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We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10−15M.

Ten mating types are known for the hypotrich ciliate Euplotes octocarinatus. The mating types are determined by four codominant alleles that control the production of four gamones. The gamones induce mature cells of certain other mating types to get ready for conjugation. That the cells respond only to gamones that they do not synthesize themselves is rationalized by assuming that mating competence is specified by gamone-specific receptors and that cells express receptors only for those gamones they do not synthesize themselves. Here we show that exconjugant clones are initially composed of cells that neither release gamones nor respond to gamones. If cells are heterozygous and therefore capable of producing two gamones, they start at a clonal age of about 15 fissions to synthesize first one gamone and five to ten fissions later a second gamone. Later still, their capacity to respond to gamones develops, first for one gamone and then five to ten fissions later for a second gamone. Cells responding in maturity to three gamones often acquire this capacity in three steps. These results support our assumption that in E. octocarinatus the gamones are perceived by gamone-specific receptors. We propose that the genes coding for these gamone receptors are derepressed individually in clonal development. The same is assumed for the derepression of the genes that code for the four gamones. We further propose that the cells have the capacity to synthesize receptors for all four gamones, but they down-regulate those receptors to which they synthesize a corresponding gamone.

Gamones 3 and 4 of the ciliate Euplotes octocarinatus were isolated and purified to chromatographic and electrophoretic homogeneity. They are secreted into the culture medium by cells of certain mating types and induce cells of other mating types to unite in pairs and exchange gametic nuclei. The purified gamones are biologically active at concentrations as low as 0.1-1 pM. Both are polypeptides with unusually low pI values of approximately 3.2, and both have a carbohydrate content of less than 2%. Gamone 3 has an Mr of 18,800 and is slightly smaller than gamone 4, which has an Mr of 23,500. Gamone 3 was isolated from the starvation medium of cells homozygous for the mating type allele mt3 and gamone 4 was isolated from the medium of cells homozygous for the mating type allele mt4. Each of the two homozygous cell lines was found to secrete one gamone only.

To determine whether the micronucleus makes essential contributions during asexual reproduction, observations were made on cells of Euplotes octocarinatus from which the micronucleus had been removed with a micropipette. Most cells underwent one postenucleation division, then became arrested in macronuclear G1, slowed down in food uptake, developed macronuclear deformations, and finally died. Such cells could be rescued if a micronucleus was reimplanted before macronuclear deformations had developed. When provided with a new micronucleus, cells initiated macronuclear DNA synthesis about 12–16 h later. The data suggest that the micronucleus is involved in the control of the cell's transition from macronuclear G1 to S, and a model is proposed which postulates that in Euplotes macronuclear DNA synthesis is initiated when a micronucleus-encoded “initiator protein” has accumulated to a critical amount.