Complementarity and Contrariety In Genomics
Majid Ali, M.D.
In 2001, the Human Genome Project Consortium estimated the number of genes in the human genome to be over 31,000, while Celera Genomics established that number to be 26,588, with 12,731 candidate genes.1-5 Implication of this work have been recently presented in special issues of Nature and Science.6-15 It is safe to predict that the knowledge of human genome—and, equally significantly, that of proteomics—will radically alter the way we think of the disease/dis-ease.disease.
A mouse genome has a few thousand less genes, and that of a fruit fly about one third of that. By the gene numbers game, a human equals a mouse plus one tenth of a fruit fly. Even a lowly roundworm (Caenorhabditis elegans) contains only one third less genes than we humans. This nematode contains a total number of 959 cells, of which 302 are neurons that make up its brain. By contrast, humans have an estimated 100 trillion cells in the body, of which 100 billion are neurons. That raises some interesting questions: With only one third additional genes, how did the human brain get so many more brain cells (100 billion less 302) than the roundworm? And, how with such a small number of additional genes, does the human genome control the generation, development, differentiation, and orderly death (apoptosis) of such an enormous number of brain cells?
We are also told that a human genome is like a chopped, churned, and rearranged mouse genome—not very flattering for the species that has long claimed to occupy the apex of the hierarchy of living beings. Evidently, neither the cellular DNA mass nor the number of genes accounts for the sophistication of human biology. If not in the number of genes and the mass of DNA, where do we look for an explanation? This is one of the areas in which nature is at its most eloquent in its mastery of molecular complementarity and contrariety.
Gene expression comprises initial transcription in the nucleus and
subsequent translation of mRNA in the cytoplasm. The cargo of gene products (proteins) is shuttled within the intracellular compartments by highly complex and finely orchestrated mechanisms involving vesicles that form by budding from the donor organelle, are transported to an acceptor organelle, dock with specific sites of delivery, and finally fuse with the target organelle for unloading. The protein families that control vesicle traffic are highly conserved through phylogeny from yeast to fruit fly to mouse to man. Some measure of the genetic complexity of vesicular traffic can be gained by simply considering the number (in hundreds) and range of proteins (extremely broad) involved with such traffic.16
Single nucleotide polymorphisms (SNPs), as is implicit in the name, are locations in the genome where individuals vary by a single genetic letter.17 It has been estimated that there are over two million SNPs in the human genome. What are the biologic implications of such a vast number of SNPs? That question probably will be never answered in full, given the accelerating rate of changes in ecologic conditions that lead to alteration in the genome.
There are three human cytoskeletal systems involved in the development, structural integrity, motility, and other diverse aspects of cell membranes: actin filaments, microtubules, and intermediate filaments. More than 70 families of actin-binding proteins, over a dozen families of microtubule-binding proteins, and over 30 human intermediate filament proteins were identified at the time of the initial reports of the human genome in February 2001.18 No one can venture at this time how many members there might be in any of those families or how many other families remain to be discovered.
Another glimpse into genetic complementarity and contrariety may be obtained by considering the genetic underpinnings of cancer. All cancers are characterized by disruptions of the genome and caused by alterations in DNA sequence. The number of oncogenes that trigger carcinogenesis is steadily increasingly. Over 30 recessive oncogenes (tumor suppressor genes) and over one hundred dominant genes were identified by February 2001.19 Undoubtedly, there will be more.
There are, of course, several other issues. The number of human mRNA species—estimated from various assemblies of expressed sequence tags (EST)—is 85,000, greater than twice that of genes.20 That discrepancy remains unresolved and may turn out to be of considerable significance. The estimated number of human cDNA species is also much larger than the proposed gene number and thought to be up to 48,000. Computer models have predicted up to 46,000 unigene EST clusters for which there was no evidence of protein-coding potential.21,22 Attempts have been made to explain the excess of cDNA/EST clusters over recognized protein-coding genes by invoking the presence of a large number of alternative forms of protein-coding transcripts along with numerous transcripts from uncharacterized, non-protein-coding “genes,” such as Xist and H19. Such genes are discovered by chance rather than by the approaches designated ab initio gene-finding programs.
At this time, the coding regions of genes, called exons, appear to account for only 3% of DNA.23 Exons are split into pieces in the genome and these pieces are separated by non-coding sequences called introns. Repeat sequences, with or without a function, form approximately 46% of the genome. The remainder of the genome contains promotes, transcriptional regulatory sequences, and almost certainly other features that remain to be discovered.
The structure, function, and evolution of genes have been examined by the following: (1) morphology of chromosomes (both normal and abnormal); (2) construction of genomic landmarks; (3) observation of genetic transmission of phenotypes; (4) study of DNA sequence variations; and (5) characterization of individual genes. Genes have been discovered by finding putative orthologues (related to a gene in another species) and paralogues (family members derived by gene duplications).
Repetitive sequences with an excess of cytosines and guanines show a tendency to clustering in the vicinity of genes while those rich in nucleotides adenine and thymine are found in abundance in the gene-poor regions (“deserts”) in chromosomes. That explains the distinctive light and dark banding patterns of chromosomes. The dark bands are generally gene-poor zones dominated by adenines and thymines, while the light bands are usually composed of gene-rich regions with a higher concentration of cytosines and guanines. The uneven gene distribution also accounts for some small chromosomes (for example, chromosome 19) having a disproportionately large number of genes. Indeed, such “ruggedness of terrain” distinguishes the human genome from genomes of some other species that show lesser degrees of gene clustering.
There is also the matter of pseudogenes, sequences that at first sight look like genes but lack paraphernalia to persuade the cells that host them to transcribe them. At present pseudogenes are so designated because matching proteins (encoded by them) are thought not to exist. This is one reason why estimates of the total number of genes in the human genome vary so much. Future work is likely to lead to recognition of many matching proteins and redesignation of sequences now considered pseudogenes as true genes.
Junk DNA or a Treasure Trove?
Until recently, much of the human DNA has been considered junk DNA because it was thought to serve no useful purpose and was found to be a nuisance in the way of identifying and classifying genes. It seemed to be an unusual display of hubris on the part of geneticists. Now we know better.
Nearly half of the human genome is at present thought to live a parasitic life, contributing nothing to the metabolic, host defense, and reproductive functions of the body.24,25 One type of such sequences is called transposons or transposable element (jumping genes, in common language). These are bits of non-coding sequences in the human DNA that appear to be the offspring of sequences that broke away from their parent DNA a long time ago but learned to duplicate themselves and re-enter the mother genome. The requirements for raw materials and energy for synthesis of transposons are similar to those of useful DNA. That creates an enigma: Since perpetuation of transposable elements makes no evolutionary sense, why is it produced at all? There are yet other mysteries about such DNA: Its amount in other species is significantly lower than in humans (about 46%). Furthermore, it is scarce in homeobox gene cluster, a region of crucial importance. It seems probable that significant functions of transposable elements will be uncovered in the future to resolve those enigmas.
Four classes of transposons have been identified: (1) fossil DNA that has the ability to replicate; (2) LTR transposon that appears to be on its way to extinction; (3) LINE (Long Interspersed Element); and (4) SINE (short interspersed element). To escape easy detection and deletion by the DNA editing systems, LINE and some other transposon sequences home in on adenine-thymine-rich and gene-poor regions of chromosomes.
LINE sequences are highly successful parasites that retain full instructions to meet its every need—from copying its DNA into intermediary RNA, copying RNA back to DNA, and re-entering its chromosomal niche. SINE sequences, like some other types of transposable elements, are not normally transcribed by the cell’s machinery to generate molecular messengers through which genes produce their results. That changes when cells come under stress and SINEs are transcribed to produce messengers that block molecules that otherwise slow down protein production. There is also evidence that several genes (over twenty so far) with useful functions are derived from transposable elements.
Enter in this picture Alu element, the perfect parasite of the parasite. These sequences run about 300 bases in length, are scattered throughout the chromosomes, and, numbering over a million, are the most abundant sequences in the human genome. Alu elements commandeer the LINE machinery to reproduce. Sometimes, they insert themselves into critical genes and cause genetic diseases. More importantly, they seem to have been co-opted by the body to modulate the immune response. For instance, in mice dunked in hot water, the rodent equivalent of human Alu element is activated, presumably to provide protection against acute thermal injury. Another line of evidence supporting their protective roles concerns their preferred residence in guanine-cytosine-rich regions, in close proximity of working genes. Furthermore, Alu elements respond to members of the nuclear receptor superfamily, which recognizes several hormones, including estrogens, thyroid hormone, retinoic acid, and others. The net effect: Alu element near genes influence their behavior by pitching the genes higher or lower, contributing to functional complementarities and contrarieties of genes. Junk is in the eye of the beholder.
From Mikonos to Munich to the Moon
Some years ago, I traveled from Mikonos in the Greek islands to Munich on my return to the United States. The plane in Mikonos was a small 16-seater. Boarding was delayed. I looked at the plane and wondered about how many discrete parts that plane might have. How do those parts interact with each other structurally and functionally?, I asked myself. I thought there was such a predictability to the behavior of those parts that I felt safe flying in it. Other passengers seemed secure in that knowledge as well. No one seemed worried that the plane might lose its way and end up in Athens. A flying machine is a miracle, albeit created by human hands.
At Munich airport, I looked out and saw a Boeing 747 jumbo jet. This monster is going to swallow me and hundreds of others milling around the gate, I told myself. How many parts does this plane have? How do they interact with each other structurally and functionally. By some miracle all those parts will do what they are designed to do and we will be disgorged in Newark, not in New York or Philadelphia. What an amazing achievement of women and men who built those machines! Later I learned that a modern jet contains about 200,000 unique parts, each of them interacting structurally with three or four others on the average.26
How is it that we can be inventive enough to build machines that operate with precision like that, but cannot control a lowly common cold virus?, my conversation with myself continued. How sublime with building planes that look like ships in flight! How clumsy with efforts to stop a viral particle from replicating! What is it that separates man from machine? I wondered.
Then my mind drifted to man’s first journey to the moon. What an astounding feat of technology that was! John Kennedy got us to the moon. How many variables were there in that journey to be contended with? How did the people at NASA ever figure out the distances and the directions to that destiny? How many corrections needed to be made? How many computer programs and interfaces had to be perfected? Next there was the journey back. How many impediments existed there? What details had to be figured out to get Neil Armstrong and his companions to return to the planet Earth? They splashed down in the ocean within miles of where they were predicted to hit the water. How was that arranged?
It occurred to me that the core difference between the living and nonliving things concerns the complementarian and contrarian roles of their molecules and cells. It is a matter of the ability of the living to “read” the conditions of their environment and to respond to them in a way that benefits the whole27. Each piece in a machine can play only the role assigned to it28. In a machine, each part is designed by man with a deterministic-reductionistic model of mechanics to subserve a specific function. A jet engine of the plane really does not care whether the landing gear functions well or not. The living beings, by contrast, “see” and “feel” and adapt and evolve to meet the challenges of the changing conditions that affect their structure or function. Each living part evolved over hundreds of millions of years in nature’s complementarity-contrariety mold.
Like virus particles, a cancer cell has a mind of its own. I do not know if Richard Nixon knew that when he ached to outdo Kennedy and declared his war on cancer in 1971. His goal was clear: win the war before the century was over. Well that century is gone. Where do we stand now? Consider the quote from a 1997 issue (Vol 33, pp 1569-74) of The New England Journal of Medicine:
In 1986, we concluded that “some 35 years of intense effort focused largely on improving treatment must be judged a qualified failure.” Now, with 12 more years of data and experience, we see little reason to change that conclusion.
Where did we go wrong?, I asked myself. Why such dismal failure in improving the results of cancer treatment?
There is yet more—much more than meets the eye—in the story of stringy and springy genes in the eternal drama of life. Consider the story of genes for interleukin-1.
Of Japanese and English Mice
Interleukin-1 and tumor necrosis factor á (TNF-á) are potent pro-inflammatory cytokines and play key roles in the pathogenesis of inflammatory autoimmune disorders, including Crohn’s colitis, rheumatoid arthritis, and vasculitis.29-34 The occupation of interleukin-1 receptors on the cell membrane of inflammatory cells by interleukin-1 initiates several pro-inflammatory molecular events, including the generation of nitric oxide, prostaglandins, chemokines (small polypeptides that are chemotactic for neutrophils, macrophages, and lymphocytes).
Interleukin-1-receptor antagonist, a member of the interleukin-1 family, is a naturally occurring inhibitor of interleukin-1-receptors. By contrast, the occupation of interleukin-1 receptors by interleukin-1-receptor antagonists prevents all those events, simply by preventing the union of the two molecules. That is an excellent example of how molecular systems operate to preserve immunity by self-regulation and collaboration among the pro- and anti-inflammatory components. In health, interleukin-1 stays ready to evoke the inflammatory response and recruit inflammatory cells when that is called for. The interleukin-1-receptor antagonist also stays prepared to oppose excessive activity of interleukin-1 and prevent unregulated and destructive inflammatory responses. In cases of runaway inflammation—in Crohn’s colitis and rheumatoid arthritis, for instance—the interleukin-1-receptor antagonist lags behind, and the inflammatory process becomes unrelenting.
Consistent with the above theoretical considerations, in two recent controlled trials administration of recombinant human interleukin-1-receptor antagonist to patients with moderately severe rheumatoid arthritis resulted in significant abatement of local inflammatory responses.35,36 However, the periods of follow-up were short and it is likely that such effects will be less impressive with time since the interleukin-1/interleukin-1-receptor antagonist system represents but one of the many known—and undoubtedly many more as yet unknown—systems of opposing molecules involved in immunity.
The story of the interleukin-1/interleukin-1-receptor antagonist system does not end there. In two recent studies, two entirely different types of inflammatory autoimmune disorders developed spontaneously in knock-out mice with targeted genetic deletion of genes for interleukin-1-receptor antagonist. In the Japanese study,37 the knock-out mice developed inflammatory arthropathy similar to rheumatoid arthritis, whereas in the English study the outcome was a lethal form of inflammatory arteritis. The arthritic mice produced large quantities of immunoglobulins, antibodies to type II collagen and double strand DNA, and a ten-fold increase in the levels of interleukin-1 messenger RNA in the joints. Extensive deformities of joints with erosions of bone and neutrophilic infiltrates developed by age 13 weeks. In the English study, the mice developed disseminated acute and chronic vasculitis with areas of marked arterial stenosis accompanied by hemorrhage and infarction in multiple organs.38 The effects on the vascular endothelium and muscularis were consistent with the known effects of interleukin on those organs. It has been speculated that the difference may be attributable to differences in the normal flora in Japan and England or genetic differences between the colonies of mice used. What may be the significance of those two entirely different outcomes in mice with deletion of the same gene? Future work is likely to show, as has been the case with the past studies, that there are many other variables in the system. It will be difficult to predict the biologic consequences of therapies based on loss-of-function and gain-of-function genetic manipulations. The advances in genetically designed therapies will exact unexpected tolls far in excess of, say, the problems caused by microbial resistance to antibiotics or pesticide treatments of crops during the last fifty years.
Genes and Aging
I include here some brief comments about genes and aging. In Oxygen and Aging, I asserted that genes positively influence the aging process and longevity only when oxygen metabolism is preserved which, in turn, is profoundly influenced by nutritional, ecologic, and stress-related factors. In his forthcoming book Aging Well, George F. Vaillant of Harvard University provides support for my view. He reports his longitudinal studies of aging in a cohort of Harvard men. The March-April issue (page 47) of Harvard Magazine includes the following quote from him:
We’re all mongrels….Genes are so—well, so heterogenous. With the passage of time, successful aging is remarkably free of genetic factors….Having better doctors and hospitals is a bit like locking the barn after the horse is out. The trick is not going to hospitals in the first place.
Genes are not generous to those who tinker with them. Notwithstanding the great excitement generated by Dolly, the first cloned ewe, the real story is very different. Consider the following quote concerning the human cloning debate from the March 19, 2001 issue of U.S. News & World Report:
And the scientists had good reason to be pessimistic. Several years of animal cloning work had taught them that most cloned animals never even make it to birth, and the rare ones that do all too frequently have problems ranging from physical deformities to life-threatening medical conditions….[such as] the lamb that scientists had to euthanize this past winter because it couldn’t stop hyperventilating because of blood vessel abnormalities.
The prospect of euthanizing human clones is not deterring proponents of such clones. And no doubt we will see much progress in that field in the future. My point here is to underscore the enormous range of complementarity and contrariety of genes and how unforgiving they can be to those who meddle with them.
Of Chimps and People
There are 26,000 to 30,000 genes in human cells. It has been proposed that each gene interacts with four or five others on the average.39,40 that were true, a human would have not been much different from a mouse. Since human biologic sophistication cannot be explained simply by figuring out the number of genes in the human genome, what else must we considered? I hold that the answer to that question has something to do with complementarity and contrariety of genes. My view is that the complexities of homeostasis as well as redox, enzymatic, and immune defenses arise from the way genes speak and act—read, sense and respond —to their changing microecologic conditions. Gene expressions are individually and finely regulated to be integrated with the whole and reflect the changing needs of the organism in its entirety.
A chimpanzee is over 99% human, a monkey only slightly less so. The place of humans in the hierarchy of the creation is something for the clergy to ascertain. But for a student of biology, the fascinating question is this: What makes us human? How do genes create love for music and painting? And for writing? At a more basic level, how do genes generate the need to understand oneself? It seems safe to conclude that humans are more than the sum of their genes. In competitive struggle and survival of the fitter, Darwin saw “grandeur in this view of life.” Perhaps there is a greater grandeur in self-organization of genes and their products under the organizing influence of some higher Presence. (Ah, the helplessness of an observer of nature striving not to intrude on the territory of the clergy!)
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