Isolation and structural characterization of cDNA clones encoding the mating pheromone Er-1 secreted by the ciliate Euplotes raikovi.

Abstract

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)....

Expression of the gene encoded by a family of macronuclear chromosomes generated by alternative DNA processing inOxytricha fallax

“…I B). However, polyadenylation sites have been identified for four other hypotrich genes, and these were determined independently of PCR (14,15,16). These three genes also lack conventional polyadenylation signals.…”

“…5). If the three introns are removed independently of each other, then cDNAs of eight different lengths would be expected, seven derived from incompletely spliced mRNAs, and one from Euplotes species it encodes 'stop' (16,18). Two facts from the If three bovine solute carriers.…”

Identification and structural characterization of a cDNA clone encoding a membrane-bound form of the polypeptide pheromone Er-1 in the ciliate protozoan Euplotes raikovi.

“…Each is synthesized as a precursor of 75 amino acids (prepropheromone) and subsequently is processed to the mature form (11,34). The secreted, native pheromones are composed of 38-40 amino acids with three intrachain disulfide bridges and occur as oligomers, apparently as homodimers or some higher-order aggregates (10,12).…”

Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein.

“…TAA and TAG are not used as stop codons; instead, they code for glutamine and glutamic acid (21). Euplotes species are the only ciliates known to use TAA as a stop codon (14,24).…”