David W Galbraith
Work Summary
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
DNA microarrays were originally devised and described as a convenient technology for the global analysis of plant gene expression. Over the past decade, their use has expanded enormously to cover all kingdoms of living organisms. At the same time, the scope of applications of microarrays has increased beyond expression analyses, with plant genomics playing a leadership role in the on-going development of this technology. As the field has matured, the rate-limiting step has moved from that of the technical process of data generation to that of data analysis. We currently face major problems in dealing with the accumulating datasets, not simply with respect to how to archive, access, and process the huge amounts of data that have been and are being produced, but also in determining the relative quality of the different datasets. A major recognized concern is the appropriate use of statistical design in microarray experiments, without which the datasets are rendered useless. A vigorous area of current research involves the development of novel statistical tools specifically for microarray experiments. This article describes, in a necessarily selective manner, the types of platforms currently employed in microarray research and provides an overview of recent activities using these platforms in plant biology.
PMID: 19675157;PMCID: PMC2754645;Abstract:
A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a g-irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways. © 2009 American Society of Plant Biologists.
PMID: 20546627;PMCID: PMC2906421;Abstract:
Background: Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly.Results: Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.Conclusion: The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays. © 2010 Zárate et al; licensee BioMed Central Ltd.
Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis.