MCB 186CIRCADIAN BIOLOGY

The cellular-molecular mechanism of

the circadian clock CLOCK MUTANTS

Lecture #4 October 18, 2006J. W. Hastings

WHAT DO YOU DO WITH A MUTANT?

Isolate the gene responsible.

How do you map a gene? Clone the gene? How do you find out what the

gene does?

CIRCADIAN CLOCK MUTANTS

WHAT PHENOTYPES to SELECT?

The first CLOCK MUTANTS: the DROSOPHILA period (per) GENE

WILD TYPE~24 h

PER short~ 18 h

PER long~ 28 h

CIRCADIAN CLOCK GENES

1) DROSOPHILAper (PERIOD)tim (TIMELESS)

2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)

3) CYANOBACTERIAkai (CYCLE IN JAPANESE)

4) HAMSTERtau (PERIOD)

5) MOUSEclk (CLOCK)per1 (PERIOD)

6) ARABIDOPSIS (CCGs)toc1 (TIMING OF CAB)

lhy (LATE ELONG HYPOCOTYL)

cca1 (CIRC CLOCK ASSOCIATED)

The clock controls an overt rhythm in developmental potential in Neurospora.

movie courtesy of Van Gooch

FRQ (frequency) GENE IN NEUROSPORADunlap et al

CIRCADIAN CLOCK BIOCHEMICAL FEEDBACK CYCLE-NEUROSPORADUNLAP, 1999

LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA

CROSTHWAITE, LOROS & DUNLAP, 1995

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

ClockProtein

ClockProtein

| |P P

ClockGene

ClockmRNA

PositiveRegulators

Other ClockProteins

| |P P

ATP

Figure 1b

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

The cyanobacterial clock

Kai genes

LUCIFERASE as a REPORTER for CIRCADIAN GENE EXPRESSION: INSERTED DOWNSTREAM from a

PROMOTER

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

DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993

BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1

BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE

Dayphase

Nightphase

Codenumbers

LUCIFERASE REPORTER RANDOMLY INSERTED IN FRONT OF PROMOTERS IN

BACTERIA.

ABOUT 800 LUMINOUS COLONIES ISOLATED

ALL EXHIBITED CIRCADIAN RHYTHMS

ACROPHASES NOT ALL THE SAME

Liu et al., Circadian orchestration of gene expression in Cyanobacteria. (1995) Genes & Develop. 9: 1469-1478

CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,1995

CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,95

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

KAI genes in SYNECHOCOCCUS and MUTANT MAPPING Ishiura et al 1998 Science 281: 1519-1523

All of ~200 mutantsisolated map to thisgene cluster

PHENOTYPES OF KAI GENE INACTIVATIONS Ishiura et al 1998 Science 281: 1519-1523

EXPRESSION OF KAI GENE CONSTRUCTS Ishiura et al 1998 Science 281: 1519-1523

EXPRESSION and ROLES of KAI genes

Cyanobacterial Clockworks 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 Science 281: 1519-1523

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

ClockProtein

ClockProtein

| |P P

ClockGene

ClockmRNA

PositiveRegulators

Other ClockProteins

| |P P

ATP

Figure 1b

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

Period of phosphorylation rhythm corresponds to mutant period

Tomita, Nakajima, Kondo & Iwasaki (2005) 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

RHYTHM of Kai C PHOSPHORYLATION in a SOLUTION CONTAINING

only ATP plus Kai A, B and C

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

IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION

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

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 in vitro P-oscillation corresponds to mutant period

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

Period of in vitro P-oscillation corresponds to mutant period

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

HOW THE TWO COMPONENTS MAY RELATE

LBP mRNA DOES NOT CYCLE IN GONYAULAX

THE SINGLE CELLED ALGA ACETABULARIA MEDITERRANEA

2 ACETABULARIA RHYTHMS:O2 EVOL & CHLOROPLAST MOVEMENTSCHWEIGER ET AL, 1981

NUCLEUS IS IN ROOT- RHYTHM CONTINUES WHEN CUT OFFBUT A NEW NUCLEUS GRAFTED ON CONFERS ITS PHASE TO HOST