– Anirudh Sharma
Illustrated by Faria Shahreen Ahmed
A key element of life on Earth is its ability to adapt to the ever-changing environment. Life forms change drastically depending on temporal and geographical variations. However, an organism’s environment also changes considerably as a day progresses. It has been known for a long time that organisms can adapt, in a periodic manner, to these daily fluctuations. Jean Jacques d’Ortous de Mairan demonstrated this by showing that mimosa plants seem to have an internal autonomous “biological clock”. Their leaves opened and faced the Sun during the day and closed at night. Mairan showed that even when these plants were kept in constant darkness they continued to show this periodic movement. It was later discovered that most organisms including humans have a version of this biological clock, named the ‘circadian rhythm’ (Ibáñez, 2017).
The circadian rhythm is an essential part of human physiology and behaviour. It regulates physiological, endocrine and metabolic functions in response to the Earth’s rotation about its axis and allows us to optimize energy expenditure. A dysfunctional circadian rhythm can cause depression and affect cognitive function in addition to causing neurological diseases (Reddy et al, 2022).
The question that now arises is; how is an organism able to maintain an internal autonomous “clock” that governs various aspects of its physiology and behaviour?
Although we have known about the existence of the circadian rhythm for a long time, its underlying mechanisms remained unknown until very recently. In 1971, Benzer and Konopka performed mutation studies on the fruit fly Drosophila melanogaster in the hopes of finding mutants that had dysfunctional or altered circadian rhythms. This would provide a genetic locus for the control of the circadian rhythm. They identified mutants that had longer, shorter or arrhythmic rhythms. Their studies suggested that all of these mutations existed on the same gene, named period, present on the X-chromosome (Ibáñez, 2017).
Molecular analysis of this locus (known as the per locus) subsequently began. An important contribution was made by Zehring et al in a paper published in 1984, wherein they collaborated to molecularly analyse the per locus and determine which RNA transcript(s) was responsible for controlling the circadian rhythm. If this could be identified, further analysis of this transcript(s) could provide groundbreaking information on how the circadian rhythm was generated, controlled and regulated.
Drosophila displays rhythmic behaviour in various aspects. Two of these are rhythmicity in interpulse intervals of the males’ courtship song, and in locomotor activity. The researchers used Drosophila mutants that did not display these behaviours for this study. In their analysis, they identified 3 regions of chromosomal aberrations that were known to alter/destroy rhythmicity in Drosophila. The RNA transcript(s) that controlled the circadian rhythm were assumed to be transcribed from DNA spanning one or multiple of these chromosomal aberrations. Segments of DNA cleaved by various DNases were mapped and the corresponding RNA transcripts were identified. These segments were cloned onto p-elements (transposable elements that can be used as vectors to deliver DNA into a target cell) and injected (using microinjection) into embryonic cells of Drosophila that were homozygous for arrhythmicity. They then screened for those segments that when cloned into arhythmic Drosophila would restore rhythmicity (Zehring et al, 1984).
Through these studies, the researchers found that two DNA segments, one 8 kb in length and the other 14.6 kb in length, when transformed into arhythmic Drosophila strains were able to restore rhythmicity. The 8 kb region was found to be located within the 14.6 kb region. The 8 kb region codes for two RNA transcripts, one 0.9 kb in length and the other 1.0 kb in length. However, a different segment of DNA which overlaps with the 8 kb region was not able to restore rhythmicity even though it could produce the 0.9 kb transcript (but not the 1.0 kb transcript). This showed that the 1.0 kb transcript was probably the key player in the period locus. In this way, the researchers were able to narrow down, using a behavioural assay, which RNA transcript produced by the per locus was likely to be integral to the circadian cycle. Note, however, that the transformation obtained by microinjection of both the 14.6 kb and 8 kb regions of DNA only partially restored rhythmic behaviour. Restoration of rhythmicity was seen in a fraction of individuals which was smaller than the fraction of wild-type individuals that display rhythmicity. In addition, transformants tended to have longer rhythmic cycles than wild-type Drosophila. This seemed to indicate that other segments of DNA located at the per locus (and therefore other RNA transcripts) were important for completely normal expression. This meant that there was a lot of room for further research. Nevertheless, the identification of the 1.0kb RNA segment as an important player in the circadian rhythm was an essential first step (Zehring et al, 1984).
This was the first time that a functioning part of an animal’s gene locus was identified by constructing a behavioural assay since no knowledge of the gene product was available and marked a key checkpoint in the quest to unravel the inner workings of the biological clock. At that point, even though the genetic basis for the circadian rhythm was known to a fair degree, scientists still had no clue about the mechanism by which this gene controlled the circadian rhythm. This required further research, now made possible by the molecular characterization of the period gene.
The work of Hall, Rosbash, Young and others provided clues for this mechanism. Several years of work led to all the pieces being linked together and the mechanism was finally elucidated. The protein PER is the gene product of period. It was seen that the abundance of PER varied in a cyclic manner over a 24-hour period in neuronal cells of Drosophila. The mRNA that codes for PER also showed a similar cyclic variation in its abundance, except that the levels of PER lagged behind the levels of the mRNA by a few hours. This led to the development of the transcription-translation feedback loop (TTFL) model, which states that the accumulation of PER suppresses the expression of the period gene. This was the first mechanism of its kind to be discovered and gave rise to a new paradigm(Ibáñez, 2017).
It was found that PER would enter the nucleus after it was synthesized, but the way in which it did so was not known. A second gene, timeless was then discovered. The gene product, TIM, binds to PER and thereby enables it to enter the nucleus. The mechanism by which period and timeless genes were regulated was still unknown. This changed with the discovery of the clock gene in mouse by Joseph Takahashi. Takahashi and others established that the gene products, CLOCK and CYCLE upregulate the expression of period and timeless
Illustrated by Faria Shahreen Ahmed
the TIM-PER complex thus formed inhibits the transcription of clock, thereby inhibiting the transcription of period. In this manner, a negative self-regulating feedback loop is generated (Darlington et al, 1998).
But how is it that the length of this cycle can adapt to external cues? This is achieved by a complex interaction of various proteins that get phosphorylated/dephosphorylated and activate/deactivate proteins of the TTFL loop. An example of this is the double-time gene. This gene codes for an enzyme called DBT, which phosphorylates, and thereby causes the degradation of PER. This leads to a delay between period mRNA expression and PER accumulation, increasing the duration of the TTFL cycle. Light and other external factors can change these protein interactions, ultimately affecting the levels of PER in cells. Thus, the circadian rhythm is an autonomous, flexible and versatile mechanism by which our cells adapt to daily environmental changes (Ibáñez, 2017).
Jeffrey C. Hall, Michael Rosbash and Michael W. Young were awarded the Nobel Prize in Physiology or Medicine in 2017 “for their discoveries of molecular mechanisms controlling the circadian rhythm” (NobelPrize.org, 2017). Through decades of work they, and many other researchers, were able to answer one of the most integral biological questions, displaying the true power of the scientific method.
Did you enjoy reading this? Let us know what you thought of this article in the comments section below!
ABOUT THE AUTHOR
Anirudh Sharma
The author is in his third year at St. Xavier’s College, studying Life Science and Biochemistry.
We would like to thank Dr. S K Tahajjul Taufique, a Post-doctoral Fellow at Soochow University,China for reviewing this article and for his valuable input.
-The Boffin Bloggers
References:
- Main reference – Zehring, W. A., Wheeler, D. A., Reddy, P., Konopka, R. J., Kyriacou, C. P., Rosbash, M., & Hall, J. C. (1984). P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster. Cell, 39(2), 369–376. https://doi.org/10.1016/0092-8674(84)90015-1
- Darlington, T.K., Wager-Smith, K., Ceriani, M.F., Staknis, D., Gekakis, N., Steeves, T.D., Weitz, C.J., Takahashi, J.S., and Kay, S.A. (1998). Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280, 1599–1603.
- Ibáñez, C. (2017). The nobel prize in physiology or medicine 2017. NobelPrize.org. Retrieved July 13, 2022, from https://www.nobelprize.org/prizes/medicine/2017/advanced-information/
- Konopka, R. J., & Benzer, S. (1971). Clock mutants of Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America, 68(9), 2112–2116. https://doi.org/10.1073/pnas.68.9.2112
- Reddy, S., Reddy, V., & Sharma, S. (2022). Physiology, Circadian Rhythm. NCBI. Retrieved July 13, 2022, from https://www.ncbi.nlm.nih.gov/books/NBK519507/
- The nobel prize in physiology or medicine 2017. NobelPrize.org. (2017). Retrieved July 13, 2022, from https://www.nobelprize.org/prizes/medicine/2017/press-release/
- Lawrence, C., & Shapin, S. (1998). Science Incarnate: Historical Embodiments of Natural Knowledge (1st ed.). University of Chicago Press.
- The Engines | Babbage Engine | Computer History Museum. (n.d.). Computer History Museum. https://www.computerhistory.org/babbage/engines/
SciXplore 