Spheroplasts were then washed as previously described and digested with 25 or 70 U of micrococcal nuclease for four minutes.
The fragments were joined as described above, digested with HindIII and BamHI, and then ligated into pK18mobsacB to generate pDImcH2.
The native RFA2 promoter was amplified as described previously, digested with SacII and NcoI, and cloned into pRS315– rfa2 -D x-Δpromoter to generate pRS315– rfa2 -D x.
For naked DNA controls, genomic DNA was extracted as previously described and digested with 0.003 0.2 mU of MN under the same conditions.
Plasmid pQE30-434CI is a pQE30 (Qiagen)- based construct, which was made by cloning of the PCR fragment (produced as described below) digested with BamHI and XmaI.
PCR amplification products were cloned into the pGEM-T-vector as described above, digested with EcoRI and BamHI, ligated into the mammalian expression vector pEGFP-C1 (Clontech, Heidelberg, Germany) and cloned according to standard protocols.
The partial CAL gene was amplified using the primers CL1 and CL2A [ 42] as described above and digested with HhaI as described elsewhere [ 29].
To generate the poly-Ser-Tag, poly-Ala-Tag, and poly-Gln-Tag constructs (Fig. 7c), the CGG interruption at the 3′ end CAG repeats of the HinDIII/XbaI fragment as described above was digested with MspA1I to remove the 3′ sequence of ExonA in the CAG direction and several stop codons immediately following the polyglutamine ORF.
Genomic DNA of the transformants was extracted as described above and digested with restriction enzymes BamHI, PstI, and NdeI (all purchased from Thermo Scientific, Rockford, IL, USA).
For Southern blot assays, DNA sample of each strains, prepared with the methods previously described [34], were digested with PstI overnight at 37°C and then electrophoresed on a 1% agarose gel with 8 mA constant current, for 16 to 18 hours and then transferred to nylon membranes [Roche] with a vacuum blotter [Bio-Rad] with manufactures' instructions.
