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MCB 186 CIRCADIAN BIOLOGY The cellular-molecular mechanism of the circadian clock CLOCK MUTANTS Lecture #4 October 18, 2006 J. W. Hastings. CIRCADIAN CLOCK MUTANTS WHAT PHENOTYPES to SELECT?. WHAT DO YOU DO WITH A MUTANT?. Isolate the gene responsible.
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MCB 186CIRCADIAN BIOLOGYThe cellular-molecular mechanism of the circadian clockCLOCK MUTANTSLecture #4 October 18, 2006J. W. Hastings
CIRCADIAN CLOCK MUTANTS WHAT PHENOTYPES to SELECT? 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?
The first CLOCK MUTANTS: the DROSOPHILA period (per) GENE WILD TYPE ~24 h PER short ~ 18 h PER long ~ 28 h
CIRCADIAN CLOCK GENES 4) HAMSTER tau (PERIOD) 5) MOUSE clk (CLOCK) per1 (PERIOD) 6) ARABIDOPSIS (CCGs) toc1 (TIMING OF CAB) lhy (LATE ELONG HYPOCOTYL) cca1 (CIRC CLOCK ASSOCIATED) 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 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
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 Luminescence Kondo, Johnson Golden et al., 1993, PNAS
BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE Day phase Night phase Code numbers
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
PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONESKONDO ET AL.1995
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
Cyanobacterial Clockworks Model -1998 Ishiura et al 1998 Science 281: 1519-1523
POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION
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 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 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
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