Effects of Chloramphenicol on the Circadian Rhythm of Neurospora crassa

Abstract

Chloramphenicol, an inhibitor of mitochondrial protein synthesis, shortened the period length of the circadian rhythm in the Timex strain of Neurospora crassa by 2 hours. Both the L(+) threo and D(-) threo optical isomers had the same effect on the period of the rhythm, whereas only the D(-) threo isomer significantly inhibited mitochondrial protein synthesis. Tetracycline, another inhibitor of mitochondrial protein synthesis, did not change the period of the circadian rhythm. The effect of chloramphenicol on the circadian rhythm is, therefore, presumably not directly related to inhibition of mitochondrial protein synthesis, suggesting that chloramphenicol has other effects.Circadian rhythms provide one type of eucaryotic cell regulation and have been described in a variety of organisms. Despite much work, very little is known about the biochemical basis of the "biological clock." One experimental approach has been to use chemicals and drugs to alter the rhythm. Heavy water (D20) (2), ethanol (5), lithium (4), and cycloheximide (6) have all been reported to increase the period of the rhythm in at least one organism. Many biological processes are presumably affected by these chemicals, and it is, therefore, difficult to make inferences regarding the basic biological mechanism of the circadian clock from such studies. There have been no previous reports of chemicals decreasing the period of a circadian rhythm.Circadian rhythms have not been clearly demonstrated in procaroytic organisms, although all studied eucaryotic organisms do exhibit a rhythm. Because only eucaryotes have mitochondria, there has been speculation that mitochondria have a role in circadian rhythms (1). We, therefore, studied the effect of an inhibitor of mitochondrial protein synthesis, chloramphenicol, on the period of the circadian rhythm in Neurospora crassa.There are L(+) threo and D(-) threo4 optical isomers of chloramphenicol. D(-) Chloramphenicol inhibits mitochondrial protein synthesis (9, 13, 19), but L(+) chloramphenicol does not (7). In this paper, we report that both isomers of chloramphenicol decrease the period length of the circadian rhythm in Neurospora crassa as expressed by periodic conidia formation. MATERIALS AND METHODSGrowth Conditions. For these experiments, we used the Timex strain of Neurospora crassa which was isolated and characterized by Sargent et al. (15,16). Glass tubes 60 cm long and 13 mm in diameter were half filled with 18 ml of medium containing Vogel's solution (20), 0.5 % sucrose, 0.5 % casamino acids, and 1.5 % bacto agar. A chloramphenicol isomer (max. 2 mg/ml) or tetracycline (max. 0.75 mg/ml) was added to the medium after autoclaving. The chloramphenicol isomers were added directly to the medium. Tetracycline was dissolved in sterile H20 which was then added to an equal volume of double strength medium at 50 C. The tubes were inoculated at one end with conidia from a 5-day-old culture. They were then placed in a constant temperature incubator inside a darkroom and were observed thereafter only...

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

“…2 shows that the addition of chloramphenicol did not alter protein synthesis patterns either in the presence or in the absence of CHX. This concentration of chloramphenicol has been shown to inhibit 60 to 80% of mitochondrial protein synthesis (7,21), which comprises about 2% of the total cellular protein synthesis (13). As expected, total protein synthesis was not measurably inhibited by chloramphenicol (Table 2).…”

Periodicity in Protein Metabolism: Time Patterns of Substrate Interaction and Utilization

“…18,19 By contrast, inhibitors of protein synthesis that attach exclusively to the 70s ribosome (such as chloramphenicol and tetracycline) have no effect. 13,18,20…”

Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa

“…There (2,5,8,9,14,19,22). Two problems arise with these experiments: (a) often the agent inhibits conidiation itself and thereby prevents assay of its effects on the rhythm (One example is cycloheximide [19], which has been used in studying many other circadian systems.…”