Figure 1.

Suppression of rat8 growth defect by ssl1 mutations. (A) Suppression of rat8-6 ts growth defect by overexpression of ssl1-t. Strain rat8-6 (PDY1) was transformed with the high-copy plasmid obtained from the library (pPD1), plasmids containing different fragments obtained from the DNA genomic insert of pPD1 (pPD2 and pPD3, see B) or a plasmid containing the full-length SSL1 gene (pPD5). The restriction fragments from the DNA genomic insert isolated from the library were subcloned in the multicopy vectors YEp13 (left) or YEp24 (right). Full-length SSL1 was obtained in plasmid pPD5 by adding to the truncated allele (ssl1-t) of plasmid pPD2 a PCR fragment containing the promoter and 5′ sequences of SSL1 ORF. As controls, the same strain was transformed with the empty vectors (YEp181 for leucine selection, or YEp24 for uracil selection) or plasmids containing the wild-type RAT8 gene (pCS8.31 in SC-leu plate, or pRAT8.31 in SC-ura plate). Cells were diluted to an OD₆₀₀ of 0.1, and 1:10 serial dilutions were spotted onto selective plate, which were incubated for 3 d at 34°C. (C) Complementation of Δssl1 by ssl1-t. The Δssl1 allele was introduced into the diploid strain FY86 × PDY1. This strain was transformed with pSSL1 (wild-type SSL1/URA3/CEN). Haploids carrying a genomic deletion of SSL1 were selected by their inability to grow on plates containing 5-FOA. Δssl1 haploids were transformed with plasmids pPD1 (ssl1-t) or YEp1ac181 (vector). To select for cells that had lost pSSL1, cells were plated on 5′-FOA plates. Growth of the different strains was checked by spotting serial dilutions (1:10) on selective media (SC-leu) and incubated at 30°C for 2 d. (D) Suppression of rat8-2 ts growth defect by ssl1 mutant alleles. Left, double mutant strains were segregants from the cross CSY550 (rat8-2) × JJ636 (ssl1-1) and CSY550 × JJ637 (ssl1-2). Serial dilutions (1:10) were spotted onto a YPD plate and incubated for 3 d at 34°C. Right, double mutant strain rat8-2 ssl1-1 was transformed with pRAT8.31 (RAT8/CEN/URA3), pSSL1 (SSL1/CEN/URA3), or YEp24 (2 μm/URA3 vector), and 1:10 serial dilutions were spotted onto selective plate (SC-ura), which were incubated for 3 d at 34°C.