MCB 186CIRCADIAN BIOLOGY

Slides Lecture 3 Clock genes & Biochemical Mechanisms

October 5, 2005

J. W. Hastings

HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?

ALTER CONDITIONS (temperature, light cycle)ISOLATE MUTANTS (select for what?)

APPLY INHIBITORS OR DRUGS (what kinds?)

TEMPERATURE HAS ONLY SMALL EFFECT ON PERIOD

DROSOPHILA PER CLOCK MUTANTS

WILD TYPE~24 h

PER short~ 18 h

PER long~ 28 h

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 GENES

1) DROSOPHILAper (PERIOD)tim (TIMELESS)

2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)

3) CYANOBACTERIAkai (CYCLE IN JAPANESE)

4) ARABIDOPSIStoc1 (TIMING OF CAB)lhy (LATE ELONG HYPOCOTYL)cca1 (CIRC CLOCK ASSOCIATED)

5) MOUSEclk (CLOCK)per1 (PERIOD)

6) HAMSTERtau (PERIOD)

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

movie courtesy of Van Gooch

FRQ (frequency) GENE IN NEUROSPORADunlap et al

Can we distinguish

CLOCK GENES from

CLOCK CONTROLLED GENES?

CIRCADIAN CLOCK MUTANTS IN ARABIDOPSIS IDENTIFIED BY FIREFLY LUCIFERASE REPORTING

MILLER, CARRE. STRAYER, CHUA & KAY, 1995

MODELING INPUT TO AND OUTPUT FROM THE CLOCK

LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION

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 180228

192

204

10000

48

Lum

ines

cenc

e

Kondo, Johnson Golden et al., 1993, PNAS

MEASURING ALL OR ONLY SOME CULTURES

BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1

DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993

LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA

CROSTHWAITE, LOROS & DUNLAP, 1995

BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE

Dayphase

Nightphase

Codenumbers

• Turntable Screening Apparatus: 12 positions for petri dishes or titer plates

Screen Display–CCD Turntable Apparatus

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

MUTANTS V.S. WILD TYPE IN DIFFERENT LD CYCLES

MUTANT TAU-28 V.S. WILD TYPE

SURVIVAL CURVE FOR FLIES KEPT IN LIGHT/DARK CYCLES WITH PERIODS 20, 24 AND 28 HOURS

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

LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION

CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,1995

CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,95

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

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

ClockProtein

ClockProtein

| |P P

ClockGene

ClockmRNA

PositiveRegulators

Other ClockProteins

| |P P

ATP

Figure 1b

COMMON ELEMENTS IN THE DESIGN OF CORE CIRCADIAN OSCILLATORS DUNLAP, 1999

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

Cyanobacterial Clockworks Model -1998

Circadian rhythms

CC

A

ABB

Protein Interactions

A B C

C

KaiC

BKaiB

A

KaiA

rhythmicmRNAs

Kai proteins

kaiBCpkaiAp

Ishiura et al 1998 Science 281: 1519-1523

Cyanobacterial Clockwork Model

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

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

Model for the posttranslational oscillator coupled with TTO

Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin 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 In vitro Science 308: 414-415

IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION

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

HOW THE TWO COMPONENTS MAY RELATE

THE 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