1. MCB 186 CIRCADIAN BIOLOGY The cellular-molecular mechanism of the circadian clock CLOCK MUTANTS Lecture #4 October 18, 2006 J. W. Hastings

2. 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? CIRCADIAN CLOCK MUTANTS WHAT PHENOTYPES to SELECT?

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 1) DROSOPHILA per ( PERIOD ) tim ( TIMELESS ) 2) NEUROSPORA frq ( FREQUENCY ) prd ( PERIOD ) 3) CYANOBACTERIA kai ( CYCLE IN JAPANESE ) 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)

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

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

7. CIRCADIAN CLOCK BIOCHEMICAL FEEDBACK CYCLE-NEUROSPORA DUNLAP, 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-COMPENSATED Q 10 ~ 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 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

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

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

20. PERIOD MUTANTS OF CYANOBACTERIA KONDO ET AL.1995

21. CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995

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

32. RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DD Tomita, 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 period Tomita, 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:O 2 EVOL & CHLOROPLAST MOVEMENT SCHWEIGER ET AL, 1981

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