Molecular cloning of the phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12

Abstract

A genomic library of Rhodosporidium toruloides DNA was constructed in bacteriophage λ1059. Recombinant phage containing phenylalanine ammonia lyase (PAL) gene sequences were identified by using 32P-labeled cDNA to partially purified PAL mRNA. The PAL gene was subcloned on an 8.5-kilobase PstI DNA restriction fragment into pUC8 to generate the recombinant plasmid pHG2. A restriction map of the PAL gene, together with its flanking regions, was constructed. Northern hybridization analysis of R. toruloides RNA with a restriction fragment encoding part of the PAL gene indicates that PAL mRNA is 2.5 kilobases in length. A single-stranded DNA hybridization probe was constructed and used to quantitate PAL mRNA levels in R. toruloides grown under different physiological conditions. PAL mRNA levels paralleled changes in functional PAL mRNA and antigen. These data are consistent with control of PAL expression being at the level of transcription.