Strains

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

Phenotype: requires elevated CO2 for photosynthetic growth

Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with Chlamydomonas amino-acid sequences at the N and C termini. This Arabidopsis large subunit has 18 amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, T391V, S398A, V399C, V418A, V428T, V439R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆-MX3312 mt+ (CC-4696), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Chlamydomonas small subunits and engineered Arabidopsis large subunits. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Phenotype: requires elevated CO2 for photosynthetic growth

Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with Chlamydomonas amino-acid sequences at the N and C termini. This Arabidopsis large subunit has 18 amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, T391V, S398A, V399C, V418A, V428T, V439R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆/rbcS1-SSAT pf2 mt+ (which lacks Chlamydomonas rbcL and rbcS, but expresses Arabidopsis rbcS), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Arabidopsis small subunits and engineered Arabidopsis large subunits, but its growth on minimal medium with 5% CO2 is much reduced relative to rbcL-LSATNC mt+ or rbcL-LSATNC-T391F mt+. Subsequent DNA sequencing revealed that the engineered Arabidopsis large subunit has a new V391F mutant substitution (GTT-TTT), which may play a role in assembly with Arabidopsis small subunits. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

Phenotype: requires elevated CO2 for photosynthetic growth

The rbcL-LSATNC-T391F gene encodes an Arabidopsis large subunit that has Chlamydomonas substitutions at the N and C termini, as well as a T391F substitution (see rbcL-LSATNC-T391F/rbcS1-SSAT mt+). This Arabidopsis large subunit has 18 amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, T391F, S398A, V399C, V418A, V428T, V439R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). Fuqiao Xu in Spreitzer’s group transformed the engineered gene into rbcL∆-MX3312 mt+ (CC-4696), and photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Chlamydomonas small subunits and engineered Arabidopsis large subunits. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with 12 Chlamydomonas amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and that lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆-MX3312 mt+ (CC-4696), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Chlamydomonas small subunits and engineered Arabidopsis large subunits. The mutant grows less than wild type on minimal medium, but the same as wild type on minimal medium with 5% CO2 in air. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Phenotype: requires elevated CO2 for photosynthetic growth

Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with 12 Chlamydomonas amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and that lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆/rbcS1-SSAT pf2 mt+ (which lacks Chlamydomonas rbcL and rbcS, but expresses Arabidopsis rbcS), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Arabidopsis small subunits and engineered Arabidopsis large subunits. On minimal medium with 5% CO2 in air, the mutant grows less than rbcL-LSATNC mt+ and rbcL-LSATNC-T391F mt+, but more than rbcL-LSATNC-T391F/rbcS1-SSAT pf2 mt+. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

Phenotype: requires elevated CO2 for photosynthetic growth

Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with 12 Chlamydomonas amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and that lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆/rbcS1-SSSO pf2 mt+ (which lacks Chlamydomonas rbcL and rbcS, but expresses spinach rbcS), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of spinach small subunits and engineered Arabidopsis large subunits. On minimal medium with 5% CO2 in air, the mutant grows less than rbcL-LSATNC mt+ and rbcL-LSATNC-T391F mt+, but more than rbcL-LSATNC-T391F/rbcS1-SSAT pf2 mt+. Western analysis indicated that the mutant strain has a decreased level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a P104A substitution (CCA-GCG) was created in the Rubisco large subunit. Because Pro-104 is hydroxylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residue in Rubisco structure or function. The P104A substitution causes a decrease in carboxylation catalytic efficiency, but has only minor effect on the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a P151A substitution (CCT-GCT) was created in the Rubisco large subunit. Because Pro-151 is hydroxylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residue in Rubisco structure or function. The P151A substitution causes a decrease in Rubisco CO2/O2 specificity, but does not affect the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), two substitutions (P104A and P151A) were created together in the Rubisco large subunit. Because Pro-104 and Pro-151 are hydroxylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residues in Rubisco structure or function. The P104A/P151A substitutions cause decreases in Rubisco CO2/O2 specificity and carboxylation catalytic efficiency, but have only minor effect on the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a C256A substitution (TGT-GCT) was created in the Rubisco large subunit. Because Cys-256 is methylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residue in Rubisco structure or function. Large-subunit Cys-256 has significant interactions with the Rubisco small subunit (Spreitzer et al. 2005). The C256A substitution causes decreases in Rubisco CO2/O2 specificity and carboxylation catalytic efficiency (Spreitzer et al., unpublished). The mutant strain grows slowly 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.

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

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a C369A substitution (TGT-GCC) was created in the Rubisco large subunit. Because Cys-369 is methylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residue in Rubisco structure or function. The C369A substitution causes a decrease in Rubisco CO2/O2 specificity, but does not affect the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), two substitutions (C256A and C369A) were created together in the Rubisco large subunit. Because Cys-256 and Cys-369 are methylated (Taylor et al. 2001), this mutant was created to investigate the role of the modified residues in Rubisco structure or function. The C256A/C369A substitutions cause decreases in Rubisco CO2/O2 specificity and carboxylation catalytic efficiency (Spreitzer et al., unpublished). The mutant strain grows slowly 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.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a C256F substitution (TGT-TTC) was created in the Rubisco large subunit. The same substitution was created previously in a different host strain (see CC-4839) (Du et al. 2003). Because Cys-256 is methylated in Chlamydomonas Rubisco (Taylor et al. 2001), but replaced by Phe in plant Rubisco (Du et al. 2003), the mutant was created to investigate the role of the modified residue in Rubisco structure or function. The C256F substitution causes a small decrease in carboxylation catalytic efficiency (Du et al. 2003), but does not affect the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). C256F has been studied in combination with other phylogenetic substitutions (Du et al. 2003; Spreitzer et al. 2005). See also rbcL-C256F/C369V mt+. 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

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

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), a C369V substitution (TGT-GTT) was created in the Rubisco large subunit. Because Cys-369 is methylated in Chlamydomonas Rubisco (Taylor et al. 2001), but replaced by Val in plant Rubisco (Du et al. 2003), the mutant was created to investigate the role of the modified residue in Rubisco structure or function. The C369F substitution causes small increases in Rubisco CO2/O2 specificity and carboxylation catalytic efficiency (Spreitzer et al., unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may alter 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

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), two substitutions (C256F and C369V) were created together in the Rubisco large subunit. Because Cys-256 and Cys-369 are methylated in Chlamydomonas Rubisco (Taylor et al. 2001), but replaced by Phe and Val in plant Rubisco (Du et al. 2003), the mutant was created to investigate the role of the modified residues in Rubisco structure or function. The C256F/C369V substitutions cause a small decrease in carboxylation catalytic efficiency, but do not affect the photosynthetic growth of the mutant cells (Spreitzer et al., unpublished). 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

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

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

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Girish Rasineni in Spreitzer’s group created four substitutions together (P104A, P151A, C256A, and C369A) in the Rubisco large subunit. Because Pro-104 and Pro-151 are hydroxylated, and Cys-256 and Cys-369 are methylated (Taylor et al. 2001), the mutant was created to investigate the role of the modified residues in Rubisco structure or function. The four substitutions cause decreases in Rubisco CO2/O2 specificity and carboxylation catalytic efficiency (Rasineni and Spreitzer, unpublished). The mutant strain grows slowly 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.

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

Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159-48164

• Locus:
• rbcL
• Chromosome:
• chloroplast

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From Susan Dutcher, Washington University in St. Loius, October 2014

fla24 is flagella-less when grown at 33 °C but has flagella at 21 °C. The mutant lesion is a missense mutation in cytoplasmic dynein gene (DHC1b), in which a T to C mutation predicts a L3242P change.

Pseudo-revertants (all the numbered strains) were selected in fla24 (CC-3866) by growth at 33 °C after mutagenesis.

Lin H, Nauman NP, Albee AJ, Hsu S, Dutcher SK (2013) New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas. Cilia 2:14

• Locus:
• DHC1b
• Chromosome:
• 6

From James V. Moroney, Louisiana State University, November 2014

Phenotype: altered CO2 assimilation

This cia5 strain was originally from Robert Togasaki who crossed CC-4427 cia5 mt+ with CC-124 137c mt- to get a mt- strain. It requires elevated CO2 for growth so it is maintained on acetate medium.

Moroney JV, Husic HD, Tolbert NE, Kitayama M, Manuel LJ, Togasaki RK (1989) Isolation and Characterization of a Mutant of Chlamydomonas reinhardtii Deficient in the CO2 Concentrating Mechanism. Plant Physiol 89:897-903

From James V. Moroney, Louisiana State University, November 2014

Phenotype: altered CO2 assimilation

This mutant was created by insertional mutagenesis of CC-4425 cw15 nit2-203 mt+ [D66] with pSP124S. The insert is at the very 5’end of Cre06.g311850. It requires elevated CO2 for growth so it is maintained on acetate medium.

Adams JE, Colombo SL, Mason CB, Ynalvez RA, Tural B, Moroney JV (2005) A mutant of Chlamydomonas reinhardtii that cannot acclimate to low CO2 has an insertion in the Hdh1 gene. Func. Plant Biol 32:55-66

• Locus:
• HAD1
• Chromosome:
• 6

From James V. Moroney, Louisiana State University, November 2014

Phenotype: altered CO2 assimilation

This mutant was created by insertional mutagenesis of CC-4425 cw15 nit2-203 mt+ [D66] with pSP124S. The insert is in the first exon of Cre04.g212350. It requires elevated CO2 for growth so it is maintained on acetate medium.

Ynalvez RA, Moroney JV (2008) Isolation and Characterization of the Novel Gene CIA7 from the BleR Insertional Mutant of Chlamydomonas reinhardtii. Funct. Plant Biol 35:373-381

• Locus:
• CIA7
• Chromosome:
• 4

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

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Boon Hoe Lim in Spreitzer’s group created an A11V substitution in the Rubisco large subunit. This mutant (also named G11) represents one of 15 “groups” of amino acids that differ between Chlamydomonas and land plants (Du et al. 2003). It was created to investigate phylogenetic differences that influence Rubisco catalysis (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

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

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, November 2014

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Boon Hoe Lim in Spreitzer’s group created three substitutions (V30E, V31T, and R32K) together in the Rubisco large subunit. This mutant (also named G30-32) represents one of 15 “groups” of amino acids that differ between Chlamydomonas and land plants (Du et al. 2003). It was created to investigate phylogenetic differences that influence Rubisco catalysis (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

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

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, November 2014

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Boon Hoe Lim in Spreitzer’s group created a D86H substitution in the Rubisco large subunit. This mutant (also named G86) represents one of 15 “groups” of amino acids that differ between Chlamydomonas and land plants (Du et al. 2003). It was created to investigate phylogenetic differences that influence Rubisco catalysis (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

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

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, November 2014

Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Boon Hoe Lim in Spreitzer’s group created two substitutions (I105L and C369V) together in the Rubisco large subunit. This mutant (also named G105-369) represents one of 15 “groups” of amino acids that differ between Chlamydomonas and land plants (Du et al. 2003). It was created to investigate phylogenetic differences that influence Rubisco catalysis (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

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

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