1. MCB 186 CIRCADIAN BIOLOGY Biochemistry of the Circadian Clock Lecture #3 October 3, 2007 J. W. Hastings

2. MODELING INPUT to and OUTPUT from THE CLOCK

3. THIS IS ONLY A MODEL DIFFERENT SYSTEMS MAY DIFFER: bacteria, plants, algae, fungi, animals and, IT MAY BE INCORRECT e.g., THERE MAY BE TWO CLOCKS or THREE or MORE

4. DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING Internnal Desynchronization

5. THREE RHYTHMS SIMULTANEOUSLY: PHASE-JUMPS ROENNEBERG & MORSE 1993 Glo FlsAgg

6. INPUT to and OUTPUT from a TWO-CLOCK MODEL

7. CORE PACEMAKER OSCILLATOR BIOCHEMICAL ELEMENTS of the CLOCK AFFECTED by SIGNAL TRANSDUCTION CLOCK GENES vs CLOCK CONTROLLED CLOCK PROTEINS vs CLOCK CONTROLLED

8. INPUT PATHWAYS SIGNAL TRANSDUCTION MANY FACTORS AFFECT THE CLOCK EFFECTS on PHASE and PERIOD DISTINCT BIOCHEMICAL PATHWAYS UNKNOWN

9. OUTPUT PATHWAYS HOW DOES the CLOCK TURN PROCESSES ON and OFF? TRANSCRIPTION: NEW mRNA, then protein TRANSLATION: REGULATE PROTEIN SYNTHEIS POST TRANSLATIONAL e.g. PHOSPHORYLATION

10. LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL Johnson et al.1984 Science 223 Western Blot

11. WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL A CLOCK CONTROLLED GENE Morse et al., 1989 PNAS 86

12. GONYAULAX CELLS AT NIGHT (LEFT) AND DAY PHASES FLUORESCENCE OF LUCIFERIN IN SCINTILLONS

13. LBP mRNA DOES NOT CYCLE in Gonyaulax LBP SYNTHESIS & ABUNDANCE are STRONGLY CIRCADIAN Morse et al., 1989 PNAS 86 LBP abundance LBP synthesis LBP mRNA

14. mRNA LEVELS ARE CONSTANT

15. SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLED In Vivo PULSE LABELING MILOS et al, 1989 MILOS ET AL, 1989 Naturwisenschaften 77

16. SYNTHESIS of PROTEINS in vitro is NOT CLOCK CONTROLLED MILOS ET AL, 1989 Naturwisenschaften 77

17. PATTERNS of CLOCK-CONTROLLED PROTEIN SYNTHESIS in Gony Markovic et al., 1996 J. Biol. Rhythms 11 p21 unknown p32 PCP p33 OEE1 p45 GAPDH p55 Rubisco II p75 Luciferin binding protein

18. ABUNDANCE vs SYNTHESIS SYNTHESIS RATE of a PROTEIN MAY EXHIBIT PRONOUNCED RHYTHM while RHYTHM in the ABUNDANCE of PROTEIN does NOT ABUNDANCE RHYTHM DEPENDS on STABILITY OF MOLECULE

19. GAPDH SYNTHESIS, ACTIVITY & ABUNDANCE RHYTHMS Fagan, Morse & Hastings, 1999

20. HALF-LIFE of PROTEIN AFFECTS AMPLITUDE of ABUNDANCE RHYTHM 2 days 12 hr

22. IS THERE a CORE CIRCADIAN OSCILLATOR? If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL CLOCK COMPONENTS? SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS

23. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRY PROTEIN synthesis inhibitors Pulses cause phase shifts PROTEIN phosphorylation inhibitors Chronically cause period changes

24. PULSES of ANISOMYCIN (protein synthesis inhibitor) CAUSE PHASE SHIFTS in Gonyaulax

25. PHASE SHIFTS BY ANISOMYCIN 0.3  M, 1 HOUR

26. VERY BRIEF ANISOMYCIN PULSES CAUSE LARGE PHASE SHIFTS

27. TYPE 1 & 0 DRCs FOR BRIEF ANISOMYCIN PULSES

28. ARHYTHMICITY AT “CRITICAL” DOSE OF PHASE SHIFTING INHIBITOR

29. D-PRC for PHASE SHIFTS by an INHIBITOR of PROTEIN SYNTHESIS

31. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRY PROTEIN synthesis inhibitors pulses cause phase shifts PROTEIN phosphorylation inhibitors chronically cause period changes KINASES

32. 6-DMAP (KINASE INHIBITOR) INCREASES Tau

33. 6_DMAP (Kinase Inhibitor) INCREASES Tau

34. 6_DMAP (KINASE INHIB) INCREASES Tau

35. NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI and HASTINGS, 1995

36. STAUROSPORINE (kinase inhibitor) INCREASES Tau

37. EFFECTS OF KINASE INHIBITORS ON PERIOD

38. 6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

39. STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

40. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRY PROTEIN synthesis inhibitors pulses cause phase shifts PROTEIN phosphorylation inhibitors chronically cause period changes PROTEIN PHOSPHATASES

41. EFFECT of OKADAIC ACID (Protein phosphatase inhibitor) on CIRCADIAN BIOLUMINESCENCE RHYTHM

42. PERIOD EFFECTS of PROTEIN PHOSPHATASE INHIBITORS

43. EFFECTS OF OKADAIC ACID AND CALYCULIN ON THE LIGHT PRC

44. EFFECT OF CREATINE (FROM DIFFERENT SOURCES) ON PERIOD

45. PRCs: LIGHT-INDUCED DELAY-PHASE SHIFTS IN an LL BACKGROUND ARE EVOKED BY CREATINE

46. IS THERE a CORE CIRCADIAN OSCILLATOR? If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL COMPONENTS? SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS

47. DROSOPHILA PERIOD GENE CLOCK MUTANTS WILD TYPE per + ARHYHMIC per o SHORT PERIOD per S LONG PERIOD per L Map location of gene Clone, sequence gene Measure mRNA Express encoded protein

48. A FEW 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 )

49. & COMPONENTS POSTULATED PATHWAYS & COMPONENTS in the REGULATION of CLOCK GENE EXPRESSION TTO TRANSCRIPTION TRANSLATION OSCILLATOR

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

51. MOLECULAR COMPONENTS of the DROSOPHILA CLOCK

52. NEUROSPORA CLOCK MUTANTS in the FREQUENCY GENE CONIDIATION RHYTHM PERIOD Short long, & arhythmic movie courtesy of Van Gooch

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

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

55. INDUCED frq in NEUROSPORA BLOCKS RHYTHM & RESETS Aronson, Johnson, Loros & Dunlap Science 1994

56. CLOCK CONTROLLED GENE & PROTEIN: GAPDH NEUROSPORA Shinohara, Loros, &Dunlap J. Biol Chem 1998

57. MOLECULAR COMPONENTS of the NEUROSPORA CLOCK

58. MOLECULAR COMPONENTS of the MOUSE CLOCK

59. MOLECULAR COMPONENTS of the PLANT CLOCK

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

61. SINGLE CELL ACETABULARIA LIVES and EXHIBITS RHYTHM with NUCLEUS REMOVED

62. NUCLEUS IS IN ROOT- RHYTHM CONTINUES WHEN CUT OFF BUT A NEW NUCLEUS GRAFTED ON CONFERS ITS PHASE TO HOST Schweiger 1964 Science 146: 658-659

63. BACTERIAL LUCIFERASE as a REPORTER of a TEMP COMPENSATED CIRCADIAN RHYTHM in a PROKARYOTE KONDO, STRAYER,KULKARNI, TAYLOR, ISHIURA, GOLDEN & JOHNSON PNAS 1993