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Leading voices in the biological sciences have called for a transformation in graduate education leading to the PhD degree. One area commonly singled out for growth and innovation is cross-training in computational science. In 1998, the University of Tennessee (UT) founded an intercollegiate graduate program called the UT-ORNL Graduate School of Genome Science and Technology in partnership with the nearby Oak Ridge National Laboratory. Here, we report outcome data that attest to the program's effectiveness in graduating computationally enabled biologists for diverse careers. Among 77 PhD graduates since 2003, the majority came with traditional degrees in the biological sciences, yet two-thirds moved into computational or hybrid (computational-experimental) positions. We describe the curriculum of the program and how it has changed. We also summarize how the program seeks to establish cohesion between computational and experimental biologists. This type of program can respond flexibly and dynamically to unmet training needs. In conclusion, this study from a flagship, state-supported university may serve as a reference point for creating a stable, degree-granting, interdepartmental graduate program in computational biology and allied areas.

Abstract Background Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs). Results Here we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings. Conclusion We conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORF-containing mRNAs and, in turn, to orchestrate plant development.

Abstract Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.

Abstract Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor.

Abstract Background Meiotically heritable epimutations affecting transgene expression are not well understood, even and in particular in the plant model species, Arabidopsis thaliana. The Arabidopsis trans-silencer locus, C73, which encodes a fusion protein between the repressor of photomorphogenesis, COP1, and green fluorescent protein (GFP-COP1), heritably modifies the expression pattern and cop1-like cosuppression phenotypes of multiple GFP-COP1 target loci by transcriptional gene silencing. Results Here we describe three additional features of trans-silencing by the C73 locus. First, the silencing phenotype of C73 and of similar complex loci was acquired epigenetically over the course of no more than two plant generations via a stage resembling posttranscriptional silencing. Second, imprints imposed by the C73 locus were maintained heritably for at least five generations in the absence of the silencer with only sporadic spontaneous reversion. Third, the pairing of two other GFP-COP1 transgene loci, L91 and E82, showed an increased tendency for epigenetic modification when L91 carried an epigenetic imprint from C73, but not when E82 bore the imprint. Conclusions The latter data suggest a transfer of trans-silencing activity from one transgene locus, C73, to another, namely L91. These results extend our operational understanding of interactions among transgenes in Arabidopsis.

In most organisms, gene expression over the course of the day is under the control of the circadian clock. The canonical clock operates as a gene expression circuit that is controlled at the level of transcription, and transcriptional control is also a major clock output. However, rhythmic transcription cannot explain all the observed rhythms in protein accumulation. Although it is clear that rhythmic gene expression also involves RNA processing and protein turnover, until two years ago little was known in any eukaryote about diel dynamics of mRNA translation into protein. A recent series of studies in animals and plants demonstrated that diel cycles of translation efficiency are widespread across the tree of life and its transcriptomes. There are surprising parallels between the patterns of diel translation in mammals and plants. For example, ribosomal proteins and mitochondrial proteins are under translational control in mouse liver, human tissue culture, and Arabidopsis seedlings. In contrast, the way in which the circadian clock, light-dark changes, and other environmental factors such as nutritional signals interact to drive the cycles of translation may differ between organisms. Further investigation is needed to identify the signaling pathways, biochemical mechanisms, RNA sequence features, and the physiological implications of diel translation. [ABSTRACT FROM AUTHOR]