NMR structure and Mg(2+) binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit

Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G•A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037–1043 and 1112–1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson–Crick/Watson–Crick (cis W.C.) G•A conformation for the tandem (G•A)(2) in the analog and a minimally perturbed helical duplex stem. Mg(2+) titration studies imply that the cis W.C. geometry of the tandem (G•A)(2) probably allows O6 of G20 and N1 of A4 to coordinate with a Mg(2+) ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg(2+) coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg(2+) coordination

Light Induces Carotenoid Biosynthesis-Related Gene Expression, Accumulation of Pigment Content, and Expression of the Small Heat Shock Protein in Apple Fruit

The coloration of the apple fruit (Malus × domestica Borkh.) depends on pigment content. Light stimulus activates a broad range of photosynthesis-related genes, including carotenoids. The effect of light on two red commercial apple cultivars, ‘Summer Prince’ and ‘Arisoo’ at the juvenile stage were examined. Apple fruits were either bagged to reduce light irradiation or were exposed to direct, enhanced sunlight (reflected). The pigment content and the expression of carotenoid metabolism genes in the peel and flesh of apple fruits were significantly different between the shaded and the reflected parts. These parameters were also different in the two cultivars, highlighting the contribution of the genetic background. Further, a combination of light and transient overexpression of carotenogenic genes increased fruit coloration and pigment content in the variety ‘RubyS’. Western blot analysis showed the expression of small heat shock proteins (smHSP) in lysates extracted from the reflected part of the fruits but not in the bagged fruits, indicating the activation of smHSP in response to heat generated by the reflected light. Therefore, the synergy between the genes and the environment dictates the color of apple fruits

Identification of Genes Associated with Nitrogen Stress Responses in Apple Leaves

Nitrogen (N) is an essential macronutrient that regulates diverse physiological processes for plant survival and development. In apple orchards, inappropriate N conditions can cause imbalanced growth and subsequent physiological disorders in trees. In order to investigate the molecular basis underlying the physiological signals for N stress responses, we examined the metabolic signals responsive to contrasting N stress conditions (deficient/excessive) in apple leaves using transcriptome approaches. The clustering of differentially expressed genes (DEGs) showed the expression dynamics of genes associated with each N stress group. Functional analyses of gene ontology and pathway enrichments revealed the potential candidates of metabolic signals responsible for N-deficient/excessive stress responses. The functional interactions of DEGs in each cluster were further explored by protein–protein interaction network analysis. Our results provided a comprehensive insight into molecular signals responsive to N stress conditions, and will be useful in future research to enhance the nutrition tolerance of tree crops

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: Crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55–59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1–6, which interact with the loop and adjacent residues 53–54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1–6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1–6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary

Antisense Expression of Apple TFL1-like Gene (MdTFL1) Promotes Early Flowering and Causes Phenotypic Changes in Tobacco

Apples (Malus × domestica Borkh.) require up to several years for flowering and bearing fruits. The transition from vegetative to reproductive phase is controlled by floral regulators such as TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT). TFL1 mediates the maintenance of vegetative phase, unlike the antagonistic function of FT to promote the transition into reproductive phase. In this study, we isolated apple TFL1-like gene (MdTFL1) to elucidate various phenotypic traits triggered by the antisense expression of MdTFL1 in tobacco apart from its floral induction function. Early flowering was observed in the tobacco line with MdTFL1 knockout, indicating the reduced time for transition to vegetative phases. Quantitative reverse-transcription PCR showed upregulation of genes involved in the regulation of floral induction, including NtAP1, NtSOC1, NFL1, and NtFTs, and downregulation of carotenoid cleavage dioxygenases (CCDs) and CEN-like genes in transgenic lines. Interestingly, transgenic tobacco expressing antisense MdTFL1 exhibited distinct morphological changes in lateral shoot outgrowth, internode length, and the development of leaves, flowers, and fruits. The results suggested that using the antisense expression of MdTFL1 gene is one of the approaches to shorten the vegetable phase and proposed improvement of plant architecture in horticultural crops

High-Throughput Analysis of Clinical Flow Cytometry Data by Automated Gating

Advancements in flow cytometers with capability to measure 15 or more parameters have enabled us to characterize cell populations at unprecedented levels of detail. Beyond discovery research, there is now a growing demand to dive deeper into evaluating the immune response in clinical trials for immune modulating compounds. However, for high-volume, complex flow cytometry data generated in clinical trials, conventional manual gating remains the standard of practice. Traditional manual gating is resource intense and becomes a bottleneck and an impractical method to complete high volumes of flow cytometry data analysis. Current efforts to automate “manual gating” have shown that computational algorithms can facilitate the analysis of daunting multi-parameter data; however, a greater degree of precision in comparison with traditional manual gating is needed for wide-scale adoption of automated gating methods. In an effort to more closely follow the manual gating process, our automated gating pipeline was created to include negative controls (Fluorescence Minus One [FMO]) to enhance the reliability of gate placement. We demonstrate that use of an automated pipeline, heavily relying on FMO controls for population discrimination, can analyze multi-parameter, large-scale clinical datasets with comparable precision and accuracy to traditional manual gating