*This pejorative name for the silent majority of DNA was coined by Suumu Ohno in the early 70’s See: Kuska B Journal of the National Cancer Institute 90, 1032 (1998)

In a basement room at Harvard University's science center one morning a few weeks ago, Dr. Gilbert was at the blackboard, describing the puzzle to nonscientists. He noted that the genes in modern animals were stored in the DNA not as nice readable texts but as choppy fragments that the cell's machinery must stitch together. That curious arrangement is one major clue. Another is that some 95 percent of a person's DNA does not code for genes at all. It seems to be just gibberish, or junk DNA.
Genes in pieces and huge stretches of junk? "At first glance, it is peculiar," Dr. Gilbert told the bleary-eyed students. "Then, at second glance, it is even more peculiar".

Dr. Gilbert and his colleagues have sketched out a theory of how it all may have happened and what the first genes were like. It was he who coined the terms for the interrupted pattern in which genes are stored on the DNA; the working parts he called "exons" and the regions in between, which the cell has to splice out, he termed "introns."...
As Dr. Philip Sharp, a molecular biologist at the Massachusetts Institute of Technology, puts it, few scientists are willing to spend time on so vexing a puzzle. "Yes," Dr. Sharp said, "it is very difficult. It may even be unsolvable. That won't stop Wally Gilbert, of course." Dr. Sharp, who shared a Nobel Prize in Physiology or Medicine in 1993 for finding introns, one of the discoveries that sent Dr. Gilbert off on this track, said the ideas that Dr. Gilbert put forward "captured the imagination of the field, and still has it, I think."

If Dr. Gilbert's approach is valid, in principle of the entire history of life on earth could be inferred from the DNA of modern go back!Cerebellum: the Graveyard of the "Junk Gene" Misnomer genes. His theory is an effort to figure out how, in the primordial waters where life began, the earliest genes were assembled. A modern gene is a chemical text with a thousand of letters. For such a structure to evolve at random would be a lengthy process. Instead, Dr. Gilbert suggests, the first genetic elements were simple modules, the forerunners of today's exons, and the exons were then mixed and matched to build up the lengthy chains that make longer and longer genes. By analyzing the structure of contemporary genes, it should be possible to discern the ancient modules within. For the modules to mix and match, Dr. Gilbert believes, they would have needed strips of extra DNA, like the leader on a reel of film, so that two modules could be spliced together without risk of cutting into their genetic message in the process .
The introns, the elements interspersed between the coding regions of genes, are in this view related to the ancient leaders that enabled the modules to be assembled. Critics say the theory leaves some awkward issues unexplained. Introns are found in the DNA of plants and animals but not in that of bacteria. So did bacteria lose their introns or never acquire them?

Dr. Jeffrey D. Palmer at Indiana University and a few other experts in molecular evolution suggest that Dr. Gilbert's theory is wrong. They say that the introns and the shuffling of genetic modules were mechanisms that did not arise until two billion years after life first began. And, they regard the introns not as clever Tinker Toy construction elements, but as just what they appear to be, pieces of junk DNA left over from billions of years of evolution. Noting that bacteria, presumably more primitive than plants and animals, do not have introns in their genes, they say it is more reasonable to assume bacteria never had introns than that they had and later lost them. But bacteria may have streamlined their genetic material by shedding their introns leading ot the "pleasantly perverse" conclusion that humans can be considered as "less evolved" than bacteria, Dr. Gilbert said with a grin. "maybe we are not in all ways at the pinnacle of evolution, as people like to think," he said. And worse, maybe nature will evolve right past humans and their junk-laden genomes in the future.

Life's Assembly Line:
In a report to be published this week in the Proceedings of the National Academy of Sciences, Gilbert's team describes how 32 three dimensional proteins are constructed from separate modules that correspond to individual exons in the gene that codes for them.
"The protein building blocks, or modules, correlate with the positions of exons separated from each other by introns," explains Sandro DeSouza, a postdoctoral affiliate in molecular and cellular biology. "The boundaries of the modules correspond to the positions of introns." During the process of protein assembly in a cell, the introns are discarded and the exon modules hooked together. The complex protein thus constructed boasts a function greater than the sum of its parts. Gilbert believes that our earliest ancestor was put together in much the same way. About 3.5 billion years ago, lifeless combinations of chemicals evolved into molecules that carried instructions for making proteins (see "Creating Life in a Lab," Sept. 12, 1996, Gazette). When such molecules, the first exons, became hooked together by introns, they could produce more complex proteins than when they existed individually. They also could be disconnected from each other and combined into new types of genes.
"Such shuffling must have speeded up evolution," notes Manyuan Long, a postdoctoral fellow. "You don't need to evolve new genes from scratch, rather you can recombine basic modules already available. That would accelerate the diversity of proteins and so of living things."

The Junk Gene Mystery
Scientists were amazed and puzzled when they found, in the 1970s, that 95 percent of the genetic material coiled up inside virtually every cell consists of DNA that apparently serves no purpose. It's not like nature to be so wasteful. In 1978, Gilbert, who won the 1980 Nobel Prize in Chemistry for his work in genetics, came up with the exon theory of genes to explain why so much junk exists in the cells of all living creatures. Although it's a tidy idea, not everyone accepts it.
To convince doubters, Gilbert has been searching for supporting evidence in the positions and structure of exons and introns. That kind of evidence became available only recently from the Human Genome Project, the herculean effort to map all the chemical bases that make up both exons and introns.
Human genes contain some 3 billion such units arranged in as many as 100,000 genes. They are often referred to as "letters" that spell out our genetic heritage. By 1990, the positions of some 60 million bases had been determined, not enough for what Gilbert wanted to do. By 1995, however, the number reached 600 million. Long took on the task of searching the entire genetic alphabet, the letters found in every creature from one-celled blobs of life to the most-gifted humans. He found that introns and exons line up in a nonrandom way in the DNA of most living things. Exons appear in the same patterns in primitive protozoans, worms, flies, and people, as do the introns that separate them.
"This fact can only be explained if introns were present from the beginning of life and not inserted later, as some biologists claim," Long insists.
"That means that ancient regions, which represent genes or portions of genes, have descended in a relatively unchanged manner from a common ancestor," Gilbert adds. "This supports the theory that introns have facilitated the shuffling of exons from the time of the origin of genes. It also means that genes are composed, not of hundreds or thousands of basic building blocks, but of a lot fewer, possibly less than a hundred."
The work on 32 common proteins, conserved from ancient times, adds further evidence in favor of the exon theory of genes.

Lost Introns
Of course, there still are objections to this neat story of life. Bacteria, which evolved before plants and animals, lack introns. If they never possessed them, that undermines Gilbert's theory. But he maintains that bacteria lost their introns during three-plus billion years of shuffling. "Losing introns would enable bacteria to reproduce faster," Long maintains. "You can think of it as a streamlining process."
Gilbert raises the fascinating possibility that bacteria may be ahead of humans and their junk-laden genes in the evolutionary race. "Maybe we're not as advanced and sophisticated as we would like to believe," he says with a smile.
Skeptics acknowledge that the Harvard team has accumulated strong evidence that new genes can arise from mixing and matching exons. However, that evidence does not prove that the common ancestor of modern life was put together in this fashion. "Since that occurred between 3 billion and 4 billion years ago, some critics say we will never be able to prove it conclusively," Long admits. He compares the situation to the accepted existence of quarks and other subatomic particles. "We cannot see them, but we believe they exist because they explain the behavior of matter so successfully," he says.
"Our theory demands certain consequences," Gilbert adds. "If we make predictions based upon it and find that the predictions are correct, then we've gone as far as we can." The practical results of successfully predicting how each protein is assembled could be staggering. Virtually every human disease and defect can be traced to abnormal or missing proteins. Having their blueprints opens up the possibility of designing drugs or replacing defective exons to correct these problems. "Determining how our common ancestor was put together doesn't promise such practical consequences; it's pure basic research," Gilbert points out. "But history has shown repeatedly that today's basic knowledge becomes tomorrow's electric motor, computer chip, or miracle material. Take flu viruses, as a possibility. New strains arise every year. That's a problem in evolution. If we can understand and predict how viruses evolve, how they shuffle their exons, we could make more effective vaccines. We can never conclusively prove the exon theory of genes, but if we can realize such practical benefits from it, we will never need to do so."

BOSTON-In a region of DNA long considered a genetic wasteland, Harvard Medical School researchers have discovered a new class of gene. Most genes carry out their tasks by making a product-a protein or enzyme. This is true of those that provide the body's raw materials, the structural genes, and those that control other genes' activities, the regulatory genes. The new one, found in yeast, does not produce a protein. It performs its function, in this case to regulate a nearby gene, simply by being turned on.
Joseph Martens, Lisa Laprade, and Fred Winston found that by switching on the new gene, they could stop the neighboring structural gene from being expressed. "It is the active transcription of another gene that is regulating the process," said Martens, HMS research fellow in genetics and lead author of the June 3 Nature study . "I cannot think of another regulatory gene such as this one," said Winston, HMS professor of genetics. The researchers have evidence that the new gene, SRG1, works by physically blocking transcription of the adjacent gene, SER3. They found that transcription of SRG1 prevents the binding of a critical piece of SER3's transcriptional machinery.
The discovery raises tantalizing questions. How does this gene-blocking occur? Do other regulatory genes work in this fashion? Does the same mechanism occur in mammals, including humans? At the same time, SRG1 provides clues to a recent puzzle. Researchers have lately begun to suspect that the long stretches of apparently useless, or junk, DNA might possess a hidden function. In the past year, evidence has been pouring in, not just from yeast but from mammals, that these apparently silent regions produce RNAs, which are associated with transcriptional activity (see Focus, March 5, 2004 http://focus.hms.harvard.edu/2004/March5_2004/biological_chemistry.html). Yet no one has found associated protein products. "For us it is easy to look at those findings and say, 'Well maybe those are examples of what is going on here in yeast,'" said Martens.
If so, the findings would carry an important message for the post-human genome era-namely, that researchers' attempts to turn the masses of data churned out by the Human Genome Project into an understanding of what actually happens in the human body may be even more complex than they anticipated. One of the main challenges for that effort is to figure out how and when genes are turned on and off during normal development and disease. Rather than look only at how genes are regulated by proteins, they would have to turn their attention to a new, and possibly more-difficult-to-detect form of control. And given that junk DNA makes up 95 percent of chromosomes, the mechanism could be fairly common. "I think if nothing else, this sends up an alert that this likely occurs in other cases," said Winston. "When people are looking to understand regulation of genes from whatever organism-humans, flies, mice, yeast-they cannot just look for proteins that are acting there. It might be that it is simply the act of transcribing that is causing regulation." Like many researchers, Winston and his colleagues may have known in the back of their minds that someday they would have to contend with junk DNA, but it was not their intention to map a new gene in those wild and relatively uncharted regions of the chromosome. If anything, the yeast SER3 gene was their lodestar.
What intrigued them about the gene, which is involved in the synthesis of the amino acid serine, was its unusual expression pattern. To be turned on, genes must first be bound by an activator molecule. A common activator in yeast is a molecule called Switch/Sniff. While most genes are turned on by Switch/Sniff, SER3 is turned off by the complex. In the course of exploring how this repression happens, Martens came across an even more surprising result. "The usual story when a gene is transcriptionally repressed is that RNA polymerase, TATA binding protein and a host of other factors associated with active transcription, will not be there," he said. He, Laprade, a research associate, and Winston conducted a series of experiments and found that the factors were all present and active, and they were located just upstream of the SER3 promoter-as was a jot of DNA needed for the onset of transcription, the TATA element. Thinking that the TATA element might signify the beginning of a new gene, one associated with both the active RNA polymerase and SER3 repression, Martens mutated it. "We no longer saw the RNA, and we found transcription of SER3 was de-repressed," he said. "That is when we thought, 'OK, we have got a new regulatory gene.'" After characterizing SRG1, which turned out to be 550 base pairs long, they tackled the question, How is it regulating SER3? They put the question on the table during a lab retreat atop a downtown skyscraper. "Everybody talks, and they are not allowed to show any data," said Winston. Out of that intellectual free-for-all, three models emerged.
The first held that RNA transcripts produced from SRG1 were being recruited to SER3 and were somehow repressing transcription. The researchers assumed that if this were true, it would not matter where the RNA came from. As it turned out, SER3 was repressed only when the RNA was produced by an adjacent SRG1. The second model, which proposed that the SRG1 promoter outcompeted the SER3 promoter for transcription factors, also did not hold up to experimental scrutiny. There had been hints all along favoring the third model. In this one, transcription of the nearby SRG1 somehow prevents an activator from binding the SER3 promotor. Using chromatin immunoprecipitation, a powerful method for imaging the location of molecules in living cells, the researchers found that this was exactly what happened: a well-known activator fell off the SER3 promotor when SRG1 was turned on. In fact, when SER3 was replaced by a reporter gene, the same thing happened-the turning on of SRG1 prevented the activator from binding to that gene as well.
As for how this interference actually occurs, one possibility is that the machinery required to transcribe SRG1 -RNA polymerase, TATA-binding proteins and other factors-somehow spills over to the nearby SER3 promotor, physically preventing it from being approached by an activator. "It is also possible that active transcription alters chromatin structure and modifies things in other ways," said Winston. As for the molecule that got them started in the first place, Switch/Sniff, the researchers now think it may activate SRG1 and in that way bring about SER3's anomalous repression. "That is our current thinking," Winston said. It is a view he expects will be revised. "Every time we thought we understood everything going on here, we have been wrong. There are additional layers of complexity."

This is a very important reason why genetic engineering is unsuitable for commercial application. It is still at a stage of early experimentation with very incomplete understanding about its consequences. According to the ethical standards of sound science, the products of such experimentation should be strictly contained in labortories, especially as released DNA may spread indefinitely in an uncontrollable way.

Therefore, scientists now generally believe that this DNA must contain some kind of coded information. But the code and its function is yet completely unknown. It has been reported that the sequences of this unknown DNA are inherited and that some repetitive patterns in it seem to be associated with increased risk for cancer.
Also, the DNA has been found to mutate rapidly for example in response to cancer. It has been speculated that this DNA may contribute to the regulation of cellular processes. Haig H. Kazazian, Jr., chairman of genetics at the University of Pennysylvania has recently found reasons to suspect they may be a key force for the development of new species during evolution. He thinks this DNA may be essential for increasing the plasticity of the hereditary substance.
Such observations have spurred an extensive research into "Junk DNA" in recent years, some of which is briefly presented below.

Various important roles of "Junk DNA" have been discovered in recent years.
In June 2004 a team at Harvard Medical School (HMS) reported, that they have, in a yeast, found a "Junk DNA" gene that regulates the activity of nearby genes. While common genes work by giving rise to proteins, this gene works by just being switched on. Then it blocks the activity of an adjacent gene.
Quote: "In a region of DNA long considered a genetic wasteland, HMS researchers have discovered a new class of gene."... "The researchers have evidence that the new gene, SRG1, works by physically blocking transcription of the adjacent gene, SER3. They found that transcription of SRG1 prevents the binding of a critical piece of SER3's transcriptional machinery." Source: "Junk DNA Yields New Kind of Gene", Focus, Harvard Medical School, June 4 2004.
Some studies have found that noncoding DNA plays a vital role in the regulation of gene expression during development (Ting SJ. 1995. A binary model of repetitive DNA sequence in Caenorhabditis elegans. DNA Cell Biol. 14: 83-85.), including:
-Development of photoreceptor cells (Vandendries ER, Johnson D, Reinke R. 1996. Orthodenticle is required for photoreceptor cell development in the Drosophila eye. Dev Biol 173: 243-255.)
-The reproductive tract (Keplinger BL, Rabetoy AL, Cavener DR. 1996. A somatic reproductive organ enhancer complex activates expression in both the developing and the mature Drosophila reproductive tract. Dev Biol 180: 311-323.)
-The central nervous system (Kohler J, Schafer-Preuss S, Buttgereit D. 1996. Related enhancers in the intron of the beta1 tubulin gene of Drosophila melanogaster are essential for maternal and CNS-specific expression during embryogenesis. Nucleic Acids Res 24: 2543-2550.).

• apolipoprotein A-II gene (Bossu JP, Chartier FL, Fruchart JC, Auwerx J, Staels B, Laine B. 1996. Two regulatory elements of similar structure and placed in tandem account for the repressive activity of the first intron of the human apolipoprotein A-II gene. Biochem J 318: 547-553.),

• the osteocalcin gene (Goto K, Heymont JL, Klein-Nulend J, Kronenberg HM, Demay MB. 1996. Identification of an osteoblastic silencer element in the first intron of the rat osteocalcin gene. Biochemistry 35: 11005-11011),

• the 2-crystallin gene (Dirks RP, Kraft HJ, Van Genesen ST, Klok EJ, Pfundt R, Schoenmakers JG, Lubsen NH. 1996. The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stagespecific expression of the rat beta B2- crystallin gene. Eur J Biochem 239: 23-32).

• the Lipoprotein Lipase gene (Ranganathan G, Vu D, Kern PA. 1997. Translational Regulation of Lipoprotein Lipase by Epinephrine Involves a Trans-acting Binding Protein Interacting with the 3' Untranslated Region. J Biol Chem 272: 2515-2519)

• glutathione peroxidase and phospholipidhydroperoxide glutathione peroxidase genes (Bermano G, Arthur JR, Hesketh JE. 1996. Role of the 3' untranslated region in the regulation of cytosolic glutathione peroxidase and phospholipidhydroperoxide glutathione peroxidase gene expression by selenium supply. Biochem J 320: 891-895),

• the luteinizing hormone/human chorionic gonadotropin receptor gene (58. Lu DL, Menon KM. 1996. 3' untranslated regionmediated regulation of luteinizing hormone/human chorionic gonadotropin receptor expression. Biochemistry 35: 12347-12353),

• the thyrotropin receptor gene (Kakinuma A, Chazenbalk G, Filetti S, McLachlan SM, Rapoport B. 1996. BOTH the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mammalian cells. Endocrinology 137: 2664-2669),

• the interleukin 1 type I receptor gene (Ye K, Vannier E, Clark BD, Sims JE, Dinarello CA. 1996. Three distinct promoters direct transcription of different 5' untranslated regions of the human interleukin 1 type I receptor: a possible mechanism for control of translation. Cytokine 8: 421-429)

The idea that a major part of our DNA is "garbage" ignored the fact that a key feature of biological organisms is optimal energy expenditure. To carry enormous amounts of unnecessary molecules is contrary to this fundamental energy saving feature of biological organisms. Increasing evidence are now indicating many important functions of this DNA, including various regulatory roles.
This means that this so-called non-coding DNA influences the behavior of the genes, the "coding DNA", in important ways. Still there is very little knowledge about the relationship between non-coding DNA and the DNA of genes. This adds to other factors making it impossible to foresee and control the effect of artificial insertion of foreign genes.