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XR-V15B/H22 (D2) is a complementing hybrid that contains two closely located but independent regions of chromosome 2, mapping to 2q36 (including the marker GNT1) and to 2q33-35 (including the marker TNP-1 (13). Subclones derived from D2 segregated the markers GNT1 and TNP-1 independently, and in every subclone examined (more than 20), radioresistance and ability to carry out V(D)J recombination cosegregated with the TNP-1 marker. D2-X-3 is a complementing subclone retaining both fragments. D2-X-5 is noncomplementing and contains only the GNT1 fragment. D2-X-38 is complementing and contains only the TNP-1 fragment. D2-X-38D is a noncomplementing derivative of D2-X-38 and does not contain detectable human sequences. Microcell transfer hybrids H22 and H27 were isolated and characterized previously (13). H22, a complementing hybrid, contains the distal third of chromosome 2, including GNT1 and TNP-1; H27, a noncomplementing hybrid, contains three small fragments, including GNT1 but excluding TNP-1. Other microsatellite markers present in D2, D2-X-3, and D2-X-38 are D2S301, D2S164, and D2S137. Markers analyzed and absent in all D2 hybrids are D2S143, D2S128, D2S334, D2S371, D2S317, D2S155, D2S154, D2S157, D2S153, D2S173, D2S163, D2S120, and those listed in (73). Primer sequences for the Ku80 3′ UTR are 5′-ACATCACAAGGGCTGCAACTGTCA-3′ and 5′-TCGCTGTGATGCTGGGAGTTCTAA-3′, and PCR conditions were as described for D2S137 (13).
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R). The accuracy of join formation and the junction modifications were analyzed by specific restriction endonuclease digestion or by direct DNA sequencing as described (10) clones were analyzed. A minimum of 20 clones were analyzed, and 100 for xrs-6 samples containing Ku80. The results of complementation of xrs-6 with Ku80 were reproducible in three independent experiments.
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note
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A 2200-Base pair sequence containing the Ku80 ORF was cloned into the expression vectors pcDNA1/Amp and p220LTR, where it was under the control of the cytomegalovirus and Rous sarcoma virus long terminal repeat (LTR) promoters, respectively. The p220LTR vector also contained a hygromycin gene (hyg), which is a dominant selectable marker in mammalian cells.
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We thank A. Kuhn for purified mouse Ku; M. Knuth, N. Thompson, R. Burgess, and J. Flint for mAbs; and W. Reeves for Ku80 cDNA. The substrates of pJH200 and pJH290 were provided by J. Hesse and M. Gellert, and the XR-1 cell line was a gift from T. Stamato. We thank G. Silverman, J. Wagstaff, A. Beggs, R. Swirski, and T. Lindahl for discussions. Research in S.P.'s laboratory is funded principally by grant SP2143/0101 from the Cancer Research Campaign (CRC). T.M.G. is supported by a CRC studentship. Research in the MRC Cell Mutation Unit was supported in part by Commission of European Communities grants F13PCT920007 and ERBSC1CT920823. Research in F.W.A.'s laboratory is supported by NIH grant A.I. 20047, the Howard Hughes Medical Institute, and a postdoctoral fellowship from Irvington Institute (G.E.T.). G.E.T. is a special fellow of the Leukemia Society of America.
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