MCB 186 CIRCADIAN 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) DROSOPHILA per ( PERIOD ) tim ( TIMELESS ) 2) NEUROSPORA frq ( FREQUENCY ) prd ( PERIOD ) 3) CYANOBACTERIA kai ( CYCLE IN JAPANESE ) 4) ARABIDOPSIS toc1 (TIMING OF CAB) lhy (LATE ELONG HYPOCOTYL) cca1 (CIRC CLOCK ASSOCIATED) 5) MOUSE clk ( CLOCK ) per1 ( PERIOD ) 6) HAMSTER tau ( PERIOD )

The clock controls an overt rhythm in developmental potential in Neurospora. movie courtesy of Van Gooch

FRQ (frequency) GENE IN NEUROSPORA Dunlap 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 Luminescence Kondo, Johnson Golden et al., 1993, PNAS

MEASURING ALL OR ONLY SOME CULTURES

BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATED Q 10 ~ 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 Day phase Night phase Code numbers

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:

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: All of ~200 mutants isolated map to this gene cluster

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

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

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

Cyanobacterial Clockworks Model Ishiura et al 1998 Science 281:

Cyanobacterial Clockwork Model Xu et al., EMBO J. 22: , 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:

NO RHYTHM OF kaiA or kaiBC mRNAs in DD Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback in circadian rhythm of KaiC phosphorylation Science 307:

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

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

Period Temperature Compensation of in vivo Rhythm of KaiC Phosphorylation in DD Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback in circadian rhythm of KaiC phosphorylation Science 307:

Period of phosphorylation rhythm corresponds to mutant period Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307:

Model for the posttranslational oscillator coupled with TTO Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback in circadian rhythm of KaiC phosphorylation Science 307:

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:

IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION Nakajima, M etal Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308:

In vitro phosphorylation rhythm is temperature compensated Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308:

In vitro phosphorylation rhythm is temperature compensated Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308:

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:

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:

HOW THE TWO COMPONENTS MAY RELATE

THE ALGA ACETABULARIA MEDITERRANEA

2 ACETABULARIA RHYTHMS:O 2 EVOL & CHLOROPLAST MOVEMENT SCHWEIGER ET AL, 1981

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