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LIMITS OF ENTRAINMENT

MCB 186 CIRCADIAN BIOLOGY Lecture 4 Drugs as probes of mechanism: Phase shifts v.s. effects on period And some basic questions October 12, 2005 J. W. Hastings. LIMITS OF ENTRAINMENT. HOW do you SPECIFY the LIMITS? ARE there EFFECTS OUTSIDE the LIMITS?.

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LIMITS OF ENTRAINMENT

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  1. MCB 186CIRCADIAN BIOLOGY Lecture 4 Drugs as probes of mechanism: Phase shifts v.s. effects on periodAnd some basic questions October 12, 2005J. W. Hastings

  2. LIMITS OF ENTRAINMENT HOW do you SPECIFY the LIMITS? ARE there EFFECTS OUTSIDE the LIMITS?

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

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

  5. MEASURING ALL OR ONLY SOME CULTURES

  6. EFFECT OF NOT MEASURING (- - - -) ON PERIOD

  7. EFFECT OF NOT MEASURING (- - - -) ON PERIOD

  8. CLOCK MUTANTS REVEAL GENES REGULATING CIRCADIAN RHYTHMS Many but not all exhibit rhythms in expression of mRNA and protein Positive elements and negative feedback result in oscillation Not established how other systems are controlled (CCGs)

  9. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

  11. CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

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

  13. CCGs in Gonyaulax are CONTROLLED by RNA (translation not transcription) mRNA levels remain constant while protein levels exhibit rhythms Synthesis of many proteins is rhythmic

  14. LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL

  15. WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL

  16. SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLED IN VIVO PULSE LABELING MILOS et al, 1989

  17. GONYAULAX CIRCADIAN PULSED PROTEIN SYNTHESIS

  18. LBP mRNA DOES NOT CYCLE IN GONYAULAX

  19. A NOVEL SEQUENCE in the LBP 3’ UTR BINDS a PROTEIN

  20. AN RNA-PROTEIN BASED FEEDBACK CLOCKCLOCK PROTEINS V.S. CLOCK CONTROLLED PROTEINS

  21. MICROARRAY ANALYSIS of EXPRESSION of ~3000 DINOFLAGELLATE GENES at TWO CIRCADIAN TIMES

  22. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CELLULAR PROCESSESPROTEIN synthesis-phase shifts-as pulses PROTEIN phosphorylation- period changes-as continuous

  23. EFFECT OF ACTINOMYCIN D (RNA synthesis) ON RHYTHM KARAKASHIAN

  24. EFFECT OF PROTEIN SYNTHESIS INHIBITORS ON RHYTHM KARAKASHIAN

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

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

  27. VERY BRIEF ANISOMYCIN PULSES CAUSE LARGE PHASE SHIFTS

  28. TYPE 1 & 0 DRCs FOR BRIEF ANISOMYCIN PULSES

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

  30. DRUG PRCs in GONYAULAX are DOSE DEPENDENT

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

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

  33. 6-DMAP (KINASE INHIBITOR) INCREASES Tau

  34. 6_DMAP (KINASE INHIB) INCREASES Tau

  35. 6_DMAP (Kinase Inhibitor) INCREASES Tau

  36. NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI

  37. STAUROSPORINE (kinase inhibitor) INCREASES Tau

  38. EFFECTS OF KINASE INHIBITORS ON PERIOD

  39. 6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

  40. STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

  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. LOSS OF RHYTHMICITYSeveral conditions, notably bright light and low temperature, lead to the loss of rhythm; has the clock stopped or is it simply not seen? Return to initial conditions results in a reappearance of rhythm at a fixed phase, CT12, independent of when the return occurs

  47. EFFECT of WHITE LIGHT INTENSITY on PERIOD and AMPLITUDE in Gonyaulax 120 fc 380 fc 680 fc

  48. EFFECT of WHITE LIGHT INTENSITYon PERIOD in Gonyaulax

  49. JCCP 1957 Fig 3 After an extended period in bright LL, with no detectable bioluminescence rhythm, transfer to DD initiates a rhythm. The phase is determined by the time of transfer, as if the clock had stopped.

  50. RHYTHM in Gonyaulax INITIATED by SHIFT from LL to DD is PHASED STARTING at CT 12

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