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cDNA clones comprising the entire coding region for the mating pheromone Er-1 of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors lambda gt10 and pUC12. The cDNA sequence contains an open reading frame of 75 amino acids that constitute pre-pro-Er-1. The amino acid sequence of secreted Er-1 starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-1 protein sequencing. The coding region of Er-1 cDNA ends with codon TAA, which specifies glutamine in other ciliates. The 5'- and 3'-noncoding regions contain, respectively, two and one inverted repeats. The 3'-noncoding-region inverted repeat, which includes the unusual polyadenylylation signal AACAAA, has been related to RNA 3'-terminus formation.

The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-11, and Er-20, secreted by cells of different mating types of the ciliated protozoa Euplotes raikovi, have been determined by automated Edman analyses of the whole proteins and germane fragments. In each case, the molecular mass was determined by plasma desorption or laser desorption mass spectrometry and was in excellent agreement with the calculated values. Where available, the determined sequences were also in accord with the corresponding segments of the precursor molecules predicted from relevant nucleic acid sequences. Of the five, two were found to be identical (Er-2 and Er-9) and one (Er-3) was identical to a pheromone previously sequenced (Er-1), even though mating pair formation was found to take place (although to a limited extent) when cells secreting those pheromones were combined in a mixture. Comparison of the five unique sequences suggested a closer relationship between Er-1 (Er-3) and Er-10 and between Er-11 and Er-20 (44% and 56% identity, respectively) than was generally observed among the other members. This pairing was also supported by hydrophobicity analyses. Interestingly, Er-20 cannot, as a rule, induce cell union in any of the other cell types, including cells secreting Er-11, despite the fact that Er-20 and Er-11 are the most similar of the five unique sequences. Thus sequence identity and secondary structure profiles are not a good indicator of biological relatedness as manifested in heterologous receptor interaction.

In the ciliate Euplotes raikovi, the same cell that secretes the pheromone Er-1, a polypeptide of 40 amino acids derived from a precursor (prepro-Er-1) of 75 amino acids, also produces a polypeptide of 130 amino acids, of which the 75 residues at the carboxyl terminus are identical to those of prepro-Er-1 and the 55 residues at the amino terminus form a new sequence. This larger Er-1 isoform is retained in membranes, where it may function as a binding site for soluble Er-1 in a mechanism of autocrine secretion. Membrane-bound and soluble Er-1 are translated from two mRNAs that apparently originate from a common micronuclear and/or macronuclear gene through alternative elimination of intervening sequences. This finding suggests that single genes responsible for the generation of isoform diversity in polypeptide hormones are present even in single-celled eukaryotes.