insertion of amino acids had not been attempted. Here, we demonstrate that a four amino

acid insertion at the b subunit carboxyl terminus does not affect the activity of the

enzyme.

 Materials and Methods

 Many of the procedures utilized in Chapter 7 can be found in detail in the previous

chapters. Many of the techniques, including recombinant DNA techniques, site directed

mutagenesis, western blotting, as well as assays of protein concentration and F1Fo ATP

synthase activity have been described in detail in Chapter 2.

Materials

 Molecular biology enzymes and mutagenic oligonucleotides were obtained from

Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England

Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from

Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific

(Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia,

C A).

Strains and Media

 The wild type b subunit expression plasmid, pKAM14, has been described

previously (193, 194, 203). The plasmids encoding the uncF(b) gene were used to

compliment E. coli strain KM2 (Ab) carrying a chromosomal deletion of the gene (218).

All strains were streaked onto plates containing Minimal A media supplemented with

succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane

preparation were grown in Luria Broth supplemented with glucose (0.2% w/v) (LBG).

Isopropyl-1 -thio-P-D-galactopyranoside (IPTG)(40 Clg/ml) and ampicillin (Ap)