Burgeoning research into circadian rhythms has led to discoveries in many different branches of biological science.  These rhythms have been found to exist in almost every walk of life, and circadian research with the Drosophila melanogaster has allowed us to better understand not only how the insect clock works, but has also given us insight as to how our own clock ticks.  This paper will discuss some of the most recent discoveries in circadian research, and the discovery of the possible “missing molecular link” between mammals and fruit flies.

Missing Link?

In nearly every living creature, scientists have found circadian rhythms.  Occurring on approximately 24-hour cycles, Circadian rhythms act like an alarm clock, and are important biological regulators that control many different aspects of life.  For mammals this circadian clock is rewound daily by exposure to sunlight. In mammals, these regulators are located in the head.  Now researchers have discovered a biochemical pathway that senses blue light, and thereby connects the sun to molecular components of the circadian clock, says Joseph Takahashi, an HHMI investigator at Northwestern University (Wilsbacher, 1998).  In insects, two new studies of cryptochrome proteins in the fruit fly drosophila have had similar findings.  In two articles published in the November 25, 1998, issue of Cell, HHMI investigator Michael Rosbash, Jeffrey Hall and colleagues at Brandeis University, and Stephen Kay at The Scripps Research Institute, confirm that cryptochrome is an important in bringing light into the circadian system (Emery, 1998.)   In addition, Rosbash says that his laboratory's work shows that without cryptochrome, "the circadian system is largely, though not entirely, dead."
 Rosbash and his colleagues first characterized the fruit fly gene, cry, and found that it is closely related to the circadian system genes, period, timeless, clock, and cycle of the Drosophila.  They then created strains of mutated Drosophila with a disabled cryptochrome gene.  These mutants lacked the normal daily oscillations in the tim and per proteins.  Nonetheless, the circadian locomotor activity remained relatively normal.  When a second mutant was formed; however with eyes that were unresponsive to light the fly exhibited no behavioral rhythms.  (Rutila, 1998)
 

What makes them tick?

Since the first discovery of circadian rhythms, scientists have wondered what makes them tick.  In the last three to four years, many discoveries in this area have been made.
One of the first of these discoveries, and one that is key to under standing  the Circadian clock is the PERIOD or (per) cycle.  “The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract.”  (Rosato,1996) Similar activity has been seen in both plants and animals, but their circadian functions are unknown.  In fruit flies, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids.  To test the coevolution hypothesis, several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster were generated, whose repetitive regions differed in length by about 150 amino acids. “The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period.”(Rosato,1996)  In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. “These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock's temperature compensation.” (Rosato, 1996)

Where heat is an important environmental factor in the regulation of per, light is also an important stimuli, and has major effects on the circadian clock.   According to Wilsbacher and Takahashi: “Light is a major environmental signal for circadian rhythms.”  Together they have identified and analyzed cry, a novel Drosophila cryptochrome gene, and have found that “All characterized family members are directly photosensitive, and include plant blue light photoreceptors. We show that cry transcription is under circadian regulation, influenced by the Drosophila clock genes period, timeless, Clock, and cycle.”  They have also shown cry protein levels are dramatically affected by light exposure because Circadian photosensitivity is increased in a cry-overexpressing strain. “These physiological and genetic data therefore link a specific photoreceptor molecule to circadian rhythmicity.  We propose that CRY is a major Drosophila photoreceptor dedicated to the resetting of circadian rhythms.”  (Emery, 1998)

In a study by Sidote, Majercak, Parith, and Edery, it was found that “Circadian rhythms are governed by endogenous biochemical oscillators (clocks) that in a wide variety of organisms can be phase shifted (i.e., delayed or advanced) by brief exposure to light and changes in temperature” (1998).  However, how these changes in temperature reset circadian timekeeping mechanisms is not known.  The researches began to try and attempt to explain this by measuring the effects of short-duration heat pulses on the protein and mRNA products from the Drosophila circadian clock genes period (per) and timeless (tim). Throughout the daily cycle, heat pulses elicited dramatic and rapid decreases in the levels of PER and TIM proteins. PER is sensitive to heat but not light, this indicates that individual clock components can markedly differ in sensitivity to environmental stimuli. Delays in the per-tim transcriptional-translational feedback loop, involve a similar resetting mechanism, which likely underlies the observation that when heat and light signals are administered in the early night, they both evoke phase delays in behavioral rhythms. Where earlier studies have shown that the light-induced degradation of TIM in the late night is accompanied by stable phase advances in the temporal regulation of the PER and TIM biochemical rhythms, the heat-induced degradation of PER and TIM, at these times in a daily cycle results in little, if any, long-term perturbation in the cycles of these clock proteins.  “Rather, the initial heat-induced degradation of PER and TIM in the late night is followed by a transient and rapid increase in the speed of the PER-TIM temporal program.”(Sidote, 1998)  The entire effect of which is similar to that of the undisturbed control group.  “These findings can account for the lack of apparent steady-state shifts in Drosophila behavioral rhythms by heat pulses applied in the late night and strongly suggest that stimulus-induced changes in the speed of circadian clocks can contribute to phase-shifting responses.”  (Sidote, 1998)

Quite a bit of work has gone on in Paul Hardin’s laboratory focusing on understanding the molecular circuitry that underlies circadian clock function in the fruit fly, Drosophila melanogaster. “Like other organisms, these flies display daily rhythms in molecular, physiological and behavioral events that are controlled by an innate, genetically encoded, circadian clock. Much of our work revolves around the period (per) gene, which appears to function within the circadian time keeping mechanism, or pacemaker.” (Qiu, 1996)  Hardin and associates have found that an important aspect of per's role within the pacemaker is related to the ability of the per protein to regulate circadian synthesis of its own mRNA (i.e. the per feedback loop).   “One goal of our studies is to understand the molecular mechanisms which govern this feedback loop. We recently defined regulatory sequences that mediate circadian expression of the per gene, and are currently determining how per protein, which does not directly bind to DNA, interacts with factors that bind these sequences to regulate circadian expression. Since per protein 1) functions in the nucleus, 2) contains a protein-protein interaction domain, 3) accumulates to high levels as per mRNA levels decline, and 4) represses per gene expression, we envision that per protein accumulates in the nucleus at certain times of the day and inactivates a transcriptional activator via protein binding. After the identity of this transcriptional activator is known, we plan to use it as a tool to identify other clock regulated genes in an effort to uncover other clock controlled processes.”(Hardin,1995)

Another Drosophila clock gene that has been identified characterized, and cloned is the cycle (cyc) gene.  Rutila and Suri report that “Homozygous cyc flies are completely arrhythmic. Heterozygous cyc/+ flies are rhythmic but have altered periods, indicating that the cyc locus has a dosage effect on period.”  (Rutila, 1998)   Their findings show that mutant flies have little or no transcription of the per and tim genes, and cloning of the gene indicates that it also encodes a bHLH-PAS transcription factor and is a Drosophila homolog of the human protein BMAL1. , consistent with its strong loss-of-function phenotype. “We propose that the CYC:CLK heterodimer binds to per and tim E boxes and makes a major contribution to the circadian transcription of Drosophila clock genes.” (Rutila, 1998)

In another study by Plautz, Kaneko, Hall, and Kay,  “Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period were used to monitor the circadian clock in explanted head, thorax, and abdominal tissues.” (Plautz, 1997)   The results of which were rather striking.  The explanted tissues still showed “rhythmic bioluminescence,” and the rhythms could still be reset by light. “The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.”  (Plautz, 1997)

Summary

 To summarize, the circadian clock is effected by many different factors, and more and more of these factors are discovered all the time.  From what is known right now heat and light exposure are key factors in circadian regulation, although it is not completely understood why.  Research into this area has shown that PER is sensitive to heat, but not light, and TIM is sensitive to light and heat.  It is suspected that Per collects in the nucleus at certain times of the day, and inactivates a transcriptional activator via protein binding.   Other proteins that have been discovered such as CYC and CLK which apparently make a major contribution to circadian transcription to, at least, Drosophila clock genes.  Additionally, it has been shown that these circadian cycles occur independently throughout the body of the Drosophila, and not just in the brain as was previously believed.  As far as the missing link theory is concerned, some striking evidence has been found that most of these systems occur in mammals and plants, but that certainly does little to prove that a missing link exists.

Conclusion

 Obviously the experiments, and discoveries discussed earlier in this paper, are just a handful of the studies that have been done in the field of circadian clocks.  For every advance we make in understanding how the circadian clock functions, we are presented with a dozen more questions that need to be answered before we will ever truly understand what makes these clocks tick.   One of these questions I feel has particular merit is that of individual cells capable of supporting their own independent clocks as suggested in Plautz, Kaneko, Hall, and Kay’s work.   Perhaps, past research has been hindered or held back by the notion that all circadian rhythms resided only in the brain or optic nerve, and now that we have broken this way of thinking perhaps even more discoveries will be made.

 Apparently, I’m not alone in my thinking.  Here in a quote form, Current Opinions in Genetic Development, Wilsbacher and Takahashi suggest that this is the direction circadian research is headed. “Much progress has been made during the past year in the molecular dissection of the circadian clock. Recently identified circadian genes in mouse, Drosophila, and cyanobacteria demonstrate the universal nature of negative feedback regulation as a circadian mechanism; furthermore, the mouse and Drosophila genes are structurally and functionally conserved. In addition, the discovery of brain-independent clocks promises to revolutionize the study of circadian biology.”  (Wilbacher, 1998)
With regard to the “missing link” theory, these discoveries in the Drosophila melanogaster certainly don’t prove anything, but have been shown to be quite useful in identifying similar trends in mammalian and plant circadian research.  Perhaps someday brain independent clocks will be found in mammals as well, thus lending more evidence to the theory that we as humans are a little more related to the world around us, though it may be decades before we ever prove any kind of  “missing link”.
 
 

References

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Hardin, P. E., Siwicki, K. (1995) The multiple roles of per in the Drosophila circadian clock. Semin. Neuro. 7, 15-25.

Hardin, P.E. (1994) Analysis of period mRNA Cycling in Drosophila Head and Body Tissues Indicates that Body Oscillators Behave Differently than Head Oscillators. Mol. Cell. Biol. 14, 7211-7218.

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