Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae

“…cerevisiae strains used were YLL302 (MATa ade2-1 his3⌬200 ura3-52 leu2-3,112 trp1⌬1 rrp2⌬::HIS3/pTOP4), derived from MES 106 (Schmitt & Clayton, 1992), and JLY1 (MATa ade2-1 his3-11,15 leu 2-3,112 trp1-1 ura3-1 can1-100 rpr1::HIS3/pB-H RNP-Ycp50 (RPR1 URA3 ); Pagan-Ramos et al+, 1996)+ Plasmid vectors used were YCplac33 (URA3 ) and YCplac22 (TRP1) (Gietz & Sugino, 1988)+ The wild-type RPR1 gene was carried by a YCplac22 derivative with 408 nt upstream of 59 end of mature P RNA and 254 nt downstream of 39 end of the mature P RNA+ Two plasmids carrying the wild-type RRP2 (alias NME1; Schmitt & Clayton, 1992) were used: pTOP4 (URA3 ) and pDK38 (TRP1)+ The pDK38 plasmid was generated by PCR amplification of genomic DNA and includes 184 bp upstream and 206 bp downstream of RRP2+ It has an A r T change in position 127 relative to the published sequence (Schmitt & Clayton, 1992), apparently generated during the PCR reaction+ The pTOP4 plasmid carries a Bgl II-EcoRI genomic fragment (Chu et al+, 1994) and has an A r G change at position 69 and a T r C change at position 265 (apparently reflecting a polymorphism in the MRP RNA sequence)+ Cells carrying a deletion of the chromosomal RRP2 gene and relying on either pDK38 or pTOP4 as the source of MRP RNA have a growth rate and a distribution of short, long, and very long 5+8S rRNAthat are indistinguishable from wild-type strains (Chu et al+, 1994; our unpubl+ results)+ For the purpose of these studies we consider both of these plasmid-encoded RRP2 genes to be wild-type+ DNA fragments containing mutated derivatives of RRP2 and RPR1 (Fig+ 1 inserts) were made using the SOE technique (Ho et al+, 1989) or standard PCR DNA technology and cloned in YCplac22+ The entire RRP2 and RPR1 genes were sequenced to confirm that they contained the desired substitutions+…”

Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

“…The y35D mutant was constructed by overlap extension using PCR [19]. Briefly, two PCR fragments were prepared with Pwo polyrnerase using the human cDNA [16] as a template and primers A+C (5' fragment) and B+D (3' fragment).…”

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

“…Site-specific mutations of EHV4 TK were generated by overlapextension PCR using overlapping oligonucleotide sets: (EHV4 TK 5 0 -gga att cca tat ggc tgc ttg cgt ac-3 0 , and EHV4 TK 3 0 -gga tcc tca gac gcc cat ctc cgc-5 0 ; EHV4 TK A143Y 5 0 -ccg gtc tact ct acc gta tgc ttt cca g-3 0 , and 3 0 -tac ggt aga gta gac cgg gtg gcg-5 0 ; EHV4 TK S144H 5 0 -gtc gcc cat acc gta tgc ttt cca gc-3 0 , and EHV4 TK S144H 3 0 -tac ggt atg ggc gac cgg gtg-3 0 ) [25]. The EHV4 TK 5 0 oligos contained a BamHI site, and the EHV4 TK 3 0 oligos contained an NdeI site for cloning into the modified pMAL c2 vector.…”