No abstract

Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures and long incubations, conditions under which most native enzymes perform poorly. Incremental increases in thermostability can be achieved by repeated rounds of mutagenesis and screening, but general strategies are needed for designing highly thermostable enzymes a priori. Here we show that enzymes can be created that can withstand temperatures ~ 30 °C higher and incubations ≥ 100 times longer than extant forms in a single step using ancestral reconstruction. We exemplify the approach with the first ancestral resurrections of two unrelated enzyme families: cytochrome P450 monooxygenases, that stereo-and regioselectively functionalize un-activated C-H bonds in pharmaceutical, flavour, fragrance and other fine chemical syntheses; and ketol acid reductoisomerases, used to make butanol-based biofuels. This shows thermostability can be designed into proteins using sequence data alone, potentially enhancing the economic feasibility of any process or product requiring a highly stable protein.

Attachment theory attempts to explain effects of social experiences, not genes, on personality development. Most studies of the development of attachment insecurities support this emphasis on social experiences rather than genes, although there are exceptions. In the present study, the authors examine associations between attachment insecurities and particular genetic polymorphisms related to emotions and social behavior. They find that (a) anxious attachment is associated with a polymorphism of the DRD2 dopamine receptor gene, (b) avoidant attachment is associated with a polymorphism of the 5HT2A serotonin receptor gene, and (c) the rs53576 A polymorphism of the OXTR oxytocin receptor gene is not associated with attachment insecurities. These findings suggest that attachment insecurities are partially explained by particular genes, although there is still a great deal of individual difference variance that remains to be explained by other genes or social experiences.

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of > or =98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species.

Trichoderma virens is a widely distributed soil fungus that is parasitic on other soil fungi. The mycoparasitic activity of T. virens is correlated with the production of numerous antifungal activities, including the secretion of a considerable repertoire of fungal cell wall-degrading enzymes. Here, we report the characterization of a diverse set of chitinase and glucanase genes from T. virens. In each case, full-length genomic clones were isolated and characterized, while sequencing of the corresponding cDNA clones and manual annotation provided a basis for establishing gene structure. Based on homology of the deduced amino acid sequences, we have identified three members of the 42Kd endochitinase gene family, two 33Kd exochitinases, two exochitinases with homology to N-acetylglucosaminidases, and three glucanase genes predicted to encode beta-1,3- and beta-1,6-proteins. The majority of these genes appear to encode signal peptides, suggesting an extracellular location for the corresponding proteins. Despite their overall similarity, the 42Kd class of chitinases can be subdivided, based on the presence of distinct N-terminal domains, suggesting that the proteins may have distinct cellular roles, while Northern blot analysis confirms that these genes possess distinct patterns of gene regulation. Similarly, one of the 33Kd chitinase genes is unique, because it is predicted to encode a protein C-terminus with high homology to the conserved family I cellulose-binding domain. The expression patterns of the chitinase genes were analyzed in both a wild-type strain and a strain disrupted for the major 42Kd chitinase gene of T. virens. The results of these transcript analyses, together with enzymatic assay of the extracellular proteins, suggest interdependent regulation of this important gene family in T. virens.

The role of extracellular chitinase in the biocontrol activity of Trichoderma virens was examined using genetically manipulated strains of this fungus. The T. virens strains in which the chitinase gene (cht42) was disrupted (KO) or constitutively over-expressed (COE) were constructed through genetic transformation. The resulting transformants were stable and showed patterns similar to the wild-type (WT) strain with respect to growth rate, sporulation, antibiotic production, colonization efficiency on cotton roots and growth/survival in soil. Biocontrol activity of the KO and COE strains were significantly decreased and enhanced, respectively against cotton seedling disease incited by Rhizoctonia solani when compared with the WT strain.

No abstract

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.