Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast -- Data Supplement - Supplemental Text -- Proceedings of the National Academy of Sciences

Supplementary material for Teige et al. (2001) Proc. Natl. Acad. Sci. USA 98 (10), 5625–5630. (10.1073/pnas.091610798)

Oligonucleotides for Generation of Rck2 Mutants

A C-terminal truncation of the last 23 amino acids of RCK2 was obtained with the primer 5'-TTTTGTCGACCTAGCGGCCGCCAATCGTGGACGTATCCAGCGTTAATTGG-3'. The NotI site is underlined. The kinase inactive RCK2 mutant (K201R) was generated by PCR using the following primers: 5'-ACCATTAATCGAGGATAgATCTGCCTTTTTGATAACTcTAAT
GGCAACAGC-3' and 5'-AGCTGTTGCCATTAGAGTTATCAAAAAGGCAGATCT
ATCCTCGATTAATGG-3'. An analytical BglII site (underlined) was introduced by silent mutagenesis in addition to the K201R mutation (lowercase letters). The phosphorylation site mutants (T379A and S520A) were also generated by PCR using the following oligonucleotides: T379A, 5'-TTTCCAAGAACACCAAGgCTCCTTGTGGTACcGTCGGTTACACTGCCCC-3' and 5'-GGGGCAGTGTAACCGACgGTACCACAAGGAGcCTTGGTGTTCTTGGAAA-3'; S520A, 5'-GAGACTCCTCGCTACTGTTTgCACCcGCgGCTGTTGCTATGCGTGACGCC-3' and 5'-GTCACGCATAGCAACAGCcGCgGGTGCAAACAGTAGCGAGGAGTCTC-3'. In addition to the mutations (lowercase letters), a KpnI and KspI site (underlined) was introduced by silent mutagenesis. The double mutant (T379A, S520A) was constructed by replacing an EcoRI-NcoI fragment of RCK2, containing the T379A mutation. All mutations were verified by DNA sequencing. For expression in yeast, all constructs were cloned in YCp33 with the endogenous 800-bp promoter.