Regulation of expression of the genes for a sex pheromone by an inducer of the sex pheromone in the Closterium peracerosum-strigosum-littorale complex

Abstract

A sex pheromone, protoplast-release-inducing protein (PR-IP), of the Closterium peracerosum-strigosum-littorale complex is known to induce the release of protoplasts from mating-type minus (Mt⁻) cells during sexual reproduction and to have two subunit polypeptides of 19 and 42 kDa. Here, we describe the regulation mechanism for the release of the PR-IP. The sex pheromone was fractionated to yield subunits of 19 and 42 kDa, respectively, and each subunit was treated with V8 protease and with CNBr. By reference to the partial amino-acid sequences of the digested polypeptides, oligo nucleotides were synthesized and used as primers for the combined reverse transcription-polymerase chain reaction. Amplified fragments of DNA, of 130 bp in the case of the 19-kDa subunit and of 330 bp in the case of the 42-kDa subunit, were obtained, sequenced, and used as probes to identify the respective transcripts. From the results of Northern hybridization, the sizes of transcripts were estimated to be 1.2 kb for the 19-kDa subunit and 1.4 kb for the 42-kDa subunit. These transcripts appeared transiently only when mating-type plus (mt⁺) cells were treated with another sex pheromone (PR-IP Inducer) for more than 4 h in the light. By immunoblotting with anti-42-kDa-subunit antiserum, it was shown that PR-IP accumulated gradually in the medium but not in the mt⁺ cells after treatment with PR-IP Inducer in the light. We suggest that PR-IP is synthesized de novo and secreted from mt⁺ cells only after the perception of PRIP Inducer that has been released from mt⁻ cells in the light during the sexual reproduction of Closterium. An analysis by genomic Southern hybridization revealed that probes for the 19-kDa and 42-kDa peptides hybridized to 6.8-kb and 5.1-kb DNA fragments, respectively, after the digestion of the genome with EcoRI. These hybridized DNA fragments were obtained not only from the genome of mt⁺ cells but also from the genome of mr cells, in which no transcripts for PR-IP could be detected by Northern hybridization. On the basis of these results, we discuss the possibility that the expression of the gene for the two subunits of PR-IP might be critically dependent upon the action of putative sex-determining genes.