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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 (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 (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

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 (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 (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