Studies on nucleic acid reassociation kinetics: V. Effects of disparity in tracer and driver fragment lengths.

Measurements are described of the kinetics of nucleic acid strand pair reassociation where the complementary strands are of different lengths and are present in different concentrations. Rate constants for the reaction of labelled fragments ("tracer") with excess complementary strands ("driver") were determined, both for driver fragment length greater than tracer fragment length and for the reverse case. Second order reactions and pseudo-first order reactions utilizing strand separated drivers and tracers were studied. The nucleic acids which served for this investigation were phiX174 DNA and RNA, plasmid RSF2124 DNA and E. coli DNA. Approximate empirical expressions relating driver and tracer fragment lengths with the observed rate constants were obtained for practical use. In long tracer-short driver reactions the observed rate constant for the tracer reaction increases proportionately with tracer length. In long driver-short tracer reactions the rate of tracer reaction is retarded. The latter result is unexpected and appears to represent a departure from standard interpretations of the renaturation reaction.     

The adaptive evolution database (TAED)

Background

The Master Catalog is a collection of evolutionary families, including multiple sequence alignments, phylogenetic trees and reconstructed ancestral sequences, for all protein-sequence modules encoded by genes in GenBank. It can therefore support large-scale genomic surveys, of which we present here The Adaptive Evolution Database (TAED). In TAED, potential examples of positive adaptation are identified by high values for the normalized ratio of nonsynonymous to synonymous nucleotide substitution rates (K_A/K_S values) on branches of an evolutionary tree between nodes representing reconstructed ancestral sequences.

Results

Evolutionary trees and reconstructed ancestral sequences were extracted from the Master Catalog for every subtree containing proteins from the Chordata only or the Embryophyta only. Branches with high K_A/K_S values were identified. These represent candidate episodes in the history of the protein family when the protein may have undergone positive selection, where the mutant form conferred more fitness than the ancestral form. Such episodes are frequently associated with change in function. An unexpectedly large number of families (between 10% and 20% of those families examined) were found to have at least one branch with high K_A/K_S values above arbitrarily chosen cut-offs (1 and 0.6). Most of these survived a robustness test and were collected into TAED.

Conclusions

TAED is a raw resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study. It can be expanded to include other evolutionary information (for example changes in gene regulation or splicing) placed in a phylogenetic perspective.     

Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.0 and 8.7. The mutant enzyme H216N has a maximal k(cat) at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. (18)O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2-HCO3- exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.     

Biological activity of Manduca sexta allatotropin-like peptides,predicted products of tissue-specific and developmentally regulated alternatively spliced mRNAs

The insect neuropeptide, allatotropin (Manse-AT), exerts multiple functions including the stimulation of juvenile hormone (JH) biosynthesis in adults and the inhibition of active ion transport across the midgut epithelium of feeding larvae. The Manse-AT gene is expressed in multiple regions of the nervous system as three mRNAs that differ by alternative splicing. The specific mRNA isoform present differs in a tissue- and developmental-specific manner thus providing a mechanism for the regulated production of peptides specific to each isoform. These peptides are predicted to include three allatotropin-like (Manse-ATL) peptides that exhibit limited structural identity to Manse-AT and overlapping biological activities.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.     

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.     

The selective inhibition of phosphatases by natural toxins: the anhydride domain of tautomycin is not a primary factor in controlling PP1/PP2A selectivity

Analogues of the potent and moderately selective PP1/PP2A inhibitor tautomycin (TM) were prepared with modifications in the C1'-C7' anhydride moiety. While all retain varying degrees of activity within a 3000-fold range of potencies, they also show remarkable constancy in their IC(50) ratios, suggesting that the anhydride moiety is not critical in controlling the selectivity of inhibition.