28] New expression vectors for identifying and testing signal structures for initiation and termination of transcription

Abstract

The chapter discusses new expression vectors for the identification and examination of signal structures for the initiation and termination of transcription. When the indicator gene is preceded by a ribosome-binding site and a translation initiation codon, the level of expression of the gene should reflect the strength of the promoter inserted before the gene. Such promoter-probe vectors with the galactokinase (galK) gene as indicator have found widespread application. The vectors described in the chapter contain pairs of divergently oriented indicator genes, either lacZ (β-galactosidase)/ galK or lacZ / phoA (alkaline phosphatase), separated by different polylinkers. The combinations are carried on the plasmids of different host ranges and copy number to further increase their versatility. The resulting plasmids are of great potential use for isolating linked or overlapping divergent promoter sequences as well as for the isolation of unidirectional promoter sequences. Salient features of the promoter-probe vectors described in the chapter are as follows: (1) polylinker sequences with multiple restriction sites, which allow for convenient cloning of DNA fragments carrying unidirectional or divergently arranged promoters, (2) translational stop codons present in all three reading frames upstream of the initiation codons, (3) terminators for transcription behind galK (t 0 from λ) and phoA , (4) a very low residual level of expression of the promoterless indicator genes, (5) the choice of vectors with different copy number and host range, and (6) the detectability of promoter activity directly on plates after transformation. The chapter mentions that the promoter-probe vectors described may be used for cloning, detection, and regulatory analyses of single as well as divergently arranged promoters.