Mutants base-recognition

Fig. 6a,b Electron density representing the inhibitor of 6-azaUMP in the active sites of base-recognition mutants, a shows the dual conformations adopted by the pyrimidine ring in S127A ODCase. b In the Q185A mutant, a chain of water molecules replaces the glutamine side chain... 

The spectra of both wild-type lac operator 14-mer d(TGTGAGCGCTCA-CA)2 (01) as well as a number of base-pair mutants, d(Ty4TGAGCGCTCATA)2 (02) and d(TGTG7GCGCACACA)2 (03 complementary sites of mutation in the palindromic operators are italicized) have been assigned (22). These symmetrical base sequences are about two-thirds the length of the 21 base-pair wild-type sequence, and the 14-mers are believed to contain most of the important recognition sites for the lac repressor protein. 

Restriction enzyme-mediated integration (REMI) is a commonly used method for random mutagenesis that has been adapted for the use in D. discoideum by Kuspa and Loomis [40]. A plasmid containing an appropriate selection marker is Hn-earized with a restriction enzyme, and the linear plasmid is transformed by electroporation along with the restriction enzyme (Fig. 5.2). It is assumed that the restriction enzyme will cut certain chromosomal loci at specific recognition sites. If a transformed plasmid would Hgate with its sticky ends to the double-strand break introduced by the restriction enzyme, mutants can be isolated based on the selection marker present on the integrated REMI plasmid. 

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