XRN1 is a Species-Specific Virus Restriction Factor in Yeasts

Paul A. Rowley; Brandon Ho; Sarah Bushong; Arlen Johnson; Sara L. Sawyer

kudriavzevii was introduced into pPAR219 between the 5' and 3' UTRs from S. 659 cerevisiae XRN1 using recombineering to produce pPAR225, pPAR226, and pPAR227, 660 respectively. As a negative control, NUP133 was cloned into the pPAR219 plasmid 661 backbone to produce pPAR221, which was used to allow growth of xrn1 on medium 662 lacking leucine without XRN1 complementation. The LEU2 gene was replaced by TRP1 663 using recombineering techniques to produce the plasmids pPAR326, pPAR327, 664 pPAR328, and pPAR329. Using PCR and recombineering, we also constructed The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/069799 doi: bioRxiv preprint sec. The dsRNA was eluted from the column by the addition of 100 l of 0.15 mM EDTA 706 (pH 7.0) and incubation at 65 o C for 5 min before centrifugation at 16,000 x g for 30 sec.