Micro-plasticity of genomes as illustrated by the evolution of glutathione transferases in 12 Drosophila species.

Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family

Epsilon GST gene cluster and adjacent region from six <i>Drosophila</i> species.

<p>Genomic maps showing the Epsilon GST gene organization in six <i>Drosophila</i> species. These examples illustrate the overall synteny of the Epsilon gene cluster as well as the micro-rearrangements that occur in the individual species. The genes are shown as arrows which represent the direction of transcription.</p

Glutathione transferases in the 12 <i>Drosophila</i> species.

<p>The numbers of genes and proteins transcribed are from FlyBase annotations and the present curation. The present curation numbers include proposed corrections for genes and protein expressions.</p><p>Glutathione transferases in the 12 <i>Drosophila</i> species.</p

Phylogenetic tree analysis.

<p>The phylogenetic tree analysis used sequences from the Omega class from the 12 <i>Drosophila</i> species. Each isoform is designated by their FlyBase symbol followed by the <i>D. melanogaster</i> ortholog name in parenthesis. For example, Dana\GF10159(O1), where O1 refers to the DmelGST Omega 1 ortholog.</p

Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates

Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates

RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection

Abstract Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines

Glutathionylation effects on dengue and Zika NS5 guanylyltransferase activity.

<p>A) DENV and Zika NS5 protein were not treated and pre-treated with 5 mM GSSG for 10 min at RT. These proteins then were used to perform the guanylyltransferase activity assay. The samples were resolved on 10% SDS-PAGE. The extent of enzyme guanylylation activity was measured by GTP-Cy5 signal tracking using Azure^™ cSeries. Gels were then stained with Coomassie Blue to normalize for protein loading. The bottom panel shows equivalent aliquots of the same samples used in a parallel gel that was transferred for Western blot and detection by anti-GSH antibody. B) The ratio of quantified guanylyltransferase bands to protein load of panel A is shown as a bar graph.</p

Immunoprecipitation result of the second method.

<p>The second method was to immunoprecipitate the dengue protein in cell lysate with specific dengue protein antibody and protein A/G beads and determine that the dengue protein was glutathionylated as shown by western blot detection with an anti-GSH antibody. The quantity of NS5 is not detectable in whole lysate. Lane 1 is DENV-infected cell lysate. Lane 2 is IP sample of NS5.</p

Amino acid alignment of flavivirus NS5 proteins.

<p>The sequences are nonstructural protein NS5 from Dengue virus 2 NP_739590.2, Dengue virus 1 NP_722465.1, Dengue virus 3 YP_001531176.2, Dengue virus 4 NP_740325.1, Zika virus AMR39834.1, Japanese encephalitis virus NP_775674.1, West Nile virus YP_001527887.1, Tick-borne encephalitis virus NP_775511.1, Yellow fever virus NP_776009.1, Murray Valley encephalitis virus NP_722539.1, Langat virus NP_740302.1. Cysteines are highlighted in yellow with the positions of the 3 glutathionylated cysteines identified in this study shown in green highlight in the alignment. Blue bars illustrate the residues of the finger subdomains, the red bars show the residues of the palm domain and the purple bars show the residues of the thumb domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193133#pone.0193133.ref030" target="_blank">30</a>]. Conservation of amino acid sequence is shown by 100 percent conserved as white on black, between 80 to 100 percent conserved as white on dark grey, between 60 to 80 percent conserved as black on light grey, and less than 60 percent conserved as black on white.</p