PMC:2195774 / 5678-7069 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2195774","sourcedb":"PMC","sourceid":"2195774","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2195774","text":"Plasmids and Additional Methods\nWild-type or mutant YDJ1 was supplied on a low copy LYS2 plasmid (Sikorski and Boeke 1991). Wild-type or mutant SIS1 was supplied on a low copy TRP1 plasmid (Yan and Craig 1999). After transformation of indicated plasmids into strain JJ1146, colonies were grown in media containing 5-fluroorotic acid (5-FOA) (USBiological) to counterselect for Ycp50-SIS1. YYS fusions (J plus G/F of Ydj1 fused to the G/M rich region and portions of the COOH terminus of Sis1) have been described (Yan and Craig 1999). The sis1-253 truncation mutant was selected in a genetic screen to isolate sis1 mutants that failed to grow in the absence of the COOH terminus of Ydj1. Sis1-253 levels expressed from a low copy vector were low (data not shown), thus this mutant was expressed on a multicopy vector in these studies. The COOH terminus of Sis1 (a.a. 170–352) was expressed under the control of the GPD promoter in p414GPD (Yan and Craig 1999) and moved to pRS317 as a Sac1-Sal1 fragment. Levels of Sis170-352 expressed from this plasmid were similar to wild-type Sis1 levels (data not shown). Plasmid Ycplac22-N134/ 121 was constructed to coexpress ydj1-N134 and sis1-121 from the same low copy plasmid (Gietz and Sugino 1988). Western blot analysis of Sis1 proteins using a polyclonal antibody raised against full-length Sis1 was performedas described (Yan and Craig 1999).","divisions":[{"label":"Title","span":{"begin":0,"end":31}}],"tracks":[]}