CC-5037 rbcL-Phylo34 mt+
$30.00
From Robert J. Spreitzer, University of Nebraska, November 2014
Phenotype: requires acetate, sensitive to light
Using plasmid p699 (GuhaMajumdar et al. 2008), Boon Hoe Lim in Spreitzer’s group performed rbcL direct mutagenesis, chloroplast transformation of wild-type 2137 mt+ (CC-4887), selection for spectinomycin resistance in the dark, and screening for a homoplasmic acetate-requiring phenotype to create 34 substitutions together in the Rubisco large subunit (A11V, V30E, V31T, R32K, M42V, C53A, D86H, I105L, V149Q, G168P, V221C, V235I, C256F, K258R, I265V, I282H, R305K, L326I, V341I, M349L, C369V, M375L, V391T, A398S, C399V, T428V, G442N, D443E, V444I, S447E, D470E, T471A, I472M, and K474T). The mutant lacks Rubisco holoenzyme (Lim and Spreitzer, unpublished). It was created to investigate phylogenetic differences that influence Rubisco catalysis (Du et al. 2003; Spreitzer et al. 2005). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or suppress light sensitivity (Spreitzer and Ogren 1983).
Du YC, Peddi SR, Spreitzer RJ (2003) Assessment of structural and functional divergence far from the large subunit active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 278:49401-49405
GuhaMajumdar M, Dawson-Baglien E, Sears BB (2008) Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii. Eukaryot Cell 7:639-646
Spreitzer RJ, Ogren WL (1983) Nuclear suppressors of the photosensitivity associated with defective photosynthesis in Chlamydomonas reinhardtii. Plant Physiol 71:35-39
Spreitzer RJ, Peddi SR, Satagopan S (2005) Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proc Natl Acad Sci USA 102:17225-17230