Oligonucleotide-mediated mutagenesi

Oligonucleotide-mediated mutagenesis was used to change the histidine codon at L173 (CAC) to a leucine codon (CTC). Photosynthetic growth assays indicate that His - Leu does not grow photosynthetically. The absorption spectrum of chromatophores from this mutant suggests that reduced levels of RCs are stably assembled in the membrane, (data not shown). RCs were purified from chromatophore membranes of His ... 

In RCs of Rb,capsulatus wild-type, mutation at amino acid residue TRP M250 was introduced by oligonucleotide-mediated mutagenesis, according to methods described in [4,5]. Quinone-titrations for determining the apparent dissociation constants in the mutant was performed as in [4]. Measurements of kg and the P Qi recombination rate k were done on RCs reconstituted with 2-methyl-1,4-naphthoquinone (NQ) in contrast to recombination studies on P H where the RCs were quinone-free. All experiments were performed on RCs in aqueous buffer/glycerol(60%) solution. Picosecond [10], nanosecond [6,7,11] and millisecond [12] transient absorption spectrometers were used for the measurements of kg, radical pair recombination parameters and k. . 

Mutations were introduced at amino acid residue Trp SO by oligonucleotide-mediated mutagenesis, according to methods previously described... 

Fig. 1. Oligonucleotide-mediated, site-directed mutagenesis using the Kunkel method. See the text (sections on site-directed mutagenesis and mutagenesis with three primers) for further details, ss, Single-stranded.

Mutations were generated in the L subunit structural gene by oligonucleotide-mediated site-specific mutagenesis as previously described for the M-side heterodimer (6). These mutations were introduced into the puf operon of pU2922 and... 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

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