Genomic screening for regulatory genes using the T-POP transposon.

The identification of a gene that activates or regulates a gene or regulon of interest often requires the artificial induction of the regulatory gene. The properties of the Tn10-derived transposon T-POP allow a simple chromosomal survey of genes that, when artificially induced from an adjacent T-POP transposon by the addition of tetracycline, can activate or inhibit the expression of virtually any gene of interest. Procedures for genome-wide screening for T-POP inducible regulatory genes are described in detail. T-POP is a derivative of transposon Tn10dTc. It encodes resistance to tetracycline, but unlike Tn10dTc, the tet(A) and tet(R) promoters do not terminate within the transposon. Instead they continue out into adjacent chromosomal DNA. When this element inserts in a gene, three things will result: (1) the target gene is disrupted by the addition of a large block of DNA (approximately 3000 bases); (2) a drug-resistance gene (tetracycline resistance) included in the inserted material is now 100% linked to the insertion mutant phenotype; and (3) the mRNA transcripts initiated at either the tet(A) or tet(R) promoters (or both) will continue out into the adjacent chromosomal DNA. Despite the fancy aspects, insertion mutants are easy to isolate and can be assayed for effects on gene regulation using simple plate tests.