The zebrafish (Danio rerio) is a useful model system for analyzing development of the inner ear. A number of mutations affecting the inner ear have been identified. Here we investigate the initial stages of otolith morphogenesis in wild-type embryos as well as in monolith (mnl) mutant embryos, which fail to form anterior otoliths but otherwise appear normal. Otolith growth is initiated at 18-18.5 h by localized accretion of free-moving precursor particles. This process, referred to as otolith seeding, is regulated by two classes of cilia: First, kinocilia of precociously forming hair cells (tether cells) bind seeding particles, thereby localizing otolith formation. Tether cells usually occur in pairs at the anterior and posterior ends of the ear. Despite the presence of functional kinocilia, tether cells initially appear immature and do not acquire the characteristics of mature hair cells until approximately 21.5 h. Second, beating cilia distributed throughout the ear agitate seeding particles, thereby inhibiting premature agglutination. Constraining particles with laser tweezers caused them to fuse into large untethered masses. Bringing such masses into contact with tethered otoliths caused them to fuse, greatly enhancing otolith growth. Selectively enhancing one otolith greatly inhibited growth of the second, creating an imbalance that persisted for many days. Seeding particles and beating cilia disappear soon after 24 h, and the rate of otolith growth decreases by nearly 90%. In mnl mutant embryos, tethers and beating cilia are distributed normally, but anterior otoliths fail to form in 80-85% of mutant ears. The binding properties of seeding particles appear normal, as shown by their ability to fuse when entrapped by laser tweezers and their binding to posterior tethers. We infer that anterior tethers have a weakened ability to bind seeding particles in mnl embryos. Immobilizing mnl embryos with the anterior end of the ear oriented downward effectively concentrated the dense seeding particles near the anterior tethers and permitted all to form anterior otoliths. However, immobilizing mnl embryos after 24 h when seeding particles were depleted did not facilitate anterior otolith formation. Together, these data demonstrate that the ability to initiate otolith formation is limited to a critical period, from 18.5 to 24 h, and that interfering with the functions of tether cell kinocilia or beating cilia impairs otolith seeding and subsequent otolith morphogenesis.

Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.

The trichocysts of Paramecium tetraurelia consitute a favorable system for studying secretory processes because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis .Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place.We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts . Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane .The trichocysts of Paramecium are secretory vesicles whose formation in the cytoplasm, migration to the cell cortex, interaction with the plasma membrane, and exocytosis are easily observable by light and electron microscopy and are also amenable to genetic dissection. A number of mutations are available that block trichocyst development and exocytosis at various stages . These mutations disclose different steps and functions that might not be suspected or identified in other systems. The trichocyst system offers two further advantages for studying secretory processes .First, in the region of contact between trichocyst and plasma membrane, organized arrays of intramembrane particles are visible on freeze-fracture replicas of both the plasma and the

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of parameciumtype telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.Foreign DNA introduced into eucaryotic cells can suffer a variety of fates depending on the sequences introduced and the system used. In mouse L cells in culture, injection of multiple copies of either supercoiled or linear DNA molecules into nuclei can result in integration into host chromosomes at a limited number of sites of head-to-tail concatamers of the input DNA (7, 18). These concatamers result from highly efficient homologous recombination mediated by host recombinational machinery. Alternatively, microinjection of a variety of different supercoiled DNA species into unfertilized Xenopus laevis eggs leads to autonomous replication of the injected DNA as supercoiled forms, apparently regardless of sequence (13,16). In the yeast Saccharomyces cerevisiae, foreign supercoiled DNA introduced by transformation replicates autonomously only if it contains distinctive sequences called autonomously replicating sequences (21), presumably reflecting specific recognition of certain sequences by host replication machinery.We describe here another fate for DNA introduced into the ciliated protozoan Paramecium tetraurelia. We have previously described a transformation system in which exogenous DNA is introduced into the macronuclei of recipients by microinjection (10). Our assay for transformation involves the synthesis of specific cell surface antigens called immobilization antigens (for a review, see reference 19). Paramecia can alter their surface antigens under different environmental conditions. In general, they express only one type at a time. Eleven different serotypes have been described for stock ...

ABSTRACT. From an intermittent stream in College Station, Texas, a Paramecium was isolated that did not appear to belong to any recognized species. On the basis of nuclear and whole‐body morphology, it can be assigned to the Paramecium aurelia species‐complex, and it can be distinguished from other members of that complex on the basis of mating‐type reactivity and isoenzyme patterns. These characteristics are felt sufficient to justify a new species assignment. The new species has been named Paramecium sonneborni n. sp. in honor of the late Dr. Tracy M. Sonneborn of Indiana University.