Sis1 activities are important for [URE3] propagation.

<p>(A) Diagram (not to scale) of Sis1 coding region. Numbers at top indicate amino acid positions. Domains, designated by abbreviations at top (see text) are indicated by variously shaded boxes. Mutations are indicated on the left. Deleted regions are shown as dashed lines and locations of point mutations H34Q and K199A as diamonds. Q is H34Q, A is K199A. Intact DnaJB1 (not diagrammed), is the closest human homolog of Sis1 and is indicated JB1 in the other panels. (B) Strain 1385 expressing wild type Sis1 from a <i>URA3</i> plasmid was transformed by single-copy <i>TRP1</i> plasmids encoding the wild type or mutant proteins (indicated on the left of each image) regulated by the <i>SIS1</i> promoter. Transformants were taken from plates selecting for [URE3] and both plasmids and streaked onto plates selecting only for the plasmids. Plates were incubated for 3 days at 30°C followed by 2 days at 25°C. [URE3] cells form small white colonies, while [ure-o] cells grow into larger red colonies. (C) [URE3] cells as in panel (B) were grown as patches on plates selecting for [URE3] and both plasmids. These were replica-plated onto medium containing uracil to allow loss of the <i>URA3</i> plasmid, and then onto medium containing limiting adenine and FOA (shown), which kills cells retaining the <i>URA3</i> plasmid. Empty vector control is indicated as ev. (D) As in (C) except adenine was omitted from all plates to ensure growth only of cells that propagate [URE3]. (E) Cells from plate in panel (C) were streaked onto similar medium. The fainter red coloration of [ure-o] cells expressing the ‘A’ mutant is due to a slightly higher amount of adenine in the medium. (F) Cells from plate in panel (D) were streaked onto medium containing or lacking adenine, as indicated. In all panels, red colonies arose from cells that lost [URE3].