Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H co...Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation ofRNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu- man orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis- sues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.展开更多
目的观察组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)对鸡胚翅芽发育中某些基因表达水平的作用,探讨不同浓度TSA(75μmol/L、750μmol/L、1.5mmol/L)对鸡胚翅芽发育的影响,提高我们对染色质结构重组和表观遗传对基因表达的调控作用,...目的观察组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)对鸡胚翅芽发育中某些基因表达水平的作用,探讨不同浓度TSA(75μmol/L、750μmol/L、1.5mmol/L)对鸡胚翅芽发育的影响,提高我们对染色质结构重组和表观遗传对基因表达的调控作用,以及对正在开发的抗癌药物的认识。方法以鸡胚翅芽作为实验模型,用TSA处理翅芽,对鸡胚胎实施原位杂交,检测与肌肉发育相关基因的表达,并采用硫酸耐尔蓝(Nile blue sulfate)法检测细胞凋亡状况。结果75μmol/L TSA能够抑制与肌肉发生相关的基因MyoD和Myf5在鸡胚翅芽的表达,经TSA(75μmol/L)处理的胚胎没有发生明显的细胞凋亡。但是随着TSA药物浓度的提高,TSA(≥750μmol/L)不仅强烈抑制相关基因的表达,而且出现翅芽外部畸形(外形改变、体积缩小)以及细胞凋亡。结论75μmol/LTSA调控某些重要的转录因子及有力地控制肌肉细胞的分化;高浓度TSA(≥750μmol/L)导致细胞凋亡和胚胎翅芽畸形;发育的鸡胚能够作为一个便利的、供体内研究药物(如HDAC抑制剂类等)的模型。展开更多
文摘Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation ofRNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu- man orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis- sues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.
文摘目的观察组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)对鸡胚翅芽发育中某些基因表达水平的作用,探讨不同浓度TSA(75μmol/L、750μmol/L、1.5mmol/L)对鸡胚翅芽发育的影响,提高我们对染色质结构重组和表观遗传对基因表达的调控作用,以及对正在开发的抗癌药物的认识。方法以鸡胚翅芽作为实验模型,用TSA处理翅芽,对鸡胚胎实施原位杂交,检测与肌肉发育相关基因的表达,并采用硫酸耐尔蓝(Nile blue sulfate)法检测细胞凋亡状况。结果75μmol/L TSA能够抑制与肌肉发生相关的基因MyoD和Myf5在鸡胚翅芽的表达,经TSA(75μmol/L)处理的胚胎没有发生明显的细胞凋亡。但是随着TSA药物浓度的提高,TSA(≥750μmol/L)不仅强烈抑制相关基因的表达,而且出现翅芽外部畸形(外形改变、体积缩小)以及细胞凋亡。结论75μmol/LTSA调控某些重要的转录因子及有力地控制肌肉细胞的分化;高浓度TSA(≥750μmol/L)导致细胞凋亡和胚胎翅芽畸形;发育的鸡胚能够作为一个便利的、供体内研究药物(如HDAC抑制剂类等)的模型。
