REGULATION OF EXPRESSION OF THE GENES FOR A SEX-PHEROMONE BY AN INDUCER OF THE SEX-PHEROMONE IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX

A sex pheromone, protoplast-release-inducing protein (PR-IP), of the C losterium peracerosum-strigosum-littorale complex is known to induce t he 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 a nd 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 reacti on. Amplified fragments of DNA, of 130 bp in the case of the 19-kDa su bunit 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 w ere estimated to be 1.2 kb for the 18-kDa subunit and 1.4 kb for the 4 2-kDa subunit. These transcripts appeared transiently only when mating -type plus (mt(+)) cells were treated with another sex pheromone (PR-I P Inducer) for more than 4h in the light. By immunoblotting with anti- 42-kDa-subunit antiserum, it was shown that PR-IP accumulated graduall y 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 PR-IP Inducer that has been released from mt(-) cells in the light during the sexual reproduction of Closterium, An analysis by genomic Southern hybridiza tion revealed that probes for the 19-kDa and 42-kDa peptides hybridize d to 6.8-kb and 5.1-kb DNA fragments, respectively, after the digestio n of the genome with EcoRI. These hybridized DNA fragments were obtain ed not only from the genome of mt(+) cells but also from the genome of mt(-) cells, in which no transcripts for PR-IP could be detected by N orthern hybridization. On the basis of these results, we discuss the p ossibility that the expression of the gene for the two subunits of PR- IP might be critically dependent upon the action of putative sex-deter mining genes.