The immobilization antigens (i-antigens) of Paramecium, large polypeptides of relative molecular mass approximately 300,000, are located on the cell surface. Each i-antigen is encoded by a different unlinked gene, and no more than one gene is expressed at a time. The proteins and the mRNAs and genes encoding them are readily isolated. Here we report the nucleotide sequence of three regions of the A i-antigen gene from stock 51 of Paramecium tetraurelia. Surprisingly, all reading frames contain TAA and TAG stop codons, even though there is evidence that one reading frame of these sequences codes for the i-antigen. We suggest that in Paramecium UAA and UAG code for amino acids, instead of serving as translational stops as they do in all other organisms.

a Hump, aboral hump-killing; MK, mate-killing; par, paralysis-killing; res, resistant to killing; sens, sensitive to killing; spin, spinning-killing; vac, vacuolization-killing.

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.

DNA processing occurs in ciliates at autogamy and conjugation when new macronuclei are formed from micronuclei and old macronuclei degrade. Processing of micronuclear DNA consists of removal of certain internal sequences, chromosomal fragmentation, addition of new telomeres, and amplification. Aside from a recent brief report, internal eliminated sequences have not been described in Paramecium. In this paper we characterize nine internal eliminated sequences found within and near the gene that codes for surface protein A in Paramecium tetraurelia. Of these nine, seven are located within the translated portion of the gene, and all include short, inverted terminal repeats. The characteristic sequence, TA, appears at the boundaries of all of the internal eliminated sequences.

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.