1887

Abstract

Recent genetic mapping of the aspartokinase II () operon of [M. Petricek. L. Rutberg & L. Hederstedt (1989) 61,85–88; N. Y. Chen. J. J. Zhang & H. Paulus (1989) 135, 2931–2940] has shown its chromosomal location to be close to the locus, the mutation of which leads to highly increased levels of aspartokinase II. In order to examine the relationship between and , we have cloned the control regions of the operon from several independent mutants and determined their nucleotide sequences. The nucleotide sequences of the mutants differed from the wild-type sequence by the substitution of one or two nucleotides at two widely separated sites in the transcribed leader region of the operon. To confirm that the observed nucleotide changes are indeed responsible for the AecA phenotype and not simply the reflection of sequence polymorphisms in different strains, we introduced the same nucleotide substitutions as those observed in the strains into the leader region of the wild-type operon by oligonucleotide-directed mutagenesis. The expression of the mutagenized genes was analysed after transcriptional or translational fusion to in a single-copy integration vector. The levels of -galactosidase were greatly elevated by the nucleotide substitutions, with similar increases observed in transcriptional and translational fusions. The high level of expression of -galactosidase in the - strains with nucleotide substitutions corresponding to the mutations was resistant to repression by L-lysine but was completely abolished by the inactivation of the promoter. These results suggest that the sites identified by nucleotide sequencing of the mutants are important regulatory sites in the operon, which exert their effect at the level of transcription involving the normal promoter.

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1991-05-01
2021-10-27
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