Stability of African swine fever virus in feed during environmental storage.

African swine fever virus (ASFV) causes high case fatality in pigs and a trade-limiting disease resulting in significant economic losses to pork production. ASFV is resistant to environmental degradation and maintains infectivity in feed ingredients exposed to transoceanic shipment conditions. As ASFV is transmissible through consumption of contaminated feed, the objective of this study was to evaluate the stability of ASFV Georgia 2007 in three feed matrices (complete feed, soybean meal, ground corncobs) exposed to three environmental storage temperatures (40°F, 68°F, 95°F) for up to 365 days. ASFV DNA was highly stable and detectable by qPCR in almost all feed matrices through the conclusion of each study. Infectious ASFV was most stable in soybean meal, maintaining infectivity for at least 112 days at 40°F, at least 21 days at 68°F and at least 7 days at 95°F. These data help define risk of ASFV introduction and transmission through feed ingredients.

The paclitaxel site in tubulin probed by site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin.

Previously, we created a paclitaxel-sensitive strain of Saccharomyces cerevisiae by mutating five amino acid residues in beta-tubulin in a strain that has a decreased level of the ABC multidrug transporters. We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of this strain to paclitaxel. We found that the change at position 19 from K (brain beta-tubulin) to A (yeast beta-tubulin) and at position 227 from H (brain beta-tubulin) to N (yeast beta-tubulin) had no effect on the activity of paclitaxel. On the other hand, the changes V23T, D26G and F270Y, drastically reduced sensitivity of AD1-8-tax to paclitaxel. Molecular modeling and computational studies were used to explain the results.

Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae.

Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.

Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression.

Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.