The Real Life of Pseudogenes

Disabled genes, molecular relics scattered across the human genomic landscape, have a story of their own to tell. And it is still unfolding

Our genetic closet holds skeletons. The bones of long-dead genes-known as pseudogenes--litter our chromosomes. But like other fossils, they illuminate the evolutionary history of today's more familiar forms, and emerging evidence indicates that a few of these DNA dinosaurs may not be quite so dead after all. Signs of activity among pseudogenes are another reminder that although the project to sequence the human genome (the complete set of genetic information in the nuclei of our cells) was officially finished, scientists are still just beginning to unravel its complexities.

It is already clear that a whole genome is less like a static library of information than an active computer operating system for a living thing. Pseudogenes may analogously be vestiges of old code associated with defunct routines, but they also constitute a fascinating record contained within the overall program of how it has grown and diversified over time. As products of the processes by which genomes remodel and update themselves, pseudogenes are providing new insights into those dynamics, as well as hints about their own, possibly ongoing, role in our genome.

Copied, Not Fake

"FALSE" GENES, which look like real genes but have no apparent function, were first recognized and dubbed pseudogenes during the late 1970s, when early gene hunters began trying to pinpoint the chromosomal locations associated with production of important molecules. For example, while seeking the gene responsible for making betaglobin, a key component of the hemoglobin protein that transports oxygen through the bloodstream, scientists identified a DNA sequence that looked like a globin gene but could not possibly give rise to a protein. Essential functional parts of the gene's anatomy were disabled by mutations, making it impossible for cellular machinery to translate the gene into a useful molecule.

Only the far more recent completion of sequencing projects covering the full genomes of humans and other organisms allowed geneticists to get an aerial view of the genomic landscape and to appreciate how riddled with such oddities it is. The human genome is made up of more than three billion pairs of nucleotides, the building blocks of DNA molecules. Yet less than 2 percent of our genomic DNA directly encodes proteins. Perhaps a third is noncoding sequences within genes, called introns. The remaining tracts between genes constitute the great majority of our DNA, and much of it is effectively genomic dark matter whose function is still largely a mystery. It is in these seemingly barren expanses that most pseudogenes are randomly scattered like rusted car parts on the landscape--and in surprising numbers.

With ongoing annotation of the human genome sequence, our research group, along with others in Europe and Japan, have identified more than 19,000 pseudogenes, and more are likely to be discovered. Humans have only an estimated 21,000 protein-coding genes, so pseudogenes could one day be found to outnumber their functional counterparts. Their sheer prevalence has raised many questions, including how they came into existence, why there are so many of them and why, if they are really useless, they have been retained in our genome for so long.

The answer to the first question is already fairly well understood. A small fraction of pseudogenes are believed to have once been functional genes that simply "died" from disabling changes to their nucleotide sequences. But most pseudogenes are disabled duplicates of working genes. They may have been dead on arrival, having suffered lethal damage during the copying process, or they may have accumulated debilitating mutations over time that collectively rendered them incapable of functioning.

Critical to a working gene is an intact anatomy that includes uninterrupted nucleotide spans called exons, which correspond to amino acid sequences in the encoded protein. Introns typically separate the exons, and at the beginning of a gene is a segment known as a promoter that serves as the starting point for cellular machinery to recognize the gene on a chromosome. When a cell expresses a gene, it first recruits essential molecular players to the promoter site, which travel down the gene's, length, transcribing it into a preliminary RNA copy. A splicing process next cuts introns out of the raw transcript and joins exonic sequences to produce an edited messenger RNA (mRNA) version of the gene. The mRNA is then read by a ribosome, a cellular machine that translates its sequence into the string of amino acids that forms a protein, the molecule that ultimately carries out the gene's function.

Pseudogenes can be born in two ways, each of which yields a distinctive facsimile of the original parent gene. Just before dividing, a cell duplicates its entire genome, and during that process, an extra copy of a gene can be inserted into the chromosomes in a new location. Alternatively, a new version of a gene can also be created through reverse transcription: during gene expression, the mRNA is copied back into a sequence of DNA that is inserted into the genome. Known as retrotransposition, this phenomenon can occur because of the activity of another type of transposable genetic actor, known as a long interspersed nuclear element, or LINE, that behaves like a genomic virus. LINEs carry their own machinery for making DNA copies of themselves to insert into the genome, and mRNA transcripts that are in the vicinity when LINEs are active can be swept up and retrotransposed as well.

These two processes, duplication and retrotransposition, are major forces that remodel genomes over the course of evolutionary time, generating new variation in organisms. They are the means by which genomes grow and diversify, because many replicated genes remain active. But if the gene copy contains disabling typos or is missing pieces of the original, such as the promoter, it will become a pseudogene. Those arising from duplication of an entire gene are recognizable because they contain both introns and exons. Pseudogenes made from mRNA lack introns and are described as processed pseudogenes.

Although the overall distribution of most pseudogenes across human chromosomes seems completely random, certain kinds of genes are more likely to give rise to pseudogenes. Geneticists organize functional genes into families based on their similarity to one another in both sequence and purpose. Only about a quarter of these family groups are associated with a pseudogene, and some families have spun off an inordinate number of copies. For example, the family of 80 human genes that produce ribosomal proteins has given rise to about 2,000 processed pseudogenes-roughly a tenth of the genome's identified total. In one extreme case, a single ribosomal protein gene known as RPL21 has spawned more than 140 pseudogene copies.

This disparity probably derives from the activity levels of different genes. Those responsible for basic cellular housekeeping functions, such as the genes in the ribosomal protein family, are abundantly expressed, providing more opportunities to create processed pseudogenes.

Because pseudogenes have been accreting this way in our genomes for so long, some are relics of genes eliminated during the course of evolution, and no functional version exists today. Others are copies of a gene that has so evolved over time, the pseudogene's sequence may reflect an older, earlier version of its parent. Consequently, intergenic regions can be seen as vast molecular fossil beds offering a silent record of events in our evolutionary past.

Family Histories

THE PRINCIPLES of natural selection appear to extend to individual genes, strongly constraining mutations in the sequences of functional genes. Beneficial mutations in a gene that improve the organism's fitness therefore tend to be preserved, whereas a sequence change that impairs a gene's function leads it to be discarded.

Once consigned to the genomic junk pile, however, pseudogenes are released from this selection pressure and are free to accumulate all kinds of mutations, including changes that would be deleterious to normal genes. Scientists can use this tendency to derive a kind of molecular clock from the nucleotide changes in pseudogenes and use it to study the overall dynamics and evolution of the genome. Tracking the evolutionary path of genes and pseudogenes helps molecular biologists to uncover instances of gene birth and death just as the study of mineral fossils tells paleontologists about the creation and extinction of species.

Our group has surveyed pseudogenes in the genomes of many forms of life, ranging from bacteria to more complex organisms, such as yeast, worms, flies and mice, and their prevalence across a wide range of creatures is striking. The number of pseudogenes in different genomes varies greatly, more so than genes, and it is not readily predictable, because it is neither strictly proportional to the size of a genome nor to the total number of genes. Comparisons of pseudogenes in related genomes can nonetheless reveal important information about the history of specific genes and the general workings of molecular evolution.

One of the largest known gene families in mammals, for example, consists of more than 1,000 different genes encoding olfactory receptors, the cell-surface proteins that confer our sense of smell. Detailed analyses of olfactory receptor (OR) genes and pseudogenes by Doron Lancet and Yoav Gilad of the Weizmann Institute of Science in Rehovot, Israel, show that humans have lost a large number of functional olfactory receptor genes during evolution, and we now have fewer than 500 of them in our genome. For comparison, versions of about 300 human olfactory receptor pseudogenes are still functional genes in the genomes of rats and mice.

This difference is not surprising given that most animals depend more for their survival on the sense of smell than humans do. In fact, humans have considerably more olfactory receptor pseudogenes than chimpanzees do, indicating that we lost many of those functional genes after our split from the ape lineage. Apes, however, have a higher proportion of olfactory receptor pseudogenes (30 to 40 percent of the OR family) than rodents or dogs do, suggesting that some influence has allowed the entire ape lineage to get by with a somewhat reduced sense of smell.

Lancet and his colleagues found in studies of apes, monkeys and other distant primate cousins that the greatest loss of olfactory receptor genes--that is, the greatest increase in OR pseudogenes--occurred in ape and monkey lineages that evolved the ability to see color in three wavelengths of visible light. The link may suggest that a sensory trade-off took place over time in the primate lineage when better eyesight made an acute sense of smell less critical.

Often, genes involved in an organism's response to its environment are subject to extensive duplication and diversification over time, leading to large gene families, such as the olfactory receptor repertoire. Many dead-on-arrival pseudogene copies are an immediate byproduct of this process. But the subsequent death of additional duplicates, which gives rise to new pseudogenes, is also frequently connected to changes in an organism's environment or its circumstances. Consequently, differences in the pseudogenes of animals offer hints about their diverse life histories that are not as easily detected in comparisons of working genes, which are strongly constrained by their function.

Analysis of the mouse genome, for example, has shown that 99 percent of human genes have a corresponding version in the mouse. Although the human and mouse lineages diverged some 75 million years ago, nearly all of the human genome can be lined up against equivalent regions, known as syntenic blocks, in the mouse genome. Yet despite this similarity in functional genes and overall genome structure, just a small fraction of the known human pseudogenes have an obvious counterpart in the mouse.

What is more, some of the specific gene families giving rise to pseudogenes differ significantly between mouse and human. Using the rate of sequence decay relative to the parent genes to determine their age, it is also clear that many pseudogenes in the human and mouse genomes have arisen at different times. These observations indicate that very disparate events have led to independent bursts of retrotransposition that created pseudogenes in each of the lineages.

Scanning and Sifting

STUDIES OF PSEUDOGENES in their own right are really just beginning, because these fossil genes were long viewed as little more than a nuisance. Early efforts to catalogue pseudogenes were largely driven by the need to distinguish them from true genes when annotating genome sequences. Identifying pseudogenes is not as straightforward as recognizing genes, however. Based on characteristic elements, pattern-seeking computer algorithms can scan DNA sequences and identify genes with moderate success. Recognition of pseudogenes, in contrast, relies primarily on their similarity to genes and their lack of function. Computers can detect similarity by exhaustively aligning chunks of intergenic DNA against all possible parent genes. Establishing a suspected pseudogene's inability to function is more challenging.

Just as a living organism can die of many different causes, a variety of deleterious mutations affecting any step in the process of making a protein can disable a copied gene, turning it into a pseudogene. But the sequence itself can offer clues to whether a mutation is debilitating. We can look for premature "stop" signs, as well as insertions or deletions of nucleotides that shift the reading frame of cellular machinery that decodes the gene's information for making a protein. These disablements cannot be tolerated by true genes and are thus typical manifestations of pseudogenes.

More subtly, the theory of neutral evolution introduced by mathematical biologist Motoo Kimura in the 1960s holds that nonfunctional DNA sequences can change freely, without the constraint of natural selection. Thus, individual nucleotide mutations can be divided into two types: those that would preserve the amino acid sequence of the protein encoded by a gene, known as synonymous changes, and nonsynonymous changes that would alter the meaning of the sequence. Because changing a protein's amino acid sequence can abolish its function, a gene under selective pressure will be more likely to contain synonymous mutations, whereas a nonfunctional DNA sequence will not be subject to that constraint.

Comparison of pseudogenes among genomes has revealed a puzzling phenomenon, however: a few pseudogenes appear to be better preserved than one would expect if their sequences were drifting neutrally. Such pseudogenes may therefore be under evolutionary constraint, which implies that they might have some function after all. One way to try to ascertain whether pseudogenes are functioning is to see whether they are transcribed into RNA. Recent experiment's by Thomas Gingeras of Affymetrix and by Michael Snyder of Yale University have found that a significant fraction of the intergenic regions in the human genome are actively transcribed. In their studies, in fact, more than half the heavily transcribed sequences map to regions outside of known genes. What is more, a number of those transcriptionally active intergenic areas overlap with pseudogenes, suggesting that some pseudogenes may have life left in them.

Our research group is part of a consortium of laboratories working to understand what is going on in the dark matter of the genome. We are now in the pilot phase of a project to create an "encyclopedia of DNA elements" (referred to as ENCODE) whose ultimate goal is to identify all of the genome's parts and their function. Previous studies as well as preliminary ENCODE data indicate that at least one tenth of the pseudogenes in the human genome are transcriptionally active. Knowing that so many pseudogenes are transcribed does not tell us their function, but together with evidence that certain pseudogenes are better preserved than background intergenic sequences, it certainly challenges the classical view of pseudogenes as dead.

One possibility is that pseudogenes play some ongoing part in regulating the activity of functional genes. Molecular biologists have come to understand in recent years that many genes in higher organisms do not code for a final protein product, but instead their RNA transcripts act to control other genes. These regulatory RNA molecules can variously activate or repress another gene or can interfere with the translation of that gene's mRNA transcript into a functional protein. And at least two examples of pseudogenes behaving in a similar manner have been documented so far.

The first was reported in 1999 by Michael O'Shea's research group at the University of Sussex in England. The investigators found that in the neurons of a common pond snail, both the gene for nitric oxide synthase (NOS) and its related pseudogene are transcribed into RNA but that the RNA transcript of the NOS pseudogene inhibits protein production from the transcript of the normal NOS gene.

Then, in 2003, Shinji Hirotsune of the Saitama Medical School in Japan traced deformities in a group of experimental baby mice to the alteration of a pseudogene. The inactivity of an important regulatory gene called Makorin1 had derailed the development of the mice, but Hirotsune had not done anything to Makorin1. He had accidentally disrupted the Makorin1 pseudogene, which affected the function of its counterpart, the Makorin1 gene.

Perhaps two dozen examples of specific pseudogenes that appear to be active in some way--often only in certain cells of an organism--have been identified, although the findings are still preliminary. Because many pseudogenes have sequences highly similar to those of their parent genes, it is very tempting to speculate that the NOS and Makorin1 pseudogenes are not just isolated cases. Yet it is hard to imagine that these two pseudogenes had the specific roles they now perform when they first arose. Instead their activity may be the result of selection preserving happy accidents or of nature having figured out an efficient way to reuse the broken parts of genes by converting them into regulatory elements.

Protogenes

AN EXCITING ERA of molecular paleontology is just beginning. We have barely scratched the surface of the pseudogene strata, and once we drill deeper, the number of identified pseudogenes will most likely grow and we may find more surprises. Large-scale pseudogene identification is a very dynamic data-mining process. Current techniques rely heavily on sequence comparison to well-characterized genes, and although they can readily identify recently generated pseudogenes, very ancient and decayed sequences are probably escaping detection. As the sequence and annotation of the human genome itself are refined and updated, characterization of pseudogenes will improve as well.

Recent hints that not all pseudogenes are entirely dead have been intriguing, and some evidence also exists for the possibility of pseudogene resurrection-a dead gene turning back into a living one that makes a functional protein product. Careful sequence comparisons have shown that one cow gene for a ribonuclease enzyme was a pseudogene for much of its history but appears to have been reactivated during recent evolutionary time. Slight differences in the pseudogene complements of individual people have also been found--for example, a few olfactory receptor pseudogenes straddle the fence: in most people they are pseudogenes, but in some they are intact, working genes. These anomalies could arise if random mutation reversed the disablement that originally produced the pseudogene. Might they account for individuals' differing sensitivities to smell? Perhaps, although it is too early to guess at the scope or significance of this unexpected source of genetic variation among humans.

Our studies have suggested, however, that in yeast, certain cell-surface protein pseudogenes are reactivated when the organism is challenged by a stressful new environment. Thus, pseudogenes may be considered not only as dead genes (which nonetheless provide fascinating new insights into our past) but also as potentially unborn genes: a resource tucked away in our genetic closet to be drawn on in changing circumstances, one whose possible roles in our present and future genomes are just beginning to unfold.

PSEUDOGENE BIRTH AND GENE DEATH
Two distinct processes can duplicate genes, and together they allow genomes to grow and diversify over evolutionary time. If errors in a copy destroy its ability to function as a gene, however, it becomes a pseudogene instead. The mutations that can kill a gene range from gross deletions (such as the loss of the promoter region that signals the start of a gene sequence) to minute changes in the DNA sequence that skew the meaning of the gene's protein-encoding segments, called exons.


GENE DEATH
Genes die and become pseudogenes when mutations generated during the gene-copying process or accumulated overtime render them incapable of giving rise to a protein. Cellular machinery reads the DNA alphabet of nucleotide bases [abbreviated A, C, G, T] in three-base increments called codons, which name an amino acid building block in a protein sequence or encode "stop" signals indicating the end of a gene. Even single-base mutations in codons can alter their amino acid meaning, and base deletions or insertions can affect neighboring codons by shifting the cellular machinery's reading frame. The alignment shown here of a partial sequence for a human gene [RPL21] against one of its pseudogene copies [ΨRPL21], along with each codon's corresponding amino acid [AA], illustrates some of the disabling mutations typically found in pseudogenes.


FLAWED COPIES
A "duplicated" pseudogene arises when a cell is replicating its own DNA and inserts an extra copy of a gene into the genome in a new location.

A "processed" pseudogene is formed during gene expression, when a gene is transcribed into RNA, then that transcript is processed into a shorter messenger RNA [mRNA]. Normally, the mRNA is destined for translation into a protein--but sometimes it can instead be reverse-transcribed back into DNA form and inserted in the genome.


Human Chromosome
PSEUDOGENE DESCENDANTS [blue] of the ribosomal protein gene RPL21 [orange] are scattered across the human chromosomal landscape. Overall distribution of pseudogenes in the human genome appears to be completely random, although some local genome regions tend to contain more pseudogenes. Those DNA regions may be analogous to certain geochemical environments that better preserve mineral fossils. Identification of genes and pseudogenes is an ongoing process, but to date more than 19,000 pseudogenes have been identified in the human genome--only slightly less than the current tally of around 21,000 human genes [inset]. About 8,000 of our pseudogenes are processed; the rest include duplicated pseudogenes and other nonprocessed subcategories.


Human Genome
Genes Pseudogenes
21,000 11,000 Non-processed
8,000 Processed


CHROMOSOMES of humans and mice carry avery similar array of functional genes [orange] but reveal distinct differences in their pseudogenes [blue], which can highlight important turning points in an organism's evolutionary history. For example, the counterpart of a mouse gene called Gulo has become a pseudogene [ΨGulo] in humans and other primates. Gulo makes an enzyme that is the last element in a biochemical pathway for synthesizing vitamin C. Most mammals possess the active gene, but the primate lineage seems to have lost it more than 40 million years ago. When the Gulo gene became a pseudogene, primates became dependent on food sources of vitamin C to avoid scurvy.

Overview/The Pseudogenome
• Pseudogenes are the molecular remains of broken genes, which are unable to function because of lethal injury to their structures.

• The great majority of pseudogenes are damaged copies of working genes and serve as genetic fossils that offer insight into gene evolution and genome dynamics.

• Identifying pseudogenes involves intensive data mining to locate genelike sequences and analysis to establish whether they function.

• Recent evidence of activity among pseudogenes, and their potential resurrection, suggests some are not entirely dead after all.

MORE TO EXPLORE
Human Specific Loss of Olfactory Receptor Genes. Yoav Gilad et al. in Proceedings of the National Academy of Sciences USA, Vol. 100, No. 8, pages 3324-3327; March 18, 2003.

Pseudogenes: Are They "Junk" or Functional DNA? Evgeniy S. Balakirev and Francisco J. Ayala in Annual Review of Genetics, Vol. 37, pages 123-151; December 2003.

Large-Scale Analysis of Pseudogenes in the Human Genome. Zhaolei Zhang and Mark Gerstein in Current Opinion in Genetics & Development, Vol. 14, No. 4, pages 328-335; August 2004.

www.pseudogene.org/

A Complex Nose with Subtle Hints of Poo

Burgundy is mad for a new kind of winemaking whose secret ingredient is cow dung

Nine years ago, the prestigious 289-year-old Burgundy winery Domaine Leflaive hosted a blind taste test of two of its wines. To be exact, the tasters, from the London wine merchant Corney & Barrow, were comparing two samples of the same wine: the 1996 Puligny-Montrachet 1er Cru Clavoillon. But one was made with organic grapes, the other with grapes grown using biodynamism, a method that's gaining popularity in wine circles -- although even its advocates will admit it sounds like something out of a Wiccan manual. Growers following one preparation are instructed to pack manure in a cow horn and bury it among the vines during the fall, dig it up in the spring and stir in rainwater vigorously for an hour, then apply it to the land after 3 p.m. The result: better grapes, apparently.

The technique doesn't sound as if it should work, but Domaine Leflaive's biodynamic Puligny-Montrachet passed the all-important taste test. Twelve of the 13 tasters preferred it to the regular wine. That year, the whole vineyard switched to biodynamism. Others have followed suit. In 1990, six grape growers in France used the method. Now, more than 100 respected wine producers in six countries practise it, including Jean-Marc Brocard, Josmeyer and M. Chapoutier in France; Benziger Family Winery in the U.S.; and the Massa Vecchia winery in Italy. North America has joined the movement late and Canada, in particular, is a slower adopter, with just one certified biodynamic grower -- Feast of Fields. But word of mouth is fuelling the trend.

The idea of biodynamism dates back to eight lectures delivered by the Austrian mystic Rudolf Steiner in the 1920s. Steiner, a well-known scientist, philosopher and self-proclaimed clairvoyant who also founded the Waldorf school, was speaking to German farmers worried about the effects of fertilizers on their crops. He advised them to nurture their land toward optimal health by applying eight basic animal-, mineral- and vegetable-based preparations, which he numbered 500 through 507, at specific times based on the moon's movement. His ideas took hold.

Biodynamism, like organic farming, rejects artificial pesticide use and tries to minimize damage to ecosystems, but, biodynamic growers also use preparations to harness natural forces. The cow horn in the preparation described above, for instance, acts as an antenna, funnelling power into the soil.

In France, biodynamism is gaining acceptance in Alsace and the Loire valley, and even in venerable Burgundy. That may seem surprising, but the latter region suffers from serious soil erosion due to centuries of grape growing and decades of pesticide use, and is losing market share to chardonnay and pinot noir from the New World and elsewhere. A method that promises to repair the soil and infuse wine with regional flavours, known as terroir, holds tremendous appeal. It's hard to get a clear count of biodynamic producers, because they don't all market it as such. Some fear being associated with a fad and others want the freedom to treat vines when necessary. In Alsace, Olivier Humbrecht, winemaker of Domaine Zind-Humbrecht, has been practising biodynamism quietly for nearly 10 years. He says it yields healthier vines and grape skins, important for wine flavour and structure. And his wine is stellar.

There's certainly resistance to biodynamism, especially among larger, wealthier producers. One director of a top Bordeaux château called biodynamic viticulture "a giant prominent intellectual swindle." Another referred to it as "a cult." Their skepticism is not unmerited. The science of biodynamism is vastly unproven; no credible study shows it changes grapes in any way, though its organic aspect may create better soil, vines and fruit than conventional farming. And some argue biodynamic growers are closer to their vines than more conventional farmers and this attention pays off.

Still, biodynamism has its appeal. It offers authenticity in an age in which wines are more generic worldwide due to reliance on commercial yeasts and a handful of grape varieties. Some consumers will choose biodynamic wine over others and perhaps even pay more for it one day. Right now, they don't have to. Despite being labour-intensive and expensive to produce, biodynamic wine doesn't cost more than conventionally produced bottles of the same quality. You can't say the same for tomatoes.

By Carolyn Hammond, Maclean's

What Makes Life Tick: Taking Apart the Living Clock

In the fourth century BC, the Greek Androsthenes recorded the first written observation of a circadian rhythm--the daily opening and nightly closing of the leaves of a tamarind tree. To Androsthenes, it must have seemed that the leaves moved in response to light or some other physical cue. For the next two thousand years, natural philosophers believed that daily rhythms of plant movement and animal behavior must be reactions to environmental signals.

But in 1729, French astronomer Jean Jacques d'Ortous de Mairan took a heliotrope plant inside a dark room and watched it continue its daily cycles of leaf movement in the absence of sunlight. While heliotrope plants--and all other circadian organisms--need environmental input to cycle at exactly 24 hours, they have "free-running" rhythms of close to that (circadian means "about a day") without any environmental cues. The circadian clock is an innate part of an organism.

Almost every organism on the planet has an internal clock: animals, plants, fungi, and cyanobacteria all have daily rhythms in behavior, physiology, and biochemistry. There are hints that purple and green bacteria and some archaebacteria may also have clocks, though there has not yet been definitive proof. The ability to anticipate, rather than respond to, daily astronomical cycles presumably gives organisms an evolutionary advantage. Those with circadian clocks can gear up metabolic processes and behaviors appropriate for a specific time of day before that time actually arrives, while those without them would meet these daily cycles unprepared.

The list of physiological processes and behaviors under circadian influence is long and diverse. Adult flies emerge from their pupae exclusively in the early morning. New asexual spores of fungi appear only at certain times of the day; the marine alga Gonyaulax radiates with bioluminescence only at night. In animals, sleep cycles, heartbeat, blood pressure, liver and kidney function, body temperature, hormonal changes, and cognitive abilities all show clear circadian rhythmicity. Even natural births and deaths peak in the early morning hours.

Over the past few decades, scientists have had considerable success deciphering the molecular mechanisms that underlie these rhythms. The particular genes that individual clocks use to generate circadian rhythms can differ dramatically between taxa. The cyanobacterial clock almost certainly evolved separately from the eukaryotic clock, says geneticist Steve Kay of the Scripps Research Institute. "Within the Eukarya, I would say the predominant view is they've arisen multiple times, certainly between fungi, plants, and animals," he says. However, "there are some design principles that are conserved, which, in a sense, is a perfect example of convergent evolution."

New studies are revealing some surprises about the molecular mechanisms that underlie these principles and about the variety of biological functions they may control. Says Kay, "The circuitry of circadian networks is turning out to be more complex than what was originally conceived."

The molecular clock
In most organisms studied so far, the generation of circadian rhythms seems to rely on self-regulating negative feedback loops in each cell--but these loops are different from those that keep body temperature or mineral content at a constant level.

"We know that there are, in biology, lots of feedback loops," says Rockefeller University geneticist Michael Young. "What makes these special is they don't close in a way that produces a homeostatic mechanism; they close in ways that produce oscillations."

In the negative feedback model for most circadian systems, core "clock" genes are first turned on, transcribed into RNA, and translated into protein. These proteins then feed back to inhibit their own production. Normally, this entire process would probably take only a few minutes. But the clock builds in lengthy delays--during which the clock proteins accumulate or degrade to certain levels--to ensure that the total cycle takes about 24 hours. "These seem to be tightly regulated processes that tend to delay feedback and cause oscillations to emerge," Young says.

At least one gene or protein in the oscillating loop must interact with photoreceptors that relay light information so the biological clock can become entrained to local time. The circadian phase can be reset by cycling inputs other than light, such as temperature or even humidity, but for most organisms, light is believed to be the major zeitgeber (German for "time giver") that triggers the shift to the circadian phase each day.

These molecular interactions account for the three fundamental characteristics of all circadian clocks: they have naturally running rhythms of close to 24 hours; they can be reset by environmental stimuli to run at exactly 24 hours; and they are temperature compensated (they do not change their speeds just because the climate becomes cooler or warmer).

Although one self-regulating loop could theoretically account for a 24-hour cycle of gene transcription, researchers are finding that most organisms have more complex oscillators than a "simple little feedback loop," says Kay Instead, many organisms seem to have multiple interconnected feedback loops, creating a network of oscillating gene and protein regulation.

Even these more complicated clock models show remarkable similarities among organisms. The clock is "constructed of multiple loops that actually look quite analogous between, for example, Drosophila and mice," Kay says. "Many of us are beginning to see the same type of multiple-loop control in plants, as well."

However, molecular genetics studies of circadian clocks have shown that the genes that make up these loops tend to be different in different organisms. Even among animals that share many of the same circadian genes, some of these genes have been co-opted for different functions in different species.

For example, the Drosophila clock gene period--the first clock gene discovered in any organism--has three homologs in the mouse, two of which appear to be classic clock genes as well. Another negatively regulated clock gene in the fly, called timeless, is found in the mouse but may not be a part of the clock. Also, the protein cryptochrome, which is the major photoreceptor for fly circadian clock entrainment, is very likely not a photoreceptor at all in mammals but rather a key molecular component of the core feedback loop itself.

Plants probably contain somewhat similar negative feedback loops, although the genes they use are completely different from those found in animals. "Although quite a bit is known about plant clocks, we really don't know nearly as much about exactly how they work, and that's partly because they look to be very complicated," says Jennifer Loros, a professor of biochemistry and genetics at Dartmouth Medical School. Plant clocks probably have more total components and more redundant components than the clocks of animals, Loros says.

Studies in Neurospora crassa, the filamentous fungus that appears on stale bread and overripe fruit, have helped to reveal circadian properties common to all organisms, such as negative feedback and light entrainment. "What we understand about animal clocks is still largely derivative from what we understand about flies and Neurospora," says Loros. As in animals and plants, the Neurospora clock consists of an auto regulatory negative feedback transcription loop, with two transcription factors driving expression of a clock gene called frequency, which, after a delay, inhibits its own transcription. One of the clock's driving transcription factors is also a photoreceptor.

Mutations in any of these core circadian genes can alter the period, entrainment, or temperature compensation of the Neurospora clock. However, fungal strains that lack functional frequency feedback loops still retain rhythms in many processes, including spore generation, gene expression, and enzyme activity, which hints that there must be other oscillators controlling those processes. These rhythms are not circadian on their own, however, so "presumably these slave oscillators are being brought under circadian control" by the central frequency oscillator, Loros says.

There is excellent evidence for two entirely separate circadian oscillators In the single-celled marine dinoflagellate Gonyaulax polyedra, says plant geneticist C. Robertson McClung of Dartmouth. Each feedback loop seems to control different aspects of metabolism and behavior and responds differently to environmental signals. There are also accumulating data that multiple clocks may exist in plants, McClung says, although it's not clear whether the oscillators are likely to be in the same cells or divided into different types of cells.

Circadian networks
Animals also contain multiple circadian oscillators, but there's no evidence that more than one oscillator resides in the same cell. Instead, they are relegated to different tissues. "If you look at gene expression across an animal, you'll find that there's great diversity in which genes are turned on in which tissues," says Vincent Cassone, a biology professor at Texas A&M University. "Similarly, you'll find different genes rhythmically expressed in different tissues."

It wasn't shown until the late 1990s, however, that mammals had any oscillators in peripheral tissues, even though peripheral rhythms had been found in Drosophila. Before that, it was widely believed that mammalian circadian rhythms were a property of a few cells in the brain--specifically, of a tiny paired cluster of neurons in the hypothalamus called the suprachiasmatic nucleus (SCN).

There was good evidence that the SCN was important for the mammalian clock: it receives direct light input from the retina, and removing the SCN destroys all behavioral and endocrine rhythms in a mammal. In an experiment that clinched the SCN's importance in mammalian circadian rhythms, neurobiologist Michael Menaker, of the University of Virginia, and his colleagues transplanted SCNs from hamsters with abnormally short circadian periods into hamsters whose SCNs had been removed. The transplant recipients immediately adopted the circadian cycles of the mutant SCNs. "The SCN is carrying with it the information," says Menaker. "The SCN is certainly very important; there's no question about that."

However, later experiments that examined molecular rhythms in gene expression in peripheral tissues, rather than organism-wide behavioral rhythms, found that these oscillations can persist in mammals without SCNs. Researchers at the University of Geneva also showed that a cell line of fibroblasts that had been in culture for 30 years could be induced to show 24-hour rhythms of gene expression.

What didn't persist in the absence of an animal's SCN was the synchrony of rhythms between cells and tissues. "The SCN sends out systemic signals to let the rest of the clocks know what's going on with the light/dark cycle," says Kay, and in this way pulls all of the body's oscillators into the same rhythm. What these systemic signals are, however, is still a mystery.

A centralized pacemaker is most likely necessary in mammals, because the retina is the only known way that light reaches the molecular clock. "In almost every case where you can identify a pacemaker tissue, that tissue is either photoreceptive itself or receives direct photoreceptive input," says Cassone. Mammals are unique in this respect; birds, reptiles, amphibians, fish, and insects all have photoreceptive cells outside of their eyes. Some scientists have suggested that the nocturnal ancestry of mammals may have caused them to lose the extraocular photoreceptors and associated pacemakers that are found in other vertebrates, he says.

Many birds, for example, have lightweight, translucent skulls that allow light to penetrate into the brain, where it is processed by many different photoreceptors. This entrainment information can therefore feed not only into the retina and the SCN but also into the pineal gland inside the brain. The retina, pineal, and SCN are considered to be separate circadian pacemakers in birds and reptiles, but the importance of each pacemaker to the total circadian system differs widely among even closely related species.

The insect network of circadian oscillators functions somewhat differently than that of vertebrates. Neurons in the Drosophila brain known as the lateral neurons are somewhat analagous to the mammalian SCN, in that normal fly locomotor rhythms disappear if the lateral neurons are destroyed. However, peripheral oscillators in Drosophila, which are present in their wings, legs, mouth, antennae, and probably elsewhere, are a step more independent than peripheral oscillators in mammals, as they entrain to light cues directly, with no input from the brain or the eyes. Cryptochrome, which was first identified as a blue-light photoreceptor in plants, is almost certainly the major photoreceptor involved in Drosophila light entrainment, although visual photoreceptors provide some input as well.

Peripheral oscillators in the weed Arabidopsis thaliana can also be entrained directly by light, through at least seven different photoreceptors, and probably more. Two are blue-light cryptochromes and five are phytochromes, which respond best to red or far red light. There are other candidate photoreceptors in Arabidopsis that look as if they feed into circadian pathways as well, says McClung. According to Kay, all of these photoreceptors "are used in an overlapping way to entrain the plant's clock, in a way that we don't understand yet."

Discovering the bacterial clock
At the end of the 1970s, significant progress had been made in understanding the clocks of eukaryotes, especially those of Drosophila and Neurospora, in which clock genes had already been identified. But it wasn't until the late 1980s that scientists showed that prokaryotes, too, have circadian clocks.

Many researchers believed that prokaryotic organisms like bacteria probably did not contain clocks. The argument was that organisms that reproduce in less than a day could not possibly have need for a 24-hour clock, says bacterial geneticist Susan Golden of Texas A&M. "There was also the idea that there wasn't enough complexity there to support the clock," she says.

Both of those ideas were put to rest in 1986 by the experiments of Tan-Chi Huang and colleagues at Academia Sinica in Taiwan, who were studying photosynthesis and nitrogen fixation in unicellular cyanobacteria found in rice paddies. The oxygen produced by photosynthesis is poisonous to a key enzyme involved in nitrogen fixation, and these researchers were trying to figure out how the bacteria managed to conduct both activities in cells with no nuclei or other partitions.

They found that the cells solved the problem by separating the two processes in time: cyanobacteria photosynthesize during the day and fix nitrogen at night. This temporal division signaled clearly to circadian researchers that these simple prokaryotes must have circadian clocks. Golden says. Other studies have suggested that cyanobacterial circadian rhythms may also prevent cells from dividing during the most light-intense hours of the day, when replicating DNA would be particularly vulnerable to ultraviolet-induced mutations.

The cyanobacterial molecular clock in the species Synechococcus elongatus has been shown to contain three central genes, called kaiA, kaiB, and kaiC (after the Japansese word for "cycle"). The transcription of these three genes creates a negative feedback loop, somewhat like the loops seen in plants, animals, and fungi. However, a study published in Science last April by Takao Kondo and his colleagues at Nagoya University has cast doubt that this transcription loop is actually critical to the circadian period in cyanobacteria.

When the Nagoya researchers placed the three Kai proteins in some buffer in a test tube, and added a little bit of ATP (adenosine triphosphate) for chemical energy, they found temperature-compensated circadian rhythms of phosphorylation of the protein KaiC. "That's fairly stunning," says Golden. From this experiment, "it's clear that you don't need these transcription/translation loops for a circadian rhythm in cyanobacteria," says McClung. "And that makes the cyanobacterial clock pretty different from the animal clocks, the fungal clocks, and. presumably, the Arabidopsis or the plant clock."

"Although the feedback loops are there, there may be other important things that these proteins are doing that enable them to act as biological timers," Young says. "Nothing about transcriptional feedback loops explains anything about these 24-hour rhythms"

More to reveal
The real significance of the cyanobacterial transcription loop isn't the only mystery left in circadian biology. Although most researchers assume it to be the case, no one has yet shown that the rhythmic oscillations seen in peripheral animal cells directly control rhythmic physiological events, says Cassone. "I strongly suspect that there will be evidence for that but, right now, there isn't," he says.

Many scientists are turning to microarray technology to try to figure out what types of physiological processes could be controlled by peripheral oscillators in different tissues. Microarrays are slides or membranes dotted with short DNA segments from different cells or tissues. When fluorescent probes for genes of interest are applied to the microarray, the amount of fluorescence reveals each gene's expression level, and these levels can be compared across the different cells and tissues.

Neurogeneticist Joseph Takahashi, whose lab at Northwestern University has led several microarray screens for circadian genes in mice, says that "as much as 10 percent of the genes we can detect in any particular tissue were under circadian control." Also, the genes that cycle rhythmically in one tissue tend not to be the same genes that cycle rhythmically in another, suggesting that many of these genes are controlled by tissue-specific oscillations.

"One of the things that came out of the microarray experiments was that most of the cycling genes were involved in fundamental metabolic pathways," says Takahashi. In the mouse liver, genes involved in glycolysis, glucose synthesis, fatty acid metabolism, and cholesterol metabolism are expressed in daily rhythms. These give "sort of a different view of what the clock is doing," Takahashi says. "It's actually much more intimately engaged in metabolism than we had really thought about before."

Loros was also surprised to find circadian expression of a glycolysis enzyme in a Neurospora microarray. For a long time, researchers "didn't tend to think of primary housekeeping genes as being clock regulated," she says.

In the past few years, there has been building evidence that, not only does the clock control metabolism, but the metabolic state of a cell can influence the clock. Golden says. Experiments in rats, for example, have shown that oscillators in the liver can entrain themselves directly to feeding cues, with no input from the SCN.

"You alter metabolic rates, you alter the clock. You alter the clock, you alter metabolic rates," Young says. "The clock becomes bigger, in a sense, than just the few simple components in a fairly significant way." He adds, "If you look harder, you find added complexity that multiplies the impact of these oscillators on the organism."

Cassone agrees that metabolism is quite likely related to the circadian clock in a fundamental way. "I think that's coming," he says. "We're going to realize that whole-body metabolic activity is also linked to clock function and is maybe one of the reasons why we have a clock in the first place."

By Melissa Lee Phillips

Melissa Lee Phillips is a freelance science writer in Seattle, Washington

Doctors are being told to use Google to help them diagnose illnesses

The internet search engine found the correct diagnosis in 58 per cent of difficult cases, according to experts.

But the idea has met with opposition from patient groups who questioned the credibility of many health sites.

And the Royal College of General Practitioners said the internet was "in no way a replacement for doctors''.

For the study, researchers at the Princess Alexandra Hospital in Brisbane, Australia, identified 26 difficult cases from the New England Journal of Medicine.

They included Creutzfeldt-Jakob disease (CJD), Cushing's syndrome (a hormonal disorder), acute chest syndrome and Churg-Strauss syndrome (an autoimmune disease).

The researchers selected three to five search terms from each case and did a Google search on each, while "blind'' to the correct diagnoses.

They then selected and recorded the three diagnoses that were ranked most prominently and which seemed to fit the symptoms and signs.

Those results were compared with the correct diagnoses as listed in the journal.

The study found that Google found the correct diagnosis in 15 of the 26 cases.

The experts said doctors would get a better result than patients, who may find the search less efficient and be less likely to reach the correct diagnosis.

They also suggested that medical students would find the tool useful.

The study, published in the British Medical Journal (BMJ), concluded: "Doctors and patients are increasingly proficient with the internet and frequently use Google to search for medical information.

How to Build an Invisibility Cloak

By Josie Glausiusz

Using strange new materials not found in nature, physicists can make an object disappear

Rare is the moment when Harry Potter fans, Star Trek aficionados, H. G. Wells enthusiasts, and theoretical physicists unite in a moment of ecstasy. But that instant came last May with a flurry of dramatic headlines. "Scientists may be able to make magic like Harry Potter," wrote the Associated Press. "Here's how to make an invisibility cloak," shouted MSNBC. "Cast no shadows," said The Economist. For Duke University physicist David Smith, though, the oddest moment was seeing his work flashed on the CNN crawl the same day it appeared in Science Express, the online edition of the journal Science. "It was surreal," he says. "The story was generating a huge splash before the scientific community had a chance to take a critical look."

Smith hardly fits the profile of a media celebrity: soft-spoken, patient, and bespectacled, he has the pale-skinned hue of a man who has perhaps spent too much time in a windowless lab fiddling with wires. All at once, however, he and his postdoc Dave Schurig became the targets of intense public interest. Reporters called from around the world, crackpots sent long letters hand-scrawled with dubious hypotheses, and a Korean television crew flew in to the leafy Duke campus, posing Schurig, graduate student Jonah Gollub, and technician Bryan Justice in lab coats in front of their intricate machinery. "They wouldn't film until we put the white coats on. We didn't even have any--we had to borrow them," recalls Schurig.

All this fuss over a theory not only unproved but so dense with equations it is all but incomprehensible to the average person. What sparked such fascination was the mind-bending notion itself: Smith, Schurig, and their coauthor John Pendry of Imperial College London proposed that by using a novel class of composite materials, they could manipulate light so as to render an object invisible to the eye. Suddenly, Harry Potter's invisibility cloak, Star Trek's Romulan ship-concealing devices, and H. G. Wells's bandaged Invisible Man seemed the stuff of testable science. In a more practical vein, the finding could have profound implications for military technology, wireless communication, and even interplanetary exploration.

In September, the journal Science accepted a paper from Smith and Schurig that proved that their method was more than just a thrilling hypothesis. They had succeeded in cloaking a small cylindrical object--shielding it not from visible light but from microwaves, a form of electromagnetic radiation with a substantially longer wavelength, which makes the cloaking effect considerably easier to achieve. The way they did it most closely recalls not Harry Potter but another fictional character: the Invisible Woman, a Marvel Comics superhero who can bend light waves at her command, rendering her body and clothing imperceptible. By tightly controlling the bending, or refraction, of microwaves as they pass through a custom-built material, the Duke researchers could force them to detour around an object so that the microwaves are neither absorbed nor reflected. If they performed the same feat with visible light, a viewer looking directly at the object would see only what lies behind it, as if the object were not even there.

To explain their technique, Smith and Schurig invoke the example of a mirage on a hot summer road. When light rays from the sky hit the hot, thin air just above the surface of the asphalt, they bend. Although light moves through a vacuum at a constant speed, it slows down when traveling through any transparent medium, like water or glass. Light travels faster in the hot, thin air close to the road than it does in the cold, dense air above, and that difference in speed is what causes it to shift direction as it crosses the boundary between the two. Rays once headed from the sky to the ground are redirected to your eye, making the road shimmer like water. In effect, the mirage is cloaking the (now invisible) road behind an image of the blue sky.

To similarly cloak something from electromagnetic radiation, Smith and Schurig must bend the incoming beam around the object in a tightly controlled manner. They managed to do so using a class of recently created "metamaterials" that possess an ability, not found in nature, to bend light at extreme angles (a property known as negative index of refraction). The team's metamaterials consist of thin, rigid sheets of fiberglass insulator stamped with neat rows of conducting metal shapes like loops, coils, or tiny rectangles. The metal circuitry is designed to direct incoming electromagnetic radiation--in this case, microwaves--so it moves in a specific way.

All electromagnetic radiation has two intertwined components: a magnetic field and an electric field. As Schurig explains, these can be redirected when they interact with a material. "Materials are made of atoms, and these atoms respond to electromagnetic waves by acting like a little tiny magnet," he says. Electrons begin moving in circles in response to the magnetic field, as well as back and forth in reaction to the electric field--and the moving charges produce fields of their own. The challenge for the Duke team was to find the right shapes and dimensions for the metal circuitry on the metamaterials so they could precisely dictate how the electrons move around, which in turn controls how the incoming radiation is bent.

To demonstrate their system in action, Smith and Schurig walk into their lab, a room lit with fluorescent bulbs and littered with wires, pliers, plugs, pulleys, flashlights, foam cladding, microscopes, computer terminals, and a lone bicycle. The object to be cloaked is just a small copper cylinder filled with black foam: 5 centimeters (2 inches) in diameter and 1 centimeter (0.4 inch) tall. For the experiment it is sandwiched between two horizontal aluminum plates, the bottom one 3 feet square and the top one 4 feet square. Leading in from the front of the apparatus is a wire that feeds microwaves toward the cylinder as it sits in the center of the bottom plate. Around it, Smith and Schurig have arranged concentric rings of metamaterials, with the empty spaces between the rings forming narrow channels. Having carefully varied the properties of the circuits on those surrounding rings, they can now bend micro waves to flow around the cylinder like water flowing around a pebble in a stream. This makes the object undetectable to an instrument downstream that measures microwaves.

According to the Duke team, this experiment shows it should be possible to make an object invisible to the human eye as well, but there are major technical hurdles. For cloaking to work, the metal shapes stamped on the metamaterial must be smaller than the wavelength of the electromagnetic radiation that is aimed at them. The wavelength of the microwaves is a little over 3 centimeters (just over an inch), and the shapes on the surface of the metamaterials are closer to 3 millimeters long. Green light, by contrast, has a wavelength of 500 nanometers--60,000 times smaller--so the shapes that could cloak it would have to be around 50 nanometers long. Theoretically, you could pattern metamaterials at that tiny scale using specialized methods like focused beams of charged atoms, but such materials would be difficult to mass-produce.

At this point, then, cloaking objects from visible light is still pie in the sky. In the meantime, the far more accessible applications of microwave cloaking have already garnered intense interest--mainly from the military. Smith is up-front as he rattles off their funding sources: DARPA (the Defense Advanced Research Projects Agency), the Air Force, the Army, the Navy, the intelligence community. One of the technique's most practical and immediate uses would be to hide obstructions that block wireless communication. But since Smith and Schurig's technique bends electromagnetic radiation in a controlled manner, it could someday also be co-opted to focus or concentrate energy in highly efficient ways. For example, it could be used to create supersensitive solar cells or even to power a Mars rover that would gather energy from a microwave beam sent by a satellite orbiting the Red Planet.

The Duke researchers are not the only ones scrambling to create cloaking devices. When their theory first appeared in the May 26 edition of Science Express, it was published alongside an independent article that outlined a similar proposal. The author of that paper, theoretical physicist Ulf Leonhardt of the University of St. Andrews in Scotland, proposed using slightly different types of engineered materials to accomplish the trick. A few weeks before that, a pair of math-loving physicists, Graeme Milton of the University of Utah and Nicolae Nicorovici of the University of Sydney in Australia, came up with yet another, drastically different scheme for making objects the size of dust specks invisible.

The Milton-Nicorovici hypothesis, which is based on rigorously proved mathematical calculations, relies on the use of a superlens, a thin transparent film that can resolve light finer than its wavelength (long considered a theoretical impossibility), producing extremely sharp images. A superlens made from a thin film of silver could have a negative index of refraction, bending light outside of its normal path. "What we found was that if you put a speck of dust near the superlens and shine light on the dust, then part of the scattered light gets trapped at the front surface of the superlens," Milton explains. "That trapped light builds up in intensity until it almost exactly cancels the incoming light," in the same way that two colliding sound waves can zero each other out. It is as if there is no light there at all, and the dust particle becomes invisible. (For an action-packed movie of this phenomenon, see Milton's Web site at www.physics.usyd.edu.au/cudos/research/plasmon.html.)

So how far off is a real invisibility device? Could such a contraption ever be used to cloak an airplane, a tank, or a ship? Smith doesn't want to be snared by such hypothetical questions. "Reporters, they call up and they just want you to say a number," he says. "Number of months, number of years. They push and push and push and then you finally say, well, maybe 15 years. Then you've got your headline, right? 'Fifteen years till Harry Potter's cloak!' So I have to resist giving you a number."

One major problem with masking objects from visible light, says Schurig, is that light is composed of a range of colors, each with its own wavelength. "We don't know how much of that spectrum we could cloak all at once," he explains. "If you could get past these fabrication issues, you could cloak one color of light, and maybe you could cloak some range of visible light. We might be able to make the cloak work for a brief time, for a microsecond at red, a microsecond at green, a microsecond at blue, and you could make it look translucent. But we don't know that you could make something 100 percent invisible to the whole spectrum simultaneously."

Milton sounds a further note of caution. Of the Duke research, he says: "I think it's a brilliant idea. But there's a certain amount of skepticism in the scientific community in so far as the time line. I remember reading claims that you could cloak some factory that would be an eyesore. I think that's a bit far-fetched. You can make some small things invisible, but making larger things invisible will be a longer time in coming."

There are other factors that neither Harry Potter fans nor the series' fabulously wealthy author, J. K. Rowling, seem to have considered. Ulf Leonhardt--the only one of the researchers who admits to enjoying the books--explains that Harry can see through his cloak, which is made of a thin material in which he can walk and climb. "The present scheme assumes you have something very rigid" surrounding the object, Leonhardt says. "It's not a cloak, it's like a suit of armor. If you want to have something flexible, then the material also has to change its properties, like a chameleon. That is also possible in principle, but with present technology we're a long way away from that."

The other, bigger problem is that to see, the eyes must absorb light--which, of course, makes them visible. "If Harry Potter wants to see through his cloak, then his eyes would be visible, because they have to see. And if they have to see, they have to be seen," Leonhardt says. "For example, a fish that camouflages itself by being transparent has eyes that are not transparent, because they have to see. Yet Harry Potter can see through the invisibility cloak. That, I think, is not possible. He would be blind behind it."

Very Interesting Fact about Pit Vipers and Boid Snakes

Reportedly New Scientist, pit vipers and boid snakes strike at prey with uncanny accuracy even when blindfolded, a feat that's been hard to explain given the rudimentary nature of their heat-sensing organs. It seems that some rather spectacular image processing may be the key.

To scout for cool shelters, and hunt in complete darkness, pit vipers and boid snakes are known to use infrared sensing organs. But their skill has amazed scientists. "In the lab, blindfolded snakes can strike a running rat behind the ears to avoid its sharp teeth," says physicist Leo van Hemmen of the Technical University of Munich in Germany. "It must be seeing more than just a warm blob."

But how, given that the snakes are saddled with very crude heat-sensing apparatus? On each side of their face, they have a pit organ that is little more than a hole with a heat-sensitive membrane stretched across it. "The eye has a lens to focus a visual image, but these holes can't do that," says van Hemmen.

Instead, the pit organs are supposed to work as "pinhole" cameras, except that the holes are too large at 1 millimetre or more in diameter. "These must produce images that are just fuzzy blurs," says van Hemmen. "So, how can the snakes strike with such precision?"

It could be down to how the snakes process the information reaching their heat sensors. Van Hemmen's team have developed a model that takes into account both the infrared "noise" created by moving prey and any errors generated by the snake's pit membrane itself.

The team recombined the inputs from the membrane's 2000-odd receptors to filter out the noise and create a sharp image. In their model, the heat signal reaching each receptor causes a neuron to fire, but the firing rate depends on the input received by all the other receptors. By tweaking how the receptors interact, the team could create precise images, even when there was a lot of background noise. "The precision is spectacular," says van Hemmen.

Nonetheless, even tiny errors generated by the pit membrane ruin this image. To minimise these errors the pit membrane would have to be no more than 15 micrometres thick — one-fifth the thickness of a sheet of paper. And this is exactly the thickness of the membrane seen in pit vipers and boids, says van Hemmen. Their work will appear in the journal Physical Review Letters.

Bruce Young, who studies snake behaviour at Washburn University in Topeka, Kansas, is impressed. "The team have addressed a fascinating question in sensory biology: what do you do with less than perfect input?" Nonetheless, he would like confirmation that this is what actually happens in real snakes.

Van Hemmen is confident that his team has uncovered the correct mechanism. "We've found a simple way that something seemingly impossible could work in the snake," he says. "If we could work it out, we're sure that nature could too"

"Blindfolded snakes can strike can strike a running rat behind the ears to avoid its teeth. It must be seeing more than just a warm blob"