cDNA

pRcCMV myc-Plk1 wt (Nigg RG6)

41160

pRcCMV myc-Plk1 wt (Nigg RG6)

Ampicillin

cDNA fragments spanning parts of the catalytic domains of human protein kinases were amplified by PCR, as described by Schultz and Nigg (1993). A 144 bp fragment (named HsPK28) showing 88% nucleotide identity to Drosophila polo was used to probe a gt 10 library prepared from a human nasopharyngeal carcinoma (Hitt et al., 1989). Approximately 500,000 plaques were screened by plaque hybridization (Sambrook et al., 1989), and 17 phage showing strong hybridization were purified. DNA was prepared using Lambdasorb (Promega Biotech, Madison, WI). One plasmid (referred to as Plk1-pGEM) with a 2143 bp insert contained the entire coding sequence for a protein kinase closely related to Drosophila polo. This insert was sequenced on both strands by the method of Chen and Seeburg (1985), using the Sequenase kit (United States Biochemicals). To express a full-length Plk1 protein, the 1998 bp fragment of Plk1-pGEM was introduced into the pRc/CMV vector (Invitrogen Corporation, San Diego), to create Plk1-CMV. An N-terminally myc-tagged Plk1-CMV was also constructed. In the resulting mycplk1 protein, the N terminus of Plk1 is extended by the oligopeptide MEQKLISEEDLNMNSCSPGS (Evan et al., 1985).

37

Unknown