Author: Siddhartha Mukherjee
Publisher: Scribner 2016
Introduction
Just like a byte is a unit of memory of computing, or an atom is a unit of matter, a gene is a fundamental unit of heredity. The entire set of genetic instructions carried by an organism is termed the genome.
A protein is created by 20 simple chemicals named amino acids. These amino acids are strung together in chains. A gene can be visualized as the director of the final configuration of a protein molecule. (Beadle and Tatum won the Nobel prize in 1958 for this discovery.) Enzymes in a cell are proteins that acted as master builders and could synthesize complex biological macromolecules out of basic precursor chemicals.
The genetic information in DNA chains must be first copied into that of complementary RNA molecules and RNA molecules must be used as “messages” to build protein.
Proteins are synthesized within cells by a specialized cellular component called ribosome.
Five nucleo-bases—adenine (A), cytosine (C), thymine (T), guanine (G) and uracil (U)—are called primary. DNA is made up of A, C, T, G and RNA is made up of A, C, U, G.
No single base—A, C, T or G—could carry sufficient genetic messages to build any part of protein. Since there are 20 amino acids in all, 4 letters could not specify twenty alternative states by themselves. But if you think of A, C, T, and G as “letters”, then a combination of these letters can create a meaningful “word”. The words ACT, CAT are made of the same letters, but carry different meanings. The code ACT in DNA becomes the code ACU in the messenger RNA. (RNA were built by stringing together A, C, G, and U. In the RNA copy of the gene ACT, the T is substituted by U.)
In the “triple code hypothesis”, we can create (\(4^3\)) sixty four three letter words from four letters. That means, there are enough combinations to create 20 amino acids and there are enough leftovers for other functions such as “starting” or “stopping” a protein chain.
The generation of an RNA copy of a gene is called transcription—referring to the rewriting of a word or sentence in a language close to the original. A gene’s code (ACGGGCC..,) was transcribed into RNA code (AUGGGCC…).
In 1959, Padree, Jacob, and Monad discovered that a gene possessed not just information to encode a protein, but also information about when and where to make that protein. (Their paper is known as Pa-Ja-Ma paper.)
Genes make proteins that regulate genes. Genes make proteins that replicate genes. Genes can make proteins that recombine genes. Genes can repair any damage to the genome. (There are the four Rs of genes.)
The flow of information can be visualize as follows.
A protein called a DNA polymerase is dedicated to copying DNA in the replication process. Like all proteins, DNA polymerase is itself a product of a gene. Built into every genome, are the codes for proteins that will allow the genome to reproduce. DNA replication can be turned on and off by other signals and regulators enabling cells to make copies only when they are ready to replicate.
When the regulators themselves go rogue, nothing can stop a cell from replicating continuously. This malfunctioning of genes is known as cancer.
There are 21,000 genes in the human genome. Smaller life forms have fewer numbers of genes in their genome. For example, the Simian Virus 40 (SV40) has only 7 genes.
Genetic Engineering
SV40 could coexist with certain kinds of infected cells. Rather than producing millions of copies after infection, and often killing the host cell as a result, SV40 could insert its DNA into the host cell chromosome, and then lapse into a non-reproducing lull. The compactness of the SV40 genome and the efficiency with which it can be delivered into cells made it an ideal vehicle to carry genes into human cells. By doing so creates a genetic chimera— a hybrid between a virus’s genes and a foreign gene. Unlike human genes that are strung along chromosomes like an open string, SV40 genes are strung into a closed circle. In order to insert a foreign gene into the circle, the circle has to be broken, insert the foreign gene, and then close the circle again. Paul Berg and Peter Lobban at Stanford in the 60s figured out how to create a genetic chimera using SV40.
DNA damage occurs routinely in cells. To repair damaged DNA, cells make enzymes called ligase (meaning to tie together) enzymes. Virtually all cells have ligase and polymerase enzymes. There is very little reason for a cell to have gene cutting enzymes. On the other hand, bacteria and viruses carry gene cutting enzymes. These proteins are called restriction enzymes.
In 1970, Berg and Lobban managed to join the entire genome of SV40 to a piece of DNA from a bacterial virus called Lambda bacteriophage (phage \(\lambda\)) and three genes from the bacterium E. Coli. Berg identified this first genetic chimera as “recombined DNA”.
E. Coli bacteria live in the human intestine.
Janet Mertz who joined the Berg term thought of inverting the process. Bacteria carry minuscule extra chromosomes called mini-chromosomes or plasmids. Plasmids have a closed circular structure just like SV40. What if we recombine SV40 with plasmids in E-Coli? Then E-Coli becomes the factory of reproducing new gene chimeras.
When Metz presented this idea at a conference at Cold Springs Harbor in 1972, one of the attendees, Robert Pollock, a virologist, called Berg urgently. Pollock argued that the danger implicit in “bridging evolutionally barriers that had existed since the last common ancestor between the bacterium and people” was far too great to continue experiment casually. (SV40 was known to cause tumors in hamsters and like I said before, E. Coli live in the human intestine. Current evidence suggests that SV40 is not “likely” to cause cancer in humans, but it was not known in the 70s.)
The National Academy of Science convened a panel of 8 scientists including Berg, Watson, Baltimore, and Zinder and they met at MIT in 1973. The panel drafted a formal letter suggesting a moratorium on certain types of recombined DNA experiments. In particular, they suggested (1) not to put toxin into E.Coli, (2) not to put drug-resistant genes into E. Coli, and (3) not to put cancer genes into E. Coli.
In 1974 a researcher at Cohen’s lab at Stanford inserted a frog gene into a bacterial cell crossing another evolutionary border. When the paper on this “frog prince” experiment was published in May 1974, it turned the heads of biochemists initially. However, slowly the media awoke to the potential impact of the study. After talking to Cohen, a newspaper reporter wrote an article with the heading “Man Made Bugs Ravage The Earth”.
Berg, Baltimore, and three other scientists organized a conference now known as the Asilomar conference in February 1975. They also invited reporters and lawyers, in addition to the leading scientists in genetic engineering. The main issue was the self imposed moratorium on recombined DNA experiments. Tensions and tempers flared. However, In the end, the self imposed moratorium was accepted.
Mutations
Sickle cell anemia was recognized by Ayurvedic practitioners as early as 6th century BC. They called it the “pandu roga” (පාඬු රෝගය). (The symptoms were pallor of the lips, skin, and fingers.) The Ga tribe in West Africa called it “chwech-weechwe (body beating) and the Ewe tribe named it “nuiduidui” (body twisting).
Every mutant gene was missing a single metabolic function corresponding to the activity of a single protein enzyme. For example, in the “sickle-cell gene” one triplet GAG has changed to another GTG.
GAG GTG
(normal code) (sickle cell code)
glutamate valine
(normal amino acid) (sickle cell causing amino acid)
The switch alters the folding of the hemoglobin chain. Rather than twisting into a neatly articulated claps-like structure, the mutant hemoglobin protein accumulated in string-like clumps within red cells. These clumps grew so large that they tugged the membrane of the red cell until a normal disk was warped into a crescent-shaped, dysmorphic “sickle cell”. Unable to glide smoothly through capillaries and veins, sickled red cells jammed into microscopic clots throughout the body, interrupting blood flow and precipitating the excruciating pain of the sickling crisis.
The factor VIII gene makes only one protein; which serves only one function: it enables blood to form clots. These type of genes are identified as “blue prints” genes. There are genes that collaborate with cascades of other genes to enable complex physiological functions. These genes are known as “recipes”. The mutated factor VIII gene fails to enable normal blood clotting, and the resulting disorder—bleeding without provocation— is known as hemophilia. (English royals had hemophilia and the last Tzar of the Russian empire had hemophilia.)
The sickle-cell anemia and hemophilia are examples of single gene—monogenic—diseases. There are other diseases that are caused by multiple genes called polygenic diseases. In Down syndrome, children are born with an extra copy of chromosome 21, which has 300 plus genes strung on it. Multiple organs are affected by the extra copy of this chromosome and only a few survived to adulthood. One positive effect of the syndrome is that these children do not possess cruelty and malice. A child born with a missing X chromosome displays the symptoms of Turner syndrome.
Another illness known since antiquity is hemochromatosis. Hemochromatosis is caused by a mutation in gene that regulates iron absorption from intestines. The skin of a patient with hemochromatosis turns bronze and then ashen gray. Organ by organ the body transforms into minerals, ultimately leading to organ failure and death.
By the mid-70s, thousands of genetic diseases had been identified. However, the actual gene or genes that cause these diseases were known only for a few diseases. Kerry Kravitz, a graduate student from University of Utah knew that two defective genes, one from each parent, are necessary to cause hemochromatosis. He wanted to identify the hemochromatosis causing gene. He suggested looking at nearby known genes of a chromosome. By studying Mormons in Utah with cascading family trees, they (Kravitz and his advisor) have discovered that the hemochromatosis was genetically linked to an immune-response gene that is in chromosome 6. The immune-response gene is like a “signpost” to identify the hemochromatosis gene. David Botstein from MIT knew that such signposts might exist for many other diseases causing mutated genes. Over the centuries of evolution, the human genome has diverged enough to create thousands of minute variations in DNA sequences. These variations are called polymorphisms. Polymorphisms may exist in DNA between genes or in introns. Looking for such polymorphisms is called linkage analysis.
By using linkage analysis methods, Wexler and Gusella mapped the Huntington’s disease- causing gene to a distant outpost of chromosome 4—4p16.3. In 1993, Gusella’s team identified a gene previously known as IT15 as the Huntington’s disease causing gene. This gene is now known as Huntingtin.
Mirror Writing
The genes get named by the mutants that cause diseases. BRCA 1 is a gene that repairs damaged DNA. However, a mutated BRCA I causes breast cancer. (The name of the gene is BRest CAncer I gene.) The gene called “wingless” encodes instruction to build wings in fruit flies. A mutated “wingless” gene stops this process leading to wingless fruit flies. All genes perform good acts that are necessary for the function of a healthy human being. The mutants of those good genes cause illnesses.
Y-chromosome
Nettie Stevens identified “sex chromosomes” by studying mealworms in 1903. Stevens' close collaborator Edmund Wilson simplified the terminology by calling the male chromosome Y and the female chromosome X. In chromosomal terms male cells were XY and female chromosomes were XX. The egg contains a single X chromosome. When a sperm carrying a Y chromosome fertilizes an egg, it results in an XY combination and when a sperm carrying an X chromosome fertilizes an egg, it results in an XX combination. The Y chromosome carries all the information to make a male embryo. The Y-chromosome, however, is an inhospitable place for genes. Unlike all other chromosomes, the Y-chromosome is “unpaired”. A mutation in a chromosome can be repaired by copying an intact gene from the paired chromosome. But, Y-chromosome cannot be repaired or recopied since it has no backup copy. Therefore, the Y chromosome has accumulated scars of mutations throughout history. The Y-chromosome is the most vulnerable spot in the human genome. For this reason, genes that are most valuable for survival have been moved to other chromosomes throughout history and the Y-chromosome has shrunk through the process and it is the smallest of all chromosomes. In genetic terms, a gene buried in the Y-chromosome must be the master regulator of maleness.
All the known genes so far were identified by their mutant versions that cause illnesses. How could the male gene be identified unless there are mutants causing illnesses? The search led to a syndrome called Swyer syndrome. Women born with Swyer syndrome were anatomically and physiologically female throughout childhood but did not achieve female sexual maturity in early adulthood. When the cells of these women were examined, geneticists discovered that they have XY chromosomes in all cells. The most likely scenario was that the master regulatory gene that specifies maleness had been inactivated by a mutation. In summer 1989, Peter Goodfellow identified the gene SRY as the master regulator gene.
Embryos
Master regulatory effector genes kick into action at specific times and determine the identities of segments and organs. The segmentation genes determine the basic architectural plan of the embryo. They are the map makers. They divide the embryo into its basic subsegments. Then they activate effector genes to start building organs.
Gene Memory
John Gurdon was not a student who received good scores in school. Once he received the lowest score for a science exam in a class of 250. But he has an aptitude for doing things on a small scale. In 1958 as a graduate student he started studying the development of frogs. Gurdon has emptied frog eggs and injected a genome of an adult frog into the empty eggs. Perfectly functioning tadpoles were born and tadpoles carried perfect replicas of adult frogs. The egg cell has everything necessary—all the factors to drive an adult genome backward through development time into a functional embryo. This process is known as the nuclear transfer. Gurdon was awarded the Nobel prize in 2012 for his discovery of nuclear transfer. Even Though Grudon was successful, the success rate was abysmal. But if genes are genes, then why was the genome of an adult cell so inefficiently coaxed backward into an embryo? Something must have been progressively imprinted on the adult cell’s gene that made it difficult for that genome to move back in developmental time. These imprints could not live in the sequence of genes but had to be etched above them. It has to be epigenetic.
(Epigenetics is the study of how your behaviors and environment can cause changes that affect the way your genes work. Unlike genetic changes, epigenetic changes are reversible and do not change your DNA sequence, but they can change how your body reads a DNA sequence.)
Mary Lyon in 1961 studied the biology of chromosomes of mice. She found out that every paired chromosome of female mice were identical except the X-chromosome pair. One of two X-chromosomes was inevitably shrunken and condensed. The shrunken X-chromosome was silent. That is, it did not generate RNA. The inactivated X-chromosome was chosen randomly. In one cell it might be the paternal X and in a nearby cell it might be the maternal X.
In cats, one gene for coat color lives in the X-chromosome. The random inactivation causes cats to have different colors in their coats. If humans carry the skin color gene, then a female child of a dark skinned and light skinned couple would be born with patches of dark and light skin.
David Allis in 1996 found another system to etch permanent marks on genes. Rather than stamping marks directly on genes, this second system placed its marks on proteins called histones that act as packaging material for genes.
The refined circular flow of biological information including epigenomes is the following.
ADA deficiency
ADA gene (adenosine deaminase) encodes an enzyme that converts adenosine into a harmless product called inosine. In the absence of ADA gene, the detoxification fails to occur and poisons T-cells. This is known as the ADA deficiency. (This is also known as the bubble-boy disease.)
Could gene therapy cure the ADA deficiency? Richard Mulligan had designed a particular strain of retrovirus—a cousin of the HIV virus—that could transfer any gene into any human cell with relative safety. In 1986, a team of gene therapists led by William Anderson and Michael Blaese at the National Institute of Health in Bethesda, Ohio decided to use variants of Mulligan’s vectors to deliver ADA genes into children with ADA deficiency.
In 1988 Ashanthi (Ashi) de Silva a two year old from Bethesda was diagnosed with the ADA deficiency. In 1990 Anderson and Blaese performed the transfer of ADA genes via an adenovirus vector to Ashi. Did it work? Ashi’s parents Raja and Van de Silva thought so. “We have seen tremendous improvement” Raja de Silva claimed. “She had runny noses and a constant cold when she was on antibiotics all the time. But by the second infusion of genes, it began to change. We noticed because we were not using so many boxes of tissues. Mulligan, the most harsh critic of the trial, was particularly incensed. “It is a sham.” “If the most ambitious gene-therapy trial attempted in humans was going to be measured in the frequency of runny noses and boxes of Kleenex, then it would be an embarrassment for the field.”
https://www.youtube.com/watch?v=IgES04-cSr8
Conclusion
We know very little about the human genome. Much of our knowledge of our genes is inferred from similar looking genes in yeast, worms, fruit flies, and mice.
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