Table 2.

Oligonucleotides for Amino Acid Substitution by PCR Mutagenesis

Name aPositions bSequence (5′ to 3′) cMutation d
Mut1f241-270CTGGCAATCTTGACTgcTGCTGCACATTTGD86A
Mut1r270-241CAAATGTGCAGCAgcAGTCAAGATTGCCAG
Mut2f250-279TTGACTGATGCTGCAgcTTTGCTTTCAGATH89A
Mut2r279-250ATCTGAAAGCAAAgcTGCAGCATCAGTCAA
Mut3f250-279TTGACTGATGCTGCAaAgTTGCTTTCAGATH89K
Mut3r279-250ATCTGAAAGCAAcTtTGCAGCATCAGTCAA
Mut4f262-291GCACATTTGCTTTCAGcTGTTGCAGGCTTTD93A
Mut4r291-262AAAGCCTGCAACAgCTGAAAGCAAATGTGC
Mut5f865-894CTGATCTGCACCgcAATCTTTTCTGTTATAL293A
Mut5r894-865TATAACAGAAAAGATTgcGGTGCAGATCAG
Mut6f886-915TCTGTTATAGTTgcGGGTACAACAATCAAAL300A
Mut6r915-886TTTGATTGTTGTACCCgcAACTATAACAGA
Mut7f907-936ACAATCAAAATGgcACGGAACATACTTGAAL307A
Mut7r936-907TTCAAGTATGTTCCGTgcCATTTTGATTGT
Mut8f928-957ATACTTGAAGTCgcGATGGAGAGCACTCCAL314A
Mut8r957-928TGGAGTGCTCTCCATCgcGACTTCAAGTAT
  • a Two partially complementary primers (f and r) were used in each PCR mutagenesis for the introduction of amino acid codon substitutions.

  • b The region of the PtdMTP1 sequence in each oligonucleotide is marked by the positions of the 5′ and 3′ nucleotides separated by a dash. The position of the 5′ oligonucleotide is shown first for each oligonucleotide. All positions are numbered in reference to the first nucleotide of the PtdMTP1 coding sequence.

  • c Uppercase letters indicate that the sequence is exactly the same as the wild-type PtdMTP1 DNA sequence. Lowercase letters indicate the mismatched nucleotides designed for specific mutations.

  • d The designated amino acid substitutions resulting from each mutagenic primer pair.