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Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.

The ciliated protozoan Paramecium provides a model system for the study of regulated secretion, featuring architecturally complex secretory storage granules-trichocysts-docked at the plasma membrane, ready to respond to an exocytotic stimulus. The trichocysts are characterized by crystalline contents that confer upon the organelle a defined shape which can be altered by single gene mutation. The crystalline trichocyst contents are built up from a heterogeneous set of small acidic polypeptides generated by proteolytic maturation of a family of precursor molecules, suggesting an important role for protein processing in this system. We have recently shown that the primary defect in several secretory mutants lacking functional trichocysts is in intracellular trafficking rather than protein processing. However, analysis of how these defects lead to altered trichocyst shape supports the notion that the protein processing is essential for morphogenesis. Preliminary results of a cloning project reveal that an extensive multigene family (approximately 100 genes) codes for the trichocyst matrix proteins. Deduced amino acid sequences of putative processing sites indicate that (at least) two distinct processing reactions are probably involved in the maturation of these proteins, and allow us to speculate that each reaction may control a key event of trichocyst biogenesis.