TABLE 1.

Correlation between the number of mutations in the PCFT coding region and function of mutants generated by random mutagenesis

The pcft coding region was amplified by PCR and random mutations were generated in this reaction. The fragments were cloned into an expression vector and transiently transfected into HeLa R1–11 cells. PCFT function was assessed by measuring MTX influx (0.5 μm) at pH 5.5 over 1 min and was normalized to wild-type PCFT. The values represent the average from three different experiments.

Mutagenesis condition	Expected mutations/1 kba	Number of clones studied	Actual mutations/1 kb	Mutations/ pcft geneb	PCFT function
					<5%	5–30%	>30%
1	2.0	8	0.97	1.3 ± 0.3	1	0	7
2	2.3	10	2.4	3.3 ± 0.5	4	3	3
3	2.7	9	3.8	5.1 ± 0.7	4	3	2
4	4.6	6	5.3	7.2 ± 1,4	5	1	0
5	8.1	5	11.7	15.8 ± 2.8	4	0	1

^a Data provided by the manufacturer.

^b The mutated pcft region encodes the whole PCFT protein except the last eight amino acid residues at the C-terminus, which does not have any functional role (28).