Subset Of Transformations Research Articles

A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Cognitive operations on space and their impact on the precision of location memory.

Learning about object locations in space usually involves the summation of information from different experiences of that space and requires various cognitive operations to make this possible. These processes are poorly understood and, in the extreme, may not occur--leading to mutual exclusivity of memories (Baguley, Lansdale, Lines, & Parkin, 2006). In this article, we investigate how the precision of location memory--evident in near-miss errors in recall--is related to different transformational processes in spatial cognition. Analyzing errors and latencies in a sequential comparative judgment task, 4 experiments show that the precision with which location is represented in memory is specifically degraded by a subset of transformations in which an object location encoded in reference to 1 anchor point is recalibrated in relation to another. We discuss the general implications of this finding for spatial learning and demonstrate that, rather than being a special case, exclusivity in memory is the extreme expression of a rational trade-off between the benefit of combining spatial information from more than 1 memory and the reduced precision that follows from the transformations required.

RNA-Mediated Gene Silencing of Superoxide Dismutase (bcsod1) in Botrytis cinerea

Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD) is a virulence factor, transformants were tested for phenotypic silencing in vitro and reduction in pathogenicity in planta. Plate-based assays with and without paraquat were performed to screen initial silencing efficiency, and a subset of transformants was used for in planta studies of virulence. Transformants exhibiting strongly decreased transcripts levels were recovered with both constructs but none of those exhibited a reduction in virulence in planta. Our investigations may help optimize a high-throughput gene silencing system useful for identifying potential gene targets for future fungal control.

Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP IAI

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1.

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

Reengenharia e transformação. Por onde começar?

Reengineering is only a sub-set of Transformation. More than a redesign of individual processes - often with the help of benchmarking data -, Transformation is the simultaneous and integrated redesign processes and systems.