TEMPORAL REGULATION IN CELLULAR PROCESSES 235 



and the time in the cycle when it is administered (Hastings and 

 Sweeney, 1960). 



The action of siicli a light pulse is presumably mediated via a 

 photochemical reaction, and our experiments were designed to at- 

 tempt to mimic this photochemical reaction in one way or another. 

 That is, instead of giving a pulse of light, miglit it not be possible 

 to obtain a phase shift bv exposing the cells for a relatively brief 

 period of time to an inhibitor or metabolite? Might it not be pos- 

 sible to inhibit (for a short while) the processes involved in the 

 rhythm and thereby see a phase shift? In this respect our proce- 

 dure differs from previous inliibitor studies, which have been gen- 

 erally carried out by adding an inhibitor but never removing it. 



The procedure and method of presenting the results are evident 

 from Fig. 9-5. A single vial, such as an uncentrifuged control shown 

 at the bottom, is traced along the horizontal. Having previously 

 been on a 12 hours light-12 hours dark schedule, the maximum glow 

 was just at the end of the dark period. The vial went into dim light 

 at 6 A.M. of the first day shown, and glow peaks occurred approxi- 

 mately every 24 hours thereafter, as indicated by the triangles. The 

 vertical lines are drawn through the time at which control peaks 

 occurred, so that phase shift in other vials, shown on other horizon- 

 tal lines, may be ascertained by noting the distance of the glow 

 peaks from the lines. 



( It should be noted here that we always do both centrif uged and 

 uncentrifuged controls and always include more than are illustrated. 

 Also, we have always continued the measurements for five or six 

 periods, but for illustrative purposes we have not shown them all. ) 



Seven-hour light pulses given at different times of day are illus- 

 trated. The most effective one starts about 10 a.m. and goes to 

 5 P.M.; the bars on the horizontal line indicate when they were ex- 

 posed. The phase shifting is evident from the glow peak. In all 

 subsequent experiments we have included a light-induced phase- 

 shift control. 



In the experiments illustrated at the top of Fig. 9-5, we have used 

 5'-fluoro-3'-deoxyuridine (FUDR). As with the other compounds 

 tested, we have given pulsed exposures; that is, we have added the 

 compound at one time of day and 8 or 9 hours later have centrifuged 

 and resuspended the cells in fresh medium. In order not to overlook 

 the possibility that the cell is sensitive at one time in the cycle but 

 not at another, we have staggered these treatments to cover the full 



