Every living creature, from pond scum bacteria to people, fine-tunesits bodily functions to the alternating light of day and dark of night.This round-the-clock metabolic beat is known as circadian rhythm.

Like an old-fashioned clock that needs to be wound once a day,mammals, microbes and the other forms of life reset their bodyclocks daily, keyed to cues of light and temperature from theenvironment.

In humans, alternating sleep and wakefulness, not to mention regularbreakfast, lunch and dinner, are crude circadian markers, but moresubtle molecular tuning forks synchronize the work of every organ,tissue and cell.

Smaller by orders of magnitude than the tiny oscillating quartzcrystal in a wristwatch, the body's master clock is a total mystery,within a black box.

Supposition says it resides somewhere, somehow, in the pinealgland, a small organ centrally located in the brain. Tradition says thatthe pineal is the remnant of a "third eye" that still receives impulsesfrom daylight, which govern a mammal's circadian rhythm.

What mainspring genes encode what cogwheel proteins that run ourbody clocks? Nobody knows, but not for lack of hunting."Researchers have had a clock gene for the fruit fly, Drosophilamelanogaster, for quite a while," observes Susan Golden of TexasA&M University, in College Station, Texas, "but that one gene isn'tenough to tell them how the clock gene works. Neither does a geneisolated in the fungus Neurospora."

Golden, who describes herself as a cyanobacteriologist, is part of athree-center consortium that is searching for circadian-rhythm genes,10,000 at a clip, in a familiar bacterium, better known by its formername, blue-green algae. Her partners in the triumvirate arebiochemist Carl Johnson, at Vanderbilt University, in Nashville,Tenn., and inventor Takao Kondo, of the National Institute for BasicBiology, in Okazaki, Japan.

Machine Screens Colonies, Measures Rhythms

What Kondo has invented, Golden told BioWorld Today, is anautomated apparatus for screening large numbers of cyanobacteriacolonies at once, measuring their circadian rhythms, and picking outthose organisms that vary from the standard 24-hour periodicity.This departure from the norm indicates that they harbor mutant clockgenes.

The device consists of a round solid aluminum turntable, about ayard in diameter and an inch thick. Twelve round wells are milledinto its circumference, to hold Petri dishes, each containing 500 to1,000 colony cultures. A cooled, high-sensitivity video camera peersdown at this array, which rotates every 45 minutes to opticallysample another culture. The camera feeds these images to acomputer, which collates and remembers what it sees.

What it sees is bioluminescence, emitted at circadian intervals byeach colony. Susan Golden's lab equipped the bacteria with thislight-emitting property by cloning into them the luciferase genesfrom a glowing, seagoing microorganism, Vibrio harveyi, andputting this transgene under the control of the bacterias'photosynthesis gene.

In nature, cyanobacteria (Synechococcus sp. str.) occupy the bottomof the planet's food chain. In saltwater and fresh (e.g., pond scum), itdevours daylight, and employs these photons to powerphotosynthesis. It also fixes atmospheric nitrogen, and thesefunctions pulse to the earth's 24-hour circadian rhythm. "Thosenormal metabolic processes were actually the first way that a clockwas discovered in cyanobacteria," Golden said.

Before Kondo's turntable went into action, she recalled, "it was verylabor-intensive to measure the clock genes." She explained:"Suppose you're looking for a mutant with a 22-hour period insteadof 24. Having your grad student taking a sample every half-hour for24 hours, you'd capture only a two-hour difference, and you couldn'tbe really sure that it was real. You'd have to run it for four or fivedays to get a convincing 10 hours out of phase."

Kondo's machine runs for six or seven days for a full cycle ofchecking every Petri dish every 45 minutes.

He and his partners in Texas and Tennessee are co-authors of a paperin the current issue of Science, dated Nov. 18, titled "Circadian clockmutants of cyanobacteria."

"In it," said Golden, "we showed that when we moved thisrecombinant luminescence gene into the cyanobacteria, that lightemission was rhythmic. It had the same 24-hour periodicity ascircadian rhythms in higher organisms: Maximum and minimumlight emissions both occurred at the same time every day."

The Hunt Was On _ For Mutants

Then the hunt was on for mutants, whose clocks didn't run on time.Kondo's computer program clued the researchers in as to where tolook for likely aberrant colonies. "We found a lot of mutants withvery altered clocks," Golden said. Somewhere the light peaked at 16hours; others, as high as 60 hours."

Next came the feat of fixing the clocks. "We took a mutant with adefective gene, added back random DNA sequences, and looked fora colony that now had its clock rescued, running normally again."They obtained that clock-repair gene by chopping up DNA fromwild-type cyanobacteria, and cloning these random fragments,ranging from 2,000 to 3,000 base pairs, into the mutant microbe, oneat a time.

"The power of having mutants," the Texas cyanobacteriologist said,"is that when you can add a gene back, and return its behavior tonormal, you know that that's the gene you want. Each sequence willrepresent a component of the clock."

Golden entertains a personal hunch that once the genes that governcircadian rhythm in cyanobacteria have been pinpointed and cloned,they will advance the search for the body clock in higher forms oflife, including mammals. "When we know what the bacterial clockis, the scales will fall from our eyes, and it will be easier to tell whatwe're looking for in humans."

Golden said this will eventually lead to practical payoffs. "The secretof being able to do anything pharmacological _ related, say, tohumans _ and how the clock affects humans, is to know what theclock is. It's a lot easier to tinker with it if you know what you'retinkering with."

She made the point that "If we know mechanically how the clockworks, an important thing would be able quickly to reset it in cases,say, of jet lag and shift work _ people needing to be awake orasleep at different times of day than their bodies were set up for."

To that practical prospect she added, There may even be somemental illnesses or emotional disorders related to problems with thecircadian clock. If we knew what it is and how it works, we might beable to tell what's wrong, and perhaps reset their clock." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.