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HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?

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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HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?

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  1. MCB 186CIRCADIAN BIOLOGYSlides Lecture 3 Clock genes & Biochemical MechanismsOctober 5, 2005J. W. Hastings

  2. HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM? ALTER CONDITIONS (temperature, light cycle) ISOLATE MUTANTS (select for what?) APPLY INHIBITORS OR DRUGS (what kinds?)

  3. TEMPERATURE HAS ONLY SMALL EFFECT ON PERIOD

  4. DROSOPHILA PER CLOCK MUTANTS WILD TYPE ~24 h PER short ~ 18 h PER long ~ 28 h

  5. 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?

  6. 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)

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

  8. FRQ (frequency) GENE IN NEUROSPORADunlap et al

  9. Can we distinguish CLOCK GENES fromCLOCK CONTROLLED GENES?

  10. CIRCADIAN CLOCK MUTANTS IN ARABIDOPSIS IDENTIFIED BY FIREFLY LUCIFERASE REPORTING MILLER, CARRE. STRAYER, CHUA & KAY, 1995

  11. MODELING INPUT TO AND OUTPUT FROM THE CLOCK

  12. LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION

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

  14. MEASURING ALL OR ONLY SOME CULTURES

  15. BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1

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

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

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

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

  20. Screen Display–CCD Turntable Apparatus

  21. PERIOD MUTANTS OF CYANOBACTERIAKONDO ET AL.1995

  22. CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995

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

  24. MUTANTS V.S. WILD TYPE IN DIFFERENT LD CYCLES

  25. MUTANT TAU-28 V.S. WILD TYPE

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

  27. 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

  28. LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION

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

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

  31. 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

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

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

  34. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

  36. CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

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

  38. Cyanobacterial Clockwork Model Xu et al., EMBO J. 22: 2117-2126, 2003

  39. 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

  40. 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

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

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

  43. 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

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

  45. 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

  46. 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

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

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

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

  50. 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

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