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Hideya Fukuzawa, et al. 2001

Ccm1 , a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO 2 availability. Hideya Fukuzawa, et al. 2001. Presented by: Arin, Artin, Judy, and Ryan. Chlamydomonas reinhardtii Strains. 5D.

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Hideya Fukuzawa, et al. 2001

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  1. Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO2 availability Hideya Fukuzawa, et al. 2001 Presented by: Arin, Artin, Judy, and Ryan

  2. Chlamydomonas reinhardtii Strains 5D • Wild type with respect to photosynthesis • Normal induction of carbon-concentrating mechanism (CCM) when grown in low CO2 conditions C16 • High-CO2-requiring mutant • Impaired induction of CCM in low CO2 conditions • Generated by gene tagging mutagenesis cia5 • High-CO2-requiring mutant • Impaired induction of CCM in low CO2 conditions • Single point mutation changes His to Tyr in exon 2

  3. Chlamydomonas reinhardtii Strains C16 Mutant: Transposon carrying Nia1 gene inserted into exon 4 of Ccm1 gene. Disrupts gene leading to high-CO2-requiring phenotype. Figure 1: Gene organization of Ccm1

  4. Chlamydomonas reinhardtii Strains cia5 Mutant: Single point mutation in exon 2 changes His to Tyr C16 Mutant: Transposon carrying Nia1 gene inserted into exon 4 of Ccm1 gene. Disrupts gene leading to high-CO2-requiring phenotype. Figure 1: Gene organization of Ccm1

  5. Isolation of a DNA Fragment That Complements the C16 Phenotype • Nia gene shown by genetic crosses to be linked to high-CO2-requiring phenotype in C16 mutants • Ccm1 gene is “tagged” by Nia1 • To isolate Ccm1 gene, oligonucleotide probes were designed from flanking regions of Nia1 gene

  6. Isolation of a DNA Fragment That Complements the C16 Phenotype • C16-5 and C16-3 oligonucleotide probes were used to screen a wild type C. reinhardtii genomic library. • A genomic library contains DNA fragments representing the entire genome of an organism. The fragments are often carried on plasmids and retained in E. coli. • Four genomic clones were detected. These plasmids were used to transform C16 mutants. • One of the genomic clones, pKI4, restored growth of mutant C16 C. reinhardtii when grown under low-CO2

  7. Isolation of a DNA Fragment That Complements the C16 Phenotype What is the smallest segment of pKI4 capable of complementing C16? SacI XhoI 3 different restriction enzymes used to digest pKI4 fragments SacI XhoI ApaI The 5.1 kb XhoI—ApaI fragment is the smallest fragment capable of complementing C16::pKI4XA transformants characterized in this paper Fig. 2A: Relative positions of a genomic clone, pKI4 and its derivatives

  8. Isolation of a DNA Fragment That Complements the C16 Phenotype • Cah1 is a gene expressed when CCM is induced, thus, it acts as an indicator of positive Ccm1 function Fig. 2B: Northern blot analyses of Cah1 expression

  9. Isolation of a DNA Fragment That Complements the C16 Phenotype • Cah1 is a gene expressed when CCM is induced, thus, it acts as an indicator of positive Ccm1 function • CCM occurs only when there are low CO2 conditions (LC), so no Cah1 expression occurs under high CO2 conditions (HC) Fig. 2B: Northern blot analyses of Cah1 expression

  10. Isolation of a DNA Fragment That Complements the C16 Phenotype • Cah1 is a gene expressed when CCM is induced, thus, it acts as an indicator of positive Ccm1 function • CCM occurs only when there are low CO2 conditions (LC), so no Cah1 expression occurs under high CO2 conditions (HC) Fig. 2B: Northern blot analyses of Cah1 expression • Both the C16 and cia5 mutants are unable to induce CCM, so no Cah1 expression is observed under LC conditions

  11. Isolation of a DNA Fragment That Complements the C16 Phenotype • Cah1 is a gene expressed when CCM is induced, thus, it acts as an indicator of positive Ccm1 function • CCM occurs only when there are low CO2 conditions (LC), so no Cah1 expression occurs under high CO2 conditions (HC) Fig. 2B: Northern blot analyses of Cah1 expression • Both the C16 and cia5 mutants are unable to induce CCM, so no Cah1 expression is observed under LC conditions • The transformants C16::pKI4, C16::pKI4Xh and C16::pKI4XA were all able to restore Ccm1 function and Cah1 was expressed. • These three clones must contain the coding region of Ccm1

  12. Isolation of a DNA Fragment That Complements the C16 Phenotype • Cah1 is a gene expressed when CCM is induced, thus, it acts as an indicator of positive Ccm1 function • CCM occurs only when there are low CO2 conditions (LC), so no Cah1 expression occurs under high CO2 conditions (HC) Fig. 2B: Northern blot analyses of Cah1 expression • Both the C16 and cia5 mutants are unable to induce CCM, so no Cah1 expression is observed under LC conditions • The transformants C16::pKI4, C16::pKI4Xh and C16::pKI4XA were all able to restore Ccm1 function and Cah1 was expressed. • These three clones must contain the coding region of Ccm1 • pKI4XA is able to complement cia5, indicating that the cia5 mutation is in the same gene as the mutation in the C16 strain, Ccm1

  13. Isolation of a DNA Fragment That Complements the C16 Phenotype With Nia1 insert (nonfunctional Ccm1) No Nia1 insert Figure 2C: Southern Blot Analysis of BamHI-digested Total Genomic DNA

  14. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics When grown at low-CO2, C16::pKI4XA transgenic strains and wild-type show similar affinities for dissolved inorganic carbon (DIC) mutant WT & transgenic Km(HCO3-)values are comparable for transgenic and wild-type: 145M (wild-type) 122M (C16::pKI4XA) Fig 3A: Relative rate of photosynthesis at various carbon concentrations of low-CO2-grown cells (pH 7.8) at 25 C

  15. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics During photosynthesis, H2O is an electron donor and O2 is released mutant Wild type and C16::pK14XA have similar slopes. Steeper slopes than C16 indicates they reach a higher relative rate of O2 evolution at lower [DIC] WT & transgenic Maximum rates were measured to be 90 (WT), 92 (C16) and 106 (C16::pKI4XA) µmol/mg of Chl/h Therefore, C16 mutant eventually reaches rate of wild-type but ONLY at higher [DIC]

  16. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics With a functional CCM, internal inorganic carbon should increase in low-CO2 environments WT & transgenic Internal carbon concentration in WT and C16::pKI4XA increases vigorously as time proceeds. Rate of inorganic carbon accumulation in the C16 mutant is much lower mutant Fig 2B: Intracellularly accumulated DIC concentration measured

  17. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics By transformation with the 5.1-kb pKI4XA fragment, acclimation to limiting CO2 and accumulation of inorganic carbon inside the cell is restored in the C16 mutant WT & transgenic mutant Accumulation of the intracellular DIC was measured using silicon-oil centrifugation method Fig 2B: Intracellularly accumulated DIC concentration measured

  18. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics This graph shows the rate of photosynthesis under limiting CO2 conditions as the time proceeds WT & transgenic mutant Note that the rate of photosynthesis of the WT and the C16::pKI4XA are approximately the same while the C16 mutant’s is very low The fragment cloned into C16::pKI4XA causes the restoration of the carbon concentrating mechanism in the transformant Fig 3C: Carbon Fixation

  19. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Figure 4: Pyrenoid Structures in WT, C16, and C16::pKI4XA Arrows indicate pyrenoid structures The chloroplast pyrenoid packages Rubisco and concentrates CO2 At low CO2 it is important in suppressing oxygenase activity and preventing wasteful photorespiration

  20. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics What is a Pyrenoid? The chloroplast pyrenoid packages Rubisco and concentrates CO2

  21. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Figure 4: Pyrenoid Structures in WT, C16, and C16::pKI4XA Arrows indicate pyrenoid structures During low-CO2 concentrations (LC), the pyrenoid expands in WT and C16:pKI4XA C16 mutant pyrenoids do not expand in low-CO2 conditions

  22. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics • When CCM is induced in low CO2 conditions, a set of CCM-related genes are expressed: • Cah1: periplasmic carbonic anhydrase • Mca: mitochondrial carbonic anydrase • Ccp2: chloroplast envelope protein LIP-36 • Lci1 • Att1: alanine -ketoglutarate aminotransferase • Transcription of some mRNAs are upregulated in low-CO2: • Cah3: chlorplastic CA • Cyp1: cyclophilin

  23. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Fig 5: RNA blot analyses of total RNA HC: high-CO2 grown cells LC: low-CO2 grown cells HL: high-light grown cells ML: moderate-light grown cells Cblp is a loading control

  24. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Activation of 5 key CCM genes in low CO2 is restored in C16::pKI4XA Fig 5: RNA blot analyses of total RNA HC: high-CO2 grown cells LC: low-CO2 grown cells HL: high-light grown cells ML: moderate-light grown cells Cblp is a loading control

  25. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Activation of 5 key CCM genes in low CO2 is restored in C16::pKI4XA No transcription of these CCM genes is observed in C16 mutant Fig 5: RNA blot analyses of total RNA HC: high-CO2 grown cells LC: low-CO2 grown cells HL: high-light grown cells ML: moderate-light grown cells Cblp is a loading control

  26. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics Activation of 5 key CCM genes in low CO2 is restored in C16::pKI4XA No transcription of these CCM genes is observed in C16 mutant Up-regulation of Cah3 and Cyp1 is observed in WT and C16::pKI4XA, but not in C16 Fig 5: RNA blot analyses of total RNA HC: high-CO2 grown cells LC: low-CO2 grown cells HL: high-light grown cells ML: moderate-light grown cells Cblp is a loading control

  27. A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics “These results strongly suggest that the 5.1-kb genomic DNA fragment in pKI4XA encodes a key regulatory gene, which controls the expression of at least seven genes and modulates physiological properties and pyrenoid-development in response to CO2 availability.”

  28. Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO2 availability Hideya Fukuzawa, et al. 2001 Presented by: Arin, Artin, Judy, and Ryan

  29. Quick Review • Identify mutants incapable of inducing carbon-concentrating mechanism • Isolate DNA fragment capable of restoring wild-type phenotype • Confirm by numerous assays that DNA fragment restores wild-type phenotype

  30. Structure and Expression of Ccm1: DNA Reverse Transcriptase-PCR isolates cDNA for ccm1 gene: • purify mRNA based on their poly-A tails • ccm1-specific primers designed from the pXI4XA fragment • Reverse transcriptase generates cDNA from mRNA • only ccm1 mRNA is reverse transcribed because of gene specific primers • cDNA is amplified using standard PCR reaction

  31. Structure and Expression of Ccm1: DNA The result: a little mRNA to LOTS of DNA!!!

  32. Structure and Expression of Ccm1: DNA • 5,128-bp cDNA isolated from low-CO2-grown WT cells • 2,097-bp ORF detected • encodes 699-aa hydrophilic protein • 176-bp 5’ UTR • 2,855-bp 3’ UTR • This cDNA named ccm1 • cDNA contains only the exons of the complete gene • derived from processed mRNA

  33. Structure and Expression of Ccm1: DNA To determine entire structure of ccm1 (including introns): • 12-kb XhoI genomic DNA is sequenced • Ccm1 mRNA is encoded by 6,491-bp region • Ccm1 is a single-copy gene in C. reinhardtii

  34. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 Fig 6A: Expression of ccm1 gene. Northern blot.

  35. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-5 Probe Fig 6A: Expression of ccm1 gene. Northern blot.

  36. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-5 Probe WT: 5.1-kb mRNA detected in high- and low-CO2 grown cells Fig 6A: Expression of ccm1 gene. Northern blot.

  37. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-5 Probe WT: 5.1-kb mRNA detected in high- and low-CO2 grown cells C16: mRNA does not accumulate Fig 6A: Expression of ccm1 gene. Northern blot.

  38. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-3 Probe Fig 6A: Expression of ccm1 gene

  39. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-3 Probe WT: 5.1-kb mRNA detected in high- and low-CO2 grown cells Fig 6A: Expression of ccm1 gene

  40. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probes, P-5 and P-3 P-3 Probe WT: 5.1-kb mRNA detected in high- and low-CO2 grown cells C16: mRNA does not accumulate Fig 6A: Expression of ccm1 gene

  41. Structure and Expression of Ccm1: RNA Northern blot analysis using ccm1-specific probe P-1 P-1 Probe 2.2-kb mRNA detected by both P-5 and P-1 Corresponds to 5’ part of mRNA, upstream of Nia1 tag No probes hybridize downstream of Nia1 Ccm1 transcription disrupted by Nia1 insert in C16 Fig 6A: Expression of ccm1 gene

  42. Structure and Expression of Ccm1: RNA “These results indicate that Ccm1 is expressed constitutively in WT but the mature Ccm1 mRNA is not accumulated in C16 because of the Nia1 insertion”

  43. Structure and Expression of Ccm1: Protein Ccm1 encodes a 699-aa polypeptide with no apparent transmembrane domains To identify CCM1 protein in the cell: • WT and C16 cells grown in [35-S]-methionine and [35-S]-cysteine • All protein is radiolabelled • CCM1 protein immunoprecipitated with anti-CCM1 antibody generated in rats • Immunoprecipitate is run on a gel

  44. Structure and Expression of Ccm1: Protein Fig 6B: Expression of Ccm1 gene. Radiolabelled protein immunoprecipitated with anti-CCM1 antibody

  45. Structure and Expression of Ccm1: Protein 76 kDa band detected in WT cells grown in high-CO2 (lane 1) and low-CO2 (lane 2) Fig 6B: Expression of Ccm1 gene. Radiolabelled protein immunoprecipitated with anti-CCM1 antibody In WT cells, CCM1 levels are not affected by CO2 levels. Perhaps CCM1 is post-translationally modified in response to CO2 availability?

  46. Structure and Expression of Ccm1: Protein C16 mutant has 80 kDA band Is this band generated by a fusion of mRNA for 5’ part of Ccm1 and 5’ part of Nia1 insert? Fig 6B: Expression of Ccm1 gene. Radiolabelled protein immunoprecipitated with anti-CCM1 antibody Lane 4 is a control for the specifity of the immunoprecipitation

  47. CCM1 Shares Significant Sequence Similarity To Zinc-Finger Domains • Amino acid sequence of CCM1 deduced from ORF and codon library • Search of protein databases showed no known sequences with a high level of similarity to CCM1 • Three characteristic sequence stretches were identified • sequence similar to C2H2-type zinc finger motif which function in DNA-protein interactions • Gln-repeat • Gly-rich domain at C-terminal region

  48. CCM1 Shares Significant Sequence Similarity To Zinc-Finger Domains Fig 7A: Amino acid sequence of CCM1 protein.

  49. CCM1 Shares Significant Sequence Similarity To Zinc-Finger Domains Fig 7A: Amino acid sequence of CCM1 protein. Putative zinc-finger motif. Possibly a DNA-binding motif typical of transcription factors?

  50. CCM1 Shares Significant Sequence Similarity To Zinc-Finger Domains Fig 7A: Amino acid sequence of CCM1 protein. Putative zinc-finger motif. Possibly a DNA-binding motif typical of transcription factors? Gln-rich repeat. Necessary for regulation of other eukaryotic transcription factors.

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