Fig 2.

Cdk9 truncation impairs growth and transcript elongation in vivo. (A) The cdk9ΔC strain was constructed by transforming a wild-type diploid strain with a PCR product containing a kanamycin resistance marker flanked by regions of homology to the cdk9⁺ genomic locus. Homologous recombination resulted in introduction of a stop codon and replacement of the region of cdk9⁺ encoding the carboxyl terminus by the marker. The heterozygous diploid strain was sporulated to produce the cdk9ΔC haploid strain. (B) Growth curves were performed by growing cells in rich medium for 10 h. Cells were counted every 2.5 h. (C) Cultures were serially diluted 10-fold and spotted on plates of the indicated media. Plates were photographed after incubation for 2 days (YES), 3 days (MPA), 4 days (EMM), or 6 days (18°C). (D) Immunoblot analysis of lysates of the indicated strains. Total Spt5 was detected by an antibody recognizing its carboxyl-terminal Myc epitope tag. Phosphorylated Spt5 was visualized with an antibody raised against an Spt5 CTD peptide phosphorylated at the Thr1 position.