To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized ...To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until M展开更多
In Xenopus laevis embryogenesis, fertilized eggs undergo 12 cycles of synchronous divisions and reach the stage called midblastula transition (MBT). It has long been believed that during the first 12 cycles of cleavag...In Xenopus laevis embryogenesis, fertilized eggs undergo 12 cycles of synchronous divisions and reach the stage called midblastula transition (MBT). It has long been believed that during the first 12 cycles of cleavage (pre-MBT stage), transcriptional activity of the zygotic nuclei is totally absent. However, heterogeneous mRNA-like RNA is synthesized in pre-MBT stage embryos, and exogenously-injected bacterial CAT genes with SV40 promoter are expressed from the cleavage stage. Nevertheless, the synthesis of rRNA as detected by rRNA-specific2’-O-methylation does not take place in pre-MBT embryos and starts only from the latter half of the MBT stage, corroborating the fact that formation of definitive nucleoli as well as the transcription of microinjected rRNA genes starts only at and after MBT stage. Thus, while mRNA-like RNA synthesis occurs from pre-MBT stage, synthesis of rRNA is controlled in the way that transcription of rRNA genes is totally silent during pre-MBT stage and is initiated only at the latter half of MBT stage. Once initiated, the rate of the synthesis of rRNA is constant throughout later stages on a per-cell basis. We searched substances which are responsible for the transcriptional silence of rRNA genes during the pre-MBT stage. Weak bases such as ammonium ion and amines selectively inhibited rRNA synthesis at the transcriptional level in post-MBT stage embryo cells. Since we found that the level of ammonia extracted from embryos is much higher in pre-MBT embryos than in post-MBT embryos, we suggest that weak bases like ammonium ion could be responsible for the transcriptional silence of rRNA genes by slightly increasing intracellular pH during the pre-MBT.展开更多
文摘To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until M
文摘In Xenopus laevis embryogenesis, fertilized eggs undergo 12 cycles of synchronous divisions and reach the stage called midblastula transition (MBT). It has long been believed that during the first 12 cycles of cleavage (pre-MBT stage), transcriptional activity of the zygotic nuclei is totally absent. However, heterogeneous mRNA-like RNA is synthesized in pre-MBT stage embryos, and exogenously-injected bacterial CAT genes with SV40 promoter are expressed from the cleavage stage. Nevertheless, the synthesis of rRNA as detected by rRNA-specific2’-O-methylation does not take place in pre-MBT embryos and starts only from the latter half of the MBT stage, corroborating the fact that formation of definitive nucleoli as well as the transcription of microinjected rRNA genes starts only at and after MBT stage. Thus, while mRNA-like RNA synthesis occurs from pre-MBT stage, synthesis of rRNA is controlled in the way that transcription of rRNA genes is totally silent during pre-MBT stage and is initiated only at the latter half of MBT stage. Once initiated, the rate of the synthesis of rRNA is constant throughout later stages on a per-cell basis. We searched substances which are responsible for the transcriptional silence of rRNA genes during the pre-MBT stage. Weak bases such as ammonium ion and amines selectively inhibited rRNA synthesis at the transcriptional level in post-MBT stage embryo cells. Since we found that the level of ammonia extracted from embryos is much higher in pre-MBT embryos than in post-MBT embryos, we suggest that weak bases like ammonium ion could be responsible for the transcriptional silence of rRNA genes by slightly increasing intracellular pH during the pre-MBT.
