Strains

From Robert J. Spreitzer, University of Nebraska, March 2014

A collection of over 350 acetate-requiring mutants (Spreitzer et al. 1988) was screened for those that could be rescued with 5% CO2 in air. This is one of seven genetically-independent mutants that was recovered. It grows on minimal medium at 25 °C, but dies on minimal medium at 35 °C. It can be rescued on minimal medium at 35 °C with 5% CO2 in air. It may be defective in the CO2-concentrating mechanism (CCM), but has not been analyzed. This strain has been maintained with acetate medium in darkness since its isolation.

Spreitzer RJ, Al-Abed SR, Huether MJ (1988) Temperature-sensitive, photosynthesis-deficient mutants of Chlamydomonas reinhardtii. Plant Physiol 86:773-777

From Laura Hilton, Quarmby lab, Simon Fraser University, April 2014

A spectinomycin resistant cassette, CrAad, was inserted within the first intron of the 5′ UTR (JGI v5.5 gene ID Cre12.g560350). The mutant has slightly longer flagella than wild-type due to a reduced rate of flagellar disassembly at steady-state length. It has been crossed to 137c four times to reduce background mutations.

Hilton LK, Gunawardane K, Kim JW, Schwarz MC, Quarmby LM (2013) The kinases LF4 and CNK2 control ciliary length by feedback regulation of assembly and disassembly rates. Curr Biol 18; 23:2208-14

Meslet-Cladière L, Vallon O (2012) A new method to identify flanking sequence tags in chlamydomonas using 3'-RACE. Plant Methods 26:21

• Locus:
• CNK2
• Chromosome:
• 12

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This is an isolate of CC-4415 that has been cloned for better growth and transformation frequency. The T60-3 mutant was recovered by transformation of cwd arg7-8 mt- (CC-3395) with pARG7.8 (Gumpel et al. 1994), which caused the deletion of the Rubisco rbcS gene family (rbcS1 and rbcS2) (Khrebtukova and Spreitzer 1996). T60-3 lacks a cell wall and flagella, and cannot be crossed. This strain has been maintained with acetate medium in darkness since its isolation.

12/21/23 – This strain was found to be wild-type for the small subunit so is not a clonal isolate of T60-3.

Gumpel NJ, Rochaix JD, Purton S (1994) Studies on homologous recombination in the green alga Chlamydomonas reinhardtii. Curr Genet 26:438-442

Khrebtukova I, Spreitzer RJ (1996) Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 93:13689-13693

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This mutant (originally named dim1) was received from Rachel Dent and Kris Niyogi in 2003. It was recovered by transformation of wild-type 4A mt+ (CC-4051) with a ble gene, which caused deletion of the Rubisco rbcS gene family (rbcS1 and rbcS2) (Dent et al. 2005). It has a cell wall and flagella, and has been used in experiments that required transformation and genetic crosses (Spreitzer et al. 2005; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness since 2003.

Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137:545-556

Genkov T, Du YC, Spreitzer RJ (2006) Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 451:167-174

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

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This pf2 mt+ progeny clone was recovered from a cross between rbcS∆-CAL005.01.13 mt+ (Dent et al. 2005) and pf2 mt-. The mutant lacks the Rubisco rbcS1 and rbcS2 small-subunit genes, and has been used in experiments that required transformation and genetic crosses (Spreitzer et al. 2005; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness since its isolation.

Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137:545-556

Genkov T, Du YC, Spreitzer RJ (2006) Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 451:167-174

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

• Locus:
• PF2
• Chromosome:
• 11

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This pf2 mt- progeny clone was recovered from a cross between rbcS∆-CAL005.01.13 mt+ (Dent et al. 2005) and pf2 mt-. The mutant lacks the Rubisco rbcS1 and rbcS2 small-subunit genes, and has been used in experiments that required transformation and genetic crosses (Spreitzer et al. 2005; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness since its isolation.

Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137:545-556

Genkov T, Du YC, Spreitzer RJ (2006) Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 451:167-174

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

• Locus:
• PF2
• Chromosome:
• 11

From Robert J. Spreitzer, University of Nebraska, June 2014

This strain was recovered by nuclear transformation of rbcS∆-T60-3 mt- with the rbcS1 gene, which encodes small-subunit 1 of Rubisco (Khrebtukova and Spreitzer 1996). Because it lacks the rbcS2 gene, and expresses half the normal amount of Rubisco, it can serve as a “wild-type” strain for comparison with engineered rbcS1 genes (Genkov and Spreitzer 2009). This strain has been maintained with acetate medium in darkness since its isolation to guard against selection for secondary mutations that may increase Rubisco expression.

Genkov T, Spreitzer RJ (2009) Highly conserved small subunit residues influence Rubisco large subunit catalysis. J Biol Chem 284:30105-30112

Khrebtukova I, Spreitzer RJ (1996) Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 93:13689-13693

• Locus:
• RBCS1
• Chromosome:
• 2

From Robert J. Spreitzer, University of Nebraska, June 2014

This strain was recovered by nuclear transformation of rbcS∆-T60-3 mt- with the rbcS2 gene, which encodes small-subunit 2 of Rubisco (Khrebtukova and Spreitzer 1996). Because it lacks the rbcS1 gene, and expresses half the normal amount of Rubisco, it can serve as a “wild-type” strain for comparison with engineered rbcS2 genes (Genkov et al. 2006). This strain has been maintained with acetate medium in darkness since its isolation to guard against selection for secondary mutations that may increase Rubisco expression.

Genkov T, Du YC, Spreitzer RJ (2006) Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 451:167-174

Khrebtukova I, Spreitzer RJ (1996) Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 93:13689-13693

• Locus:
• RBCS2
• Chromosome:

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This Rubisco rbcL knock-out mutant was created by Genhai Zhu by replacing chloroplast rbcL of wild-type 2137 mt+ (CC-3269) with aadA (Satagopan and Spreitzer 2004; Zhu et al. 2010). It has been cloned and maintained with acetate medium in darkness since its isolation.

Satagopan S, Spreitzer RJ (2004) Substitutions at the Asp-473 latch residue of Chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO2/O2 specificity. J Biol Chem 279:14240-14244

Zhu G, Kurek I, Liu L (2010) Engineering photosynthetic enzymes involved in CO2–assimilation by gene shuffling. In CA Rebeiz, C Benning, H Bohnert, H Daniell, JK Hoober, HK Lichtenthaler, AR Portis, BC Tripathy, eds, The Chloroplast: Basics and Applications, Springer, pp 307-322

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light, zeocin and spectinomycin resistant

This mutant was isolated as progeny clone 1A from a cross between rbcLΔ-MX3312 mt+ and rbcSΔ-CAL005.01.13 mt- (dim1). It lacks photosynthesis due to the loss of all Rubisco genes. The strain has been maintained with acetate medium in darkness since its isolation in 2011.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light, zeocin and spectinomycin resistant

This mutant was isolated as progeny clone 7C from a cross between rbcLΔ-MX3312 mt+ and rbcSΔ-CAL005.01.13 mt- (dim1). It lacks photosynthesis due to the loss of all Rubisco genes. The strain has been maintained with acetate medium in darkness since its isolation in 2011.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light, zeocin and spectinomycin resistant

This mutant was isolated as progeny clone 23C from a cross between rbcLΔ-MX3312 mt+ and rbcSΔ-CAL005.01.13 mt- (dim1). It lacks photosynthesis due to the loss of all Rubisco genes. The strain has been maintained with acetate medium in darkness since its isolation in 2011.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This 25B1 mt+ progeny clone, which is green in the dark, was recovered from a cross between 25B1 mt+ (CC-2803) and pf2 mt-. The 25B1 Rubisco rbcL knock-out mutant was created by chloroplast transformation and insertional mutagenesis (Newman et al. 1991). This strain has been used as a host for engineering the Rubisco large subunit (Zhu and Spreitzer 1994, 1996; Larson et al. 1997). It has been maintained with acetate medium in darkness since its isolation.

Larson EM, O'Brien CM, Zhu G, Spreitzer RJ, Portis AR Jr (1997) Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033-17037

Newman SM, Gillham NW, Harris EH, Johnson AM, Boynton JE (1991) Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Molec Gen Genet 230:65-74

Zhu G, Spreitzer RJ (1994) Directed mutagenesis of chloroplast ribulose-bisphosphate carboxylase/oxygenase: Substitutions at large subunit asparagine 123 and serine 379 decrease CO2/O2 specificity. J Biol Chem 269:3952-3956

Zhu G, Spreitzer RJ (1996) Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Loop-6 substitutions complement for structural stability but decrease catalytic efficiency. J Biol Chem 271:18494-18498

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, June 2014

Phenotype: requires acetate, sensitive to light

This 25B1 pf2 mt- progeny clone, which is green in the dark, was recovered from a cross between 25B1 mt+ (CC-2803) and pf2 mt- (Zhu and Spreitzer 1994). The 25B1 Rubisco rbcL knock-out mutant was created by chloroplast transformation and insertional mutagenesis (Newman et al. 1991). This strain has been maintained with acetate medium in darkness since its isolation.

Newman SM, Gillham NW, Harris EH, Johnson AM, Boynton JE (1991) Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Molec Gen Genet 230:65-74

Zhu G, Spreitzer RJ (1994) Directed mutagenesis of chloroplast ribulose-bisphosphate carboxylase/oxygenase: Substitutions at large subunit asparagine 123 and serine 379 decrease CO2/O2 specificity. J Biol Chem 269:3952-3956

• Locus:
• rbcL, PF2
• Chromosome:
• chloroplast,11

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create a P89R substitution (CCA to CGT) in the Rubisco large subunit, which alters the species specificity of the interaction between Rubisco and Rubisco activase (Larson et al. 1997). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve interactions between activase and Rubisco.

Larson EM, O'Brien CM, Zhu G, Spreitzer RJ, Portis AR (1997) Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033-17037

Spreitzer RJ, Goldschmidt-Clermont M, Rahire M, Rochaix JD (1985) Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82:5460-5464

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

This is an mt+ progeny clone obtained from a cross between rbcL-P89R mt+ (Larson et al. 1997) and pf2 mt-. The P89R substitution in the Rubisco large subunit alters the species specificity of the interaction between Rubisco and Rubisco activase (Larson et al. 1997). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve interactions between activase and Rubisco.

Larson EM, O'Brien CM, Zhu G, Spreitzer RJ, Portis AR (1997) Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033-17037

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

This is a pf2 mt- progeny clone obtained from a cross between rbcL-P89R mt+ (Larson et al. 1997) and pf2 mt-. The P89R substitution in the Rubisco large subunit alters the species specificity of the interaction between Rubisco and Rubisco activase (Larson et al. 1997). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve interactions between activase and Rubisco.

Larson EM, O'Brien CM, Zhu G, Spreitzer RJ, Portis AR (1997) Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033-17037

• Locus:
• rbcL, PF2
• Chromosome:
• chloroplast,11

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create a P89A substitution (CCA to GCT) in the Rubisco large subunit, which alters the species specificity of the interaction between Rubisco and Rubisco activase (Ott et al. 2000). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve interactions between activase and Rubisco.

Ott CM, Smith BD, Portis AR, Spreitzer RJ (2000) Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Nonconservative substitution in the large subunit alters species specificity of protein interaction. J Biol Chem 275:26241-26244

Spreitzer RJ, Goldschmidt-Clermont M, Rahire M, Rochaix JD (1985) Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82:5460-5464

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create a D94K substitution (GAC-AAA) in the Rubisco large subunit, which alters the species specificity of the interaction between Rubisco and Rubisco activase (Ott et al. 2000). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve interactions between activase and Rubisco.

Ott CM, Smith BD, Portis AR, Spreitzer RJ (2000) Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Nonconservative substitution in the large subunit alters species specificity of protein interaction. J Biol Chem 275:26241-26244

Spreitzer RJ, Goldschmidt-Clermont M, Rahire M, Rochaix JD (1985) Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82:5460-5464

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create a K356Q substitution (AAA-CAA) in the Rubisco large subunit to investigate the interaction between Rubisco and Rubisco activase (Larson et al. 1997). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter the interactions between activase and Rubisco.

Larson EM, O'Brien CM, Zhu G, Spreitzer RJ, Portis AR (1997) Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033-17037

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create a D86R substitution (GAT-CGT) in the Rubisco large subunit to investigate the interaction between Rubisco and Rubisco activase (Ott et al. 2000). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter the interactions between activase and Rubisco.

Ott CM, Smith BD, Portis AR, Spreitzer RJ (2000) Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Nonconservative substitution in the large subunit alters species specificity of protein interaction. J Biol Chem 275:26241-26244

Spreitzer RJ, Goldschmidt-Clermont M, Rahire M, Rochaix JD (1985) Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82:5460-5464

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create a D94C substitution in the Rubisco large subunit to investigate the interaction between Rubisco and Rubisco activase (Portis and Spreitzer, unpublished). This is the original mutant strain created in 2002. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter the interactions between activase and Rubisco.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create an N95D substitution in the Rubisco large subunit to investigate the interaction between Rubisco and Rubisco activase (Portis and Spreitzer, unpublished). This is the original mutant strain created in 2002. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter the interactions between activase and Rubisco.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create a K466V substitution in the Rubisco large subunit to investigate the interaction between Rubisco and Rubisco activase (Portis and Spreitzer, unpublished). This is the original mutant strain created in 2002. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter the interactions between activase and Rubisco.

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create a C172S substitution (TGT-TCT) in the Rubisco large subunit to investigate stress-induced degradation of Rubisco (Moreno and Spreitzer 1999). The x-ray crystal structure of the mutant protein has been determined (Garcia-Murria et al. 2008). This is the original mutant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco stability.

Garcia-Murria MJ, Karkehabadi S, Marin-Navarro J, Satagopan S, Andersson I, Spreitzer RJ, Moreno J (2008) Structural and functional consequences of the replacement of proximal residues Cys-172 and Cys-192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii. Biochem J 411:241-247

Moreno J, Spreitzer RJ (1999) Cys-172 to Ser substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulosebisphosphate carboxylase/oxygenase. J Biol Chem 274:26789-26793

Spreitzer RJ, Goldschmidt-Clermont M, Rahire M, Rochaix JD (1985) Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82:5460-5464

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Phenotype: requires acetate

Directed mutagenesis and chloroplast transformation of rbcL-W66Amber mt+ (Spreitzer et al. 1985) were used to create C449S (TGT-AGC) and C459S (TGT-TCT) substitutions in the Rubisco large subunit to investigate stress-induced degradation of Rubisco (Marin-Navarro and Moreno 2006). This strain was received from Joaquin Moreno in 2011. Although this mutant was originally recovered on minimal medium and could grow photosynthetically (Marin-Navarro and Moreno 2006), a check of its phenotype in Spreitzer’s laboratory revealed that it now requires acetate for growth. Some photosynthesis-competent Rubisco mutants have been previously observed to become acetate-requiring after many years of culture in darkness.

Marin-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction–oxidation conformational changes of ribulose 1·5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898-908

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Girish Rasineni in Spreitzer’s group recovered this mutant by transforming rbcL∆-MX3312 mt+ (CC-4696) with plasmid pLS-C449S/C459S (Marin-Navarro and Moreno 2006) obtained from Joaquin Moreno in 2011. This Rubisco large-subunit mutant can grow photosynthetically on minimal medium like the original rbcL-C449S/C459S mutant previously described by Marin-Navarro and Moreno (2006).

Marin-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction–oxidation conformational changes of ribulose 1·5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898-908

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Girish Rasineni in Spreitzer’s group performed directed mutagenesis of plasmid pLS-C449S/C459S (Marin-Navarro and Moreno 2006), obtained from Joaquin Moreno in 2011, to produce an additional D470C substitution (GAT-TGT). The triple-mutant plasmid was then transformed into rbcL∆-MX3312 mt+ (CC-4696) by standard procedures (Zhu and Spreitzer 1996), and photosynthesis-competent colonies were selected on minimal medium. After many rounds of cloning for homoplasmicity, this strain was retained for future study. It is maintained with acetate medium in darkness to prevent selection of other mutations.

Marin-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction–oxidation conformational changes of ribulose 1·5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898-908

Zhu G, Spreitzer RJ (1996) Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Loop-6 substitutions complement for structural stability but decrease catalytic efficiency. J Biol Chem 271:18494-18498

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Girish Rasineni in Spreitzer’s group performed directed mutagenesis of plasmid pLS-C449S/C459S (Marin-Navarro and Moreno 2006), obtained from Joaquin Moreno in 2011, to produce an additional K474C substitution (AAA-TGT). The triple-mutant plasmid was then transformed into rbcL∆-MX3312 mt+ (CC-4696) by standard procedures (Zhu and Spreitzer 1996), and photosynthesis-competent colonies were selected on minimal medium. After many rounds of cloning for homoplasmicity, this strain was retained for future study. It is maintained with acetate medium in darkness to prevent selection of other mutations.

Marin-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction–oxidation conformational changes of ribulose 1·5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898-908

Zhu G, Spreitzer RJ (1996) Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Loop-6 substitutions complement for structural stability but decrease catalytic efficiency. J Biol Chem 271:18494-18498

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Robert J. Spreitzer, University of Nebraska, July 2014

Using plasmid pSS1, which contains rbcS1 that encodes Rubisco small-subunit 1, and rbcS∆-T60-3 mt- (CC-4415) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to create a C41S substitution in the Rubisco small subunit (Moreno and Spreitzer, unpublished). The mutant may be useful for studying redox modulation of Rubisco (reviewed by Moreno et al. 2008). This strain has been maintained with acetate medium in darkness since being isolated in 2002.

Khrebtukova I, Spreitzer RJ (1996) Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 93:13689-13693

Moreno J, García-Murria MJ, Marín-Navarro J (2008) Redox modulation of Rubisco conformation and activity through its cysteine residues. J Exp Bot 59:1605-1614

• Locus:
• RBCS1
• Chromosome:
• 2