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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?).
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MCB 186CIRCADIAN BIOLOGYSlides Lecture 3 Clock genes & Biochemical MechanismsOctober 5, 2005J. 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?)
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 4) ARABIDOPSIS toc1 (TIMING OF CAB) lhy (LATE ELONG HYPOCOTYL) cca1 (CIRC CLOCK ASSOCIATED) 5) MOUSE clk (CLOCK) per1 (PERIOD) 6) HAMSTER tau (PERIOD) 1) DROSOPHILA per (PERIOD) tim (TIMELESS) 2) NEUROSPORA frq (FREQUENCY) prd (PERIOD) 3) CYANOBACTERIA kai (CYCLE IN JAPANESE)
The clock controls an overt rhythm in developmental potential in Neurospora. movie courtesy of Van Gooch
CIRCADIAN CLOCK MUTANTS IN ARABIDOPSIS IDENTIFIED BY FIREFLY LUCIFERASE REPORTING MILLER, CARRE. STRAYER, CHUA & KAY, 1995
Cyanobacterial in vivo Rhythm with Bacterial Luciferase as a Reporter Luminescence Kondo, Johnson Golden et al., 1993, PNAS
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
PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONESKONDO ET AL.1995
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 ISOLATEDALL EXHIBITED CIRCADIAN RHYTHMSACROPHASES NOT ALL THE SAME Liu et al., Circadian orchestration of gene expression in Cyanobacteria. (1995) Genes & Develop. 9: 1469-1478
KAI genes in SYNECHOCOCCUS and MUTANT MAPPING Ishiura et al 1998 Science 281: 1519-1523 All of ~200 mutants isolated map to this gene 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
COMMON ELEMENTS IN THE DESIGN OF CORE CIRCADIAN OSCILLATORS DUNLAP, 1999
Cyanobacterial Clockworks Model -1998 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 OSCILLATIONSIN CYANOBACTERIATomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin 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 feedback in circadian rhythm of KaiC phosphorylation Science 307: 251-254
NO RHYTHM OF KaiC PROTEIN in DDTomita, Nakajima, Kondo & Iwasaki (2005)Science 307: 251-254
RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DDTomita, 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 feedback in 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 feedback in 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