The water-borne protein signals (pheromones) of the Antarctic ciliated protozoan Euplotes nobilii: structure of the gene coding for the En-6 pheromoneThis article is one of a selection of papers in the Special Issue on Polar and Alpine Microbiology.

Abstract

The marine Antarctic ciliate, Euplotes nobilii, secretes a family of water-borne signal proteins, denoted as pheromones, which control vegetative proliferation and mating in the cell. Based on the knowledge of the amino acid sequences of a set of these pheromones isolated from the culture supernatant of wild-type strains, we designed probes to identify their encoding genes in the cell somatic nucleus (macronucleus). The full-length gene of the pheromone En-6 was determined and found to contain an open-reading frame specific for the synthesis of the En-6 cytoplasmic precursor (pre-pro-En-6), which requires 2 proteolytic cleavages to remove the signal peptide (pre) and the prosegment before secretion of the mature protein. In contrast to the sequence variability that distinguishes the secreted pheromones, the pre- and pro-sequences appear to be tightly conserved and useful for the construction of probes to clone every other E. nobilii pheromone gene. Potential intron sequences in the coding region of the En-6 gene imply the synthesis of more En-6 isoforms.

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate, Euplotes crassus

“…DNA was prepared from 1 _ 2 d food‐deprived cultures following a standard protocol (La Terza et al. ), and used as a template for polymerase chain reaction (PCR) amplifications with primers synthesized by Invitrogen (Van Allen Way, Carlsbad, CA). Amplifications were performed in an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany), using 50‐μl reaction mixtures containing 0.5 μg of DNA with 0.2 μM of each primer, 1 U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 0.2 mM dNTP, and 2 mM MgSO 4 .…”

Phylogeographical pattern ofEuplotes nobilii, a protist ciliate with a bipolar biogeographical distribution

“…Purified DNA preparations were obtained, following an optimized protocol (La Terza et al . ), from cell pellets of cultures previously fed only with bacteria for not less than 1 month. The selected genes were PCR‐amplified by adding aliquots (100 ng) of purified DNA to 50 μL reaction mixtures containing 2 m m MgCl 2 , 250 m m of dNTP, one unit of Taq DNA polymerase (Polymed, Florence, Italy) and 0.2 m m of each primer.…”

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

“…All of them were obtained by a PCR strategy based on: (i) the knowledge of the sequence of the pheromone gene en-ant6 that, as reported elsewhere [13], was cloned earlier using a genetic approach developed in relation to the structural characterization of the pheromone En-6 specified by this gene [10]; (ii) the assumption that the sequences of the E. nobilii pheromone genes, like those of the E. octocarinatus pheromone genes [14,15] equivalent to the sub-telomeric sequences of the known pheromone gene en-ant6.…”