A TETRACYCLINE-INDUCIBLE GENE-EXPRESSION SYSTEM IN ENTAMOEBA-HISTOLYTICA

We have developed an episomal inducible gene expression system in Enta moeba histolytica based on the TetR repressor. The tetR gene was place d under control of 5' and 3' ferredoxin (fdx) regulatory sequences on a plasmid encoding the hygromycin resistance gene directed by 5' and 3 ' hgl sequences. The reporter luciferase constructs were introduced on a second episome bearing the neomycin resistance gene controlled by 5 ' and 3' actin sequences. The reporter constructs were driven by the h gl5 promoter in which the tetO sequence was introduced. We found that the optimal tetO location for induction by tetracycline was +4 from th e start of transcription. The efficiency of repression and the inducti on ratio could be improved by increasing hygromycin levels, presumably by increasing tetR plasmid levels. Under these conditions, maximal in duction of reporter luciferase could be effected with 5 mu g/ml tetrac ycline in 18 h. This system permits regulated expression of the report er gene over two orders of magnitude and should be useful in the analy sis of gene function. Copyright (C) 1997 Elsevier Science B.V.