Clock Genetics - A Short History

As I have mentioned before, there was quite an angst in the field of chronobiology around 1960s about the lack of undestanding of circadian and other rhythms at cellular and subcellular levels. Experiment involved manipulation of the environment (e.g,. light cycles) and observing outputs (e.g., wheel-running rhythms), while treating the clock, even if its anatomical location was known, as a "black box". Breaking into the black box was one of the most important goals of the field, and, until recently, it was a tough box to break. Is the molecular clock running with 24 hour cycles? Or is it a sum of a series of shorter (ultradian) cycles? Or is it a sum of activities of numerous coupled cells?

Much of the early research on the biochemistry of circadian rhythms was performed in Protists, e.g, Euglena, Paramecium, Acetabularia and Gonyolax polyedra, yet only one clock mutant was discoverd in Chlamidomonas - the protists' genomes proved to tough to crack. When I took my introductory chronobiology course in Spring of 1994., only three clock mutations were known: the tau-mutant in hamsters (Mesocricetus aureatus), the period (per) mutation in fruit-flies (Drosophila melanogaster), and the frequency (frq) mutation in the bread mold (Neurospora crassa).

I have written more about the tau-mutant in hamsters before. While wild-type hamsters have a natural freerunning period in constant darkness almost exactly 24 hours, the tau-mutant has a 20-hour day, while in heterozygotes (crosses between the wild type and mutants) it is somewhere in between: 22 hours. However, it took decades before the real nature of the mutation was cracked. The tau-mutant hamster actually proved to be a good animal model for a familial human circadian disorder (extreme "larkiness"), as it is the mutation in the same gene - Caseine Kinase 1 epsilon - that is responsible for both.

The bread mold (Neurospora crassa) grows mycelia inside glass tubes. Every 24 hours or so it stops growing, grows hyphae and conidiae for a little while, then goes on with mycelial growth. The "freak" (frq) mutation in Neurospora has several different variants with different periods (or lack of rhythm altogether). This mutation has been traced to a gene on the chromosome VII R.

In fruitflies, the period ("per") mutation had three variants. While wild-type Drosophila cycles with about 24 hours period, the perS (short) freeruns with a period of about 19 hours, perL (long) with a period of about 29 hours, and per0 (null) is completely arrhythmic. The mutation was localized to the X chromosome. If a mosaic fruitfly is constructed in which one side of the brain possesses perL and the other side perS, the fly as a whole expresses both rhythms of activity: the 19-hour and the 29-hour rhythm cross over each other on the actograph. If a perS brain is transplanted into the abdomen of the Per0 fly, the previously arrhythmic animal develops a circadian rhythm with perS specific period of 19 hours.

These three mutants were discovered in the 1980s and 1990s, all three quite serendipitously, and not much happened for a while. In the meantime, various models were designed to account for the behaviors of circadian clocks, including in clock mutants. For instance, Ehret came up with a chronon model. In this model, a series of genes induce each other's expression, i.e., protein A induces trasncription of gene B, protein B induces expression of gene C and so on until the last protein in the series, about 24 hours later, induces expression of gene A again. This model is, actually, not that far from the currently understood mechanism of interlocking trasncription/translation feedback loops (see the Dunlap paper I linked to a couple of days ago).

Another interesting model from the past was put forward by Njus. According to his cell-membrane model, daily oscillations in ion concentrations feed back on transport proteins in the membrane. Discarded after the discovery of clock-genes, this model is now getting a second look, as some of the recent data suggest involvement of the membrane (or at least a non-genomic activity of the cell) in circadian rhythm generation in some organisms. I plan to write a whole long post soon about the old and new data that challenge the gene-only model for the circadian clock.

So, everything I wrote above was known in Spring of 1994. The following year, the explosion starts. In 1995, Amita Seghal discovers the second clock gene in the fruitfly: timeless (tim). As the molecular techniques got more and more sophisticated, discovery of new clock genes became a small cottage industry. Discovery of the Clock (clk) gene in the mouse by Joe Takahashi opened up mammalian genome to investigation. Soon, it was seen that the fruitfly and the mouse have very similar molecular players involved in generation of circadian rhythms: there is Period in mammals, Clock in fruitflies, Cycle in fruitflies is the same as Bmal in mammals, both have Cryptochrome (cry) as a component of the system, Caseine kinase 1 epsilon is known in flies as Doubletime.

A couple of dozen genes involved in generation or modulation of circadian rhtyhms in mice and flies have been discovered since then and most have been shown to be working in an almost identical ways in fish, amphibians, reptiles and birds, as well as a number of invertebrates. The other organisms followed suit. White-collar (wc, sometimes refered to as "water closet" in the halls of conferences) in Neurospora, KaiA, B and C in the cyanobacterium Synechococcus, Toc in plants and others soon became known. By about the year 2000 this progress was noted two years in a row by Science Magazine when it gave the field runner-up position in its annual list of most exciting findings of the year. Currently, it is agreed that the circadian clock is the best understood behavioral system at the genetic level. For details, read Dunlap's paper I linked below..