Protective Antigens Against Glanders Identified by Expression Library Immunization

Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens’ proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine

High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning

Genome-Wide Identification of Alternatively Spliced mRNA Targets of Specific RNA-Binding Proteins

BACKGROUND: Alternative splicing plays an important role in generating molecular and functional diversity in multi-cellular organisms. RNA binding proteins play crucial roles in modulating splice site choice. The majority of known binding sites for regulatory proteins are short, degenerate consensus sequences that occur frequently throughout the genome. This poses an important challenge to distinguish between functionally relevant sequences and a vast array of those occurring by chance. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used a computational approach that combines a series of biological constraints to identify uridine-rich sequence motifs that are present within relevant biological contexts and thus are potential targets of the Drosophila master sex-switch protein Sex-lethal (SXL). This strategy led to the identification of one novel target. Moreover, our systematic analysis provides a starting point for the molecular and functional characterization of an additional target, which is dependent on SXL activity, either directly or indirectly, for regulation in a germline-specific manner. CONCLUSIONS/SIGNIFICANCE: This approach has successfully identified previously known, new, and potential SXL targets. Our analysis suggests that only a subset of potential SXL sites are regulated by SXL. Finally, this approach should be directly relevant to the large majority of splicing regulatory proteins for which bonafide targets are unknown

Drosophila polypyrimidine-tract binding protein (PTB) functions specifically in the male germline

The mammalian polypyrimidine-tract binding protein (PTB), which is a heterogeneous ribonucleoprotein, is ubiquitously expressed. Unexpectedly, we found that, in Drosophila melanogaster, the abundant PTB transcript is present only in males (third instar larval, pupal and adult stages) and in adult flies is restricted to the germline. Most importantly, a signal from the somatic sex-determination pathway that is dependent on the male-specific isoform of the doublesex protein (DSX(M)) regulates PTB, providing evidence for the necessity of soma–germline communication in the differentiation of the male germline. Analysis of a P-element insertion directly links PTB function with male fertility. Specifically, loss of dmPTB affects spermatid differentiation, resulting in the accumulation of cysts with elongated spermatids without producing fully separated motile sperms. This male-specific expression of PTB is conserved in D.virilis. Thus, PTB appears to be a particularly potent downstream target of the sex-determination pathway in the male germline, since it can regulate multiple mRNAs

High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis

Fold changes for selected candidates (ratio of RNA-Seq reads—mutant versus control).

<p>Fold changes for selected candidates (ratio of RNA-Seq reads—mutant versus control).</p

Alternative splicing of the <i>Mlc1</i> gene in the <i>heph</i>^<i>2</i> mutant.

<p>RNA Seq pileups were generated using UCSC Genome Browser, y-axis is auto-scaled to show differences in isoform fractions, alternative exon is highlighted by the box. <b>(A)</b> RNA-Seq read pileup across the <i>Mlc1</i> skipped exon in control (top) and <i>heph</i>^<i>2</i> mutant (bottom) flies, showing significantly increased exon skipped in the <i>heph</i>^<i>2</i> mutant. (<b>B)</b> RT-PCR analysis of <i>Mlc1</i> for exon skipping, using primers in the flanking exons shown by the arrows.</p

Effect of the <i>heph</i>^<i>2</i> mutation on fold-changes in gene expression.

<p>Histogram of fold-changes in the <i>heph</i>^<i>2</i> mutants of genes called significant by Cufflinks (note that Cufflinks did not call any genes significant below 0.78 log₂ change). The list is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150768#pone.0150768.s002" target="_blank">S1 Table</a>. (B). Pie chart representing gene ontology categories over-represented in the genes that are significantly affected in the <i>heph</i>^<i>2</i> mutant.</p

RT-PCR analysis reveals differentially expressed transcripts.

<p>RNA from the wild type control and the <i>heph</i>^<i>2</i> mutant was analyzed by RT-PCR analysis. Gene-specific primer pairs were used for amplification of several randomly picked genes that showed significant differential expression. For <i>Tequila</i>, primer pairs were designed to test alternative 5’ ends (transcription start site usage).</p