DNA-SEQUENCE REQUIREMENTS FOR THE REGULATION OF IMMOBILIZATION ANTIGEN-A EXPRESSION IN PARAMECIUM-TETRAURELIA

The Paramecium surface proteins (immobilization antigens) are expresse d in a mutually exclusive manner; only one antigen is found on the cel l surface at a time. Expression of these proteins is regulated in resp onse to environmental cues such as temperature and pH. This reg ulatio n has been shown to be controlled at the level of mRNA abundance by tr anscriptional and post-transcriptional mechanisms. Here, we have studi ed the transcription and regulated expression of the immobilization an tigen A gene in Paramecium tetraurelia by transforming an A-deficient strain, d12, with cloned portions of the A gene via microinjection. Th e A gene is approximately 8 kilobases (kb) long with the transcription start site at position -9 or -8 and the start of translation at posit ion +1. Paramecia transformed with cloned DNA containing A-gene sequen ces beginning at position -264 and ending 63 base pairs (bp) past the gene's polyadenylation site show properly regulated expression of immo bilization antigen A. Lines derived from paramecia transformed with a plasmid containing A-gene sequences starting at position -211, however , show markedly reduced A-gene mRNA levels, and rarely express the A a ntigen. Nevertheless, cells that do express the A protein exhibit mutu al exclusion and normal responses to environmental stimuli. Thus, the 54 bp between -264 and -211,while important for transcription, are not involved in the control of mutual exclusion and responses to environm ental changes. Further deletion to position -151 yields similar, but m ore extreme, results. Therefore, the start of the A-gene promoter lies within the region -264 to -211,with additional sequences affecting tr anscriptional regulation present between base pairs -211 and -151. Seq uences controlling environmental responses and mutual exclusion must b e located downstream of position -211. Thus, we have defined regions o f DNA necessary for immobilization antigen A expression and have locat ed the approximate position of the A-gene promoter in Paramecium. This work paves the way for a precise mutational analysis of these regions and the first detailed molecular characterization of a Paramecium pro moter. (C) 1994 Wiley-Liss, Inc.