1. MCB 186 CIRCADIAN BIOLOGY Slides Lecture 3 Clock genes & Biochemical Mechanisms October 5, 2005 J. 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 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 )

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

8. FRQ (frequency) GENE IN NEUROSPORA Dunlap et al

9. Can we distinguish CLOCK GENES from CLOCK 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-COMPENSATED Q 10 ~ 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 CYANOBACTERIA KONDO ET AL.1995

22. CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995

23. PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONES KONDO 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 ISOLATED ALL EXHIBITED CIRCADIAN RHYTHMS ACROPHASES 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 OSCILLATIONS IN CYANOBACTERIA Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback in 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 DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254

42. RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DD Tomita, 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 period Tomita, 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

51. 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

52. HOW THE TWO COMPONENTS MAY RELATE

53. THE ALGA ACETABULARIA MEDITERRANEA

54. 2 ACETABULARIA RHYTHMS:O 2 EVOL & CHLOROPLAST MOVEMENT SCHWEIGER ET AL, 1981

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