ABSTRACT
This chapter describes the beginning of a series of in vitro site-directed mutagenesis experiments, the purpose of which is to increase our understanding of the Dihydrofolate reductase (DHFR) molecule in particular, and of enzyme-protein structure in general. The enzyme DHFR, is found in every kind of organism, from bacteria to mammals, and in large amounts in rapidly dividing cell lines. It catalyzes the nicotinamide adenine dinucleotide phosphate-dependent reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, which in turn plays a central metabolic role as a carrier of one-carbon units in the biosynthesis of thymidylate, purines, and some amino acids. Oligonucleotides designed to produce the desired mutations were then used as primers on the single-stranded phage DNA. In addition to the DHFR gene, the plasmid pCV29 was shown by sequence analysis to contain a point mutation in the DHFR promoter that results in overproduction of the enzyme.
