The mutagenic PCR conditions were as follows: 50 mM KCl, 10 mM Tris (pH 8.3 at 25uC), 7 mM MgCl 2 , 0.3 mM MnCl 2 , 1 mM dCTP and dTTP each, 0.2 mM dGTP and dATP each, 0.2 mM of each primer, 20 pM of template DNA and 10 units of Taq polymerase in a 10 ml reaction volume in 10 aliquots. The PCR was performed for 30 cycles at 94uC for 1 min, 50uC for 1 min, and 72uC for 1 min in a conventional thermal cycler. Three overlapping ,300-500 bp N-, central-and C-terminal segments of the TEF1 gene were amplified separately by PCR using primer pairs: #2767 (GTT-TCAGTTTCATTTTTCTTGTTC)/#2788 (GAGTCCATCT-TGTTGACAG), #2787 (CATCAAGAACATGATTACTGGT-AC)/#2790 (GACGTTACCTCTTCTGATTTC) and #2789 (CGGTGTCATCAAGCCAGGT/#2771, (TTCGGTTAGAGC-GGATGTGG), respectively.