In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement.

In the ciliate Euplotes raikovi, the same cell that secretes the pheromone Er-i, 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-i isoform is retained in membranes, where it may function as a binding site for soluble Er-i in a mechanism of autocrine secretion. Membrane-bound and soluble Er-i 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.

Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding.

ABSTRACTcDNA clones comprising the entire coding region for the mating pheromone Er-i of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors Agt1O 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-i starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-i protein sequencing. The coding region of Er-i 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.Since Sonneborn's discovery of mating types in Paramecium aurelia (1), numerous species of ciliates have been found capable of controlling cell-cell recognition phenomena and cell transformation for mating by complexes of genetically determined mating types (for reviews, see refs. 2-6). However, the chemical substances determining mating types have been purified to homogeneity and partially characterized in only a few species (for reviews, see refs. 6 and 7; also see refs. 8-11), and nothing is yet known about their genetic regulation at the molecular level. In Euplotes raikovi, four ofthese substances-referred to as mating pheromones or euplomones r and abbreviated as Er (12)-have been shown to be small soluble, acidic proteins, biologically active at concentrations of _10-12 M (11, 12). According to data from Mendelian genetic analysis, their genetic control is carried out, as in Euplotes patella (13,14) and Euplotes octocarinatus (15), by a series of codominant multiple alleles at the highly polymorphic mating type (mat) locus (7, 16). Each mating pheromone segregates in one-to-one correspondence with one mat allele and confers one specific molecular phenotype (mating type) to one cell class (16).Here we describe the molecular cloning and sequencing of cDNAs encoding the mating pheromone Er-1, which is secreted by E. raikovi of mating type I, honmozygous for the allele mat-iJ. The cDNA sequence specifies an Er-1 precursor of 75 amino acids, including the 40-amino acid sequence of mature Er-1 near the carboxyl terminus (17).Knowledge of the molecular structure and genetic control of E. raikovi mating pheromones will broaden the concept of mating type function in ciliates and at the same time yield opportunities for seeking molecular details of cell-cell recognition phenomena in unicellular eukaryotes.MATERIALS AND METHODS Cells and Culture Conditions. E. raikovi of clone 1aF113 homozygous for the allele mat-i and secreting the mating pheromone Er-1 was the source of RNA and DNA. Cells were maintained as described (16). For RNA extraction logarithmically growing cells were harvested, resuspended for 4 hr in starvation medium, and pelleted by centrifugation at 800 x g for 3 min.RNA Extraction. Total RNA was extracted by the LiCi method (18)....

No abstract