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Note for: Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

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Note for: Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40 (doi:  10.1098/rstb.2000.0755) DT40; - transformed B-lymphocyte cell line - infected with leucosis virus - high targeted/random ratio - result from the property of cell itself which has diversification properties to produce various Ig through DNA recombination - Making this cell line easy to generate the gene-targeted mutant Strong similarities phenotype between DT40 and murine of gene involving in DNA "recombination" From theirs phenotype similarities --> suitable for studying DNA "recombination" Gene-targeting normally done in; 1. murine embryonic stem cell 2. human cell lines efficiency is normally low-->10^-2-10^-5 Reason behind why DT40 having high targeted/random integration is unknown. There are three from chicken B-lymphocyte cell lines which have high targeted integration; two avian (ALV)-transformed cell l

Note for: Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair

Note for: Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair (doi: 10.1074/jbc.M106295200) DNA-PK domains; 1. Ku70/80 binding domain Ku70/Ku80 2. Catalytic subunit Previous study showed Ku70(-/-) --> Rad54-dependent radio-resistant on S-G2 phase cells. Ku70(-/-)Rad54(-/-) showed radioresistance. DSB repairs in vertebrates have two "complementary" DSB repair; NHEJ and HR. HR requires Rad52 epistasis group -- Rad51 and Rad54 NHEJ requires DNA-PK, XRCC4, and Lig4. Ku70/80 binds to DSB --> recruit DNA-PK-->kinase activity occurs Mice lacking DNA-PK-->increase radiosensitivity and defect in V(D)J recombination. Though Ku involved with NHEJ but defect of Ku in mice showed more effect that DNA-PK(-/-)-->conferring the other important functions Ku comparing with DNA-PKs (less effect when KO). Observation has been raised from the complementary to

Note for: The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair

Note for: The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair (doi: 10.1128/mcb.20.17.6476-6482.2000) Rad51 family in vertebrate; 7 members 1. Rad51 2. Dmc1 3. 5 Rad51 paralog DNA double-stranded break repair pws are conserved from yeast to human. HRR is mainly used in yeast but in vertebrate use both. Rad51 = RecA in E. coli Function - 1. binding to ssDNA (from db strand break product) 2.searching for the homologous sequence 3.DNA pairing 4.initiate strand exchange In yeast; KO-Rad51 - cell is still viable but in vertebrate --> rapid chromosomal aberration --> leading to cell death Complete genome sequence of yeast reveals --> Rad51,Rad55,Rad57, and Dmc1 In mammal; 7 members Rad51, Dmc1, XRCC2, XRCC3,Rad51B (Rad51L1/hRec2),Rad51C,and Rad51D (Rad51L3) Paralogs -- 50%-20% compare to Rad51 (original) The appearance of paralogs is thought to play roles as accessory factors. But having them sound to me they a

Note for: Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation

00609_Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation (doi: 10.1038/sj.onc.1203172) Points - belong to phosphatidylinositol-3 kinase family - Using DT40 to study the function of ATM DT40 does not express p53 - ATM(-/-) - retard in growth, defect in G2/M checkpoint, radio-resistant DNA synthesis - sensitive to ionizing radiation - higher in spontaneous chromosomal aberrations - higher in radiation-induced chromosomal aberrations - slight reduction in targeted integration frequency - all above properties indicated p53-independent ATM functions p53 is the key player for 1. cell cycle checkpoint control 2. apoptosis 3. DNA repair (work through the downstream of ATM phosphorylation -- p53 is the substrate of ATM) DT40 lacks p53 expression, but ATM(-/-)DT40 - defect in 1. cell cycle checkpoint control 2. DNA repair (this means there is another p53 independent mechanisms co

Note for: Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Note for: Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells (doi:  10.1093/emboj/17.18.5497) Main points of this paper; 1. investigate two proteins; Ku70 and Rad54 2. each responsible for DSB and complement to each other. 3. both repair pw. play role in genetic maintenance DSB -- in lower organisms has been majorly fixed by HR. But in the higher eukaryote, ike in veterbrate - favour NHEJ to fix the DSB (my guess would be the cell is not divided anymore) 3 components important for NHEJ; 1.DNA-PK 2.XRCC4 3.DNA ligase IV Advantage of DT40; 1. high homologous recombination - easy to generate knockout 2.stable phenotype -easy to compare phenotype of the effect genes Reason of this study; no one studies these two genes (KU70 and Rad54) on DSB repair in higher eukaryote except in yeast. In yeast, HR is much preferr

Note for Increased Ratio of Targeted to Random Integration after Transfection of Chicken B Cell Lines

Note for: Increased Ratio of Targeted to Random Integration after Transfection of Chicken B Cell Lines (doi: 10.1016/0092-8674(91)90581-I ) Viral infection cell line; 1.avian leukosis virus-induced chicken B cell line 2. 4 cell line were used to check the targeted integration but only B-cell had the highest rate of targeted integration compare to non-B cell Factor contributes to the ratio of targeted integration/random (mammalian); 1.length of homology - the longer is better 2.transcription rate of target loci normal rate - 1/100-1/10,000 (in mammalian genome) There were the attempts to increase the rate of targeted in mammalian - modified the input DNA or synchronize the cells Main point of this paper; 1.Higher ratio of targeted integration in DT40 vs mammalian 2.Compare with other types of chicken cell lines, B-cell lines had a higher frequency of targeted integration. 3.Higher ratio of targeted integration in DT40 does not rely on the transcripti