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

The Paramecium surface proteins (immobilization antigens) are expressed in a mutually exclusive manner; only one antigen is found on the cell surface at a time. Expression of these proteins is regulated in response to environmental cues such as temperature and pH. This reg ulation has been shown to be controlled at the level of mRNA abundance by transcriptional and post-transcriptional mechanisms. Here, we have studied the transcription and regulated expression of the immobilization antigen A gene in Paramecium tetraurelia by transforming an A-deficient strain, d12, with cloned portions of the A gene via microinjection. The A gene is approximately 8 kilobases (kb) long with the transcription start site at position -9 or -8 and the start of translation at position +1. Paramecia transformed with cloned DNA containing A-gene sequences beginning at position -264 and ending 63 base pairs (bp) past the gene's polyadenylation site show properly regulated expression of immobilization 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 antigen. Nevertheless, cells that do express the A protein exhibit mutual 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 environmental changes. Further deletion to position -151 yields similar, but more extreme, results. Therefore, the start of the A-gene promoter lies within the region -264 to -211,with additional sequences affecting transcriptional regulation present between base pairs -211 and -151. Sequences controlling environmental responses and mutual exclusion must be located downstream of position -211. Thus, we have defined regions of DNA necessary for immobilization antigen A expression and have located 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 promoter. (C) 1994 Wiley-Liss, Inc.