3.2 M ETHODS
3.2.2 Construction of cDNA Clones
3.2.2.9 Construction of domain deletion mutants
The deletion of single extracellular cadherin motifs, the transmembrane domain, the C-terminal PDZ-binding domain or the cytosolic tail was performed by oligonucleotide-directed mutagenese using loop out PCR. Here, the first part of the designed oligonucleotide primer binds to 15 - 20 base pairs upstream of the targeted
domain, whereas the rear part attaches to a sequence downstream of the target. By replication with the polymerase, the targeted region is “lopped out” and the new synthesized DNA strand lacks the distinct sequence.
For deletion of the single cadherin repeats, the transmembrane domain and the cytosolic tail, the sequence encoding the domain was replaced by a spacer coding for six glycines in order to avoid interference with the secondary protein structure.
For the PCR, 4 U of proof-reading Isis-DNA polymerase (QBiogene, Heidelberg, Germany) with buffers and dNTP as described above (cp. 3.2.2.1) were set up with primers, listed below, at 5 pmol. Following cycling protocol was applied: 95°C for 10 min, then 20 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 24 minutes. Finally, the last extension step was 72°C for 20 min. Subsequently, the reaction mix was incubated for 1 hour at 37°C with DpnI, an endonuclease which is capable to digest DNA at the palindromic site “GATC” only if it is methylated. That means that plasmids which have been synthesized by bacteria and therefore bear methylated adenine residues are destroyed, whereas plasmids newly synthesized by PCR are still intact. The remaining plasmids were transformed and colonies isolated as described above.
Screening the singles clones of the cadherin-motif-deletion constructs were performed by restriction fragmentation with enzymes affected by the deletion of the distinct repeat (listed in 4.4.1).
Primers for site-directed mutagenesis deleting discrete cadherin motifs (bold: glycine spacer).
PLKC∆1 forward
5’ (base 74) (base 339) 3’
G TTC CTA GCC AAC ATG ACG GGA GGA GGA GGA GGA GGA GGA AGG GAG ATG CTG G TG ATT G
PLKC∆1 reverse
5’ (base 339) (base 74) 3’
C AAT CAC CAG CAT CTC CCT TCC TCC TCC TCC TCC TCC TCC CGT CAT GTT GGC TAG GAA C
PLKC∆2 forward
5’ (base 366) (base 699) 3’
GTT TTC CAG AAC ACC GCT TTC GGA GGA GGA GGA GGA GGA GGA CTG TCC ATC TCC GTG GTG GAC C
PLKC∆2 reverse
5’ (base 699) (base 366) 3’
G GTC CAC CAC GGA GAT GGA CAG TCC TCC TCC TCC TCC TCC TCC GAA AGC GGT GTT CTG GAA AAC
PLKC∆3 forward
5’ (base 714) (base 1041) 3’
CCC CAG TTT GTC AGG GAG TTT GGA GGA GGA GGA GGA GGA GGA GTG AGA GTG ATG GAC GTC AAT G
PLKC∆3 reverse
5’ (base 1041) (base 714) 3’
C ATT GAC GTC CAT CAC TCT CAC TCC TCC TCC TCC TCC TCC TCC AAA CTC CCT GAC AAA CTG GGG
PLKC∆4 forward
5’ (base 1431) (base 1751) 3’
CCC ACG TTT CCC CAG AGC TTG GGA GGA GGA GGA GGA GGA GGA GAC ATC AAC GAC AAT GCA CCC
PLKC∆4 reverse
5’ (base 1751) (base 1431) 3’
GGG TGC ATT GTC GTT GAT GTC TCC TCC TCC TCC TCC TCC TCC CAA GCT CTG GGG AAA CGT GGG
PLKC∆5 forward
5’ (base 1746) (base 2078) 3’
GCA CCC GTG GTT AGC GGC TCC GGA GGA GGA GGA GGA GGA GGA GAC ATC AAT GAT AAC CTG CCC
PLKC∆5 reverse
5’ (base 2078) (base 1746) 3’
GGG CAG GTT ATC ATT GAT GTC TCC TCC TCC TCC TCC TCC TCC GGA GCC GCT AAC CAC GGG TGC
PLKC∆6 forward
5’ (base 2076) (base 2412) 3’
CCC ATC TTC AAT CAG TCC AGC GGA GGA GGA GGA GGA GGA GGA GTG AAA GAC GTG AAC GAC AAT C
PLKC∆6 reverse
5’ (base 2412) (base 2076) 3’
G ATT GTC GTT CAC GTC TTT CAC TCC TCC TCC TCC TCC TCC TCC GCT GGA CTG ATT GAA GAT GGG
PLKC∆7 forward
5’ (base 2777) (base 3148) 3’
C TAT TTT CTG CCT GAG AAT AAG GGA GGA GGA GGA GGA GGA GGA GAC CAG AGT TAC CGC TCG CG
PLKC∆7 reverse
5’ (base 3148) (base 2777) 3’
CG CGA GCG GTA ACT CTG GTC TCC TCC TCC TCC TCC TCC TCC CTT ATT CTC AGG CAG AAA ATA G
For verification of the correct deletion of the transmembrane domain, forward and reverse primers contained an artificial silent mutation (AAG Æ AAA, underlined), leading to a deletion of a HindIII-cutting site but no alteration in the amino acid sequence.
PLKC∆TM forward
5’ (base 3432) (base 3555) 3’
GAGTCA GAC CTGTCG AAA CAGGGAGGAGGAGGAGGAGGAGGAGTG CGG AAG AGC TAC AAC CGG AAA CTT C
PLKC∆TM reverse
5’ (base 3555) (base 3432) 3’
G AAG TTT CCG GTT GTA GCT CTT CCG CAC TCC TCC TCC TCC TCC TCC TCC CTG TTT CGA CAG GTC TGA CTC
The oligonucleotides designed for deletion of the PDZ-binding motif contain a deletion of a cutting site for BamHI (CGG Æ CGA, underlined). The PDZ-recognition sequence consisting of four amino acids was replaced by base triplets encoding four glycines (bold).
PLKC∆PDZ forward
5’ (base 3896) (base 3951) 3’
C TAC ACC AAC GCT GGC CTG GAC GGA GGA GGA GGA TCA CCG CGG GCC CGA GAT CCA C
PLKC∆PDZ reverse
5’ (base 3951) (base 3896) 3’
G TGG ATC TCG GGC CCG CGG TGA TCC TCC TCC TCC GTC CAG GCC AGC GTT GGT GTA G
By replacement of the DNA fragment coding for the cytosolic tail of PLKC, a HindIII cutting site is deleted which served the screening for positive clones (bold = glycines spacer).
PLKC∆Cyt forward
5’ (base 3509) (base 3986) 3’
G ACC ATG GCC TTC GTG TGT GGA GGA GGA GGA GGA GGA GGA ATG GTG AGC AAG GGC GAG GAG CTG
PLKC∆Cyt reverse
5’ (base 3986) (base 3509) 3’
CAG CTC CTC GCC CTT GCT CAC CAT TCC TCC TCC TCC TCC TCC TCC ACA CAC GAA GGC CAT GGT C
Finally, in order to generate an untagged version of PLKC, the original stop-codon in the pEYFP-PLKC construct was re-introduced by mutagenese-PCR with following primers (bold = introduced stop codon, underlined deletion of a SacII cutting side for the identification of positive clones):
PLKC∆YFP forward
5’ (base 3495) (base 3538) 3’
GC CTG GAC ACC ACG GAC CTG TGA CCA CGG GCC CGG GAT CCA CC
PLKC∆YFP reverse
5’ (base 3538) (base 3495) 3’
GG TGG ATC CCG GGC CCG TGG TCA CAG GTC CGT GGT GTC CAG GC
3.2.3 Cell culture