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ABSTRACTCiliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged a1 tubulin minichromosomes into the macronucleus of Stylonychia lemnaeas a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnaeminichromosomes, whereas the USEs appear to be specific for the a1 tubulin minichromosome. The a2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of a-tubulin expression in cells treated with concanavalin A activated the a2 but not the a1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnaeoccurs at the level of transcription initiation on the basis of structurally different promoters.

Ciliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged 1 tubulin minichromosomes into the macronucleus of Stylonychia lemnae as a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnae minichromosomes, whereas the USEs appear to be specific for the 1 tubulin minichromosome. The 2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of -tubulin expression in cells treated with concanavalin A activated the 2 but not the 1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnae occurs at the level of transcription initiation on the basis of structurally different promoters.