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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

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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]


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

A 5.1-kb Genomic DNA Fragment Complements the C16 Characteristics

What is a Pyrenoid?

The chloroplast pyrenoid packages Rubisco and concentrates CO2


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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.”


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

Structure and Expression of Ccm1: DNA

The result: a little mRNA to LOTS of DNA!!!


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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.


Hideya fukuzawa et al 2001

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.


Hideya fukuzawa et al 2001

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.


Hideya fukuzawa et al 2001

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.


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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”


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

Structure and Expression of Ccm1: Protein

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


Hideya fukuzawa et al 2001

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?


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

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


Hideya fukuzawa et al 2001

CCM1 Shares Significant Sequence

Similarity To Zinc-Finger Domains

Fig 7A: Amino acid sequence of CCM1 protein.


Hideya fukuzawa et al 2001

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?


Hideya fukuzawa et al 2001

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.


Hideya fukuzawa et al 2001

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.

Gly-rich region (underlined)


Hideya fukuzawa et al 2001

CCM1 Shares Significant Sequence

Similarity To Zinc-Finger Domains

Fig 7B: Sequence comparison of putative zinc-finger motif of CCM1 with zinc finger motifs in other organisms

Regions of CCM1 show high sequence similarity to other known zinc fingers


Hideya fukuzawa et al 2001

CCM1 Shares Significant Sequence

Similarity To Zinc-Finger Domains

Characteristic zinc finger showing Cys and His binding Zn ligand

Zinc fingers binding DNA. A frequent motif in eukaryotic transcription factors.


Hideya fukuzawa et al 2001

Identification of a Mutation Site of Ccm1 in the High-CO2-Requiring Mutant, cia5

  • When cia5 mutant is transformed with pKI4XA, inducibility of CCM is restored

  • Mutation site in cia5 identified by sequencing Ccm1 gene

  • cia5 has single point mutation from T to C in second exon


Hideya fukuzawa et al 2001

Identification of a Mutation Site of Ccm1 in the High-CO2-Requiring Mutant, cia5

  • Base substitution causes His-54 in putative zinc finger motif to be replaced by Tyr

  • Loss of function in cia5 mutant strongly suggests that His-54 in CCM1 plays a critical role in regulation of carbon-concentrating mechanism

  • His residues favor zinc binding. No His, no zinc finger?


Hideya fukuzawa et al 2001

Discussion

  • What does CCM1 do?

  • A ccm1 gene encoding a hydrophilic nuclear regulatory protein was isolated

  • The gene product CCM1 controls the induction of CCM:

    • the seven CCM-related genes mentioned earlier

    • active Dissolved Inorganic Carbon (DIC) transport

    • The development of the pyrenoid (site for DIC accumulation) structure in chloroplasts


Hideya fukuzawa et al 2001

Discussion

What is not controlled by CCM1?

Pyrenoid starch sheath formation is not controlled by CCM1 since the mutant cia5 is shown to form the starch sheath. This suggests that the starch sheath is not involved in CCM.


Hideya fukuzawa et al 2001

Discussion

How does CCM1 know when CO2 levels are low?

The nature of the signal that induces responses to ambient CO2 levels is unclear.

Candidates include the total inorganic carbon level, the ratio of

[CO2]/[O2] or [CO2(aq)].

Since CCM1 has 14 putative phosphorylation sites, the signal may be transduced via a phosphorylation cascade.


Hideya fukuzawa et al 2001

Discussion

What type of nuclear regulatory gene is Ccm1?

The CCM1 may be a C2H2-type zinc finger transcription factor.

CCM1 lacks a second His usually required in zinc fingers to interact with zinc. Instead, CCM1 has an Asp residue.


Hideya fukuzawa et al 2001

Discussion

What type of nuclear regulatory gene is Ccm1?

Asp serves as a zinc ligand in other subcellular components, so it is possible that Asp ligands to zinc in CCM1. It is also possible that other His or Cys residues around the motif serve as a zinc ligand.

The identification of a mutation site in cia5 revealed that His-54 in the putative zinc-finger motif is essential to CCM1’s function.

In addition, other AA residues and the -sheet structure in this domain of CCM1 are highly conserved with other Zn-containing transcription factors.


Hideya fukuzawa et al 2001

Discussion

Is His-54 and the zinc finger all that is needed for CCM1 to function?

No, the region that codes for this domain is expressed in the mutant C16 but the CO2 responsive regulation of CCM is impaired.

The C-terminal region is lacking in the C16 mutant.

Therefore, the glutamine-repeat stretch on the C-terminal region of CCM1 is also required for CCM1 function.


Hideya fukuzawa et al 2001

Discussion

What affects CCM expression besides [CO2]?

CCM and the Cah1 expression seem to be affected by the light regime and the phase of the cell cycle.

Therefore, Ccm1 transcription and modification may also depend on the light regime and cell phase.


Hideya fukuzawa et al 2001

Discussion

What happens to the level of CCM1 protein during CO2 acclimation?

As [CO2] varies, the levels of CCM1 protein and ccm1 mRNA in wild-type cells do not change.

This indicates that modifications of CCM1 protein under specific conditions (high or low [CO2]) occurs post-translationally.

Again, this could be influenced by 14 putative phosphorylation sites on CCM1


Hideya fukuzawa et al 2001

Discussion

Are there different CCM1’s?

Yes, during the isolation of CCM1 a 3bp shorter cDNA of 5,125bp encoding another CCM1 protein – CCM1-B was isolated.

Sequence comparisons between the two different cDNA’s and Ccm1 revealed that the shorter cDNA was made by alternative splicing between the third and fourth exons causing the 3nt deletion.


Hideya fukuzawa et al 2001

Discussion

Are there different CCM1 Proteins?

This shorter cDNA encodes a 698-aa CCM1-B which has a substitution of Glu-183 instead of Asp-183 in CCM1-A and a deletion of Arg-184 present in CCM1-A.

Thus, it is possible that heterologous CCM1 proteins are translated simultaneously from alternatively spliced mRNA transcripts.

Affect of heterogeneity in the CCM1 proteins on their regulatory functions is not yet known.


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