Transcription and Translation involved in Uniparental Inheritance and Cell Nuclei Fusion in the Chlamydomonas reinhardtii Zygote

Transcription

1 1997 The Japan Mendel Society Cytologia 62: , 1997 Transcription and Translation involved in Uniparental Inheritance and Cell Nuclei Fusion in the Chlamydomonas reinhardtii Zygote Lena Suzuki 1, 2, *, Yasuhito Yuasa1 and Tsuneyoshi Kuroiwa 2 Department of Hygiene and Oncology, Tokyo Medical and Dental University School of Medicine, Yushima, Tokyo 113, Japan Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113, Japan Accepted November 12, 1997 The heterothallic isogamous green alga Chlamydomonas reinhardtii has been used as a model organism for the study of the uniparental inheritance of chloroplast genes (Sager 1954). Observation of 4',6-diamidino-2-phenylindole (DAPI) stained zygotic cells with high-resolution epifluorescent microscopy revealed that the mechanism of uniparental inheritance is the preferential digestion of the male-derived chloroplast DNA (cp-dna), 40 min after mating (Kuroiwa et al. 1982). Treatment with actinomycin D and cycloheximide, inhibitors of transcription and translation in the cytoplasm, inhibits the preferential digestion of the male-derived cp-dna. This suggests that de novo synthesis of mrna and proteins is required for this process (Kuroiwa et al. 1983a, b). This led to the identification of seven zygote specific peptides, 94 kda(a), 94 kda(Ĉ), 94 kda(y, 52 kda, 50 kda, 38 kda and 20 kda, which play roles in uniparental inheritance (Nakamura et al. 1988), five zygote specific cdna clones, zysla, zys1b, zys2, zys3 and zys4 (Uchida et al. 1993), and six zygote specific cdna clones, class I, class II, class III/ezy-1, class IV/zsp-1, class V and class VI/zsp-2 (Ferris and Goodenough 1987). The complete molecular mechanism of uniparental inheritance is still unknown. Fusion of the two cell nuclei follows the preferential digestion of the cp-dna (Cavalier-Smith 1970). This was observed 1 hr or more after mating, in DAPI stained zygotic cells (Kuroiwa et al. 1982). Karyogamy is the most significant process in mating, but it is not as well studied as the preferential digestion of cp-dna in this system. To elucidate the relationships between the aforementioned events and peptides or genes, it is necessary to understand the time schedule for the expression of these genes in detail. The aims of this study are to estimate when transcription and translation play a role in the preferential digestion of male-derived cp-dna and fusion of the cell nuclei, by treating zygotes with transcription and translation inhibitors at various times after mating, and to observe the effects of inhibitors on these two events. Material and methods Strain and culture conditions Wild-type strains of C. reinhardtii, 137c mt+ and mt-, were used. Cells of the two mating types were grown on Medium I plates (Sager and Granick 1954) containing 1.2% agar. Five days after plating, the cells were collected with a glass spreader, suspended in mating buffer (0.6 mm MgCl2, 1.2 mm HEPES (ph 6.8)) at a density of approximate 2-5 ~ 107 cells per ml, induced to form gametes, and then mixed to allow mating, as described in a previous report (Nakamura et al. Corresponding author: Lena Suzuki. Tel: , ext Fax: lena@biol.s.utokyo.ac.jp.

2 428 Lena Suzuki, Yasuhito Yuasa and Tsuneyoshi Kuroiwa Cytologia ). Aliquots of the cell suspension at appropriate stages were used for the analyses described below. Observation of cp-dna (nucleoid) and cell nuclei Aliquots of the cell suspension were fixed with 1% glutaraldehyde, and then stained with 1 mg/ml 4',6-diamidino-2-phenylindole (DAPI) in TAN buffer (20 mm Tris-HC1 (ph 7.6), 0.5 mm EDTA, 1.2 mm spermidine, 7 mm 2-mercaptoethanol and 0.4 mm PMSF). The cells were sandwiched between a cover slip and a slide glass, then squashed by applying gentle pressure to the surface of the cover slip. They were observed under an epifluorescence microscope (Olympus BHS- RFK, Olympus Optical Co.). cp-dna and cell nuclei were observed at the same time. More than 100 zygotic cells were counted for each treatment at each time, and were classified as Types I to V, according to Kuroiwa's criteria (Kuroiwa et al. 1985) illustrated in Fig. 1. Treatment with inhibitors Actinomycin D (Sigma and rifampicin (Wako, Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/ml. Cycloheximide (Sigma) and chloramphenicol (Wako, Osaka, Japan) were dissolved in ethanol at concentrations of 10 and 34 mg/ml, respectively. Stock solutions of these inhibitors were stored at -20 Ž until use, and then added to the cell suspensions in mating buffer to give the final concentrations used in previous studies (Kuroiwa et al. Type I II III IV V Fig. 1. The stages in the preferential digestion of male-derived cp-dna and fusion of the cell nuclei. DAPI stained cells were observed under an epifluorescence microscope. Zygotic cells were classified into Types I-V according to Kuroiwa's (1985) classification: fused gametes (Type I), beginning (Type II) and completion (Type III) of the preferential digestion of male-derived cp-dna, and beginning (Type VI) and completion (Type V) of fusion of the cell nuclei. These are shown above the graph. The graph shows the accumulated number of each type of zygote after mating. The number of each type of cell includes cells that have passed through a given stage.

3 1997 Zygote Specific Gene Expressions (2) a, b) (actinomycin D, 20ƒÊg/m1; cycloheximide, 10ƒÊg/ml; rifampicin, 80 ktg/ml and chloramphenicol, 80 ƒêg/ml). Each inhibitor was added to the mating medium at eight different stages: 30 min before the gametes were mixed, and then 5 min, 10 min, 20 min, 40 min, 1 hr, 2 hr and 3 hr after mating. The cp- DNA and cell nuclei were observed at 5 min, 10 min, 20 min, 40 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr and 8 hr after mating for each inhibition series. Results The cells observed were classified into five cell types: fused gametes (Type I), beginning (Type II) and completion (Type III) of the preferential digestion of the male-derived cp-dna, and beginning (Type VI) and completion (Type V) of fusion of the cell nuclei. With the passing of time, the five types of zygotic cells appear successively in the order listed. The results without an inhibitor are shown in Fig. 1. This is also the control for the experiments with antibiotics that follow. The effects of four different inhibitors on the gametes were tested. Rifampicin and chloramphenicol inhibit transcription and translation, respectively, in the organelles, while actinomycin D and cycloheximide inhibit these activities in the cytoplasm. The effects of these inhibitors on the appearance of Types I-V zygotic cells were observed up to 8 hr after mating. None of the inhibitors affected cell fusion (Type I). The organelle inhibitors had no effect on the preferential digestion of male-derived cp-dna (Types II and III), while the cytoplasmic inhibitors interrupted the preferential digestion of male-derived cp-dna (Table 1). The effects on the appearance of Types IV and V, which are in the process of nuclear fusion, were the same as for Types II and III. Only the two inhibitors of cytoplasmic transcription and translation interrupted nuclear fusion (Table 1). This indicates that cytoplasmic transcription and translation are necessary for both the preferential digestion of cp-dna and fusion of the cell nuclei after mating. The effects of actinomycin D and cycloheximide, given at various times after the gametes were mixed, are shown separately for each of the five cell types in Fig. 2. Neither inhibitor affected cell fusion (Type I). The progression from Types II to V was not affected by the addition of inhibitors. No irregular cells that appeared to have skipped a stage in the progression were observed with inhibition by either actinomycin D or cycloheximide. The times at which the inhibitors were added influenced the appearance of the different cell types. When they were added immediately after the mating period, they had the same, full effect as when added to the gametes. This is referred to as the sensitive period. This is followed by a period Table 1. Inhibition of transcription/translation in the cytoplasm and the organelles Inhibitors were given to gametes 30 min before mating, and observed at 8 hr after mating. Relative percentages against the control values are shown. See Fig. 1 about the definition of zygotic cell types. a) More than 120 zygotic cells were counted for each inhibitors. b) The same concentrations given with chloramphenicol and rifampicin as a solvent of the stock solutions, respectively.

4 430 Lena Suzuki, Yasuhito Yuasa and Tsuneyoshi Kuroiwa Cytologia 62 Actinomycin D treatment Cychloheximide treatment Fig. 2. The effects of antibiotics on the digestion of male-derived cp-dna and fusion of the cell nuclei. Either 20 Đg/ml actinomycin D or 10 Đg/ml cycloheximide was added to the gametes before mixing or to the zygotes 5, 10, 20 and 40 min and 1, 2 and 3 hr after mixing the gametes. The cumulative totals for each cell type are shown separately, with plots for the different addition times for the inhibitors. The control values, shown in Fig. 1, are indicated by thicker lines for comparison. Some overlapping curves were omitted, unless they were significant. For example, only the curve for the earliest treatment with an inhibitor was shown when several curves overlapped the control line.

5 1997 Zygote Specific Gene Expressions (2) 431 Fig. 3. Minimum transcription and translation periods for the early-zygotic-events genes. This figure summarizes the transcription and the translation periods for the genes required for the preferential digestion of male-derived cp-dna, and fusion of the cell nuclei, as estimated by the treatment with inhibitors. of partial sensitivity (Fig. 2). These periods of partial sensitivity to actinomycin D and cycloheximide, affecting transcription and translation respectively, indicate that there is some accumulation of mrna or gene products involved in these processes (summarized in Fig. 3). The transcription of genes leading to the preferential digestion of male-derived cp-dna (Types II and III) begins immediately after cell fusion, while there is a time lag after mating before the start of transcription leading to fusion of the cell nuclei (Types IV and V). The effect of cycloheximide on the appearance of Types II and III cells showed a remarkable inhibitory pattern (Fig. 2). When cycloheximide was added during the period of partial sensitivity, between 10 and 40 min after mixing, cells inhibited as Type II never became Type III cells. Without exception, 8 hr after mating, the male-derived cp-dna remained partially digested. Actinomycin D had the same effect as cycloheximide, although the period of partial sensitivity was shorter. Furthermore, the beginning of the period of partial sensitivity starts before the midpoint of the period required to complete digestion. These results suggest that the expression of additional gene(s) is required to complete the preferential digestion once it has begun. Discussion mrna and protein synthesis is involved in the preferential digestion of male-derived cp-dna The mrna synthesis required for the preferential digestion of the male-derived cp-dna starts as soon as the cells fuse. The mrna synthesis period also suggests that the minimum amount of essential mrna for uniparental inheritance is synthesized within the first hour after mating. All the peptides that are candidates for roles in uniparental inheritance are expressed within this transcription period: 94 kda(a), 94 kda(Ĉ), 94 kda(y), 52 kda, 50 kda, 38 kda and 20 kda peptides (Nakamura et al. 1988), and young zygote specific genes (Ferris and Goodenough 1987, Uchida et al.

6 432 Lena Suzuki, Yasuhito Yuasa and Tsuneyoshi Kuroiwa Cytologia , Armbrust et al. 1993). In previous studies (Kuroiwa et al. 1983a, b), completion of the preferential digestion of cp- DNA was observed with inhibitor treatment, i.e. cell Type III appeared. In this study, the two cell types representing the beginning (Type II) and completion (Type III) of digestion were considered separately. Two possibilities were considered to explain the additional translation necessary to complete preferential digestion of the cp-dna. First, the expression of an additional gene or genes after the expression of the first zygotic gene is required to complete the preferential digestion of cp- DNA. In this case, there are at least two steps that control the uniparental inheritance related genes, and the gene group expressed first includes a transcription factor for the induction of new genes. Second, there is a continuous signal promoting cp-dna digestion that must not be interrupted. An essential gene, which is already expressed before the beginning of the preferential digestion of cp- DNA, may have a quick turnover, e.g., calcium-dependent nuclease C (Kuroiwa 1985, 1991) and its promoting factor. mrna and protein synthesis involved in fusion of the cell nuclei Actinomycin D and cycloheximide treatments also affected fusion of the cell nuclei. In previous studies (Kuroiwa et al. 1983a, b), both inhibitors affected the preferential digestion of male-derived cp-dna, but did not affect fusion of the cell nuclei. It is felt that different cell conditions used in these studies affected cell sensitivity to the inhibitors. Fusion of the cell nuclei requires a longer period of mrna and protein synthesis than the preferential digestion of cp-dna. The progression of gene expression in fusion of the cell nuclei is not as synchronous as in the destruction of malederived cp-dna. It is difficult to determine the minimum periods required for mrna and protein synthesis. If this is possible, it will help us to determine which of the zygote specific genes are involved in fusion of the cell nuclei. Summary The preferential digestion of male-derived chloroplast-dna (cp-dna) and fusion of the cell nuclei were observed in zygotes of the alga Chlamydomonas reinhardtii, when transcription (actinomycin D) and translation (cycloheximide) inhibitors were given at various times after mating. The minimum periods required to complete cytoplasmic transcription and translation were estimated. Genes playing a role in uniparental inheritance are transcribed from the moment the gametes fuse until about 1 hr after mating, but fusion of the cell nuclei requires a longer period of gene expression. Observations of two cell types indicating the beginning and completion of the preferential digestion of male-derived cp-dna suggested two possibilities. (1) Expression of additional genes after expression of the first zygotic genes is required to complete the process. (2) The presence of a continuous signal promoting cp-dna digestion is necessary for completion. Acknowledgments The authors would like to thank Dr. H. Takano and Dr. A. Sakai for their helpful discussions and advice. This work was supported by a Grant for Special Promoted Research (project no ) to T.K. from the Ministry of Education, Science, Sports and Culture of Japan. References Armbrust, E. V, Ferris, P. J. and Goodenough, U. W A mating type-linked gene cluster expressed in Chlamydomonas zygotes participates in the uniparental inheritance of the chloroplast genome. Cell 74: Cavalier-Smith, T Electron microscopic evidence for chloroplast fusion in zygotes of Chlamydomonas reinhardii.

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