Molecular Cloning of a Novel Sex Pheromone Responsible for the Release of a Different Sex Pheromone in Closterium peracerosum-strigosum-littorale Complex

Abstract

A sex pheromone, protoplast-release-inducing protein (PR-IP) inducer, of the Closterium peracerosum-strigosum-littorale complex is known to induce the release of PR-IP, from mating-type plus (mt+) cells during sexual reproduction. The purified PR-IP inducer was treated with trypsin to obtain internal peptides for determination of partial amino acid sequences. Using these sequences, oligonucleotides were synthesized and used as primers for the combined reverse transcription-PCR. A 296 bp cDNA fragment was amplified, permitting the cloning of corresponding full length cDNA (CpPI; Closterium peracerosum-strigosum-littorale complex PR-IP inducer). The deduced amino acid sequence of CpPI encodes a protein of 212 amino acid residues of M(r) 23,071 whereas portion of the peptide secreted is predicted to have 142 amino acid residues of M(r) 15,717 and shows no significant similarity with known proteins. The predicted protein has three possible consensus sequences for asparagine-linked glycosylation site. The CpPI gene was expressed when mating-type minus (mt-) cells were incubated at a low cell density in the light. Nitrogen deprivation from the medium enhances expression of the CpPI gene. An analysis by genomic Southern hybridization revealed that the cDNA probe hybridized to several DNA fragments obtained from both the genome of mt- and mt+ cells. However, in mt- cells, transcripts for the PR-IP inducer could not be detected by Northern hybridization.

SEX PHEROMONES THAT INDUCE SEXUAL CELL DIVISION IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (CHAROPHYTA)1

“…This pheromone was purified and its partial amino acid sequences revealed significant similarity to the sequence of PR‐IP Inducer (Fukumoto et al 2002). These results suggested that the primary structure of PR‐IP Inducer was related to that of SCD‐IP‐minus in C. psl c. These compounds might constitute a multigene family of Closterium pheromones (Sekimoto et al 1998). Alternatively, both the induction of SCD and the release of PR‐IP might be triggered by PR‐IP Inducer itself.…”

Isolation of a differentially expressed gene in separate mating types of the dinoflagellateAlexandrium tamarense

“…Secretory proteins with a signal peptide have been isolated from other unicellular microalgae. The gamete lytic enzyme was identified from Chlamydomonas, and pheromone proteins were isolated from Closterium (Sekimoto et al, 1990(Sekimoto et al, , 1998Kinoshita et al, 1992;Nojiri et al, 1995). The signal peptide regions of these molecules are rich in hydrophobic residues, but there is no similarity among amino acid sequences (data not shown).…”

Sexual Reproduction of a Unicellular Charophycean Alga, Closterium peracerosum-strogosum-littorale Complex

“…PR-IP Inducer is released constitutively from mt − cells in the presence of light and directly induces the production and release of PR-IP from mt + cells. cDNAs encoding the subunits of PR-IP (Sekimoto et al 1994a , b ) and PR-IP Inducer (Sekimoto et al 1998 ) have been isolated. A computer search using the nucleotide sequences and deduced amino-acid sequences failed to reveal any homologies to known proteins.…”

“…A computer search using the nucleotide sequences and deduced amino-acid sequences failed to reveal any homologies to known proteins. Genes for these pheromones can be detected in cells of both mating types using genomic Southern hybridization analysis, but they are only expressed in cells of the respective mating type, suggesting the presence of sex-specifi c regulation of gene expression by sex-limited trans -acting factors (Sekimoto et al 1998 ;Sekimoto et al 1994c ;Endo et al 1997 ).…”