Diurnal rhythms or circadian rhythms are biological rhythms which are present in eukaryotes, bacteria, etc. that regulate biological processes in a 24-hour cycle which responds to light and darkness in an organism's environment. Circadian system ensures adaptation to daily environmental changes and functions on different levels-from gene expression to behaviour (Roenneberg and Merrow, 2005). The rhythms are generated by natural factors inside the body, however, they are also affected by indicators from surroundings. The major cue influencing the rhythms is light which turns on and off genes which controls internal clocks. Circadian clocks provide temporal coordination by synchronizing internal biological processes with daily environmental cycles (Xiaodong, et al., 2010). All circadian clocks have one autonomous circadian oscillator at least at their core. These oscillators consist of elements which are positive and negative and forms autoregulatory feedback loops. Circadian clock is constituted of transcriptional/translational feedback loops. Circadian system contains a biological clock located in suprachiasmatic nucleus (SCN) which is present in the hypothalamus. SCN contains 20,000 neurons and controls the rhythms. It controls the production of a hormone called melatonin that makes an individual sleepy. Melatonin increases during darkness while decreases during the day. Since SCN is located above the optical nerves it obtains information about arriving light. The information is transmitted by the optic nerves from the retina to the brain. When there is darkness the SCN informs the brain to make more melatonin.
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By mutagenesis the first circadian clock mutant discovered which was per. Mammalian circadian oscillator requires the activity of several components: negative elements period 1 and 2 (PER1 and PER2) and cryptochrome 1 and 2 (CRY1 and CRY2) and the positive-acting proteins CLOCK and BMAL1 (Pedersen, et al., 2005). Genetic information which are encoded in these genes are converted to mRNA which are transported to the cytoplasm. The mRNA then generates protein products which are encoded by circadian clock genes. In mammals, there are two loops; the first loop includes per while the second bmal1as seen in figure 1. clk and bmal1 are transcription factors that regulate the expression of per and cry. per and cry genes is driven by accumulating CLOCK and BMAL1 heterodimers, which in turn bind to consensus E-box elements (Xiang, et al., 2008). CLOCK:BMAL1 controls a gene called Wee-1, which consists of three E boxes in its promoter. It encodes a protein kinase which phosphorylates leading to inactivation of CDC2/Cyclin B1 complex. This hold-up or averts entrance into mitosis. Mutations in kinases modifies normal rhythmicity.
The dimers of CLOCK protein and BMAL1 transcription factors turn on the light induced promoters. clk and bmal1 are inhibited by the products in a negative feedback loop. In SCN cells per gene expression is started by darkness. In the cytoplasm PER1 and PER2 are phosphorylated by CKIε protein. PER and CRY mRNAS are translated in the cytoplasm where then the proteins enter the nucleus. The 24-hour cycle completes as the BMAL1 and clock proteins provoke increased production of Per and Cry proteins. CRY and PER then create a heterodimer which is translocated into the nucleus and inhibits their own genes being transcripted and are then degraded in proteasomes leading to a decline in protein levels. CLK/BMAL1 embody the transcription activator of per and cry promoter. As levels of PER and CRY decrease, CLOCK and BMAL1 will dimerize mPer (murine Pers1-3) and mCry genes. During night, transcription of genes per1 and cry2 increases. However, in constant darkness the expression of per3 and cry1 genes dampens then eradicates. CK1Ñ” is a protein that by destabilizing per protein helps to regulate clock protein levels. By binding ROREs REV-ERBα represses bmal1 transcription. A mutation in the human per2 gene changes one amino acid in a protein which makes a regulatory phosphate not able to get attached to a kinase. This leads to advanced sleeping patterns. A gene, TIM, was identified in mammals, however, its function is not yet known.
Figure 1 : Proposed model for interdependent feedback loops in mammals circadian loops (Berger, 2004)
In plants, the circadian clock directs processes such as flowering, photosynthesis, etc. Mutants of Arabdiposis circadian clock indicated that there are three feedback loops and two Myb domain transcription factors, circadian clock associated (CCA1) and late elongated hypocotyls (LHY). Timing of cab1 (TOC1) closes one loop, while three TOC1 paralogs, PRR5, PRR7, and PRR9, close a second loop (Clung, 2006). Overexpression of PRR5 causes repression of PRR7 and PRR9, suggesting an antagonistic role for PRR5 in the regulation of morningphased genes (Jones, 2009). The third loop is lux arrythmo (LUX). All three genes encode a Myb transcription factor. Mutation on TOC1 affects numerous circadian outputs. Overexpression of CCA and LHY leads to decreased levels of transcripts which signifies they are involved in negative feedback loops. Plants perceive light with proteins cryptochromes and phytochromes which are involved in setting the clock. SPA1, a protein, regulates a phytochrome PhyA. Light detection with circadian clock system is connected by this protein. Circadian clock regulates the gene encoding elements of light harvesting chlorophyll a/b complex (LHC). LHC transcription and its protein synthesis occur during noon. Towards the end of the night, the positive element, TOC1 is involved in inducing the expression of CCA1 and LHY (Yakir, 2006). CCA1 and LHY encode transcription factors which is phosphorylated and bind the TOC1 promoter. TOC1 expression increases when it reaches evening time whereby CCA1 and LHY levels drop, therefore feedback loop is completed. Such an arrangement is likely to be important for conferring stability to the oscillator and is part of the mechanism ensuring that the circadian system is able to function accurately under a range of environmental conditions (Locke, et al., 2005).
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More recently, additional genes, including early flowering 4 (ELF4), gigantean (GI) and LUX and feedback loops have been identified suggesting that the oscillator is more complex and may be composed of several interlocking feedback loops (Paltiel, et al., 2006). ELF4 is a positive regulator of CCA1. ELF4 is a core-clock gene working in the evening phase of the circadian cycle to drive morning expression of CCA1 and LHY. A seize of the elf4 oscillator after one cycle can be observed since elf4 loss-of-function mutant has low CCA1 expression. Mutants are found to influence rhythms. For plants held in the light, elf3-1, causes arrhythmicity however, it does not occur in plants held in dark. Independent of light input tej, a mutant, causes a rise in period length. ztl, a mutant, has a light-dependent functionality (Kim, et al., 2007). gi-2, concurrently elongates a few clock output whilst shortening some. CRY1/CRY2 double mutants demonstrate vigorous rhytmicity which specifys that cryptochromes in plants do not play an oscillator role as it is in animals. Mutations in TOC1, independent of light input, causes a short-period phenotype PIF3, a light-signalling element, binds to promoters of CCA1 and LHY which indicates playing a role in the resetting clock of light-mediation since it.
Chemical agents were used to introduce mutations in fungi Neurospora crassa, which lead to the identification of the main gene frequency (frq). In negative feedback loop, a complex of FRQ and FRH (FFC) forms the negative limb of the loop, whereas two PER-ARNT-SIM (PAS) domain-containing transcription factors, WC-1 and WC-2, are the positive elements (Cheng, et al., 2005). WC-1 and WC-2 bind to frq promoter and are accountable for the increase in frq transcription. Any mutations in frq, wc-1 or wc-2 can change entrainment, period or temperature compensation. WC-1 and WC-2 in darkness form a heterodimeric complex (D-WCC). D-WCC binds into frq promoter to the C-box (Clock box) which leads to the activation of frq transcription. In the late day frq mRNA reaches maximum, whereas FRQ protein levels do not until some hours later. FRQ protein dimerizes with itself after it has been synthesised and forms a complex with FRH. Around midday, there is a decrease in nuclear frq levels which is due to FFC inhibiting D-WCC activity. FRQ is phosphorylated by numerous kinases and by two phosphatises it is dephosphorylated. When FRQ becomes extensively phosphorylated, it interacts with FWD-1, an F box/WD-40 repeat-containing protein and the substrate-recruiting subunit of an SCF-type ubiquitin ligase complex, resulting in the ubiquitination and degradation of FRQ by the proteasome system (Liu and Pedersen, 2006). FRQ levels fall at darkness and when the level drops below a certain threshold, FFC no longer inhibits D-WCC. Late at night the cycle repeats after frq transcription is reactivated.
In conclusion, circadian system plays a major role in eukaryotes. However, even though there are substantial advances in understanding the molecular and cellular foundation of circadian rhythms, research still continues as mechanisms to control rhythms are yet to unfold. Challenges are faced such as to find the definite mechanism of how cryptochrome works, elements that permits communication between oscillators, rhythm gene expression in embryos, why CRY is engaged in light detection in plants while in animals it is more concerned in the operational of the clock.