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First published online 31 May 2005
doi: 10.1242/jcs.02359

Journal of Cell Science 118, 2735-2741 (2005)
Published by The Company of Biologists 2005
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Developmentally and environmentally regulated expression of gamone 1: the trigger molecule for sexual reproduction in Blepharisma japonicum

Mayumi Sugiura1,2, Seiko Kawahara1, Hideo Iio3 and Terue Harumoto1,2,*

1 Department of Biological Science, Faculty of Science, Nara Women's University, Kitauoyanishi-machi, Nara, 630-8506, Japan
2 Department of Biological Science and Environment, Graduate School of Human Culture, Nara Women's University, Kitauoyanishi-machi, Nara 630-8506, Japan
3 Department of Material Science, Graduate School of Science, Osaka City University, Sugimoto-cho, Osaka 558-8585, Japan



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  		Fig. 1. Detection of the gamone 1 gene transcript. (A) Diagram of gamone 1 and synthesized probes. The box represents the gamone 1 ORF (from +1 to +915). The signal peptide cleavage site (+49) and the putative N-terminus of mature gamone 1 (+100) are indicated. Probes were prepared by labeling the fragment (probe 1) and full-length gamone 1 cDNA (probe 2) with DIG. (B) Transcription of gamone 1. Total RNA from mating type I cells was loaded in lanes 1-4 (2, 5, 11, 11 µg, respectively) and lane 5 (6 µg), and separated on a 1% denaturing agarose gel, transferred to a nylon membrane and then hybridized with probe 1 (lanes 1-4) or probe 2 (lane 5). A single band of about 1000 bases can be detected in each lane (arrowhead). The size of RNA markers (Novagen) is indicated on the left-hand side.

 


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  		Fig. 2. Selective expression of gamone 1 in B. japonicum cells. (A) The transcriptional levels of the gamone 1 gene were examined by northern analysis using probe 2. Total RNA was extracted from mating type I (I) and II (II) cells and was separated by electrophoresis (6 µg/lane). Gamone 1 mRNA (about 1000 bases) was detected only in samples prepared from mating type I cells. The graph shows the relative amounts of gamone 1 mRNA with respect to that of rRNA calculated from the results of the northern hybridisation above. (B) The secreted mature gamone 1 protein was detected by SDS-PAGE and silver staining. (C) The biological activity of secreted mature gamone 1 is represented as the index of pair formation (black bar) and in units (cross-hatched bar).

 


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  		Fig. 3. Influence of sexual maturation on the expression of gamone 1 during development. (A) The level of transcription of gamone 1 was examined in a progeny clone at each clonal age and in sexually mature type I cells (mature). The relative amounts of gamone 1 mRNA are indicated in the graph. For cells of clonal age of 10, 2.2 µg/lane was loaded, and for the others, 10 µg/lane. The extent of maturation at each clonal age is represented by the maturation index. These results suggested that the transcription of gamone 1 and maturation were strictly linked. (B) The biological activity of secreted mature gamone 1, represented in units.

 


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  		Fig. 4. Influence of the complementary mating type pheromone, gamone 2, on the expression of gamone 1. (A) The transcriptional level of gamone 1 was examined by northern blotting of the moderately starved cells of both types stimulated with gamone 2 (+G2) or unstimulated (–G2). Total RNA was prepared from each cell type (6 µg/lane). The level of the transcription of gamone 1 was increased when starved type I cells were stimulated with gamone 2. Type II cells did not transcribe gamone 1 mRNA, irrespective of the presence of gamone 2. The graph shows the relative amounts of gamone 1 mRNA. (B) The secreted mature gamone 1 protein was detected by SDS-PAGE and silver staining. (C) The biological activity of secreted mature gamone 1 was measured and is represented by the index of pair formation.

 


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  		Fig. 5. Effect of gamone 2 on the promotion of gamone 1 transcription. (A) We examined the transcriptional levels of gamone 1 in type I cells in the early stationary phase after the addition of gamone 2 (G2). Total RNA was prepared and separated (6 µg/lane) from cells that had been incubated with gamone 2 for 0, 2, 4, 6, 8 or 20 hours, and gamone 1 mRNA was detected by northern hybridization using probe 2. A peak of transcriptional activity was reached in cells stimulated for 4-6 hours with gamone 2. The graph indicates the relative amounts of gamone 1 mRNA. (B) SDS-PAGE analysis and (C) biological activity tests of CFFs collected from individual cell suspensions.

 


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  		Fig. 6. Influence of nutrient deprivation on the expression of the gamone 1 gene. (A) Total RNA was extracted from type I cells in the log phase [I (L)] and in the early stationary phase [I (1D)]. Northern hybridization showed that cells in the log phase did not transcribe the gamone 1 gene, but that early stationary phase cells started to transcribe it when they were moderately starved (1D). (B) The transcriptional level was compared for type I cells in the early stationary phase (1D) and cells starved in SMB– for 5 days (5D). In both cells, the response to gamone 2 was also examined (G2–, untreated; G2+, treated with gamone 2). The transcription of gamone 1 was promoted in cells that had been starved longer (5D) and was accelerated by gamone 2 treatment. The graph shows the relative amounts of gamone 1 mRNA.

 


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  		Fig. 7. Schematic illustration of asymmetric cell-cell interactions mediated by gamones during conjugation in B. japonicum. When mature type I cells receive the food-deprivation stimulus (step 1), they start transcribing the gamone 1 gene and synthesizing gamone 1 (steps 2-3). Secreted gamone 1 is recognized by starved mature type II cells through the putative gamone 1 receptor (step 4), inducing them to synthesize gamone 2 from tryptophan (steps 5-6). The gamone 1 gene is inactive in mating type II cells. Secreted gamone 2 is recognized by type I cells through the putative gamone 2 receptor (step 7). Gamone 2 signal is transmitted to the nucleus, promoting further transcription of the gamone 1 gene (step 8). I, mating type I cell; II, mating type II cell; G1, gamone 1; G1-R, hypothetical receptor for gamone 1; G2, gamone 2; G2-R, hypothetical receptor for gamone 2; E, enzyme(s) for production of gamone 2; Trp, tryptophan.

 

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© The Company of Biologists Ltd 2005