gain in total bipmass. In addition to selection for yield, it is suggested that efficient breeding for higher yield requires simultaneous selection for yield's three major, genetically controlled physiological components. The following are needed: (1) a superior rate of biomass accumulation. (2) a superior rate of actual yield accumulation in order to acquire a high harvest index, and (3) a time to harvest maturity that is neither shorter nor longer than the duration of the growing season. That duration is provided by the environment, which is the fourth major determinant of yield. Simultaneous selection is required because genetically established interconnections among the three major physiological components cause: (a) a correlation between the harvest index and days to maturity that is usually negative; (b) a correlation between the harvest index and total biomass that is often negative, and (c) a correlation between biomass and days to maturity that is usually positive. All three physiological components and the correlations among them can be quantified by yield system analysis (YSA) of yield trials. An additive main effects and multiplicative interaction (AMMI) statistical analysis can separate and quantify the genotype × environment interaction (G × E) effect on yield and on each physiological component that is caused by each genotype and by the different environment of each yield trial. The use of yield trials to select parents which have the highest rates of accumulation of both biomass and yield, in addition to selecting for the G × E that is specifically adapted to the site can accelerate advance toward the highest potential yield at each geographical site. Higher yield for many sites will raise average regional yield. Higher yield for multiple regions and continents will raise average yield on a world-wide basis. Genetic and physiological bases for lack of indirect selection for biomass from exclusive selection for yield are explained.     

The cytoplasmic tail of CD4 targets chimeric molecules to a degradative pathway.

Many different cell surface receptors undergo endocytosis via coated pits. Once having entered the cell, the receptors are sorted into diverse pathways. Which path a given receptor will follow is determined by signals inherent in the receptor's structure. The nature of these structural features is not yet known. In this study, we have taken the approach of constructing chimeric molecules to localize the domain of the T-cell surface molecule CD4 which is responsible for targeting it for degradation. Chimeric molecules bearing the cytoplasmic domain of CD4 and the extracellular domain of either the low-density lipoprotein receptor or a major histocompatibility complex (MHC) class I molecule were both internalized in response to phorbol 12-myristate 13-acetate and were subsequently degraded, indicating that the cytoplasmic tail of CD4 contains all the information required for both processes. The ability to modulate the level of MHC class I molecules on the cell surface offers an approach to investigating quantitative aspects of antigen presentation, the initial possibilities of which are explored herein.     

Phytoplankton composition and spatial distribution of copper and zinc in the Fal Estuary (Cornwall,UK)

In the estuaries near Falmouth (Cornwall, UK) levels of dissolved copper and zinc are high, due to drainage of copper and tin mines. Phytoplankton species composition in the autumn of 1989 deviated in the metal-contaminated Restronguet Creek from that in other estuarine branches,viz. Fal, Tresillian and Percuil. In the riverine part of Restronguet Creek (Carnon River)Euglena mutabilis, known as an acidophilic (pH 3) metal-resistant flagellate, occurred at micromolar Cu and Zn, whereas in the clean riversChlamydomonas sp. andOocystis sp. occurred at nanomolar Cu and Zn. An ordination analysis revealed the following patterns in Cu, Zn and phytoplankton species composition in the poly- and euhaline waters. Seston-bound Zn and dissolved Zn were in equilibrium,Katodinium rotundatum is Zn-tolerant, andSkeletonema costatum occurred in water with high contents of Cu in seston. However, none of the variables in these patterns correlated at a significant level (p>0.05). The results show that algae-metal interactions are complicated, and that statistical correlations foundin situ need experimental verification.Communication no. 542 of the Delta Institute for Hydrobiological Research.