Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers

The same strategy as described for the construction of A2-101 was cartied out for the A16-101 mutant (p63 with a deletion of amino acids 16 to 101), except for the 5' primer that was GCC CGC CAT GCC CTC GC~ CAA ACA AAG ~ CTC CAA GC.K3 CC~ CCA CGG CC_~ CCG CAG GCT CGG CAG ~ GCT CAA. All other mutations were introduced by the overlap extension technique (Ho et al., 1989) using bp 774-791 of the p63 sequence and bp 170-193 of the Bluescript KS-vector as downstream and upstream flanking primers. An appropriate partial complementary pair of oligonucleotides in which the desired mutation had been incorporated was chosen as internal primers. The final PCR products were digested with BamHI and used to replace the BamHI-BamHI fragment ofpBKS-AI6-101 (AI6-101 introduced in EcoRI site of pBKS). Sequencing and further subcloning were as described above. Alternatively, final PCR products were directly subcloned into the Sinai site of the pECE vector and sequenced.