采用 RPA方法对番茄乙烯过表达单基因突变体 Epi和野生型 VFN8中 L e ETR2 m RNA的表达特征进行了研究 .结果表明 ,在所有被检组织中 Le ETR2 m RNA均表达 ,其表达丰度在叶组织中呈发育调节模式 ,但不受内源乙烯含量的影响 ;而在果实成...采用 RPA方法对番茄乙烯过表达单基因突变体 Epi和野生型 VFN8中 L e ETR2 m RNA的表达特征进行了研究 .结果表明 ,在所有被检组织中 Le ETR2 m RNA均表达 ,其表达丰度在叶组织中呈发育调节模式 ,但不受内源乙烯含量的影响 ;而在果实成熟后期受乙烯的轻微诱导 . Le ETR2的这种表达模式明显有别于其他乙烯受体基因 .为了进一步研究 Le ETR2的功能 ,构建了 Le展开更多
Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-...Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.展开更多
Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The...Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD 2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1 3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD 2 mRNAs in both constitutive and inducible types.展开更多
基金Supported by National Natural ScienceFoundation of China( 39870 5 12)
文摘采用 RPA方法对番茄乙烯过表达单基因突变体 Epi和野生型 VFN8中 L e ETR2 m RNA的表达特征进行了研究 .结果表明 ,在所有被检组织中 Le ETR2 m RNA均表达 ,其表达丰度在叶组织中呈发育调节模式 ,但不受内源乙烯含量的影响 ;而在果实成熟后期受乙烯的轻微诱导 . Le ETR2的这种表达模式明显有别于其他乙烯受体基因 .为了进一步研究 Le ETR2的功能 ,构建了 Le
文摘Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.
文摘Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD 2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1 3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD 2 mRNAs in both constitutive and inducible types.
