Summary• One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage.• A microarray of 10 400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process.• White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants.• Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

Background: The sequencing and analysis of ESTs is for now the only practical approach for largescale gene discovery and annotation in conifers because their very large genomes are unlikely to be sequenced in the near future. Our objective was to produce extensive collections of ESTs and cDNA clones to support manufacture of cDNA microarrays and gene discovery in white spruce (Picea glauca [Moench] Voss).

Background: High-throughput genotyping technologies represent a highly efficient way to accelerate genetic mapping and enable association studies. As a first step toward this goal, we aimed to develop a resource of candidate Single Nucleotide Polymorphisms (SNP) in white spruce (Picea glauca [Moench] Voss), a softwood tree of major economic importance.

Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N-and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k cat ∕K M by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k cat ∕K M by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.mutually exclusive folding | design | HIV protease | molecular switch | barnase Z ymogens are inactive enzyme precursors that are activated by proteolytic cleavage. Natural zymogens play essential roles in apoptosis, blood coagulation, digestion, viral maturation, and other cellular processes. Artificial zymogens are of broad interest, with potential applications that include diagnostic tools and toxins that attack pathogens or diseased cells. Here we introduce a unique technique (alternate frame protein folding, or AFF) to convert the cytotoxic ribonuclease barnase (Bn) into an artificial zymogen that is activated by HIV-1 protease (PR). The significance of AFF is that it is general and can thus be applied to many enzymes, including those whose activities are not regulated by any existing mechanism.Several groups have previously engineered artificial zymogens. Raines and colleagues circularly permuted ribonuclease A (RNase A) and bridged the original N-and C-termini with a peptide linker that contained cleavage sequences for PR, the NS3 protease from hepatitis C, and plasmepsin II from Plasmodium falciparium (1-3). The linkers inhibited enzymatic activity by partially occluding the active site. Cleavage with the respective protease increased k cat ∕K M by approximately 100-fold. In a recent study we took a related approach by permuting Bn with a short linker peptide intended to force the termini together, thereby distorting the active site or unfolding the enzyme (4). Cleaving the linker with a chemical reagent relieved conformational strain and increased RNase activity. Although these strategies successfully created artificial zymogens, they rely on specific properties of the target enzyme and are therefore not general. Cyclic permutation typic...

The tissue-specific etiology of aging and stress has been elusive due to limitations in data processing of current techniques. Despite that many techniques are high-throughput, they usually use singular features of the data (e.g. whole fluorescence). One technology at the nexus of fluorescence-based screens is large particle flow cytometry (“biosorter”), capable of recording positional fluorescence and object granularity information from many individual live animals. Current processing of biosorter data, however, do not integrate positional information into their analysis and data visualization. Here, we present a bioanalytical platform for the quantification of positional information (“longitudinal profiling”) of C. elegans, which we posit embodies the benefits of both high-throughput screening and high-resolution microscopy. We show the use of these techniques in (1) characterizing distinct responses of a transcriptional reporter to various stresses in defined anatomical regions, (2) identifying regions of high mitochondrial membrane potential in live animals, (3) monitoring regional mitochondrial activity in aging models and during development, and (4) screening for regulators of muscle mitochondrial dynamics in a high-throughput format. This platform offers a significant improvement in the quality of high-throughput biosorter data analysis and visualization, opening new options for region-specific phenotypic screening of complex physiological phenomena and mitochondrial biology.