Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

Abstract

The continuous sequence of 2.3 kilobases in 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the phage M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selectd CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption 2/3 of the way through the nucleotide sequence of the structural gene. The 5''- and 3''-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the 2nd coding region of the synthase is not incorporated into the final protein product. Structural evidence confirming the presence of the intervening sequence in the phase genome was obtained by restriction and hybridization analysis. Support for the presence of the intron was also obtained at the functional level by enzyme expression studies using selected td gene fragments. The termination codon for the dihydrofolate reductase gene and the triplet initiating thymidylate synthase overlap by a 4-base stretch, A-T-G-A. The implications of this unusual gene arrangement are discussed.