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发布于:2020-11-27 09:52:50  访问:0 次 回复:0 篇
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Accumulating evidence suggests that ATM functions upstream of the p tumor suppressor protein. Functional ATM is required for optimal p induction and activation following cellular exposure to agents that induce DNA doublestrand breaks. First, p induction are observed in AT cell lines after UV irradiation.When considered in light of recent reports concerning ATM, these results suggest that ATR and ATM play both overlapping and independent roles in the phosphorylation of p during cellular exposure to genotoxic stress.The stabilization of p levels by SV large T therefore facilitates the interpretation of immunoblotting results obtained with phosphospecific antibodies directed against p.Nonetheless, these results support the conclusion that ATR kinase activity is not required for the activation of ATM in girradiated cells.ATR was immunoprecipitated from testis extracts using either preimmune rabbit serum. Phosphorylation of p by ATR requires a functional kinase domain.We also noted that ATR catalytic activity was present in AT cells.ATR that was immunoprecipitated from the AT cell line ATBI retained the ability to phosphorylate p in vitro, indicating that ATR catalytic activity does not require coexpression of a functional ATM polypeptide. The model that ATM and ATR function sequentially during the response to damaged DNA has an interesting precedent in the process of meiotic recombination, during which ATR and ATM are selectively associated with asynapsed and synapsed regions of meiotic chromatin. Collectively, the available data strongly support the conclusion that ATM and ATR carry out both overlapping and distinct signaling functions during cellular responses to different types of genotoxic stress.In addition, immunoprecipitated ATR phosphorylates the aminoterminal region of p, suggesting that the interaction between ATR and p is direct.This hypothesis is supported by two experimental observations.The sequential recruitment of ATM and ATR into the p phosphorylation pathway may be a consequence of dynamic changes in the initiating lesion during the cellular response to gradiationinduced DNA damage.The primary genetic lesions induced by gradiation may activate ATM efficiently, but subsequent processing of the lesions may be required for the recognition of altered DNA structures by ATR.The methodology used to generate the apphosphoserine antibody. The resulting plasmid encodes the aminoterminal amino acids of p fused to GST.For transient transfections, K cells were subjected to a V msec electropulse in the presence of g of plasmid DNA using a BTX square wave electroporator. The transfected cells were then cultured for hr prior to harvest.The homogenate was centrifuged at, g and fractionated with ammonium sulfate as described previously. For ATR kinase assays, ATR was immunoprecipitated with g of aATR from mg of cell or testis extract.The immunoprecipitates were resuspended in l of kinase buffer containing mM dithiothreitol. ATM kinase assays were performed by a similar procedure.GM cells were harvested by trypsinization, washed with PBS, and subjected to two freeze thaw cycles.The publication costs of this article were defrayed in part by payment of page charges.Science. Microbiology. Science. Oncogene. Oncogene. Science. Cold Spring Harbor Laboratory Press In eukaryotic cells, DNA damage and stalled replication activate an evolutionarily conserved checkpoint. Three genes that belong to the last group, MEC, RAD SPK, and CHK, encode buy Abiraterone Acetate protein kinases, which become activated in response to DNA damage or stalled replication.
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