Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.     

Beta-Arrestins: new roles in regulating heptahelical receptors' functions

The last few years have seen a marked expansion in appreciation of the diversity of roles played by the betaArrestins in regulating GPCR functions. Originally discovered as molecules that desensitize such receptors, the roles of betaArrestins have expanded to include acting as signalling adapters or intermediates that recruit other key molecules to the GPCRs in an agonist-regulated fashion. For example, interactions with components of the endocytic machinery, such as clathrin, the adapter protein AP-2 and the N-ethylmaleimide sensitive fusion protein (NSF), demonstrate the ability of betaArrestins to act as adapters to facilitate the clathrin-mediated endocytosis of certain members of the GPCR family. BetaArrestins have also been shown to serve as signalling molecules. The Ras-dependent activation of ERK1/2 may involve the betaArrestin-dependent recruitment of c-Src to the beta2-adrenergic receptor (beta2-AR). More recently, betaArrestins have been shown to act as molecular scaffolds that coordinate the assembly of certain MAP kinase complexes that lead to the stimulation of either ERK1/2 or JNK3. Finally, long-term accumulation of arrestin-rhodopsin complexes, in photoreceptor cells has been shown to trigger apoptosis.     

Cellular uptake and in situ binding of a peptide agonist for calmodulin

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells.     

QTL mapping of temperature sensitivity reveals candidate genes for thermal adaptation and growth morphology in the plant pathogenic fungus Zymoseptoria tritici

Different thermal environments impose strong, differential selection on populations, leading to local adaptation, but the genetic basis of thermal adaptation is poorly understood. We used quantitative trait locus (QTL) mapping in the fungal wheat pathogen Zymoseptoria tritici to study the genetic architecture of thermal adaptation and identify candidate genes. Four wild-type strains originating from the same thermal environment were crossed to generate two mapping populations with 263 (cross 1) and 261 (cross 2) progeny. Restriction site-associated DNA sequencing was used to genotype 9745 (cross 1) and 7333 (cross 2) single-nucleotide polymorphism markers segregating within the mapping population. Temperature sensitivity was assessed using digital image analysis of colonies growing at two different temperatures. We identified four QTLs for temperature sensitivity, with unique QTLs found in each cross. One QTL had a logarithm of odds score >11 and contained only six candidate genes, including PBS2, encoding a mitogen-activated protein kinase kinase associated with low temperature tolerance in Saccharomyces cerevisiae. This and other QTLs showed evidence for pleiotropy among growth rate, melanization and growth morphology, suggesting that many traits can be correlated with thermal adaptation in fungi. Higher temperatures were highly correlated with a shift to filamentous growth among the progeny in both crosses. We show that thermal adaptation has a complex genetic architecture, with natural populations of Z. tritici harboring significant genetic variation for this trait. We conclude that Z. tritici populations have the potential to adapt rapidly to climate change and expand into new climatic zones.     

Synergistic and additive effects of kinetin and ethrel on the release of seed dormancy

Dormancy in scarified Indian rice grass seeds was partially released by kinetin. Germination percentage was highly dependent on the concentration of kinetin and peaked at 0.1 mM. Additional promotion, however, was observed in the presence of Ethrel. A synergistic or additive effect on germination by the two growth regulators was observed irrespective of whether they were applied in acetone solution or in water. ATP content, but not the activity of α-amylase or alkaline phosphatase, in the germinating seeds (24 hr) was enhanced by kinetin, ethrel or a combination of both. A synergistic effect of these two hormones on the release of dormancy in cocklebur seeds was also observed.