MCB 186CIRCADIAN BIOLOGY

Clock genes & Biochemical Mechanisms: Kai genes

October 10, 2007

J. W. Hastings

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

TEMPORAL SEPARATION OF CELL BIOCHEMICAL ACTIVITIES IN CYANOBACTERIA (OXYGEN, NITROGENASE ETC) MITSUI ET AL 1986

CIRCADIAN RHYTHM of CYANOBACTERIAL GENE mRNA

Kondo et al. 1993PNAS

LUCIFERASE REPORTER

• Turntable Screening Apparatus: 12 positions for

petri dishes or titer plates

BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE

Kondo et al. 1993PNAS

Cyanobacterial in vivo Rhythm with Bacterial Luciferase as a Reporter

24016814412096720

Hours in Constant Light

2000

4000

6000

8000

12 60 84 108 132 156 180

228

192

204

10000

48

Lu

min

esce

nce

Kondo, Johnson Golden et al., 1993, PNAS

EFFECT OF DARK EXPOSURES ON PERIOD

In LL during ----

In light but with 3 min in dark every 30 min

DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993

TEMPERATURE has SMALL EFFECT on PERIOD of BACTERIAL RHYTHM

PERIOD MUTANTS OF CYANOBACTERIA KONDO ET AL.1995

CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995

PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONES KONDO ET AL.1995

LUCIFERASE REPORTER RANDOMLY INSERTED IN FRONT OF PROMOTERS IN

BACTERIA.

ABOUT 800 LUMINOUS COLONIES ISOLATED

ALL EXHIBITED CIRCADIAN RHYTHMS

ACROPHASES NOT ALL THE SAME

ACROPHASE CLASSES OF CLOCK-CONTROLLED GENES LIU ET AL ,1995

KAI genes in SYNECHOCOCCUS and MUTANT MAPPINGISHIUR

PHENOTYPES OF KAI GENE INACTIVATIONS ISHIURA ET AL 1998

EXPRESSION OF KAI GENE CONSTRUCTS ISHIURA ET AL 1998

Cyanobacterial Clockworks TTO Model -1998

Circadian rhythms

C

CA

ABB

Protein Interactions

A B C

C

KaiC

B

KaiB

A

KaiA

rhythmicmRNAs

Kai proteins

kaiBCpkaiAp

Ishiura et al., 1998

Cyanobacterial Clockwork Model TRANSCRIPTIONAL CONTROL OF MANY GENES 2003

Xu et al., EMBO J. 22: 2117-2126, 2003

Kai C PHOSPHORYLATION RHYTHM

in vitro

in a SOLUTION CONTAINING only ATP with Kai A, B and C

Phase is not determined by light Nakajima et al, Science, 308: 414-415 (2005 )

IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION

In vitro phosphorylation rhythm is temperature compensated

Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

In vitro phosphorylation rhythm is temperature compensated

Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

Period of phosphorylation rhythm corresponds to mutant period

Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254

Tau of in vitro P-oscillation corresponds to mutant tau

Nakajima, M et al Kondo,T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

Simplified model for the posttranslational oscillator Nakajima, M et al Kondo,T (2005) Science 308: 414-415

EVIDENCE THAT TRANSCRIPTION and TRANSLATION are not

NECESSARY for CIRCADIAN OSCILLATIONS

IN CYANOBACTERIA Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback

in circadian rhythm of KaiC phosphorylation Science 307: 251-254

NO RHYTHM OF kaiA or kaiBC mRNAs in DD

Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254

NO RHYTHM OF KaiC PROTEIN in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254

RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254

Period Temperature Compensation of in vivo Rhythm of

KaiC Phosphorylation in DD

Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254

Model for the posttranslational oscillator coupled with TTO

Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translationfeedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254

Tau of in vitro P-oscillation corresponds to mutant tau

Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415