目的:利用酵母双杂交系统筛选与人POT1(human protection of telomeres 1,hPOT1)相互作用的蛋白。方法:以hPOT1的300～634氨基酸片段为诱饵,在人乳腺cDNA文库中筛选能与hPOT1相互作用的蛋白质;运用营养缺陷型培养基和X-α-Gal实验排除...目的:利用酵母双杂交系统筛选与人POT1(human protection of telomeres 1,hPOT1)相互作用的蛋白。方法:以hPOT1的300～634氨基酸片段为诱饵,在人乳腺cDNA文库中筛选能与hPOT1相互作用的蛋白质;运用营养缺陷型培养基和X-α-Gal实验排除假阳性,并对阳性克隆进行序列测定和比对。结果:经过酵母双杂交筛选,发现7个与hPOT1相互作用的蛋白;选取NM23B与hPOT1通过GST-pull down和免疫共沉淀进行进一步的验证,结果证明它们确实存在相互作用。结论:hPOT1能与NM23B发生相互作用,在此实验基础上可以进一步研究NM23B与hPOT1相互作用的生物学意义。展开更多
目的:建立SYBR Green Ⅰ实时荧光PCR定量检测人POT1基因的方法。方法:依据人POT1基因及参照基因TBP基因序列设计引物,以人类乳腺癌细胞系MCF-7总RNA逆转录合成的cDNA为模版,以TBP基因为参照基因,应用SYBR GreenI实时荧光定量PCR检测POT...目的:建立SYBR Green Ⅰ实时荧光PCR定量检测人POT1基因的方法。方法:依据人POT1基因及参照基因TBP基因序列设计引物,以人类乳腺癌细胞系MCF-7总RNA逆转录合成的cDNA为模版,以TBP基因为参照基因,应用SYBR GreenI实时荧光定量PCR检测POT1基因的表达。结果:POT1和TBP基因熔解曲线为单峰,无杂峰及二聚体。POT1基因及参照基因TBP基因扩增效率相同(直线斜率0.0171<0.1)。POT1基因批内变异与批间变异分别为1.3%和2.1%;TBP基因批内变异与批间变异分别为1.2%和2.6%。结论:建立了SYBR Green Ⅰ实时定量检测人类POT1基因的方法,该方法简单快速,经济,灵敏度高,特异性和重复性好,可用于人类POT1基因的定量分析。展开更多
Deregulated telomere length is a causative factor in many physiological and pathological processes,including aging and cancer.Many studies focusing on telomeres have revealed important roles for cooperation between th...Deregulated telomere length is a causative factor in many physiological and pathological processes,including aging and cancer.Many studies focusing on telomeres have revealed important roles for cooperation between the Shelterin protein complex and telomerase in maintaining telomere length.However,it remains largely unknown whether and how aging-related stresses,such as deregulated protein homeostasis,impact telomere length.Here,we explored the possible roles of aminoacyl tRNA synthetases(AARSs),key enzymes catalyzing the first reactions in protein synthesis,in regulating telomere length and aging.We selected seryl tRNA synthetase(SerRS)since our previous studies discovered expanded functions of SerRS in the nucleus in addition to its canonical cytoplasmic role in protein synthesis.In this study,we revealed that overexpression of SerRS promoted cellular senescence and inhibited the growth of cervical tumor xenografts in mice by triggering the senescence of tumor cells.In the nucleus,SerRS directly bound to telomeric DNA repeats and tethered more POT1 proteins to telomeres through a direct interaction between the UNE-S domain of SerRS and the OB1 domain of POT1.We further demonstrated that SerRS-induced enrichment of POT1 prevented the recruitment of telomerase to telomeres,resulting in progressive telomere shortening.Our data suggested a possible molecular link between protein synthesis and telomere length control,the deregulation of which may be associated with aging and cancer.展开更多
目的建立人端粒保护蛋白1(protection of telomeres 1,POT1)rs1034794位点多态性的检测方法。方法应用创造酶切位点法(created restriction site PCR,CRS-PCR)根据单碱基突变位点的碱基替代情况设计PCR引物。其中1条引物根据突变位点邻...目的建立人端粒保护蛋白1(protection of telomeres 1,POT1)rs1034794位点多态性的检测方法。方法应用创造酶切位点法(created restriction site PCR,CRS-PCR)根据单碱基突变位点的碱基替代情况设计PCR引物。其中1条引物根据突变位点邻近序列设计,引入错配碱基,使得引物3’端和单碱基突变的一种突变型在PCR扩增后形成1个酶切位点,PCR产物用PCR-限制性片段长度多态性(PCR restriction fragment length polymorphism,PCRRFLP)法进行分析。结果设计1对特异引物,其中正向引物3’末端与多态相邻,倒数第三位碱基为错配碱基A可在PCR扩增后与多态T等位基因及相邻片段形成AGCT结构,使用限制性内切酶Alu I酶切效果较好。结论应用CRS-PCR方法创造的酶切位点可以有效而简捷地检测POT1(rs1034794)基因多态性。展开更多
目的检测端粒保护蛋白POT1(protection of telomeres 1)m RNA和TPP1(POT1-interacting protein 1)m RNA在膀胱尿路上皮癌中的表达。分析两者表达的相关性及与膀胱尿路上皮癌的病理分级、临床分期之间的关系。方法收集32例膀胱尿路上皮...目的检测端粒保护蛋白POT1(protection of telomeres 1)m RNA和TPP1(POT1-interacting protein 1)m RNA在膀胱尿路上皮癌中的表达。分析两者表达的相关性及与膀胱尿路上皮癌的病理分级、临床分期之间的关系。方法收集32例膀胱尿路上皮癌组织标本,17例癌旁组织标本,12例正常膀胱粘膜组织标本。提取各组标本的总RNA,采用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-q PCR)检测端粒保护蛋白POT1 m RNA和TPP1m RNA在各组标本中的表达。结果 POT 1 m RNA、TPP 1 m RNA在膀胱尿路上皮癌组织组中的相对表达量,均低于其在癌旁组织组中的相对表达量,组间差异有统计学意义(P<0.01);且均低于其在正常膀胱粘膜组织组中的相对表达量,组间差异有统计学意义(P<0.01);而POT 1 m RNA、TPP 1 m RNA的相对表达量在癌旁组织组与正常膀胱粘膜组织组相比较,组间差异无统计学意义。在各组标本中,POT 1 m RNA与TPP 1 m RNA相对表达量之间成线性相关。膀胱尿路上皮癌组织中POT1 m RNA、TPP 1 m RNA的相对表达量在各病理分级间差异无统计学意义,而膀胱尿路上皮癌组织中POT1m RNA、TPP 1m RNA的相对表达量在临床分期间的差异有统计学意义,并且随着临床分期的增加而升高。结论端粒保护蛋白POT1 m RNA和TPP1 m RNA在膀胱尿路上皮癌组织中的表达低于癌旁组织及正常粘膜组织。POT1 m RNA和TPP1 m RNA的表达之间线性相关。端粒保护蛋白POT1和TPP1的表达可能与膀胱肿瘤的发生及病理分级、临床分期存在相关性。展开更多
文摘目的:建立SYBR Green Ⅰ实时荧光PCR定量检测人POT1基因的方法。方法:依据人POT1基因及参照基因TBP基因序列设计引物,以人类乳腺癌细胞系MCF-7总RNA逆转录合成的cDNA为模版,以TBP基因为参照基因,应用SYBR GreenI实时荧光定量PCR检测POT1基因的表达。结果:POT1和TBP基因熔解曲线为单峰,无杂峰及二聚体。POT1基因及参照基因TBP基因扩增效率相同(直线斜率0.0171<0.1)。POT1基因批内变异与批间变异分别为1.3%和2.1%;TBP基因批内变异与批间变异分别为1.2%和2.6%。结论:建立了SYBR Green Ⅰ实时定量检测人类POT1基因的方法,该方法简单快速,经济,灵敏度高,特异性和重复性好,可用于人类POT1基因的定量分析。
基金This work was supported by grants from the National Natural Science Foundation of China(No.81772974)the Natural Science Foundation of Tianjin City(18JCQNJC12600)+1 种基金the Project of Science and Technology Assistance in Developing Countries(KY201501006)the Ph.D.Candidate Research Innovation Fund of Nankai University.
文摘Deregulated telomere length is a causative factor in many physiological and pathological processes,including aging and cancer.Many studies focusing on telomeres have revealed important roles for cooperation between the Shelterin protein complex and telomerase in maintaining telomere length.However,it remains largely unknown whether and how aging-related stresses,such as deregulated protein homeostasis,impact telomere length.Here,we explored the possible roles of aminoacyl tRNA synthetases(AARSs),key enzymes catalyzing the first reactions in protein synthesis,in regulating telomere length and aging.We selected seryl tRNA synthetase(SerRS)since our previous studies discovered expanded functions of SerRS in the nucleus in addition to its canonical cytoplasmic role in protein synthesis.In this study,we revealed that overexpression of SerRS promoted cellular senescence and inhibited the growth of cervical tumor xenografts in mice by triggering the senescence of tumor cells.In the nucleus,SerRS directly bound to telomeric DNA repeats and tethered more POT1 proteins to telomeres through a direct interaction between the UNE-S domain of SerRS and the OB1 domain of POT1.We further demonstrated that SerRS-induced enrichment of POT1 prevented the recruitment of telomerase to telomeres,resulting in progressive telomere shortening.Our data suggested a possible molecular link between protein synthesis and telomere length control,the deregulation of which may be associated with aging and cancer.
文摘目的建立人端粒保护蛋白1(protection of telomeres 1,POT1)rs1034794位点多态性的检测方法。方法应用创造酶切位点法(created restriction site PCR,CRS-PCR)根据单碱基突变位点的碱基替代情况设计PCR引物。其中1条引物根据突变位点邻近序列设计,引入错配碱基,使得引物3’端和单碱基突变的一种突变型在PCR扩增后形成1个酶切位点,PCR产物用PCR-限制性片段长度多态性(PCR restriction fragment length polymorphism,PCRRFLP)法进行分析。结果设计1对特异引物,其中正向引物3’末端与多态相邻,倒数第三位碱基为错配碱基A可在PCR扩增后与多态T等位基因及相邻片段形成AGCT结构,使用限制性内切酶Alu I酶切效果较好。结论应用CRS-PCR方法创造的酶切位点可以有效而简捷地检测POT1(rs1034794)基因多态性。
文摘目的检测端粒保护蛋白POT1(protection of telomeres 1)m RNA和TPP1(POT1-interacting protein 1)m RNA在膀胱尿路上皮癌中的表达。分析两者表达的相关性及与膀胱尿路上皮癌的病理分级、临床分期之间的关系。方法收集32例膀胱尿路上皮癌组织标本,17例癌旁组织标本,12例正常膀胱粘膜组织标本。提取各组标本的总RNA,采用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-q PCR)检测端粒保护蛋白POT1 m RNA和TPP1m RNA在各组标本中的表达。结果 POT 1 m RNA、TPP 1 m RNA在膀胱尿路上皮癌组织组中的相对表达量,均低于其在癌旁组织组中的相对表达量,组间差异有统计学意义(P<0.01);且均低于其在正常膀胱粘膜组织组中的相对表达量,组间差异有统计学意义(P<0.01);而POT 1 m RNA、TPP 1 m RNA的相对表达量在癌旁组织组与正常膀胱粘膜组织组相比较,组间差异无统计学意义。在各组标本中,POT 1 m RNA与TPP 1 m RNA相对表达量之间成线性相关。膀胱尿路上皮癌组织中POT1 m RNA、TPP 1 m RNA的相对表达量在各病理分级间差异无统计学意义,而膀胱尿路上皮癌组织中POT1m RNA、TPP 1m RNA的相对表达量在临床分期间的差异有统计学意义,并且随着临床分期的增加而升高。结论端粒保护蛋白POT1 m RNA和TPP1 m RNA在膀胱尿路上皮癌组织中的表达低于癌旁组织及正常粘膜组织。POT1 m RNA和TPP1 m RNA的表达之间线性相关。端粒保护蛋白POT1和TPP1的表达可能与膀胱肿瘤的发生及病理分级、临床分期存在相关性。
