DNA sequence requirements for the regulation of immobilization antigen a expression in Paramecium tetraurelia

Abstract

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 regulation 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.

“…In the ciliated protozoan Tetrahymena, mRNA stability plays a major role in the expression of the variable surface antigens (27). This contrasts with Paramecium surface antigen expression, which appears to be controlled primarily at the level of transcription (17,19,20).…”

“…A number of these genes have been cloned and completely or partially sequenced, including 51A (10), 51B (11), 51C (10), 51D (15), and 51H (16 (14,18). Recent studies have shown that surface antigen expression is regulated at the transcriptional level and, under certain circumstances, may involve posttranscriptional mechanisms (17,19,20 (20). In this paper, we show that at least some of the elements necessary to control mutually exclusive expression are located in the coding region of the surface antigen gene.…”

RNA-Mediated Programming of Developmental Genome Rearrangements in Paramecium tetraurelia

“…Wild-type A29 and A51 transgenes were constructed by cloning a 10.2-kb SalI-XhoI macronuclear fragment containing the gene and regulatory sequences (22,25) into the pCRScriptAmpSK ϩ vector (Stratagene). The plasmids were injected either as circular molecules or after linearization in the vector sequence.…”

Homology-dependent Gene Silencing inParamecium

“…The injected DNA is lost at autogamy, along with the macronuclear chromosomes (Godiska et al, 1987;Gilley et al, 1988;Bourgain and Katinka, 1991). This phenomenon has generally been studied in the context of complementation experiments: expression of the microinjected sequences was evaluated in cells in which the functional product of the corresponding endogenous gene was absent due to choice of geographic strain (Meyer, 1992), deletion (Godiska et al, 1987;Gilley et al, 1988;Martin et al, 1994), or mutation (Kanabrocki et al, 1991;Haynes et al, 1996;Skouri and Cohen, 1997).…”