Unraveling the DNA Myth

Biology once was regarded as a languid, largely descriptive discipline, a passive science that was content, for much of its history, merely to observe the natural world rather than change it. No longer. Today biology, armed with the power of genetics, has replaced physics as the activist Science of the Century, and it stands poised to assume godlike powers of creation, calling forth artificial forms of life rather than undiscovered elements and subatomic particles. The initial steps toward this new Genesis have been widely touted in the press. It wasn't so long ago that Scottish scientists stunned the world with Dolly¹, the fatherless sheep cloned directly from her mother's cells; these techniques have now been applied, unsuccessfully, to human cells. ANDi², a photogenic rhesus monkey, recently was born carrying the gene of a luminescent jellyfish. Pigs now carry a gene for bovine growth hormone and show significant improvement in weight gain, feed efficiency, and reduced fat.³ Most soybean plants grown in the United States have been genetically engineered to survive the application of powerful herbicides. Corn plants now contain a bacterial gene that produces an insecticidal protein rendering them poisonous to earworms.⁴

Our leading scientists and scientific entrepreneurs (two labels that are increasingly interchangeable) assure us that these feats of technological prowess, though marvelous and complex, are nonetheless safe and reliable. We are told that everything is under control. Conveniently ignored, forgotten, or in some instances simply suppressed, are the caveats, the fine print, the flaws and spontaneous abortions. Most clones exhibit developmental failure before or soon after birth, and even apparently normal clones often suffer from kidney or brain malformations.⁵ ANDi, perversely, has failed to glow like a jellyfish. Genetically modified pigs have a high incidence of gastric ulcers, arthritis, cardiomegaly (enlarged heart), dermatitis, and renal disease. Despite the biotechnology industry's assurances that genetically engineered soybeans have been altered only by the presence of the alien gene, as a matter of fact the plant's own genetic system has been unwittingly altered as well, with potentially dangerous consequences.⁶ The list of malfunctions gets little notice; biotechnology companies are not in the habit of publicizing studies that question the efficacy of their miraculous products or suggest the presence of a serpent in the biotech garden.

The mistakes might be dismissed as the necessary errors that characterize scientific progress. But behind them lurks a more profound failure. The wonders of genetic science are all founded on the discovery of the DNA double helix-by Francis Crick and James Watson in 1953-and they proceed from the premise that this molecular structure is the exclusive agent of inheritance in all living things: in the kingdom of molecular genetics, the DNA gene is absolute monarch. Known to molecular biologists as the "central dogma," the premise assumes that an organism's genome-its total complement of DNA genes---should fully account for its characteristic assemblage of inherited traits.⁷ The premise, unhappily, is false. Tested between 1990 and 2001 in one of the largest and most highly publicized scientific undertakings of our time, the Human Genome Project, the theory collapsed under the weight of fact. There are far too few human genes to account for the complexity of our inherited traits or for the vast inherited differences between plants, say, and people. By any reasonable measure, the finding (published last February) signaled the downfall of the central dogma; it also destroyed the scientific foundation of genetic engineering and the validity of the biotechnology industry's widely advertised claim that its methods of genetically modifying food crops are "specific, precise, and predictable"⁸ and therefore safe. In short, the most dramatic achievement to date of the $3 billion Human Genome Project is the refutation of its own scientific rationale.

Since Crick first proposed it forty-four years ago, the central dogma has come to dominate biomedical research. Simple, elegant, and easily summarized, it seeks to reduce inheritance, a property that only living things possess, to molecular dimensions: The molecular agent of inheritance is DNA, deoxyribonucleic acid, a very long, linear molecule tightly coiled within each cell's nucleus. DNA is made up of four different kinds of nucleotides, strung together in each gene in a particular linear order or sequence. Segments of DNA comprise the genes that, through a series of molecular processes, give rise to each of our inherited traits.

Guided by Crick's theory, the Human Genome Project was intended to identify and enumerate all of the genes in the human body by working out the sequence of the three billion nucleotides in human DNA. In 1990, James Watson described the Human Genome Project as "the ultimate description of life." It will yield, he claimed, the information "that determines if you have life as a fly, a carrot, or a man." Walter Gilbert, one of the project's earliest proponents, famously observed that the 3 billion nucleotides found in human DNA would easily fit on a compact disc, to which one could point and say, "Here is a human being; it's me!"⁹ President Bill Clinton described the human genome as "the language in which God created life."¹⁰ How could the minute dissection of human DNA into a sequence of 3 billion nucleotides support such hyperbolic claims? Crick's crisply stated theory attempts to answer that question. It hypothesizes a clear-cut chain of molecular processes that leads from a single DNA gene to the appearance of a particular inherited trait. The explanatory power of the theory is based on an extravagant proposition: that the DNA genes have unique, absolute, and universal control over the totality of inheritance in all forms of life.

In order to control inheritance, Crick reasoned, genes would need to govern the synthesis of protein, since proteins form the cell's internal structures and, as enzymes, catalyze the chemical events that produce specific inherited traits. The ability of DNA to govern the synthesis of protein is facilitated by their similar structures-both are linear molecules composed of specific sequences of subunits. A particular gene is distinguished from another by the precise linear order (sequence) in which the four different nucleotides appear in its DNA. In the same way, a particular protein is distinguished from another by the specific sequence of the twenty different kinds of amino acids of which it is made. The four kinds of nucleotides can be arranged in numerous possible sequences, and the choice of any one of them in the makeup of a particular gene represents its "genetic information" in the same sense that, in poker, the order of a hand of cards informs the player whether to bet high on a straight or drop out with a meaningless set of random numbers.

Crick's "sequence hypothesis" neatly links the gene to the protein: the sequence of the nucleotides in a gene "is a simple code for the amino acid sequence of a particular protein."¹¹ This is shorthand for a series of well-documented molecular processes that transcribe the gene's DNA nucleotide sequence into a complementary sequence of ribonucleic acid (RNA) nucleotides that, in turn, delivers the gene's code to the site of protein formation, where it determines the sequential order in which the different amino acids are linked to form the protein. It follows that in each living thing there should be a one-to-one correspondence between the total number of genes and the total number of proteins. The entire array of human genes-that is, the genome must therefore represent the whole of a person's inheritance, which distinguishes a person from a fly, or Walter Gilbert from anyone else. Finally, because DNA is made of the same four nucleotides in every living thing, the genetic code is universal, which means that a gene should be capable of producing its particular protein wherever it happens to find itself, even in a different species.

Crick's theory includes a second doctrine, which he originally called the "central dogma" (though this term is now generally used to identify his theory as a whole). The hypothesis is typical Crick: simple, precise, and magisterial. "Once (sequential) information has passed into protein it cannot get out again."¹² This means that genetic information originates in the DNA nucleotide sequence and terminates, unchanged, in the protein amino acid sequence. The pronouncement is crucial' to the explanatory power of the theory because it endows the gene with undiluted control over the identity of the protein and the inherited trait that the protein creates. To stress the importance of this genetic taboo, Crick bet the future of the entire enterprise on it, asserting that "the discovery of just one type of present-day cell" in which genetic information passed from protein to nucleic acid or from protein to protein "would shake the whole intellectual basis of molecular biology."¹³

Crick was aware of the brashness of his bet, for it was known that in living cells proteins come into promiscuous molecular contact with numerous other proteins and with molecules of DNA and RNA. His insistence that these interactions are genetically chaste was designed to protect the DNA's genetic message-the gene's nucleotide sequence-from molecular intruders that might change the sequence or add new ones as it was transferred, step by step, from gene to protein and thus destroy the theory's elegant simplicity.

Last February, Crick's gamble suffered a spectacular loss. In the journals Nature and Science and at joint press conferences and television appearances, the two genome research teams reported their results. The major result was "unexpected."¹⁴ Instead of the 100,000 or more genes predicted by the estimated number of human proteins, the gene count was only about 30,000. By this measure, people are only about as gene-rich as a mustard-like weed (which has 26,000 genes) and about twice as genetically endowed as a fruit fly or a primitive worm-hardly an adequate basis for distinguishing among "life as a fly, a carrot, or a man." In fact, an inattentive reader of genomic CDs might easily mistake Walter Gilbert for a mouse, 99 percent of whose genes have human counterparts.¹⁵

The surprising results contradicted the scientific premise on which the genome project was undertaken and dethroned its guiding theory, the central dogma. After all, if the human gene count is too low to match the number of proteins and the numerous inherited traits that they engender, and if it cannot explain the vast inherited difference between a weed and a person, there must be much more to the "ultimate description of life" than the genes, on their own, can tell us.

Scientists and journalists somehow failed to notice what had happened. The discovery that the human genome is not much different from the roundworm's led Dr. Eric Lander, one of the leaders of the project, to declare that humanity should learn "a lesson in humility."17 In the New York Times, Nicholas Wade merely observed that the project's surprising results will have an "impact on human pride" and that "human self-esteem may be in for further blows" from future genome analyses, which had already found that the genes of mice and men are very similar.¹⁶

The project's scientific reports offered little to explain the shortfall in the gene count. One of the possible explanations for why the gene count is "so discordant with our predictions" was described, in full, last February in Science as follows: "nearly 4096 of human genes are alternatively spliced."¹⁸ Properly understood, this modest, if esoteric, account fulfills Crick's dire prophecy: it "shakes the whole intellectual basis of molecular biology" and undermines the scientific validity of its application to genetic engineering.

Alternative splicing is a startling departure from the orderly design of the central dogma, in which the distinctive nucleotide sequence of a single gene encodes the amino acid sequence of a single protein. According to Crick's sequence hypothesis, the gene's nucleotide sequence (i.e., its "genetic information") is transmitted, altered in form but not in content, through RNA intermediaries, to the distinctive amino acid sequence of a particular protein. In alternative splicing, however, the gene's original nucleotide sequence is split into fragments that are then recombined in different ways to encode a multiplicity of proteins, each of them different in their amino acid sequence from each other and from the sequence that the original gene, if left intact, would encode.

The molecular events that accomplish this genetic reshuffling are focused on a particular stage in the overall DNA-RNA-protein progression. It occurs when the DNA gene's nucleotide sequence is transferred to the next genetic carrier-messenger RNA. A specialized group of fifty to sixty proteins, together with five small molecules of RNA-known as a "spliceosome"-assembles at sites along the length of the messenger RNA, where it cuts apart various segments of the messenger RNA. Certain of these fragments are spliced together into a number of alternative combinations, which then have nucleotide sequences that differ from the gene's original one. These numerous, redesigned messenger RNAs govern the production of an equal number of proteins that differ in their amino acid sequence and hence in the inherited traits that they engender. For example, when the word TIME is rearranged to read MITE, EMIT, and ITEM, three alternative units of information are created from an original one. Although the original word (the unspliced messenger RNA nucleotide sequence) is essential to the process, so is the agent that performs the rearrangement (the spliceosome).¹⁹

Alternative splicing can have an extraordinary impact on the gene/protein ratio. We now know that a single gene originally believed to encode a single protein that occurs in cells of the inner ear of chicks (and of humans) gives rise to 576 variant proteins, differing in their amino acid sequences.²⁰ The current record for the number of different proteins produced from a single gene by alternative splicing is held by the fruit fly, in which one gene generates up to 38,016 variant protein molecules.²¹

Alternative splicing thus has a devastating impact on Crick's theory: it breaks open the hypothesized isolation of the molecular system that transfers genetic information from a single gene to a single protein. By rearranging the single gene's nucleotide sequence into a multiplicity of new messenger RNA sequences, each of them different from the unspliced original, alternative splicing can be said to generate new genetic information. Certain of the spliceosome's proteins and RNA components have an affinity for particular sites and, binding to them, form an active catalyst that cuts the messenger RNA and then rejoins the resulting fragments. The spliceosome proteins thus contribute to the added genetic information that alternative splicing creates. But this conclusion conflicts with Crick's second hypothesis-that proteins cannot transmit genetic information to nucleic acid (in this case, messenger RNA)--and shatters the elegant logic of Crick's interlocking duo of genetic hypotheses.²²

The discovery of alternative splicing also bluntly contradicts the precept that motivated the genome project. It nullifies the exclusiveness of the gene's hold on the molecular process of inheritance and disproves the notion that by counting genes one can specify the array of proteins that define the scope of human inheritance. The gene's effect on inheritance thus cannot be predicted simply from its nucleotide sequence-the determination of which is one of the main purposes of the Human Genome Project. Perhaps this is why the crucial role of alternative splicing seems to have been ignored in the planning of the project and has been obscured by the cunning manner in which its chief result has been reported. Although the genome reports do not mention it, alternative splicing was discovered well before the genome project was even planned-in 1978 in virus replication²³, and in 1981 in human cells.²⁴ By 1989, when the Human Genome Project was still being debated among molecular biologists, its champions were surely aware that more than 200 scientific papers on alternative splicing of human genes had already been published.²⁵ Thus, the shortfall in the human gene count could-indeed should-have been predicted. It is difficult to avoid the conclusion-troublesome as it is that the project's planners knew in advance that the mismatch between the numbers of genes and proteins in the human genome was to be expected, and that the $3 billion project could not be justified by the extravagant claims that the genome-or perhaps God speaking through it would tell us who we are.²⁶

Alternative splicing is not the only discovery over the last forty years that has contradicted basic precepts of the central dogma. Other research has tended to erode the centrality of the DNA double helix itself, the theory's ubiquitous icon. In their original description of the discovery of DNA, Watson and Crick commented that the helix's structure "immediately suggests a possible copying mechanism for the genetic material." Such self-duplication is the crucial feature of life, and in ascribing it to DNA, Watson and Crick concluded, a bit prematurely, that they had discovered life's magic molecular key.²⁷

Biological replication does include the precise duplication of DNA, but this is accomplished by the living cell, not by the DNA molecule alone. In the development of a person from a single fertilized egg, the egg cell and the multitude of succeeding cells divide in two. Each such division is preceded by a doubling of the cell's DNA; two new DNA strands are produced by attaching the necessary nucleotides (freely available in the cell), in the proper order, to each of the two DNA strands entwined in the double helix. As the single fertilized egg cell grows into an adult, the genome is replicated many billions of times, its precise sequence of three billion nucleotides retained with extraordinary fidelity.²⁸ The rate of error-that is, the insertion into the newly made DNA sequence of a nucleotide out of its proper order-is about one in 10 billion nucleotides. But on its own, DNA is incapable of such faithful replication; in a test-tube experiment, a DNA strand, provided with a mixture of its four constituent nucleotides, will line them up with about one in a hundred of them out of its proper place. On the other hand, when the appropriate protein enzymes are added to the test rube, the fidelity with which nucleotides are incorporated in the newly made DNA strand is greatly improved, reducing the error rate to one in 10 million. These remaining errors are finally reduced to one in 10 billion by a set of "repair" enzymes (also proteins) that detect and remove mismatched nucleotides from the newly synthesized DNA.²⁹

Thus, in the living cell the gene's nucleotide code can be replicated faithfully only because an array of specialized proteins intervenes to prevent most of the errors-which DNA by itself is prone to make-and to repair the few remaining ones. Moreover, it has been known since the 1960s that the enzymes that synthesize DNA influence its nucleotide sequence. In this sense, genetic information: arises not from DNA alone but through its essential collaboration with protein enzymes-a contradiction of the central dogma's precept that inheritance is uniquely governed by the self-replication of the DNA double helix.

Another important divergent observation is the following: in order to become biochemically active and actually generate the inherited trait, the newly made protein, a strung-out ribbon of a molecule, must be folded up into a precisely organized ball-like structure. The biochemical events that give rise to genetic traits-for example, enzyme action that synthesizes a particular eye-color pigment-take place at specific locations on the outer surface of the three-dimensional protein, which is created by the particular way in which the molecule is folded into that structure. To preserve the simplicity of the central dogma, Crick was required to assume, without any supporting evidence, that the nascent protein-a linear molecule-always folded itself up in the right way once its amino acid sequence had been determined. In the 1980s, however, it was discovered that some nascent proteins are on their own likely to become misfolded-and therefore remain biochemically inactive-unless they come in contact with a special type of "chaperone" protein that property folds them.

The importance of these chaperones has been underlined in recent years by research on degenerative brain diseases that are caused by "prions," research that has produced some of the most disturbing evidence that the central dogma is dangerously misconceived.30 Crick's theory holds that biological replication, which is essential to an organism's ability to infect another organism, cannot occur without nucleic acid. Yet when scrapie, the earliest known such disease, was analyzed biochemically, no nucleic acid-neither DNA nor RNA-could be found in the infectious material that transmitted the disease. In the 1980s, Stanley Prusiner confirmed that the infectious agents that cause scrapie, mad cow disease, and similar very rare but invariably fatal human diseases are indeed nucleic-acid-free proteins (he named them prions), which replicate in an entirely unprecedented way. Invading the brain, the prion encounters a normal brain protein, which it then refolds to match the prion's distinctive three-dimensional shape. The newly refolded protein itself becomes infectious and, acting on another molecule of the normal protein, sets up a chain reaction that propagates the disease to its fatal end.³¹

The prion's unusual behavior raises important questions about the connection between a protein's amino acid sequence and its biochemically active, folded-up structure. Crick assumed that the protein's active structure is automatically determined by its amino acid sequence (which is, after all, the sign of its genetic specificity), so that two proteins with the same sequence ought to be identical in their activity. The prion violates this rule. In a scrapie-infected sheep, the prion and the brain protein that it refolds have the same amino acid sequence, but one is a normal cellular component and the other is a fatal infectious agent. This suggests 'that the protein's folded-up configuration is, to some degree, independent of its amino acid sequence and therefore determined, in part, by something other than the DNA gene that governed the synthesis of that sequence. And since the prion protein's three-dimensional shape is endowed with transmissible genetic information, it violates another fundamental Crick precept as well-the forbidden passage of genetic information from one protein to another.* Thus, what is known about the prion is a somber warning that processes far removed from the conceptual constraints of the central dogma are at work in molecular genetics and can lead to fatal disease.**

** In 1997, when Prusiner was awarded the Nobel Prize, several scientists publicly denounced the decision because that the prion, through infectious, is a nucleic-acid-free protein contradicted the central dogma and was too controversial to warrant the award. This bias impeded not only scientific progress but human health as well. Although Prusiner's results explained why the prion's structure resists them, conventional sterilization procedures were nevertheless relied on to fight mad cow disease in Britain, with fatal results.

By the mid 1980s, therefore, long before the $3 billion Human Genome Project was funded, and long before genetically modified crops began to appear in our fields, a series of protein-based processes had already intruded on the DNA gene's exclusive genetic franchise. An array of protein enzymes must repair the all-too-frequent mistakes in gene replication and in the transmission of the genetic code to proteins as well. Certain proteins, assembled in spliceosomes, can reshuffle the RNA transcripts, creating hundreds and even thousands of different proteins from a single gene. A family of chaperones, proteins that facilitate the proper folding- and therefore the biochemical activity-of newly made proteins, form an essential part of the gene-to-protein process. By any reasonable measure, these results contradict the central dogma's cardinal maxim: that a DNA gene exclusively governs the molecular processes that give rise to a particular inherited trait. The DNA gene clearly exerts an important influence on inheritance, but it is not unique in that respect and acts only in collaboration with a multitude of protein-based processes that prevent and repair incorrect sequences, transform the nascent protein into its folded, active form, and provide crucial added genetic information well beyond that originating in the gene itself. The net outcome is that no single DNA gene is the sole source of a given protein's genetic information and therefore of the inherited trait.

The credibility of the Human Genome Project is not the only casualty of the scientific community's stubborn resistance to experimental results that contradict the central dogma. Nor is it the most significant casualty. The fact that one gene can give rise to multiple proteins also destroys the theoretical foundation of a multibillion-dollar industry, `the genetic engineering of food crops. In genetic engineering it is assumed, without adequate experimental proof, that a bacterial gene for an insecticidal protein, for example, transferred to a corn plant, will produce precisely that protein and nothing else. Yet in that alien genetic environment, alternative splicing of the bacterial gene might give rise to multiple variants of the intended protein-or even to proteins bearing little structural relationship to the original one, with unpredictable effects on ecosystems and human health.

The delay in dethroning the all-powerful gene led in the 1990s to a massive invasion of genetic engineering into American agriculture, though its scientific justification had already been compromised a decade or more earlier. Nevertheless, ignoring the profound fact that in nature the normal exchange of genetic material occurs exclusively within a single species, biotech-industry executives have repeatedly boasted that, in comparison, moving a gene from one species to another is not only normal but also more specific, precise, and predictable. In only the last five years such transgenic crops have taken over 68 percent of the U.S. soybean acreage, 26 percent of the corn acreage, and more than 69 percent of the cotton acreage.³²

That the industry is guided by the central dogma was made explicit by Ralph W.F. Hardy, president of the National Agricultural Biotechnology Council and formerly director of life sciences at DuPont, a major producer of genetically engineered seeds. In 1999, in Senate testimony, he succinctly described the industry's guiding theory this way: "DNA (top management molecules) directs RNA formation (middle management molecules) directs protein formation (worker molecules)."³³ The outcome of transferring a bacterial gene into a corn plant is expected to be as predictable as the result of a corporate takeover: what the workers do will be determined precisely by what the new top management tells them to do. This Reaganesque version of the central dogma is the scientific foundation upon which each year billions of transgenic plants of soybeans, corn, and cotton are grown with the expectation that the particular alien gene in each of them will be faithfully replicated in each of the billions of cell divisions that occur as each plant develops; that in each of the resultant cells the alien gene will encode only a protein with precisely the amino acid sequence that it encodes in its original organism; and that throughout this biological saga, despite the alien presence, the plant's natural complement of DNA will itself be properly replicated with no abnormal changes in composition.

In an ordinary unmodified plant the reliability of this natural genetic process results from the compatibility between its gene system and its equally necessary protein-mediated systems. The harmonious relation between the two systems develops during their cohabitation, in the same species, over very long evolutionary periods, in which natural selection eliminates incompatible variants. In other words, within a single species the reliability of the successful outcome of the complex molecular process that gives rise to the inheritance of particular traits is guaranteed by many thousands of years of testing, in nature.

In a genetically engineered transgenic plant, however, the alien transplanted bacterial gene must properly interact with the plant's protein-mediated systems. Higher plants, such as corn, soybeans, and cotton, are known to possess proteins that repair DNA miscoding;³⁴ proteins that alternatively splice messenger RNA and thereby produce a multiplicity of different proteins from a single gene;³⁵ and proteins that chaperone the proper folding of other, nascent proteins.³⁶ But the plant systems' evolutionary history is very different from the bacterial gene's. As a result, in the transgenic plant the harmonious interdependence of the alien gene and the new host's protein-mediated systems is likely to be disrupted in unspecified, imprecise, and inherently unpredictable ways. In practice, these disruptions are revealed by the numerous experimental failures that occur before a transgenic organism is actually produced and by unexpected genetic changes that occur even when the gene has been successfully transferred.³⁷

Most alarming is the recent evidence that in a widely grown genetically modified food crop-soybeans containing an alien gene for herbicide resistance-the transgenic host plant's genome has itself been unwittingly altered. The Monsanto Company admitted in 2000 that its soybeans contained some extra fragments of the transferred gene, but nevertheless concluded that "no new proteins were expected or observed to be produced."³⁸ A year later, Belgian researchers discovered that a segment of the plant's own DNA had been scrambled. The abnormal DNA was large enough to produce a new protein, a potentially harmful protein.³⁹

One way that such mystery DNA might arise is suggested by a recent study showing that in some plants carrying a bacterial gene, the plant's enzymes that correct DNA replication errors rearrange the alien gene's nucleotide sequence.⁴⁰ The consequences of such changes cannot be foreseen. The likelihood in genetically engineered crops of even exceedingly rare, disruptive effects of gene transfer is greatly amplified by the billions of individual transgenic plants already being grown annually in the United States.

The degree to which such disruptions do occur in genetically modified crops is not known at present, because the biotechnology industry is not required to provide even the most basic information about the actual composition of the transgenic plants to the regulatory agencies. No tests, for example, are required to show that the plant actually produces a protein with the same amino acid sequence as the original bacterial protein. Yet this information is the only way to confirm that the transferred gene does in fact yield the theory-predicted product. Moreover, there are no required studies based on detailed analysis of the molecular structure and biochemical activity of the alien gene and its protein product in the transgenic commercial crop. Given that some unexpected effects may develop very slowly, crop plants should be monitored in successive generations as well. None of these essential tests are being performed, and billions of transgenic plants are now being grown with only the most rudimentary knowledge about the resulting changes in their composition. Without detailed, ongoing analyses of the transgenic crops, there is no way of knowing if hazardous consequences might arise. Given the failure of the central dogma, there is no assurance that they will not. The genetically engineered crops now being grown represent a massive uncontrolled experiment whose outcome is inherently unpredictable. The results could be catastrophic.

Crick's central dogma has played a powerful role in creating both the Human Genome Project and the unregulated spread of genetically engineered food crops. Yet as evidence that contradicts this governing theory has accumulated, it has had no effect on the decisions that brought both of these monumental undertakings into being. It is true that most of the experimental results generated by the theory confirmed the concept that genetic information, in the form of DNA nucleotide sequences, is transmitted from DNA via RNA to protein. But other observations have contradicted the one-to-one correspondence of gene to protein and have broken the DNA gene's exclusive franchise on the molecular explanation of heredity. In the ordinary course of science, such new facts would be woven into the theory, adding to its complexity, redefining its meaning, or, as necessary, challenging its basic premise. Scientific theories are meant to be falsifiable; this is precisely what makes them scientific theories. The central dogma has been immune to this process. Divergent evidence is duly reported and, often enough, generates intense research, but its clash with the governing theory is almost never noted.

Because of their commitment to an obsolete theory, most molecular biologists operate under the assumption that DNA is the secret of life, whereas the careful observation of the hierarchy of living processes strongly suggests that it is the other way around: DNA did not create life; life created DNA.⁴¹ When life was first formed on the earth, proteins must have appeared before DNA because, unlike DNA, proteins have the catalytic ability to generate the chemical energy needed to assemble small ambient molecules into larger ones such as DNA. DNA is a mechanism created by the cell to store information produced by the cell. Early life survived because it grew, building up its characteristic array of complex molecules. It must have been a sloppy kind of growth; what was newly made did not exactly replicate what was already there. But once produced by the primitive cell, DNA could become a stable place to store structural information about the cell's chaotic chemistry, something like the minutes taken by a secretary at a noisy committee meeting. There can be no doubt that the emergence of DNA was a crucial stage in the development of life, but we must avoid the mistake of reducing life to a master molecule in order to satisfy our emotional need for unambiguous simplicity. The experimental data, shorn of dogmatic theories, points to the irreducibility of the living cell, the inherent complexity of which suggests that any artificially altered genetic system, given the magnitude of our ignorance, must sooner or later give rise to unintended, potentially disastrous, consequences. We must be willing to recognize how little we truly understand about the secrets of the cell, the fundamental unit of life.

Why, then, has the central dogma continued to stand? To some degree die theory has been protected from criticism by a device more common to religion than science: dissent, or merely the discovery of a discordant fact, is a punishable offense, a heresy that might easily lead to professional ostracism. Much of this bias can be attributed to institutional inertia, a failure of rigor, but there are other, more insidious, reasons why molecular geneticists might be satisfied with the status quo; the central dogma has given them such a satisfying, seductively simplistic explanation of heredity that it seemed sacrilegious to entertain doubts. The central dogma was simply too good not to be true.

As a result, funding for molecular genetics has rapidly increased over the last twenty years; new academic institutions, many of them "genomic" variants of more mundane professions, such as public health, have proliferated. At Harvard and other universities, the biology curriculum has become centered on the genome. But beyond the traditional scientific economy of prestige and the generous funding that follows it as night follows day, money has distorted the scientific process as a once purely academic pursuit has been commercialized to an astonishing degree by the researchers themselves. Biology has become a glittering target for venture capital; each new discovery brings new patents, new partnerships, new corporate affiliations. But as the growing opposition to transgenic crops clearly shows, there is persistent public concern not only with the safety of genetically engineered foods but also) with the inherent dangers in arbitrarily overriding patterns of inheritance that are embedded in the natural world. through long evolutionary experience. Too often those concerns have been derided by industry scientists as the "irrational" fears of an uneducated public. The irony, of course, is that the biotechnology industry is based on science that is forty years old and conveniently devoid of more recent results, which show that there are strong reasons to fear the potential consequences of transferring a DNA gene between species. What the public fears is not the experimental science but the fundamentally irrational decision to let it out of the laboratory into the real world before we truly understand it.

I'm behind on my reading? No, you are behind on your reporting. You made the assertion. Back it up. And try to avoid being snide.

Tu quoque. (My doctoral thesis work is in molecular/cell biology.) Here are two sources that relate to differential splicing and the multiplier effect it has on the apparent number of 30-35,000 human genes. Their citations should also provide lots of background. A quick precis of all this is as follows:

Based on protein diversity and the idea of one "gene" per protein, there was an expected 100,000 or so human genes. Genes became defined as DNA stretches flanked by untranslated regions. Defined this way, there are, as of now, an estimated 30-35,000 genes. However, if "gene" is defined as a stretch of DNA which will give rise to a functional protein of biological significance (some virtually identical proteins do different things in different tissues or at different times of development), then the original estimates of gene number in the human genome are probably on the low side. It is now becoming apparent that a single stretch of DNA formerly called a single gene is, in actuality, capable of coding for two, three, or more functionally-different proteins. Alternate or differential splicing, once thought to be rare, is now seen to be commonplace.

1.

Commentary

March 2001 Volume 19 Number 3 p 196

Piecing together the significance of splicing

Rotem Sorek & Mor Amitai

Rotem Sorek is a scientist (e-mail: rotem.sorek@cgen.com) and Mor Amitai (e-mail: mor.amitai@cgen.com) is the president and chief executive officer at Compugen, 72 Pinchas Rosen St., Tel-Aviv, Israel, 69512.

Alternative splicing increases protein diversity by allowing multiple, sometimes functionally distinct, proteins to be encoded by the same gene. It can be specific to tissues, stress conditions, and developmental and pathological states. In many cases, it serves as an on/off regulation mechanism by introducing a premature stop codon¹. We still do not understand how alternative splicing is regulated, but the following fact is now quite clear: in metazoans, it happens very often.
Computational analysis of expressed sequences can teach us a great deal about alternative splicing, because aligning expressed sequences of different splice variants of the same gene usually results in a typical gapped pattern of alignment. The recent information explosion in nucleotide databases gives us the possibility of analysis at the transcriptome (the set of messenger RNAs) level. We can take the entire human expressed sequence tags (ESTs) database (http://www.ncbi.nlm.nih.gov/dbEST/) (currently containing more than 3.1 million ESTs), together with the known complementary DNAs and genomic sequence data, and cluster them by alignment overlaps.

Such large-scale analyses were conducted in the past few years by four independent groups using different methods^2-5. They estimated that 33–59% of human genes have at least two splice variants, with the highest estimation being the most recent one². All four groups also pointed out that these figures are probably an underestimation, because the EST database does not cover the entire repertoire of tissues or developmental states, and precautions taken to avoid false positives were extremely stringent. Because the latest estimation of the total number of human genes is 30,000–40,000 (ref. 2), one must bear in mind that at least 10,000 of our genes, and probably many more, undergo alternative splicing.
Understanding alternative splicing and gaining knowledge of the transcriptome are crucial for the design and interpretation of expression profiling experiments, in particular DNA chip experiments. Such experiments enable comparisons between transcriptomes of different cell types or under different conditions. Designing DNA chips that will effectively report on the transcriptional levels of genes must take into account their alternative splicing patterns, even if alternative splicing is not the subject being studied.
To demonstrate this, let us assume a putative gene X that has three exons (A, B, and C) and two splice variants, ABC and AC. To design a chip that will measure the transcriptional levels of gene X in different tissues, we must use a probe from exons A or C to which both variants will hybridize in the assay. Taking the probe from exon B will cause the hybridization of variant ABC only, and will not correctly measure the transcriptional level of the gene. The accuracy of the experiment can be increased by measuring the level of each variant separately; hence, two probes will have to be taken, one from exon B to measure variant ABC, and one from exon A or C to measure both variants.
Another illustration of the importance of awareness of alternative splicing comes from the field of gene prediction. One thing gene prediction programs do not predict is alternative splicing, because sequences that regulate alternative splicing are generally unknown. Because alternative splice sites often correspond weakly to the splice consensus sites, gene prediction programs will probably frequently fail to identify alternative exons or introns.
Alternatively spliced genes are likely to take center stage as drug targets, therapeutic agents, and diagnostics markers in the next decade. First, there are many splice variants of pharmaceutically important genes that have been detected but not yet studied in depth. The function of the known variant gives us a clue to the function of the new variant, especially if we know which domain was added or removed. For example, we have identified some 60 kinase enzymes that undergo alternative splicing that eliminates their catalytic domains (E. Levanon et al., unpublished data). Although many of them have not been biochemically studied, our educated guess is that they function as competitive inhibitors of the known kinases.
Second, it has been estimated that 15% of the point mutations that cause genetic diseases in humans alter the normal splicing pattern⁶. Splice variants that are disease specific can be excellent diagnostic markers for these and other human diseases, being easily identifiable by PCR reactions.
Alternative splicing is no longer considered an esoteric twist of nature. Articles with the phrase "alternative splicing" or "splice variants" in their title or abstract are published at the rate of two a day (according to Medline query). In many ways, the concept is breaking our iron-clad rules: exons are not always exons, and introns are sometimes expressed.
Indeed, the very definition of "gene" should be reconsidered in light of discoveries of unusual alternative splicing events, such as the one yielding a novel splice variant of PSA (prostate specific antigen)—the standard prostate cancer marker⁷. This variant shares with PSA only the first exon, which encodes only a signal peptide, leaving the two mature proteins with no common protein sequence. The only connection between them is that they are coded by the same genomic region and probably share the same transcriptional regulation (A. David et al., unpublished data).
Even more extreme is the example of the p19 and p16 protein products of the INK4a/ARF locus⁸. The two transcripts are synthesized from different promoters and have different first exons, but share exons 2 and 3, and are encoded in two distinct reading frames, in a process that yields two entirely different protein products. Although the p19 and p16 proteins are clearly the products of the same genomic locus, can we say that these two unusual and entirely distinct splice variants are coded by the same gene?
Clearly, increasing protein diversity does not simply correlate with increasing gene number. It is dependent both on the number of genes in the genome and on the rate of alternative splicing of those genes. Work is now needed to characterize in greater detail the molecular basis for this process and its regulation. This will likely uncover a host of new targets for drug discovery, yield new diagnostic markers for disease, and perhaps even help us unravel the mechanisms underlying biological complexity.

REFERENCES

Smith, C.W. & Valcarcel, J. Trends Biochem. Sci. 25, 381-388 (2000). | PubMed | ISI |
International Human Genome Sequencing Consortium. Nature 409, 860-921 (2001). | Article | PubMed |
Brett, D. et al. FEBS Lett. 474, 83-86 (2000). | Article | PubMed | ISI |
Mironov, A.A., Fickett, J.W. & Gelfand, M.S. Genome Res. 9, 1288-1293 (1999). | Article | PubMed | ISI |
Croft, L. et al. Nat. Genet. 24, 340-341 (2000). | Article | PubMed | ISI |
Cooper, T.A. & Mattox, W. Am. J. Hum. Genet. 61, 259-266 (1997). | PubMed | ISI |
Diamandis, E.P. Trends Endocrinol. Metab. 9, 310-316 (1998). | Article | ISI |
Sharpless, N.E. & DePinho, R.A. Curr. Opin. Genet. Dev. 9, 22-30 (1999). | Article | PubMed | ISI |

2. I had to reformat this since it didn't come across via source in double columns. The figures and tables didn't appear, either. For figures, tables, and legends, see the original article.

Nature Genetics 30 * January 13 2002

A genomic view of alternative splicing
Barmak Modrek & Christopher Lee
Departments of Chemistry and Biochemistry,
University of California Los Angeles,
Los Angeles, California 90095-1570, USA.
Correspondence should be addressed to C.L.
(e-mail: leec@mbi.ucla.edu).

Recent genome-wide analyses of alternative splicing indicate that 40–60% of human genes have alter- native splice forms, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Here we review these recent results from bioinformatics studies, assess their reliability and consider the impact of alternative splicing on biological functions. Although the `big picture' of alternative splicing that is emerging from genomics is exciting, there are many challenges. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. We recommend a community-based effort to discover and characterize alternative splice forms comprehensively throughout the human genome.

Introduction
The sequencing of the human genome has raised important questions about the nature of genomic complexity. It was widely anticipated that the human genome would contain a much larger number of genes (estimates based on expressed-sequence clustering ran as high as 150,000 genes) than Drosophila (14,000 genes) or Caenorhabditis elegans (19,000 genes) 1–3 . The report of only 32,000 human genes thus came as a surprise 4,5 . This basic disparity indicated that the number of human expressed- sequence (mRNA) forms was much higher than the number of genes, suggesting a major role for alternative splicing in the production of complexity. Many groups have recently presented genomic analyses of alternative splicing that strongly support this hypothesis, raising intriguing questions about the identification, functional roles and regulation of alternative splice forms across the whole genome. The study of alternative splicing has long been a valuable subfield of molecular biology, but has received comparatively little attention compared with major fields such as the discovery of new genes or transcriptional regulation. Only several hundred alternatively spliced genes have been identified so far by molecular biologists (see Table 1 for database resources). After the discovery of exons and introns in the Adenovirus hexon gene in 1977 (ref. 6), Walter Gilbert proposed that different combinations of exons could be spliced together (`alternative splicing') to produce different mRNA isoforms of a gene 7 . By the early 1980s, alternative splicing was well documented in several genes 8,9 , and researchers estimated that 5% of genes in higher eukaryotes might have alternative splicing 10 . A range of processes from sex determination to apoptosis use alternative splicing 11,12 . Its regulatory mechanisms have recently been discovered in several genes 11,13 .

Genome-scale analyses of alternative splicing
High-throughput sequencing of the human genome and especially of expressed sequence tag (EST) sequences has enabled a completely different approach based on bioinformatics. Because ESTs are derived from fully processed mRNA (after 5 capping, splicing and polyadenylation), they provide a broad sample of mRNA diversity. This diversity can be analyzed computationally. In the last two years, bioinformatics studies have identified an order of magnitude more alternatively spliced genes than were found in the past 20 years and are beginning to provide a global view of alternative splicing in humans. We will first describe these studies and then assess the evidence. Bioinformatics approaches. Most bioinformatics studies 4,14–18 (Table 2) rely on identifying ESTs that come from the same gene and looking for differences between them that are consistent with alternative splicing, such as a large insertion or deletion in one EST (Fig. 1 a ). Each candidate splice can be fur- ther assessed by aligning the ESTs exactly to their gene sequence in the draft genome (Fig. 1 b ). This reveals candidate exons (matches to the genomic sequence) separated by candidate splices (large gaps in the EST-genomic alignment; Fig. 1 b ). As intronic sequences at splice junctions are highly conserved (99.24% of introns have a GT-AG at their 5 and 3 ends, respectively), they can be used to verify candidate splices 19 . In the earliest large-scale discovery of new alternative splicing, Mironov et al. 14 aligned ESTs to genomic sequence for 392 known genes and found alternative splicing in 133 of these genes 14 . Croft et al . 15 took a different approach that did not rely on aligning ESTs to the complete genomic sequence: they created a database of individual intron sequences annotated in GenBank and searched for EST sequences that matched intronic sequence. They found matches to introns from 582 genes, suggesting an alternative splice. Brett et al . 16 looked for insertions or deletions in ESTs relative to a set of known mRNAs, indicative of alternative splices, but without EST alignment to the genomic sequence. This work identified 3,011 alternatively spliced genes 16 . The International Human Genome Sequencing Consortium reported 145 alternatively spliced genes from a comprehensive analysis of chromosome 22 based on aligning ESTs to the genomic sequence 4 . Modrek et al . 18 aligned available human EST and mRNA sequences (2.1 million) to the whole draft genome, applying strict matching, splice site and alternative splice detection criteria, to identify 6,201 alternative splices in 2,272 genes.

Alternative splicing frequency.
These studies have consistently reported a high rate of alternative splicing in the human genome, with 35–59% of human genes showing evidence of at least one alternative splice form 4,14,16–18 . Moreover, given that only a few ESTs have been sequenced for most genes, it seems possible that even more alternative splicing exists that is not yet detectable in the available ESTs. These studies indicate that alter- native splicing is far more abundant, ubiquitous and functionally important than previously thought. And there are more types of mRNA isoforms. For example, bioinformatics studies have reported that about 25% of genes have alternative polyadenylation forms, that is, mRNAs that are cleaved and polyadenylated at different sites 4,20 .

Functional impact.
How do these newly discovered alternative mRNA forms affect protein function? Despite an early report that most alternative splices occur within the 5 untranslated region 14, recent studies indicate that 70–88% of alternative splices change the protein product 4,17,18 . The majority of these changes appear to be functionally interesting, such as replacement of the amino or carboxy terminus, or in-frame addition and removal of a functional unit (Fig. 2 b ) 18 . Only 19% of the alternative protein forms were shortened due to frameshift 18 . Fig. 2 c shows an alternative isoform of a new FC receptor -like protein, whose C-terminal transmembrane domain (TM) and cytoplasmic tail (important for signal transduction in this class of receptors) is neatly replaced with a new TM domain and tail by alternative polyadenylation 18 . What is the functional pattern of alternative splicing across the genome? A random sample of 50 alternatively spliced genes showed that over three-quarters were involved in signaling and regulation (such as receptors, signal transduction, transcription factors, and so on). Moreover, the systemic categories most highly represented in this sample were genes specific to the immune and nervous systems 18 . This should be interpreted cautiously, as the overall breakdown of gene functions in the whole genome is still unclear. However, alternative splicing may be most important in complex systems where information must be processed differently at different times (such as immune tolerance, or development) or a very high level of diversity is required (such as axonal guidance). Notable examples of combinatorial alternative splicing of multiple cassettes of exons, generating up to 40,000 isoforms of a single gene, have recently been discovered in the nervous system, including Dscam (axonal guidance receptor in Drosophila ) and neurexin (neuropeptide receptor) 21 .

Fig. 2 Types of alternative splicing and possible effects on protein. a , Alternative splicing can lead to either the inclusion or exclusion of an exon, use of a different 5 site, or use of a different 3 site. b , Alternative splicing can lead to use of a different site for translation initiation (alternative initiation), a different translation termination site due to a frameshift (truncation or extension), or the addition or removal of a stop codon in the alternative coding sequence (alternative termination). Alternative splicing can also change the internal region because of an in-frame insertion or deletion. c , Alternative splicing of Hs.11090, a putative FC receptor chain homolog: genomic structure and two alternative spliced (and polyadenylated) mRNA forms. The differential RNA processing results in substitution of one transmembrane domain instead of another. However, one form has a different cytoplasmic tail (involved in signaling in this family), whereas the other does not.

Bioinformatics evidence for alternative splicing.
It is essential that biologists understand the forms of evidence and problems that underlie this new `big picture' view of alternative splicing. Bioinformatics is an automated analysis of high-through-put experimental data and follows a very different process than traditional molecular biology. It can be simultaneously `more rigorous' (much more detailed, mathematical measures of evidence are required for a computer to do this analysis at all) and much less rigorous (bioinformaticists typically cannot order a new set of experimental tests for all the isoforms they detect, as is common in molecular biology labs studying a specific isoform). Two kinds of problems must be distinguished: (i) a false negative, the failure to detect a real splice form, and (ii) a false positive, a reported result that is not a true, functional splice form. Analyzing the causes of these problems during cDNA library construction, EST sequencing and sequence comparison suggests many interesting questions for the next stage of this research (Table 3). Detection of alternative splicing through bioinformatics depends on finding deviant EST forms within the mass of data produced by undirected EST sequencing, raising a fundamental question: when an analysis is used to look for some form of deviation in a very large data set, other causes of deviation, even if infrequent, could add up to a substantial fraction of the result. How can we be sure this is real alternative splicing? The bioinformatics studies have tried carefully to screen out many possible sources of false positives. Simple forms of EST deviation, such as random variation in where a given EST sequence begins or ends within a gene, and potential vector contamination at the ends of ESTs, are excluded. The most important screen is provided by mapping (aligning) ESTs to the draft human genome sequence. Chimeric ESTs can be easily excluded by requiring that each EST align completely to a single genomic locus. The genomic location found by homology search and alignment can often be checked against radiation hybrid mapping data. As the genomic regions that match the ESTs should be exons and the alignment gaps between them should be introns, the putative splice sites at their boundaries can be carefully checked. Because the splice-site motifs (GT-AG, polypyrimidine tract, and so on) are primarily in the intron, this provides a validation that is independent of the EST evi- dence. Reverse transcriptase artifacts or other problems causing imperfect cDNA construction may be screened out in this way. Improper inclusion of genomic sequence in ESTs (due to either mRNA purification problems or incomplete splicing) can also be excluded by requiring pairs of mutually exclusive splices in different ESTs. Observing a given splice in one EST but not in a second EST may be insufficient, because the latter could be an un-spliced EST rather than a biologically significant intron inclusion. This problem can be eliminated by focusing on mutually exclusive splices, two different splices seen in different ESTs, that overlap in the genomic sequence. One can make this even stricter by requiring that the two splices share one splice site but differ at the other. This approach detects the classic forms of alternative splicing, such as alternative exon usage and alternative 5 or 3 splicing (Fig. 2 a ). Detection of valid intron inclusions will probably require further statistical analysis. The presence in the human genome of many pseudogenes and paralogous genes resembling other genes greatly complicates the problem. Correct alternative splice detection depends on clustering the EST data into separate groups representing individual genes. EST clustering (such as UniGene) is well known to have both exces- sive `splitting' of genes (there are 80,000 UniGene clusters, versus the estimate of 32,000 human genes) and excessive `lumping', in which paralogous gene sequences are mixed together 4,22 . This mixing can suggest spurious alternative splices that are actually just differences between similar but distinct genes 23 . Methods that map the ESTs onto genomic sequence with a high level of identity (95–98%) probably exclude much of this paralog mixing, but not all. Ultimately, mapping ESTs to their unique gene location in the genomic sequence is the only way to sort out paralogs. Requiring that the consensus sequence for an EST cluster match completely, over its full length, to its genomic contig can help exclude artifacts where the genomic sequence has been misassembled. Instead of getting false positives (incorrect alternative splices), this may cause false negatives due to refusing to map the EST cluster at all. A high rate of false negatives is the greatest disadvantage of methods that require mapping ESTs to the draft genome sequence. Despite these sources of uncertainty, the agreement among many studies on a high frequency of alternatively spliced genes (35–60%) suggests that this result is valid. These studies support each other persuasively, because they differ not only in the sets of genes sampled (ranging from well-characterized mRNAs, to specific chromosomes, to a whole-genome study), but also in their specific criteria for reporting an alternative splice. It is important, however, to emphasize that there has only been one study so far verifying alternative splices detected by bioinformatics. Twenty genes with putative alternative splices were amplified from a multiple tissue cDNA panel by RT–PCR, with primers flanking the alternative splice (Fig. 3 a ). Sixteen were confirmed to be alternatively spliced, although thirteen of them were already recognized in the literature 16 .

Future Challenges

High-throughput validation.
Large-scale experimental verification of alternative splicing will be needed to assess the accuracy of the bioinformatics-based analyses. One promising technology is inkjet printing of long probes (up to 60 nt) to make rapidly customizable microarrays. Shoemaker et al . 24 used this technology to monitor the coordinate expression of 8,183 exons annotated on chromosome 22q. This technology could easily be adapted to detect alternative splicing, by designing probes that span specific exon–exon junctions. As alternative splicing of a given gene creates different exon–exon junctions, it can be detected by measuring hybridization of mRNA samples from different tissues to these probes (Fig. 3 b ). Whereas the hybridization ratios of most exon–exon junction probes for a given gene will be constant, alternative splicing will cause some junctions to be up- or down- regulated in different tissues. Rapid printing of such `splicing chips' will enable cataloging of splice forms for all genes, in different tissues, developmental states and conditions. Com- bined with the human genome sequence, this data can in turn be used to identify cis elements that regulate these forms. Recently, the Affymetrix microarray design has also been used to identify potential alternative splices within the rat genome. The Affymetrix array uses 20 probe pairs (25 nt) representing different exons of a gene. Whereas the intensities of most probes for a gene varied together in different tissues, probes for certain exons were anomalously depressed in some tissues, indicating potential alternative splices 25 . Other methodologies that use microarray technology to assess alternative splicing have also been developed (X.-D. Fu and M. Ares Jr, personal communication).

Rigorous measures of evidence.
It should be emphasized that microarray approaches will not settle the question of identifying alternative splices independent of bioinformatics analysis. If any- thing, these data are likely to increase the need for bioinformatics, to measure rigorously the strength of the evidence for alternative splices in all the raw experimental data (ESTs, microarrays, and so on). For example, the original inkjet microarray paper treated differences in probe hybridization among exons in a gene as indicators that low-expressed probes were not real exons but simply gene prediction errors. By con- trast, the Affymetrix study treated such differences as evidence of alternative splicing. The assessment of both competing interpretations is a bioinformatics analysis problem. This will require moving beyond simple `rules' for filtering out potentially misleading data to probabilistic measurement of the relative strength of the evidence for the competing interpretations.

Cataloguing alternative splice forms.
Although the new bioinformatics results are based on data from the whole genome, it is important to understand they are highly incomplete. They detect many new splice forms but miss many known isoforms. This is a result of both the incompleteness and fragmentation of the EST and genomic sequence data, as well as many causes of false negatives in the bioinformatics methods (Table 3). In Modrek et al . 18 , at least 50% of the EST data (and their potential alter- native splices) were excluded by these problems. These studies are just the beginning of an accelerating process of mRNA isoform discovery. The EST sequence data are growing rapidly, the draft genome sequence is being completed and new streams of high-throughput data (such as splice-detection microarrays) are beginning. Thus, a worthwhile goal is simply to build a catalog of alternative splice forms, just as the human genome sequence is being used to build a catalog of the genes. The development of new high-throughput technologies for detecting the protein products of alternative splicing will be needed to streamline this process.

What is truly functional?
Although bioinformatics and high- throughput experiments can have a key role in building a catalog, in our view this can only succeed as a community annotation process involving all molecular biology researchers. For example, how can one prove that a particular splice form is actually carrying out an important biological function? Even with strong evidence that a form is real (that it was actually made by the spliceosome in a living cell), it does not seem safe to assume that it has a biological function. If the spliceosome had a 0.1% rate of mis-splicing, it could produce over 4,000 meaningless `alternatively spliced' ESTs among the approximately 4 million ESTs. Bioinformatics can partly address this by discerning that a large subset of alternative splice forms (47%) are observed in multiple ESTs (often from different libraries) and thus are unlikely to be low-frequency error products 18 . At the same time, it is also not safe to dismiss a given form as `functionless' simply because it has no obvious function. For example, even an alternative splice form that causes early translational termination (and an inactive protein product) can act as an important form of regulation of biological activity 13 . Only detailed functional studies can resolve these questions. Bioinformatics can infer likely functional impacts, however, by detecting the addition or removal of known domains, and can predict how experimenters could verify the presence of these forms and their likely disease or tissue specificity. Biologists interested in some of these putative forms could then use a variety of techniques (PCR, northern and western blots) to test these predictions. This process will be best served by a central repository for both the bioinformatics predictions and subsequent experimental verification and functional studies, which would act as a community annotation database (Fig. 4). We hope this process can evolve rapidly into an active partnership between prediction and experiment.

Alternative splicing regulation.
One intriguing new area is the study of alternative splicing regulation. Regulation of splicing could be involved in 15% of genetic diseases 26 and may contribute to cancer by missplicing of exon 18 in BRCA1 , which is caused by a polymorphism in an exonic enhancer 27 . If alternative splicing is as widespread as bioinformatics studies indicate, how different splice forms are turned on and off may become a major research area, like transcriptional regulation. So far, molecular biology has identified some cis regulatory elements (such as exonic splicing enhancers) and trans factors (SR proteins, PTB, and so on) 11,13 . Bioinformatics could make important contributions, for example, in the identification of cis regulatory elements 28–31 . Recently, Brudno et al . 31 analyzed intronic sequence upstream and downstream of 25 alternatively spliced brain specific exons. They detected the motif UGCAUG at a much higher frequency downstream of alternatively spliced exons (relative to constitutive exons), for both brain-specific and muscle-specific alternative splicing 31 . This motif had previously been implicated in the alternative splicing of several genes including c-src, fibronectin, calcitonin/CGRP, and nonmuscle myosin II heavy chain-B 32–35 , so this result is very suggestive. It bodes well for genome-wide studies that combine the flood of new alternative splicing data with complete genome sequences for multiple organisms.

Acknowledgments
We are grateful to D. Black, S. Galbraith and K. Ke for their critical comments and suggestions. C.L. was supported by a grant from the Department of Energy. B.M. was supported by National Science Foundation Integrative Graduate Education and Research Training award. Received 16 August; accepted 20 November 2001.

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Dear Phaedrus:

What an interesting thread! Thanks for posting the story at the top. What I'm most reminded of, by the Human Genome Project's results thus far, is the persistent failure of the "science" of alchemy to transmute dross metal into gold. In the Mediaeval ages and later (and maybe sooner), some of the most brilliant and penetrating minds were devoted to facilitating this object. But nobody ever got anywhere. After centuries of trying, the answer always came up: "No, you can't do that. It is in the nature of things that that should be so." So gambling is all you've got left to satisfy such cravings…. Some passages from Voegelin on the general context in which the present hush may be whispered: * * * * * * We must remind the reader that at the end of the sixteenth century Giordano Bruno had formulated clearly the issue between speculation on the infinite substance of the cosmos and a mathematized science of the "accidences of accidences." Bruno's speculation, on the one hand, found no immediate succession. The "accidences of accidences," on the other hand, had become the absorbing interest of scholars as well as of a wider public in the centuries of the rising natural sciences. The impressive spectacle of the advancement of science and of the Newtonian system created attitudes and sentiments that have become a decisive ingredient in modern man and modern civilization. One element in this new complex of sentiments … [is] scientism: the belief in mathematized science as the model science to the methods of which all other sciences should conform. We must now deal with the complex as a whole, and we shall call it phenomenalism in order to indicate the preoccupation of man with the phenomenal aspects of the world, as they appear in science, and the atrophy of awareness of the substantiality of man and the universe. Phenomenalism has nothing to do with the method of the advancement of science itself; the term is supposed to designate sentiments, imaginations, and speculations, as well as patterns of conduct determined by them, which originate on occasion of the advancement of mathematized science. Furthermore, we must beware of the assumption that the advancement of science is the one and only cause of the rise of phenomenalism. The new sentiments and attitudes, while hardly conceivable without the prodigious advancement of science, are not necessitated by it. That phenomenalism could gain the importance that it actually has is primarily due to the atrophy of Christian spirituality and the growth of intramundane sentiments. The advancement of science is a contributing factor in the process, insofar as its success is apt to fortify intramundane sentiments; and insofar as phenomenalism, grafted on science, has become an important instrument for their expression. [Eric Voegelin, The History of Political Ideas, Volume VII: The New Order and Last Orientation. Columbia: University of Missouri Press, 1999. ] * * * * * * EV is daunting. (Personally, I almost had a heart attack the first time I encountered his term, "hypostasization of reality.") Maybe some notes on the above text might be helpful. (Please beware, this according to my interpretation.) First, Giordano Bruno was an "Italian philosopher, b. at Nola in Campania, in the Kingdom of Naples, in 1548; d. at Rome, 1600. At the age of eleven he went to Naples, to study "humanity, logic, and dialectic", and, four years later, he entered the Order of St. Dominic, giving up his worldly name of Filippo and taking that of Giordano. He made his novitiate at Naples and continued to study there. In 1572 he was ordained priest." [Catholic Encyclopaedia on-line] In 1600, however, he was burned at the stake as a heretic of pantheist and Unitarian persuasions. "The infinite substance of the cosmos" refers ultimately to the life of God and its manifestation in man and nature. It grapples with the questions, "why does anything exist? And why is a given thing the way it is, and not some other way?" A student of culture and history knows that such ultimate questions have resonated with intelligent human beings for millennia by now. They constitute the formal philosophical discipline called ontology: the study of Being. I conclude that "being" and "substance" are virtually synonymous terms in the contexts of Bruno – and EV. With the Greeks and the Christians, the result of such questions has been the development of a "science of man," an anthropology, that is premised on man being a "natural creature," but also a "spiritual creature." That is to say, man lives in the space-time dimension that conditions empirical reality; but he is not completely contained, constrained, or determined by empirical conditions. (This is why man is said to have Free Will. But again, this development deduces from classical/Christian premises.) The "intramundane man" has extension into the infinite; that is, there is a native capacity for transcendence in the nature of man. He lives in at least two time orders, the "natural," spatio-temporal order in which we all "naturally" live; and also an order that is "outside" or "beyond" time. The mystery is that both orders interleave, or "play" more or less simultaneously, whether we are specifically conscious of this or not. (But this would be the subject of a whole 'nother thread. I'd love to get back to it some time; but right now, we're out of time and bandwidth.) "Accidences of accidences" is Bruno's quaint way of signifying a chain of causation that never reaches out beyond the intramundane dimension. That is, it confines itself to the study of causal relations among observable phenomena – the way science must. But that supposition suggests to me that a very great deal of the human picture must be deliberately erased in order to make this "understanding" turn out "right." If you properly understand the point of the phenomenalist exercise, you know it seeks to account for man and the universe without reference to anything lying beyond time and space such that sensory perception can register. Its basic definition (it doesn't even have an anthropology) is that man is abstract individual, with no ties to the past, the most tenuous ties to the present (living in TV land and admiring hard-core sophistry as much as he seems to do) no expectations of the future, and no interest in understanding his own existence as having extension and expression beyond a world which itself is condemned to intramundane existence. I figure you get "accidences of accidences" problems anytime you get your fundamental premise wrong. As arguably, the Human Genome Project has done exactly this. But then, the point and purpose of the Human Genome Project from the beginning was to factually establish the theory of "accidences of accidences," not to refute it. It's late. Must be time to stop. Anyone wants to continue with anything above, please just give me a yell. Thank you for a wonderful discussion, Phaedrus. Peace and love, bb.

#73 posted by betty boop

ME TOO!!!! YEAH, YEAH!!!!! me too!!

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Ahhhh yes, - NO one can refute BB on Voegelin, because NO one can understand the man to begin with.

He is a master of gibberish, bombast & grandiloquent nonsense. -- A rhetorical fraud, imo.

And please, -- run off another few hundred words, telling me about his 'brilliant' career. -- I love this sort of pretentious BS.

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