FIG. 1.

Deletion of the Spt5 CTD affects cells growth and morphology. (A) Heterozygous spt5⁺/spt5-(1-800)::ura4⁺ diploids were sporulated, and asci were dissected. Individual spores (A, B, C, and D) from two tetrads (1 and 2) were germinated on YES agar at 30°C for 4 days (upper panel). The four haploid progeny of tetrad 2 were grown in liquid culture. The cultures were diluted to attain an A₆₀₀ of 0.1, and aliquots (3 μl) of serial 10-fold dilutions were spotted on YES agar medium. The plates were photographed after incubation for 3 days at 30°C (lower panel). The spt5-ΔC haploids 2A and 2D are ura⁺; the spt5⁺ haploids 2B and 2C are ura⁻. (B) spt5⁺ and spt5-ΔC cells grown in YES medium at 30°C were examined by light microscopy. Bars, 10 μm. (C) The lengths of 270 spt5⁺ cells and 330 spt5-ΔC cells were measured and sorted into length bins as specified. The percentage of cells in each bin is represented in a bar graph. (D) Dosage suppression of the spt5-ΔC phenotype by mammalian capping enzyme Mce1. spt5-ΔC[spt5-(1-800)::ura4⁺] cells were transformed with LEU2 plasmids as specified. Serial dilutions of spt5-ΔC strains harboring a pREP81x-Spt5 plasmid (positive control), an empty vector plasmid (negative control), or plasmids for expression of Mce1 under the transcriptional control of an intermediate-strength (41x) or a low-strength (81x) nmt promoter were spotted on Leu⁻ agar medium. The plates were photographed after incubation for 6 days at 25°C or 3 days at 30°C. (E) Dosage suppression of spt5-ΔC by fission yeast capping enzymes. The spt5⁺ and spt5-ΔC [spt5-(1-800)::kanMX] strains harbored two plasmids marked with ura4⁺ and LEU2, respectively. These were either empty vector plasmids or plasmids for expression of S. pombe pct1⁺ (RNA triphosphatase) and pce1+ (RNA guanylyltransferase) under the transcriptional control of the low-strength nmt (81x) promoter. Cultures were grown at 30°C in minimal medium lacking uracil and leucine. Aliquots (3 μl) of serial 10-fold dilutions were spotted on Ura⁻ Leu⁻ agar medium and incubated at 25°C (4 days), 30°C (3 days), or 34°C (3 days) as specified. (F) Overexpression of Mce1 partially rescues the elongated phenotype of spt5-ΔC cells. The lengths of 300 to 500 individual cells were measured. The percentages of cells in each of the four size categories are represented by vertical bars.