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15844386464
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in preparation; E. W. May and N. L. Craig, In preparation
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C. Nolan Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2
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D114 of TnsA corresponds to a conserved position in this protein's putative DD-35-E motif, a domain common to many transposases and implicated as the catalytic site (13). The mutation was introduced into the TnsA gene by a polymerase chain reaction (PCR)-based procedure. Oligonucleotides were synthesized such that a PCR reaction with pEM279 [a derivative of pGST-A (8)] as a template would generate a small fragment containing the point mutation flanked by two cloning sites. The product of the PCR was isolated and treated with appropriate restriction enzymes The mutagenic insert was gelpurified and then ligated into pEM279 Unless otherwise noted, all molecular and genetic protocols were from J Sambrook, E F Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, C. Nolan Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2, 1989).
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21
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2+-coordinated column (12, 13) (P. L. Sharpe and N. L. Craig, unpublished data). TnsC was prepared as described by P Gamas and N. L. Craig [Nucleic Acids Res 20, 2525 (1992)].
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15844374561
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note
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2, 14 mM NaCI, 21 mM KCl, 120 μM CHAPS, 4 μM PMSF, 1.2% glycerol, 24 nM TnsA, 3 nM TnsB, 8 nM TnsC, and 6 nM TnsD. Reactions were stopped with 25 mM HDTA.
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25
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15844421662
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note
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r). These in vivo experiments were conducted with the Tns(ABCE) pathway of transposition; consistent results were obtained with the Tns(ABCD) pathway
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15844399819
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note
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We thank P. Eckhoff for help in the preparation of this manuscript; J Boeke and members of the Craig laboratory for critical reading of the manuscript; P. Gary, P. Sharpe, B Sarnovsky, and A. Stellwagen for useful reagents; and M. Biery for developing several critical methods.
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