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WHAT DO YOU DO WITH A MUTANT?

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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WHAT DO YOU DO WITH A MUTANT?

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  1. MCB 186CIRCADIAN BIOLOGYThe cellular-molecular mechanism of the circadian clockCLOCK MUTANTSLecture #4 October 18, 2006J. W. Hastings

  2. 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?

  3. The first CLOCK MUTANTS: the DROSOPHILA period (per) GENE WILD TYPE ~24 h PER short ~ 18 h PER long ~ 28 h

  4. 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)

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

  6. FRQ (frequency) GENE IN NEUROSPORADunlap et al

  7. CIRCADIAN CLOCK BIOCHEMICAL FEEDBACK CYCLE-NEUROSPORADUNLAP, 1999

  8. LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA CROSTHWAITE, LOROS & DUNLAP, 1995

  9. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

  10. CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

  11. The cyanobacterial clock Kai genes

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

  13. Cyanobacterial in vivo Rhythm with Bacterial Luciferase as a Reporter Luminescence Kondo, Johnson Golden et al., 1993, PNAS

  14. DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993

  15. BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1

  16. BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE Day phase Night phase Code numbers

  17. 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

  18. CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,1995

  19. CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,95

  20. PERIOD MUTANTS OF CYANOBACTERIAKONDO ET AL.1995

  21. CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995

  22. PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONESKONDO ET AL.1995

  23. 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

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

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

  26. EXPRESSION and ROLES of KAI genes

  27. Cyanobacterial Clockworks Model -1998 Ishiura et al 1998 Science 281: 1519-1523

  28. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

  29. 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

  30. 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

  31. NO RHYTHM OF KaiC PROTEIN in DDTomita, Nakajima, Kondo & Iwasaki (2005)Science 307: 251-254

  32. RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DDTomita, Nakajima, Kondo & Iwasaki (2005)Science 307: 251-254

  33. 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

  34. Period of phosphorylation rhythm corresponds to mutant periodTomita, Nakajima, Kondo & Iwasaki (2005)Science 307: 251-254

  35. 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

  36. 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

  37. IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

  38. In vitro phosphorylation rhythm is temperature compensated Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

  39. In vitro phosphorylation rhythm is temperature compensated Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415

  40. 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

  41. 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

  42. HOW THE TWO COMPONENTS MAY RELATE

  43. LBP mRNA DOES NOT CYCLE IN GONYAULAX

  44. THE SINGLE CELLED ALGA ACETABULARIA MEDITERRANEA

  45. 2 ACETABULARIA RHYTHMS:O2 EVOL & CHLOROPLAST MOVEMENTSCHWEIGER ET AL, 1981

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

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