Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5 ' and 3 ' regulatory elements

Down regulation of gene expression by antisense RNA is one of the ways to i nvestigate the specific contribution of certain components to the physiolog y and activities of a cell. A successful inhibition of gene expression in E ntamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promoters that produce transcripts which do n ot bind to polysomes. Different promoters were found to be required for Ent amoeba histolytica or Entameoba dispar. In E. histolytica one of the two co pies (g34) of the gene coding for ribosomal protein L21 was previously foun d to be transcribed but not translated. Inhibition of gene expression was o btained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhib it amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 prom otor of RP-L21, which is known to be translated, did not accumulate antisen se transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' a nd 3' untranslated regions of the amoebapore gene. In E. dispar the opposit e situation was found, plasmid constructs containing the promotor regions o f the gLE3 copy, which were shown to be poorly translated, were more effici ent in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element. (C) 2000 Elsevier Science B.V. Al l rights reserved.