Strains

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create a K258R substitution (AAA-CGT) in the Rubisco large subunit (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create a I265V substitution (ATT-GTA) in the Rubisco large subunit (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create C256F (TGT-TTC) and K258R (AAA-CGT) substitutions in the Rubisco large subunit, which cause a decrease in carboxylation catalytic efficiency (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create C256F (TGT-TTC) and I265V (ATT-GTA) substitutions in the Rubisco large subunit, which cause decreases in carboxylation catalytic efficiency and CO2/O2 specificity (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create K258R (AAA-CGT) and I265V (ATT-GTA) substitutions in the Rubisco large subunit, which cause decreases in carboxylation catalytic efficiency and CO2/O2 specificity (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Directed mutagenesis and chloroplast transformation of rbcL∆-25B1 mt+ (CC-4700) were used to create C256F (TGT-TTC), K258R (AAA-CGT), and I265V (ATT-GTA) substitutions in the Rubisco large subunit, which cause decreases in carboxylation catalytic efficiency and CO2/O2 specificity (Du et al. 2003). This is the original mutant strain. It was created to investigate phylogenetic differences near large-subunit residue Leu-290 (see rbcL-L290F) (Chen et al. 1988; Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

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:
• rbcL
• Chromosome:
• chloroplast

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

A plasmid containing the rbcL-C256F/K258R/I265V gene (Du et al. 2003) was transformed into rbcL∆-MX3312 mt+ (CC-4696) (Spreitzer et al. 2005). This strain was created for isogenic comparison with rbcL-V221C/V235I/C256F/K258R/I265V mt+ (Penta) (Spreitzer et al. 2005). The strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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:
• rbcL
• Chromosome:
• chloroplast

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

Using a plasmid containing the rbcL-C256F/K258R/I265V gene (Du et al. 2003), directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) were used to create V221C (GTT-TGT), V235I (GTT-ATT), C256F (TGT-TTC), K258R (AAA-CGT), and I265V (ATT-GTA) substitutions in the Rubisco large subunit (Spreitzer et al. 2005). The addition of the V221C and V235I substitutions increases the CO2/O2 specificity of rbcL-C256F/K258R/I265V Rubisco (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.

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

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:
• rbcL
• Chromosome:
• chloroplast

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

To create the penta/ABSO mutant, rbcL-V221C/V235I/C256F/K258R/I265V mt+ (Penta) was crossed with rbcS∆-CAL005.01.13 mt- (CC-4693), and an mt+ acetate-requiring progeny clone was recovered (Spreitzer et al. 2005). That strain was then transformed with an rbcS1-ABSO plasmid, which encodes a Rubisco small subunit containing the spinach betaA-betaB loop (Karkehabadi et al. 2005). Penta/ABSO Rubisco has increased CO2/O2 specificity and other kinetic properties similar to plant Rubisco enzymes (Spreitzer et al. 2005). 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 expression.

Karkehabadi S, Peddi SR, Anwaruzzaman M, Taylor TC, Cederlund A, Genkov T, Andersson I, Spreitzer RJ (2005) 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:
• rbcL, RBCS1
• Chromosome:
• chloroplast,2

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

R116-1B was recovered as a photosynthesis-competent revertant after methyl-methanesulfonate mutagenesis of rbcL-L290F mt+ (68-4PP) (Hong and Spreitzer 1997). It results from a mutation in rbcS2 that causes an N54S substitution (AAC-AGC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Du et al. 2000; Genkov et al. 2006). Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This is the original revertant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

R116-1B was recovered as a photosynthesis-competent revertant after methyl-methanesulfonate mutagenesis of rbcL-L290F mt+ (68-4PP) (Hong and Spreitzer 1997). It results from a mutation in rbcS2 that causes an N54S substitution (AAC-AGC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Du et al. 2000; Genkov et al. 2006). Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This strain was recovered from a cross between the original revertant R116-1B mt+ and pf2 mt-. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

R116-1B was recovered as a photosynthesis-competent revertant after methyl-methanesulfonate mutagenesis of rbcL-L290F mt+ (68-4PP) (Hong and Spreitzer 1997). It results from a mutation in rbcS2 that causes an N54S substitution (AAC-AGC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Du et al. 2000; Genkov et al. 2006). Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This strain was recovered from a cross between the original revertant R116-1B mt+ and pf2 mt-. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2, PF2
• Chromosome:
• chloroplast,2,11

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

R116-10C was recovered as a photosynthesis-competent revertant after methyl-methanesulfonate mutagenesis of rbcL-L290F mt+ (68-4PP) (Hong and Spreitzer 1997). It results from a mutation in rbcS2 that causes an A57V substitution (GCC-GTC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Du et al. 2000; Genkov et al. 2006). Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This is the original revertant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

R116-10C was recovered as a photosynthesis-competent revertant after methyl-methanesulfonate mutagenesis of rbcL-L290F mt+ (68-4PP) (Hong and Spreitzer 1997). It results from a mutation in rbcS2 that causes an A57V substitution (GCC-GTC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Du et al. 2000; Genkov et al. 2006). Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This strain was recovered from a cross between the original revertant R116-10C mt+ and pf2 mt-. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2, PF2
• Chromosome:
• chloroplast,2,11

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

R88-5A was recovered as a spontaneous photosynthesis-competent revertant of rbcL-L290F mt+ (68-4PP) (Hong 1996; Hong and Spreitzer 1997). It results from a transversion mutation in rbcS2 that causes a C65S substitution (TGC-TCC) in the Rubisco small subunit, which increases the CO2/O2 specificity of the original mutant enzyme (Genkov et al. 2006). See also S88-5A. Gene-centromere mapping indicated that rbcS2 is 20 map units from its centromere (Du et al. 2000). This is the original revertant strain. It has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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

Hong S (1996) Nuclear mutations affect the catalysis, stability, and expression of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. Ph. D. thesis, University of Nebraska

Hong S, Spreitzer RJ (1997) Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase. J Biol Chem 272:11114-11117

• Locus:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

Using standard methods of directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690), a C65S substitution (TGC-AGC) was created in the Rubisco small subunit (Genkov et al. 2006). This is the small-subunit intergenic-suppressor substitution that complements the large-subunit L290F substitution in photosynthesis-competent revertant R88-5A (rbcL-L290F/rbcS2-C65S). The small-subunit C65S substitution alone improves the thermal stability of wild-type Rubisco (Genkov et al. 2006). This strain has been maintained with acetate medium in darkness since its creation.

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:
• RBCS2
• Chromosome:

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

Using standard methods of directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690), a C65A substitution (TGC-GCG) was created in the Rubisco small subunit (Genkov et al. 2006). This mutant was created to investigate rbcS suppression of an rbcL mutation. See also R88-5A (rbcL-L290F/rbcS2-C65S). This strain has been maintained with acetate medium in darkness since its creation.

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:
• RBCS2
• Chromosome:

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

Using standard methods of directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690), a C65P substitution (TGC-CCG) was created in the Rubisco small subunit (Genkov et al. 2006). This mutant was created to investigate rbcS suppression of an rbcL mutation. See also R88-5A (rbcL-L290F/rbcS2-C65S). The C65P substitution in the Rubisco small subunit causes a decrease in CO2/O2 specificity but improves holoenzyme thermal stability (Genkov et al. 2006). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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:
• RBCS2
• Chromosome:

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

Phenotype: requires acetate, sensitive to light

This strain was recovered from a cross between rbcL-L290F mt+ (CC-4833) and rbcS∆-CAL005.01.13 pf2 mt- (CC-4693) (Genkov et al. 2006). It was used to investigate Rubisco rbcS2 small-subunit substitutions that complement an rbcL large-subunit substitution in the absence of the rbcS1 gene (Du et al. 2000; Genkov et al. 2006). The strain has been maintained with acetate medium in darkness since its recovery.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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:
• rbcL
• Chromosome:
• chloroplast

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

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

This strain was created by Todor Genkov in Spreitzer’s group by transforming rbcL-L290F/rbcS∆-CAL005.01.13 mt+ with the pSS1 plasmid that contains the Rubisco rbcS1 small-subunit gene (Khrebtukova and Spreitzer 1996). Like the original rbcL-L290F mutant (Chen et al. 1988), it can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The mutant was created for comparison with rbcL-L290F/rbcS1-L66G mt+. It 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

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:
• rbcL, RBCS1
• Chromosome:
• chloroplast,2

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

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

This strain was created by transforming rbcL-L290F/rbcS∆-CAL005.01.13 mt+ with the rbcS2 small-subunit gene (Genkov et al. 2006). Like the original rbcL-L290F mutant (Chen et al. 1988), it can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C. The mutant was created for comparison with rbcS suppressors of rbcL-L290F (Genkov et al. 2006). It 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

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:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

Plasmid pSS2-N54S (Du et al. 2000) was used to transform rbcL-L290F/rbcS∆-CAL005.01.13 mt+ (Genkov et al. 2006). This strain is like revertant R116-1B (Du et al. 2000), but it lacks the wild-type rbcS1 gene. The small-subunit N54S substitution increases the CO2/O2 specificity of the original large-subunit L290F mutant enzyme (Du et al. 2000; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

Plasmid pSS2-A57V (Du et al. 2000) was used to transform rbcL-L290F/rbcS∆-CAL005.01.13 mt+ (Genkov et al. 2006). This strain is like revertant R116-10C (Du et al. 2000), but it lacks the wild-type rbcS1 gene. The small-subunit A57V substitution increases the CO2/O2 specificity of the original large-subunit L290F mutant enzyme (Du et al. 2000; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

Plasmid pNESS2-C65S was used to transform rbcL-L290F/rbcS∆-CAL005.01.13 mt+ (Genkov et al. 2006). This strain is like revertant R88-5A (Genkov et al. 2006), but it lacks the wild-type rbcS1 gene. The small-subunit C65S substitution increases the CO2/O2 specificity of the original large-subunit L290F mutant enzyme (Du et al. 2000; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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:
• rbcL, RBCS2
• Chromosome:
• chloroplast,2

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

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

Using standard methods of directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690) (Genkov and Spreitzer 2009), Todor Genkov in Spreitzer’s group created an L66F substitution (CTG-TTC) in the Rubisco small subunit. This mutant was created to see whether an L66F substitution in the nuclear-encoded small subunit would mimic the biochemical defects that arise from an L290F substitution in the chloroplast-encoded large subunit (Chen et al. 1988). Like mutant rbcL-L290F (68-4PP), the rbcS1-L66F mutant has a temperature-conditional phenotype. It grows on minimal medium at 25 °C, but dies on minimal medium at 35 °C (Genkov and Spreitzer, unpublished). L66F Rubisco also has decreases in CO2/O2 specificity and holoenzyme stability in vivo and in vitro (Genkov and Spreitzer, unpublished). See also rbcS1-L66F pf2 mt+ (CC-4737). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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, August 2014

Using standard methods of directed mutagenesis and nuclear-gene transformation of rbcS∆-T60-3 mt- (CC-4690) (Genkov and Spreitzer 2009), Todor Genkov in Spreitzer’s group created an L66G substitution (CTG-GGC) in the Rubisco small subunit. This mutant was created to see whether an L66G substitution in the nuclear-encoded small subunit could suppress the biochemical defects that arise from an L290F substitution in the chloroplast-encoded large subunit (Chen et al. 1988). The L66G substitution increases the thermal stability of wild-type Rubisco (Genkov and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness since its creation.

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

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, August 2014

By employing standard methods (Genkov et al. 2006), Todor Genkov in Spreitzer’s group used plasmid pSS1-L66G to transform rbcL-L290F/rbcS∆-CAL005.01.13 mt+. The small-subunit L66G substitution suppresses the temperature-conditional phenotype of rbcL-L290F (68-4PP), and increases the CO2/O2 specificity and thermal stability of the original large-subunit L290F mutant enzyme (Genkov and Spreitzer, unpublished). Other small-subunit suppressor substitutions include N54S, A57V, and C65S (Du et al. 2000; Genkov et al. 2006). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Du YC, Hong S, Spreitzer RJ (2000) RbcS suppressors enhance the CO2/O2 specificity and thermal stability of rbcL-mutant ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 97:14206-14211

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:
• rbcL, RBCS1
• Chromosome:
• chloroplast,2

From Mary Porter, U of MN, August 2014

Song K, Awata J, Tritschler D, Bower R, Witman GB, Porter ME, Nicastro D (2015) In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography. J Biol Chem. 290:5341-53

• Locus:
• PF2
• Chromosome:
• 11

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

Phenotype: requires acetate, sensitive to light

Using plasmid p699 (GuhaMajumdar et al. 2008), Sriram Satagopan in Spreitzer’s group performed rbcL direct mutagenesis, chloroplast transformation of wild-type 2137 mt+ (cloned), selection for spectinomycin resistance in the dark, and screening for a homoplasmic acetate-requiring phenotype to create a K175C substitution (AAA-TGT) in the Rubisco large subunit. The K175C substitution causes a 90% decrease in Rubisco holoenzyme and a 99% decrease in Rubisco carboxylase activity (Lim and Spreitzer, unpublished). This mutant was created to investigate the role of active-site Lys-175 in catalysis (Harpel et al. 2002). It has been maintained with acetate medium in darkness since its creation.

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

Harpel MR, Larimer FW, Hartman FC (2002) Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants. Biochemistry 41:1390-1397

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Phenotype: requires acetate, sensitive to light

Using plasmid p699 (GuhaMajumdar et al. 2008), Sriram Satagopan in Spreitzer’s group performed rbcL direct mutagenesis, chloroplast transformation of wild-type 2137 mt+ (cloned), selection for spectinomycin resistance in the dark, and screening for a homoplasmic acetate-requiring phenotype to create a K175G substitution (AAA-GGA) in the Rubisco large subunit. The K175G substitution causes a 90% decrease in Rubisco holoenzyme and a 99% decrease in Rubisco carboxylase activity (Lim and Spreitzer, unpublished). This mutant was created to investigate the role of active-site Lys-175 in catalysis (Harpel et al. 2002). It has been maintained with acetate medium in darkness since its creation.

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

Harpel MR, Larimer FW, Hartman FC (2002) Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants. Biochemistry 41:1390-1397

• Locus:
• rbcL
• Chromosome:
• chloroplast