Strains

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nuclear-gene transformation were used to create a C83S substitution in the Rubisco small subunit (Moreno and Spreitzer, unpublished).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

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 C84S substitution in the Rubisco large subunit by standard procedures (Du and Spreitzer 2000), and photosynthesis-competent colonies were selected on minimal medium. After many rounds of cloning for homoplasmicity, this strain was retained for future study (Moreno and Spreitzer, unpublished). The mutant may be useful for studying redox modulation of Rubisco (reviewed by Moreno et al. 2008). It has been maintained with acetate medium in darkness since being isolated in 2002.

Du Y, Spreitzer RJ (2000) Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/ oxygenase. J Biol Chem 275:19844-19847

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

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:
  • 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 C284S substitution in the Rubisco large subunit by standard procedures (Du and Spreitzer 2000), and photosynthesis-competent colonies were selected on minimal medium. After many rounds of cloning for homoplasmicity, this strain was retained for future study (Moreno and Spreitzer, unpublished). The mutant may be useful for studying redox modulation of Rubisco (reviewed by Moreno et al. 2008). It has been maintained with acetate medium in darkness since being isolated in 2002.

Du Y, Spreitzer RJ (2000) Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/ oxygenase. J Biol Chem 275:19844-19847

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:
  • 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 C427S substitution in the Rubisco large subunit by standard procedures (Du and Spreitzer 2000), and photosynthesis-competent colonies were selected on minimal medium. After many rounds of cloning for homoplasmicity, this strain was retained for future study (Moreno and Spreitzer, unpublished). The mutant may be useful for studying redox modulation of Rubisco (reviewed by Moreno et al. 2008). It has been maintained with acetate medium in darkness since being isolated in 2002.

Du Y, Spreitzer RJ (2000) Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/ oxygenase. J Biol Chem 275:19844-19847

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:
  • 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-4690) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to create an I58A substitution (ATC-GCC) in the Rubisco small subunit (Esquivel et al. 2013). This strain has been maintained with acetate medium in darkness since its isolation.

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

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, July 2014

Using plasmid pSS1, which contains rbcS1 that encodes Rubisco small-subunit 1, and rbcS∆-T60-3 mt- (CC-4690) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to create an I58E substitution (ATC-GAG) in the Rubisco small subunit (Esquivel et al. 2013). This strain has been maintained with acetate medium in darkness since its isolation.

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

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, July 2014

Using plasmid pSS1, which contains rbcS1 that encodes Rubisco small-subunit 1, and rbcS∆-T60-3 mt- (CC-4690) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to create an I58K substitution (ATC-AAG) in the Rubisco small subunit (Esquivel et al. 2013). This strain has been maintained with acetate medium in darkness since its isolation.

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

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, July 2014

Using plasmid pSS1, which contains rbcS1 that encodes Rubisco small-subunit 1, and rbcS∆-T60-3 mt- (CC-4690) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to create an I58W substitution (ATC-TGG) in the Rubisco small subunit (Esquivel et al. 2013). This strain has been maintained with acetate medium in darkness since its isolation.

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

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, July 2014

Phenotype: requires acetate at 35 °C, temperature-conditional

Using plasmid pSS1, which contains rbcS1 that encodes Rubisco small-subunit 1, and rbcS∆-T60-3 mt- (CC-4690) as the host strain (Khrebtukova and Spreitzer 1996), directed mutagenesis and nuclear-gene transformation were used to replace Ile-58 with three Trp residues (ATC-TGGTGGTGG) in the Rubisco small subunit (Esquivel et al. 2013). This strain has been maintained with acetate medium in darkness since its isolation.

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

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, July 2014

Phenotype: requires acetate at 35 °C, temperature-conditional

Using plasmid pI58W3 (Esquivel et al. 2013), which would replace Ile-58 with three Trp residues (ATC-TGGTGGTGG) in the Rubisco small subunit, Todor Genkov in Spreitzer’s group transformed rbcS∆-CAL005.01.13 pf2 mt+ (CC-4692) (Dent et al. 2005). In contrast to the I58W3 mutant described previously (Esquivel et al. 2013), this strain has a cell wall and can be used in genetic crosses. It 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

Esquivel MG, Genkov T, Nogueira AS, Salvucci ME, Spreitzer RJ (2013) Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynth Res 118:209-218

  • Locus:
  • RBCS1, PF2
  • Chromosome:
  • 2,11

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create an R59A substitution (CGC-GCC) in the betaA-betaB loop of the Rubisco small subunit (Spreitzer et al. 2001; Spreitzer 2003). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create an R59E substitution (CGC-GAG) in the betaA-betaB loop of the Rubisco small subunit, which causes a decreases in Rubisco holoenzyme stability (Spreitzer et al. 2001; Spreitzer 2003). This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Using a plasmid that would create an R59E substitution (CGC-GAG) in the betaA-betaB loop of the Rubisco small subunit (Spreitzer et al. 2001; Spreitzer 2003), Kelsey Swartz in Spreitzer’s group transformed rbcS∆-CAL005.01.13 pf2 mt+ (CC-4692) (Dent et al. 2005). In contrast to the R59E mutant described previously (Spreitzer et al. 2001), this strain has a cell wall and can be used in genetic crosses. This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1, PF2
  • Chromosome:
  • 2,11

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create a Y67A substitution (TAC-GCC) in the betaA-betaB loop of the Rubisco small subunit, which causes a decrease in Rubisco holoenzyme stability (Spreitzer et al. 2001; Spreitzer 2003; Esquivel et al. 2006). Mutant Y67A has also been studied with respect to enhanced hydrogen production (Pinto et al. 2013). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Esquivel MG, Pinto TS, Marin-Navarro J, Moreno J (2006) Substitution of tyrosine residues at the aromatic cluster around the betaA-betaB loop of Rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. Biochemistry 45:5745-5753

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

Pinto TS, Malcata FX, Arrabaça JD, Silva JM, Spreitzer RJ, Esquivel MG (2013) Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production. Appl Microbiol Biotechnol 97:5635-5643

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create a Y68A substitution (TAC-GCC) in the betaA-betaB loop of the Rubisco small subunit, which causes decreases in Rubisco carboxylation catalytic efficiency and holoenzyme stability (Spreitzer et al. 2001; Spreitzer 2003; Esquivel et al. 2006). Mutant Y68A has also been studied with respect to enhanced hydrogen production (Pinto et al. 2013). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Esquivel MG, Pinto TS, Marin-Navarro J, Moreno J (2006) Substitution of tyrosine residues at the aromatic cluster around the betaA-betaB loop of Rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. Biochemistry 45, 5745-5753

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

Pinto TS, Malcata FX, Arrabaça JD, Silva JM, Spreitzer RJ, Esquivel MG (2013) Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production. Appl Microbiol Biotechnol 97:5635-5643

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create a D69A substitution (GAC-GCC) in the betaA-betaB loop of the Rubisco small subunit, which causes a decrease in Rubisco carboxylation catalytic efficiency (Spreitzer et al. 2001; Spreitzer 2003). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create an R71A substitution (CGC-GCC) in the betaA-betaB loop of the Rubisco small subunit, which causes decreases in Rubisco CO2/O2 specificity and holoenzyme stability (Spreitzer et al. 2001; Spreitzer 2003). This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Using a plasmid that would create an R71A substitution (CGC-GCC) in the betaA-betaB loop of the Rubisco small subunit (Spreitzer et al. 2001; Spreitzer 2003), Todor Genkov in Spreitzer’s group transformed rbcS∆-CAL005.01.13 pf2 mt+ (CC-4692) (Dent et al. 2005). In contrast to the R71A mutant described previously (Spreitzer et al. 2001), this strain has a cell wall and can be used in genetic crosses. This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141-149

Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit betaA-betaB loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615-5621

  • Locus:
  • RBCS1, PF2
  • Chromosome:
  • 2,11

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (Khrebtukova and Spreitzer 1996) were used to create a Y72A substitution in the betaA-betaB loop of the Rubisco small subunit, which causes decreases in Rubisco carboxylase activity and holoenzyme stability (Esquivel et al. 2006). Mutant Y72A has also been studied with respect to enhanced hydrogen production (Pinto et al. 2013). This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Esquivel MG, Pinto TS, Marin-Navarro J, Moreno J (2006) Substitution of tyrosine residues at the aromatic cluster around the betaA-betaB loop of Rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. Biochemistry 45:5745-5753

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

Pinto TS, Malcata FX, Arrabaça JD, Silva JM, Spreitzer RJ, Esquivel MG (2013) Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production. Appl Microbiol Biotechnol 97:5635-5643

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Directed mutagenesis and nuclear-gene transformation of rbcS∆-CAL005.01.13 pf2 mt+ (CC-4692) (Dent et al. 2005; Genkov et al. 2006) were used to create an L66F substitution (CTG-TTC) in the betaA-betaB loop of the Rubisco small subunit (Genkov and Spreitzer, unpublished), which mimics the biochemical defects of chloroplast Rubisco mutant rbcL-L290F (Chen et al. 1988). This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. It has a cell wall and can be used in genetic crosses. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Chen Z, Chastain CJ, Al-Abed SR, Chollet R, Spreitzer RJ (1988) Reduced CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 85:4696-4699

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

  • Locus:
  • RBCS1, PF2
  • Chromosome:
  • 2,11

From Claire Remacle, Universite de Liège, Belgium, July 2014

This mutant has gene deletions that encompass cob and the 3′ end of nd4.

Cardol P, Matagne RF, Remacle C (2002) Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme. J Mol Biol. 319:1211-21

Cardol P, Gloire G, Havaux M, Remacle C, Matagne R, Franck F (2003) Photosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration. Plant Physiol 133:2010-20

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Genetic engineering and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to replace the betaA-betaB loop of the Chlamydomonas Rubisco small subunit with the loop from Synechococcus Rubisco, which causes a decrease in Rubisco CO2/O2 specificity and holoenzyme stability (Karkehabadi et al. 2005a). The mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Karkehabadi S, Peddi SR, Anwaruzzaman M, Taylor TC, Cederlund A, Genkov T, Andersson I, Spreitzer RJ (2005a) Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 44:9851-9861

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Genetic engineering and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to replace the betaA-betaB loop of the Chlamydomonas Rubisco small subunit with the loop from spinach Rubisco, which causes a decrease in Rubisco carboxylation catalytic efficiency (Karkehabadi et al. 2005a; 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 stability.

Karkehabadi S, Peddi SR, Anwaruzzaman M, Taylor TC, Cederlund A, Genkov T, Andersson I, Spreitzer RJ (2005a) Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 44:9851-9861

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:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Using standard methods of genetic engineering and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690) (Genkov et al. 2010), the betaA-betaB loop of the Chlamydomonas Rubisco small subunit was replaced with the loop from Galdieria partita Rubisco, which causes a decrease in Rubisco holoenzyme stability (Genkov and Spreitzer, unpublished). The mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Genkov T, Meyer M, Griffiths H, Spreitzer RJ (2010) Functional hybrid Rubisco enzymes with plant small subunits and algal large subunits: Engineered rbcS cDNA for expression in Chlamydomonas. J Biol Chem 285:19833-19841

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Using standard methods of genetic engineering and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690) (Genkov et al. 2010), the betaA-betaB loop of the Rubisco small subunit was replaced with the small-subunit betaA-betaB loop from Galdieria partita, and residues 128 through 140 were replaced with the small-subunit betaE-betaF loop (residues 105 through 138) from Galdieria. These changes cause a decrease in Rubisco CO2/O2 specificity and holoenzyme stability (Genkov and Spreitzer, unpublished). The mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Genkov T, Meyer M, Griffiths H, Spreitzer RJ (2010) Functional hybrid Rubisco enzymes with plant small subunits and algal large subunits: Engineered rbcS cDNA for expression in Chlamydomonas. J Biol Chem 285:19833-19841

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to change the codon for Phe-132 (TTC) in the Rubisco small subunit to an ocher nonsense codon (TAA), which causes the deletion of nine carboxyl-terminal residues. The mutant Rubisco has decreases in carboxylation catalytic efficiency and thermal stability. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

Esquivel MG, Anwaruzzaman M, Spreitzer RJ (2002) Deletion of nine carboxyl-terminal residues of the Rubisco small subunit decreases thermal stability but does not eliminate function. FEBS Lett 520:73-76

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to create an S16A substitution (TCC-GCC) in the Rubisco small subunit, which causes a decrease in Rubisco holoenzyme stability (Genkov and Spreitzer 2009). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Phenotype: requires acetate at 35 °C, temperature-conditional

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to create an L18A substitution (CTG-GCC) in the Rubisco small subunit, which causes a decrease in Rubisco holoenzyme stability (Genkov and Spreitzer 2009). This mutant can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to create a P19A substitution (CCT-GCC) in the Rubisco small subunit, which causes a decrease in Rubisco holoenzyme stability (Genkov and Spreitzer 2009). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

  • Locus:
  • RBCS1
  • Chromosome:
  • 2

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

Directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4415) were used to create a Y32A substitution (TAC-GCC) in the Rubisco small subunit, which causes a decrease in Rubisco CO2/O2 specificity and holoenzyme stability (Genkov and Spreitzer 2009). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function or stability.

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

  • Locus:
  • RBCS1
  • Chromosome:
  • 2