kaiC Clock Mutant Rescue

Figure 1A: Rescue of Clock Mutant C44a

• A long-period clock mutant (C44a, period = 44 hours) was transformed utilizing a wild-type (WT) genomic DNA library to restore normal circadian rhythms
• The mutation of the kaiC gene was responsible for the altered period
• Restoring a wild-type copy corrected the rhythm
• The clock mutant C44a (44-hour period) was transformed using a plasmid DNA library containing wild-type genomic fragments
• Colonies underwent screening for restored 25-hour rhythms, similar to the wild-type
• A rescued clone (plasmid p44N) was found to restore the normal period
• Reintroducing the plasmid into the mutant confirmed the correction
• Rescued clones presented a 25-hour period, like the wild-type
• It was suggested that the mutant had a recessive loss-of-function mutation in kaiC
• The rescue of clock mutant C44a
• Wild-type kaiC expression restores circadian rhythms in a clock mutant
• C44a mutant (44-hour period) was transformed with a wild-type plasmid
• Rescued clones demonstrated a normal 25-hour period, proving that the kaiC mutation disrupted rhythmicity

Figure 1B: Map of the kaiABC Gene Cluster

• The genetic map of the kaiABC gene cluster, essential for circadian rhythms in cyanobacteria
• Confirmed that three genes (kaiA, kaiB, kaiC) are responsible for generating rhythm
• The genomic DNA fragment (4.7 kb EcoRI segment) responsible for rescuing the mutant was mapped
• DNA sequencing revealed six open reading frames (ORFs)
• The three clock genes (kaiA, kaiB, kaiC) were identified to form a single cluster
• The kaiA, kaiB, kaiC genes reside adjacently in the cyanobacteria genome
• kaiC had the mutation in the mutant strain C44a
• Southern blot analysis affirmed that there is only one copy of this wild-type Synechococcus gene cluster
• The kaiABC gene cluster encodes core circadian clock components
• A sequenced 4.7 kb DNA fragment contained clock genes.
• Clock genes kaiA, kaiB, and kaiC are clustered and essential for rhythm regulation

Figure 1C: Deduced Amino Acid Sequences of Kai Proteins

• The amino acid sequences of KaiA, KaiB, and KaiC proteins show key functional motifs
• The predicted amino acid sequences of KaiA (284 residues), KaiB (102 residues), and KaiC (519 residues) were analyzed
• Key motifs in KaiC Include:
  • Walker A & B motifs (ATP/GTP binding sites)
  • Catalytic glutamate residues (critical for ATP hydrolysis)
  • DXXG motifs (common in GTP-binding proteins)
• KaiC is an ATP-binding protein, similar to other clock proteins
• KaiA and KaiB do not possess already known enzymatic domains but are critical for rhythm regulation
• Structural features suggest KaiC plays a key role in the feedback loop
• KaiC possesses ATPase motifs and is structurally similar to clock proteins in other organisms
• Predicted amino acid sequences of Kai proteins underwent analysis, critical conserved motifs being identified
• KaiC possesses Walker motifs (ATP-binding), thus suggesting an enzymatic role in rhythm regulation

Figure 1D: Mapping of Clock Mutations

• Mutations present in kaiA, kaiB, and kaiC in circadian rhythm mutants were mapped
• Mutations in any of the three kai genes disrupts rhythms
• Kai genes' sequencing occurred in 19 clock mutants, some having long/short periods or arrhythmic phenotypes
• The exact mutation site in each mutant underwent mapping
• 14 mutants had mutations in kaiC, meaning this gene is likely a key regulator
• 3 mutants had mutations in kaiA, and 2 had mutations in kaiB
• Even a single amino acid change in kaiC completely stopped rhythms
• keiABC is critical for the cyanobacterial clock
• Mutations to kaiABC genes disrupt circadian rhythms
• Kai genes sequencing occurred in 19 clock mutants
• Most mutations presented in kaiC (14/19), designating it as the key regulator of circadian rhythms

Summary of Figure 1

• Subfigure Key Takeaways:
  • Figure 1A: Restored wild-type kaiC rescues the long-period mutant (44h → 25h)
  • Figure 1B: The kaiABC gene cluster regulates rhythm
  • Figure 1C: KaiC has ATP-binding motifs, and may have an enzymatic role
  • Figure 1D: With the majority of mutations occurring in kaiC, it may be the core clock component

Figure 2A: Wild-Type (WT) Circadian Rhythms

• Wild-type Synechococcus shows strong bioluminescence rhythms (aprox. 25h period)
• Wild type as a control to compare against kai gene knockouts
• Wild-type Synechococcus was transformed with a lux bioluminescence reporter system
• Synchronized cells, in darkness, for 12 hrs, then placed in constant light (LL)
• Rhythmic bioluminescence was recorded over 96 hrs
• Wild-type cells show robust, around 25 hours circadian rhythms, making the reporter system functional
• Synechococcus Wild-type cells show normal (aprox. 25h) bioluminescence rhythms
• A lux reporter system was experimentally set up, synchronized in darkness and measured in LL
• Strong circadian oscillations were a control for kai gene knockouts

Figure 2B: Deletion of kaiABC (ΔkaiABC) Causes Arrhythmicity

• Removal of the whole kaiABC cluster halts rhythmicity, so these genes are critical for the clock
• Through homologous recombination, kaiABC genes were deleted, creating a ΔkaiABC mutant strain
• Bioluminescence was recorded under LL conditions
• A ΔkaiABC strain shows no oscillatory rhythm and a gradual increase in bioluminescence
  • Growth remained normal
• Deleting kaiABC disrupts the clock, causing arrhythmia
• An experimental ΔkaiABC strain had its bioluminescence measured in LL
• Loss of rhythmicity asserts kaiABC genes are essential for the circadian clock

Figure 2C: Restoring kaiABC Rescues Rhythmicity

• By reintroducing kaiABC into the ΔkaiABC strain, normal (aprox. 25h) rhythms are restored, meaning these genes are both critical and sufficient for circadian function
  • The kaiABC cluster was reintroduced into the ΔkaiABC strain
• Bioluminescence rhythms were measured
• Rhythm returned to Wild-type levels (aprox. 25 hours period)
• kaiABC is proved necessary and sufficient to make rhythm
• kaiABC is essential and sufficient for circadian rhythms
• After experiments where kaiABC was reintroduced into ΔkaiABC strain that measured rhythms
• Normal oscillations were established, reinforcing that kaiABC is the core clock system

Figures 2D, 2E, 2F: Individual Kai Gene Knockouts

• Removal of a single kai gene (kaiA, kaiB, kaiC) stops circadian rhythms, so all three are critical
• kaiA, kaiB, and kaiC were individually knocked out with targeted gene deletions
• Bioluminescence rhythms were measured under LL conditions.

Summary of Figure 2

• Subfigure Key Takeaways:
  • Figure 2A: Wild type Synechococcus presents aprox. 25 bioluminescence rhythms
  • Figure 2B: The ΔkaiABC mutant is completely arrhythmic, validating kaiABC is essential
  • Figure 2C: After restoring kaiABC, normal (aprox. 25h) rhythms can be rescued
  • Figure 2D-F: Knockout of kaiA, kaiB, or kaiC halts rhythmicity -KaiABC gene cluster is proven critical for circadian rhythms
  • The deletion of the genes results in arrhythmic phenotypes

Figure 3A: Bioluminescence Rhythms from kai Promoters

• Presentation of rhythmic expression of kaiA, kaiB, kaiC using lux reporters
• kaiC expression is arrhythmic, and may have post-transcriptional regulation
• By linking kai promoters, fusion constructs (USRkaiA::lux, USRkaiB::lux, USRkaiC::lux) were created to a lux bioluminescence reporter
• Bioluminescence rhythms were measured under LL conditions.

Figure 3B: Bioluminescence from kaiABC::lux Reporter:

• By fusing luxAB to the kaiABC operon, a kaiABC::lux reporter was constructed
• Bioluminescence rhythms were measured in LL
• The kaiABC operon shows circadian oscillations (aprox. 25h period)
• kaiA/kaiB can cause transcriptional rhythms
• kaiC has post-transcriptional control

Figure 3C: Bioluminescence from psbAI::lux Reporter

• psbAI shows strong circadian rhythms
• This result can validate lux reporter
• psbAI is attached to lux to track circadian oscillations
• Oscillations are confirmed to be approx.. 25 hours
• Confirms the validity of the bioluminescence system.
• PsbAI oscillations can serve as a control
• The experiments confirm the robustness of the bioluminescence reporter system

Figure 3D: Northern Blot of kai Transcripts

• Can confirm the transcription factors of kaiA, kaiB, kaiC being produced
• KaiC transcript is larger (2.3 kb), and may have to undergo post-transcriptional regulation
• Rna was extracted from wild-type Synechococcus
• By using kaiA, kaiB, kaiC-specific, it can be concluded
• kaiA, kaiB, kaiC are able to be transcribed at the same time
• KaiC transcripts are very large
• KaiC is larger at aprox. 2.3kb

Figure 3E: Circadian Expression of kaiA and kaiC mRNA

• kaiA and kaiC show mRNA levels oscillating at aprox. 25 hours
• Northern blot analysis was performed, using the RNA samples collected over 48h
• kaiA* and kaiC probes are used to measure MRNA Rhytyms
• kaiA and kaiC show rhythmic (aprox. 25hour period)mRNA accumulation
• Can be used to to demonstrate transcriptional control
• KaiA and kaiC levels of mRNA are shown to oscillate following a 25 hours schedule
• Results can be shown to follow to the same level
• Results match up, and circadian rhythm follows to the same level.
• Results follow regulation on a transcription level

Summary of Figure 3

• Figure 3A Shows : the kaiA and kaiB expressions oscillate
• -Figure 3B Confirms this

##Figure 3C

• psbsI does not show oscillatory Rhythms--it says it measures circadian rhythms Figures 3D

Figure 4A-D: Specific Mutations Effects

• of Bioluminescence Rhythms
• KaiC (mutations in kaiC *(A30a, B22a, C28a)) show consistent Rhythm
• The CLAD Mutation
• CLAds Mutation is able to disrupt Rhythms that is proving role of clock function.
• Mutants from CLAB are analyzed through Biolum conditions

Figure 4E-I: KaiC Overexpression Disrupts Rhythmicity

• Over expression allows the rhythms to be ABOLISHED
• KaiC can be given at a certain dosage
• PTG is to over express the rhythmicity of the KAI gene
• Control shows and addition by water can alter the Rhythyms

Figure 4J-N: Timing of KaiC Overexpression Altered the Phase

• By Using/Over Expression through KaiC it can be proven to alter rhythms of Cicada
• PTOG WAS to overexpress at certain levels
• PTG was added to see where rhythmicity was at

Figure 4O: KaiC Regulation

• All the protein can assist with Maintanence
• Interactions can generate feedback thru rythmicity
• 4O ( Kai regulation occurs where the loops can regulate) .

Conclusion 4

• The most important is KAI which allows all the functions to be used correctly.
• kaiC must be TIGHTLY controlled
• The process allow feedback for certain phases