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Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4. / Dissertation (MSc)--University of Pretoria, 2013. / Biochemistry / unrestricted

Mass production of translationally optimized bacteriophages (hereafter referred to as phages) is the need of the hour in the application of phages to therapy. Understanding translational efficiency of phages is the major preliminary step for mass producing efficient phages. The objective of this thesis is to understand factors affecting translational efficiency of phages. In chapter two, we hypothesized that weak translation initiation efficiency is responsible for weak codon concordance of Escherichia coli lambdoid phages with that of their hosts. We measured the strength of translation initiation using two indices namely minimum folding energy (MFE) and proportion of Shine-Dalgarno sequence (PSD). Empirical results substantiate our hypothesis suggesting lack of strong selection for improving codon adaptation in these phages is due to their weak translation initiation. In chapter three, we measured codon usage concordance between GC-rich and GC-poor Aeromonas phages with their GC-rich host Aeromonas salmonicida. We found low codon usage concordance in the GC-poor Aeromonas phages. We were interested in testing for the role of tRNAs in the GC-poor phages. We observed that the GC-poor phages carry tRNAs for codons that are overused by the phages and underused by the host. These findings suggest that the GC-poor Aeromonas phages carry their own tRNAs for compensating for the compositional difference between their genomes and that of their host. Previously several studies have reported observed avoidance of stable secondary structures in start site of mRNA in a wide range of species. We probed the genomes of 422 phage species and measured their secondary structure stability using MFE. We observed strong patterns of secondary structure avoidance (less negative MFE values) in the translation initiation region (TIR) and translation termination region (TTR) of all analyzed phages. These findings imply selection is operating at these translationally important sites to control stable secondary structures in order to maintain efficient translation.

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Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. / Russian Foundation for Basic Research / Revisión por pares

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No description available.

Short height and high yield per unit energy in controlled environments are essential to the success of a food production system for spaceflight. Temperature and light quality can be manipulated in controlled environments to reduce plant height and increase yield. Although the effects of temperature on height and yield are well studied at ambient CO2, temperature effects at elevated CO2 with a hydroponic root zone are not well characterized. We studied soybean yield and height under two lamp types over a broad range of temperatures. Temperature had little effect on yield or height, but lamp type had a significant effect on canopy height. This first study highlighted the importance of understanding spectral quality in controlling plant growth, especially canopy height. Numerous studies have compared lamp types and suggested that profound differences in leaf area, canopy height, yield, and total dry mass responses were due to blue light differences. Unfortunately, the most energy-efficient light sources have the least blue light. We have a poor understanding of the specific morphological and histological effects of blue light on leaves and stems. Three species, soybeans, wheat, and lettuce, were grown at five blue light fractions (0, 2, 6, 12, and 26%) and two light levels (200 and 500 μmol m-2 s-1). Phytochrome photoequilibria were constant among treatments. Blue light responses were species dependent. Wheat leaf area, dry mass, and stem length were insensitive to blue light fraction. Increasing blue light to 26% decreased soybean stem length, but leaf area was greatest at 6% blue. Lettuce leaf area, stem length, and dry mass were highly sensitive to blue light fraction between 0% and 6% under high pressure sodium lamps, but were insensitive between 6% and 26% under metal halide lamps. These results may be complicated by sensitivity to other wavelengths . The decrease in soybean stem length with increasing blue light was caused by an inhibition of cell division, while the decrease in leaf area was caused primarily by a decrease in cell expansion. Increased lettuce leaf area with increasing blue light fraction was caused by both cell division and expansion. This research indicates that lamps high in blue photons are not only energetically wasteful, but do not benefit, and in some cases reduce, plant growth. However, some blue light is necessary for controlling plant height in soybean and even required for proper growth and development in lettuce.