Background
Genome
Science is the search for answers to mysteries not the validation or negation of proofs. One of the most fascinating scientific mysteries is the quest to understand the human genome, why each cell functions in certain capacities, and how to manipulate those capacitates for the betterment of mankind. There are about 50 trillion cells in each human body 1. Inside each cell are multiple smaller functioning pieces called organelles. One organelle, the nucleus, contains two sets of the human genome (sex cells are the exception); one set came from the mother's egg, the other from the father's sperm. On each set there are between 20,000 and 25,000 genes 2. The sets are not exactly identical physically or chemically. Physically, one arm of the pair is slightly larger, called the long arm to the short arm. Chemically, the exact gene encoding may vary as the father may pass on the gene for curly hair and the mother for straight. However these refined differences become one complete set of twenty-three pairs of chromosomes, and the genes within these chromosomes define (to a large extent) the individual.
If you were to spread out models of all twenty-three pairs of chromosomes on a table you'd first notice that each pair is of different size. It is their size in fact, from largest to smallest that provides the organization for the numbering the chromosomes. Chromosome 1 is thereby the largest, carrying about 249 million base pairs and eight percent the total DNA 3. As being the largest it might also be considered the oldest. Not in that it is created first, but in that all species that have DNA have a Chromosome 1, and all Chromosome 1s are similar. On each chromosome are genes, genes are made up of DNA or long chains of coded instructions.
To better understand the structure of the human genome. I offer Matt Ridley's analogy from Genome: The Autobiography of a Species in 23 Chapters.
"Imagine the genome is a book. There are twenty three chapters called chromosomes. Each chapter contains several thousand stories called genes. Each story is made up of paragraphs called exons, which are interrupted by advertisements called introns. Each paragraph is made up of words called codons. Each word is written in letters called bases."
Thus, the genome is made up of pairs of chromosomes given from two parental sources. Each set of chromosomes stores a massive amount of genes. The genes are sectioned into exons and separated by introns. Each individual combination of bases is called codons. These bases are made up of four letters. Those letters form a very long chain. So long in fact, the genome is as tall as 80 stacked bibles and if read out loud continuously at a rate of two words per second would take approximately 47 ½ years 4. The four letter alphabet of genomes is made up of A, C, G, and T or adenine, cytosine, guanine, and thymine. These bases are attached as rungs to long chains of sugar and phosphate molecules. This structure which looks like a long spiral ladder is called DNA. Both supporting sides of the ladder are made up of a sugar and phosphate chain. The rungs of the ladder are a base which is paired with the base's compliment to make the step part of the ladder. In DNA, A only pairs with T, and C only pairs with G. A pairs as the complement to T, as T pairs as the compliment to A and so on.
Diagram i – Cell with Nucleus, Chromosome Pair, Gene, DNA, Base Pairs – Chromosomal material is located within the nucleus of the cell. A chromosome set is synched in by a centromere. Each gene is made up of thousands of genes. Genes are made of DNA. The thick curved outside lines represent the sugar and phosphate bods of the ladder's side structure. The straight vertical inner lines connected to the side represent the location a base which is connected to its compliment which in turn is connected to the opposite side.
DNA is fascinating in that it has the ability to both reproduce and read itself. Reproduction is as simple as copying a compliment and then rewriting the original. For example, the arrangement CGTAC becomes GCATG (compliment) then records back to CGTAC. The reading of DNA is a much more sophisticated process. First a copy must be made, but all thymine molecules are replaced with uracil (U). This "edited" copy is called messenger RNA or mRNA. The RNA travels outside the nucleus and forms a partnership with another cellular organelle called a ribosome. The ribosome reads triplicate combinations of bases called codons. These are then translated into amino acids which are put together to form protein chain. Proteins are used for various functions all over the human body. From digestion to hair growth, all possible from protein synthesis, every protein is a translated gene 5. For example:
This same formula or reading is true for every living thing. The codon UCG isused to specify the amino acid serine in humans, rattle snakes, chickens, fruit flies, fern trees, and corn.
Put more simplistically, DNA is simply a database of information, coded upon genes using base pairs. These combinations of codons write the recipes for proteins. The gene's code, although quite long, is easily read by ribosomes which in turn create long chains of protein polymer chains that operate our bodies. Each gene creates one specific protein, although not all cells use every gene. For example the gene that expresses Aliases or insulin is located on chromosome 11 6. Every skin cell has a copy of chromosome 11 but doesn't utilize that gene as insulin isn't required in skin repair or replacement. This elaborate process of gene expression, and regulation of gene expression, happens in every living cell, every day.
Biological information must be expressed. DNA molecules store the instructions for building the proteins. So how does gene expression turned into protein define life? One correctly assembled protein can go to work. For example, the protein that makes red pigment, this protein is an enzyme catalyst that speeds up a chemical reaction that produces a red color 7. This color could result in many things including red hair on a human, or the red petals of a rose. Red pigment is an observable trait or phenotype. Phenotypes are determined by genotypes or the codes written on genes inherited from parent cells. For example, in dogs a German Shepard's ears naturally stand straight up whereas a Labradors flop over and do not stand up. The uprightness is the phenotype, the genetic codes that cause the uprightness is the genotype. Interestingly, phenotype can be can also be effected by the environment. To illustrate, we often think of flamingos as pink, however their color is environmentally dependent. It has a gene that expresses a pink coloration; however that enzyme only acts in the presence of certain foods. Thus, the color of a flamingo is determined by its diet 8. To summarize, the genotype codes for a specific trait or phenotype, however phenotype can also be impacted by environmental factors.
This process is seamless; it happens without the organism knowing unless cells are replicating to heal something the organism is finitely aware of (like the healing of a cut or bruise on skin), and can be ignored unless there is a change or error in the replication, reading, or protein production. Many errors, or mutations, in these processes are uneventful and harmless.
The human genome is 23 sets of chromosomes, thousands of genes, and millions of bases that code for the diverse proteins that operate our bodies. The same is true in dogs, except instead of twenty three chromosomes all dog breeds have 78 chromosomes. Mice have 40, chimpanzees have 48, fruit flies only have eight, and rice has 24 9. Some species have a varying number of chromosomes. Strawberry plants for instance, have anywhere from 14 to 70 chromosomes depending on the plant. While most species of strawberries are diploid, meaning they have 2 pieces in each pair of chromosomes, some are polyploid, meaning they have multiple copies of the same chromosome (many plants have this characteristic). Strawberry species and hybrids can be diploid, tetraploid, pentaploid, hexaploid, heptaploid, octoploid, or decaploid (having 2, 4, 5, 6, 7, 8, or 10 sets of the seven strawberry chromosomes, respectively).
All species have a varying number of genes written to their independent sets of chromosomes. Clearly there are both heaps of commonalities and differences in genetic material among different species. However, since most organisms have DNA one could easily identify it as the common thread and building blocks of life. What if we could edit the make-up of those threads to maximize performance? What would that look like? How far could we extend that process? Is it ethical? Contrary to popular belief, these ideas didn't start in a laboratory. The idea of transitioning or modifying the make-up of food sources has been in practice since humans started farming 11,000 years ago. It has evolved to the lab, and extended its practices across academic and medical fields.
Beginnings in Genetic Engineering
To understand how we have manipulated the genetic make-up of organisms we have to understand the evolution of food production and the hunter gatherer's background, as simplistic forms of genetic modification are directly linked to husbandry. Food production (especially on a massive level) is a recent development. Hunter-gathering societies have existed for four million years 10. Producing food has been in existence .3% of that time (or about 11,000 years). This change was a subtle one. Similar to the time it takes for most social constructs to change, the process was gradual. Early farming was not a stable leisure life, nor was hunting-gathering a miserable meander of shiftless searching. In fact, comparisons of early farmers and concurrent hunter-gatherers show no disparity in nutritional equality 11. Instead of viewing a differentiated society of either food producer or hunter-gatherer, many early food producing societies depended upon both producing and gathering 12. No group would risk concentrating on a limited number of crops and/or herding because failure would only result in starvation.
Food availability and population are clearly proportional. With abundant supplies of food populations can increase and without they can potentially become extinct. Whether increased population inspired innovation or innovation inspired increased populations is unclear however it has been calculated that without any form of agriculture, the Earth's surface could only sustain a population of 20 to 30 million people 13. Clearly with a growing population of over seven billion people 14 food production has played a pivotal role in the conception of the societies we see today.
How did we become a planet with over seven billion inhabitants? Lots of factors added to this, but food production was an especially significant contribution. Once inaugurated, food production spread rapidly. It took only 4,000 years to spread from South-west Asia to Western Europe 15. Earliest settlements were along waterfronts and nutrient rich areas. Most primitive communities may have planted seeds that were found in other habitats, planted crops that tended to survive harsh weather, or planted for taste preferences. Once people began farming they could be choosy. They could select and plant seeds of only the best-tasting, most nutritious plant foods. They could also breed two slightly different plants together to produce a third, called a hybrid. Primitive herd manipulation would not have required extensive tools. Animals that had previously migrated may have been restrained or confined. Although these simplistic changes do nothing to impact the organisms' genotype, these simple steps are the precursors to the earliest genetic modifications.
Initial domestication of any animal generally results in a decrease in size 16. By limiting the movement you also limit the interactions of the animal and therefore its potential mating peers. This could also be a response to malnourishment from crowding. Early domesticators would have encouraged breeding amongst the strongest, biggest, fastest, or whichever other desired phenotype to increase their food supply or potential bartering commission. Thereby encouraging, limiting, or suppressing certain features breeders automatically impact the genotype of the organism.
Additionally, cultivation with the element of human selectivity, whether purposeful or not, naturally leads to genetic changes. These modifications are labeled domesticated. For example, today, there are no wild maize (corn) crops. The domestication of maize took over 3,500 years, as breeding and cultivation crisscrossed from wild genotypes to those specifically bred for phenotypes. Maize is wind pollinated so sustaining a pure breed raises significant difficulties. However after considerable effort these favorable genetic qualities stabilized in about 1500 BC. Modern maize Modern corn is completely dependent on man.
Early farmers thus housed animals, planted seeds with purpose, selecting for preferable phenotypes, and bred animals for size and use. Other prehistory examples of purposeful control of genetics include controlled breeding of humans through royal dynasties, which were kept pure by incest. Practical information derived from these breeding techniques was transmitted by word of mouth to families with similar interests in genetic purity 17.
Fast-forward to the early 1400s in Western Europe where nothing at the time was more valuable than wool. The insignia of the Order of the Golden Fleece came to be known as an economic powerhouse. Of all of the domesticated animals, none were more purposefully and experimentally altered than sheep. Because of its connection and symbolism of wealth, who can blame breeders for attempting more efficiency? Over the next several hundred years, breeders acted on their own initiative, making decisions on an empirical basis. In the 18 th century Robert Bakewell of Dishley in Leicestershire, England became the benchmark sheep breeder. He targeted his attentions to maximum growth and maximum edible tissue. He encouraged and led a mini scientific revolution of the breeding community that had both designed experimental and abstract concepts. These sheep farmers ignored conventions and religious banter and followed an economic drive to create a more sellable sheep. Bakewell expressed his findings and best practices in a document called the 'Sheepish Doctrine 18.' Part joke, part instructional manual his findings were labeled both ingenious and heresy. The document circulated eventually outside of England and was adapted for local needs.
Bakewell was inspired by his horses; built for strength and stamina. He knew that the public had a demand for cheap meat and envisioned providing that by breeding his sheep for specific purposes. By maximizing both the sheathing process for wool and the slaughter process for food production he could exploit profitability. He needed animals that would grow quickly on minimum food, whose meat was tender with sufficient fat content 19. He began gaining his genetic knowledge though making environmental changes and careful observation. He found through experimentation that castrated rams ate more and bulked up quickly. He created controls and variables, collected data and analyzed the results choosing which animals to breed specifically for their worthy phenotypes and discarding the rest. He did all of this without any scientific training. This process is now known as selective breeding.
Despite the work of generations of farmers who purposely produced plants with desirable phenotypes, and some of the work of Bakewell and herders like him who tried to establish a dictionary of heredity, the actual scientific evidence of gene inheritance and potential manipulation is the life's work of a Central European monk named Gregor Mendel. Mendel's work although published in 1866, wasn't recognized until 1900 20. He systematically observed the breeding habits and inherited traits of pea plants. He recognized a regularity and specificity to how phenotypes are determined and related it back to the breeding process and the plant's genotype. From Mendel we learned that all genes are inherited from parents and that each parent provides an allele for each gene. Often times an offspring inherits two dominant alleles or one dominant and one recessive allele in this case the dominant phenotype will be observed. The only way for a recessive phenotype to be observed is through the inheritance of two recessive alleles. Mendel also noticed the possibility of a blending of phenotypes, called incomplete dominance, whereas a red and yellow flower may daughter an orange flower. And he noted a co-dominate feature where there was two dominant alleles as in AB blood type.
Mendel's findings were eloquent, extensive, and elaborately detailed. Although pushed under the rug for quite some time, once the information was accepted in the scientific community and penned for the common farmer, selective breeders had the scientific proof and mathematical back up to make specified decisions with predictably dependable results. Thus thousands of years of farming experiments were finally summed up into a concise definition of genotype combinations and phenotype expectations.
Modern Genetic Modifications
Mendel's principles are still effective and informatively used in selective breeding today. In 1900, three European botanists rediscovered Mendel's work. This was the beginning of genetics as a modern science and for the next 50 years many scientists focused on discovering exactly what the genetic material was. They assumed it was within the nucleus, and were especially interested in the structure. Then, in 1953, British scientist Francis Crick and U.S. scientist James Watson showed that the substance had a long, double spiral structure. They discovered the structure of DNA.
The following 60 years after the structure of DNA was defined by Crick and Watson involved a whirlwind of discoveries into finally the mapping of the human genome which was completed in 2003 21. However, a complete map was not necessary to consider laboratory exploration of genetic modification. The structure of DNA gave scientists the clues needed to show how DNA reproduced itself, and how genetic information in the DNA affected characteristics and traits of the organism. As soon as the structure was unharnessed, scientists could now envision the manipulation of genes without selective breeding.
The biggest disadvantage to selective breeding is the time involved. The heart of Mendel's work focused on pea plants. They are quick to reproduce, and quick to flower, two of the specific reasons he used them in his research. However, Bakewell's sheep may have taken two to three years before displaying a specific acquired trait. Additionally, breeding to achieve desired traits may have also bred in undesired traits. Historically royal families often interbreed to keep their blood purely royal. Queen Elizabeth of England (1837-1901) was a carrier of the gene for hemophilia (a mutation of the F8 gene that codes a protein that causes blood to clot, it mostly effects male offspring of female carriers). She had four sons, one of whom died as a young man of hemophilia. However, of her five daughters, two were also carriers who wed to royalty in Spain and Russia 22. The hemophilia gene then infected their royal lineage. By not providing diversity in the gene pool these royal families kept their pedigree, but also inherited the same genetic problems.
As scientists gained an understanding of DNA structure and were gathering more information daily on specific genes responsible for desired traits, industries demanded research into expedient methods of gene manipulation. In order for such to be possible a few genetic breakthroughs were necessary. First, scientists had to have a way to artificially replicate DNA to practice with and study it. Next, they needed methods to unravel and ravel it again. Third, they needed methods of splicing or cutting of the genome, and replacement of required sections. Finally they needed host cells to incubate the modified DNA.
Development of procedures and processes took time. Many methods of solving some of these needs involved identifying an appropriate enzyme. For example, one of the first enzymes that researchers managed to isolate was DNA polymerase. This is the essential tool to the DNA replication process. In nature, the two stands of the DNA double helix unzip or untwist. DNA polymerase then whips into action and makes a strand complementary to each side. Since the new DNA is complementary to the old each strand can reconnect with the newly written code. The end result is two independent copies of the exact same strands of DNA.
Once identified, scientists were able to utilize DNA polymerase to make multiple copies of any strand of DNA they wished to study. They simply heated the samples (this weakens the bonds of the DNA molecule) then added the DNA polymerase. An additionally useful enzyme find was the discovery of ligase. Ligase acts as a repairing agent to damaged DNA. It can repair the ends of two pieces of DNA that had been cut through 23.
The effectiveness of polymerase was amplified when in 1983 a U.S. Scientist named Kary Mullis invented a process called polymerase chain reaction or PCR 24. PCR allows for the infinite replication of a tiny amount of DNA. This process has become extremely important in forensic studies, and the tracking of pathogens, and modern genome sequencing.
With PCR and polymerase as effective replication techniques and ligase for repair, researchers needed methods for cutting DNA. Enter an additional family of enzymes called restriction enzymes. First located in bacteria as a defensive mechanism, restriction enzyme managed to attack the DNA of predator virus, leaving the virus indefensible by chopping its DNA to little bits. Much like a video game plot, restriction enzymes act as the weapon of use for the bacteria, cutting like scissors the virus' DNA molecule apart through several sections. However, unlike a video game, restriction enzymes don't make random wounds. They cut at specific coded locations. The cut isn't always made in a straight line either (please see picture below). If using identical strands of DNA, since restriction enzyme cuts in the same place, it produces pieces of DNA that are the exact same size.
With the ability to cut specific sections of DNA, researchers can eliminate what isn't needed and focus on single genes or single sections of genes. One the section needed is identified, scientists can use a restriction enzyme specific to that coding (or something nearby) to isolate the needed DNA. They then can replicate millions of copies of this specific section using PCR. Now with many copies of that specific section or that specific codon, or protein building chain, they cannot simply inject those back into a cell and expect it to grow. Replication and expression of genes requires a scaffold with the appropriately placed signals to allow for control of replication and activation of transcription. As the first step, scientists use bacteria and a particular kind of gene scaffold, called a plasmid.
Many bacteria have a natural and distinct organelle called a plasmid. Plasmids are circular pieces of DNA that are independent of their chromosomes. They are additional pieces of DNA that help bacteria survive in certain conditions 25. Bacteria will also take on additional plasmids from their environment if conditions are right. This is why they can be used in the manipulation process. First a plasmid is isolated outside the bacterial cell. Then a restriction enzyme is used to cut the plasmid open at a desired location. Then the human DNA is cut using the same restriction enzyme. This way the human gene can be replaced inside the bacterial plasmid because they have the same pattern of cut-ends. If this is done correctly the bacterial plasmid will re-enter the bacteria and start synthesizing for the human gene. With the correct conditions not only will the bacteria begin to make human proteins it will also begin to replicate itself including the replication of the inserted plasmid and will thereby have made multiple human protein producing bacterium.
This process is essential to the making of many modern medicines including human insulin. Many diabetes patients are insulin dependent as their pancreas cells have stopped or slowed the process of making insulin. By replacing the genetic code for insulin manufacturing into a plasmid and then reintroducing that plasmid to bacterium, the plasmid will begin to successfully fabricate human insulin in a bacteria cell. If then given further advantageous conditions, the bacteria making insulin can duplicate making many insulin producing bacterium. The insulin can then be removed from the bacteria and harnessed for inoculations for insulin dependent individuals. This same genetic manipulation technique is used to make human growth hormone, interferon, and tissue plasminogen activator, a drug given to stoke victims 26.
Another use of genetic modification outside biomedical practices is the modification of food sources. Old agricultural traditions have been upgraded with this modern technology. Although, as discussed preciously, farmers had been using genetic engineering principles for centuries, it was not until the twentieth century that farmers begin to understand that successes and failures had to do with genes. Genetically Modified Organisms or GMOs can be created using several technological advancements.
Advancement in the technologies of food included the creation of canola oil in 1974, which was developed by painstakingly examining the seed of thousands of varieties of the plant rape. These rape seeds were assessed individually for low erucic acid levels. Previously cultivated rape seed oil was especially high in erucic acid which was linked to heart disease. By examining a portion of the seed and testing for low erucic levels, scientists slowly weeded through until creating a finalized hybrid plant with seeds with very low erucic acid levels that created oil with low levels of saturated fats. This is the same canola oil we use today. Apples could be said to be far more unnatural than that. As apples are the product of one tree grafted onto the trunk and roots of a completely different tree. Apple growers proliferate this by cutting the branches into slips and grafting them onto root stocks. Thus by planting an apple seed found in your forbidden fruit you may not grow a plant identical to its parent. As seeds are a form of sexual reproduction, grafting is a low technology form of cloning 27. But, these are hardly the genetic modifications in question today.
Many foods consumed today are either genetically modified whole foods, or contain ingredients derived from gene modification technology. This is a billion dollar business that is surrounded in controversy. Critics believe that applying genetic modifications to human food production could have several adverse consequences. For these critics the potential effects far outweigh increased food production and improved food quality. Specific issues raised include:
- A consumer's right to know what is in their food.
- Countries' rights to regulate agriculture.
- Potential "food terrorism" and the security of genetically modified food crops.
- The natural spread of modified plant seeds to non-modified farm land.
- Coincidental rise of food related allergies.
- The possible growth of insects resistant to modified plant toxins.
Supporters of genetic modification include many farmers and the industries and companies that financially benefit from the modification. Some farmers are especially supportive as genetic modification can lead to faster growing, disease resistant, weather resistant, and pest-resistant crops, crops that tolerate large amounts of pesticides and herbicides. Crops are also modified for taste, convenience, nutrition, and preservation. Additionally, because of poor dietary quality and malnutrition in developing countries, genetic modification of crops may assist in increasing the health and wellness of socioeconomically disadvantaged adults and children. As good access to affordable and safe food supply is linked to a productive workforce, genetic modification of crops could produce a plentiful crop and therefore increase that country's GDP. Additionally with an expected human population of over nine billion by 2050, something has to be done to sustain food production and nutritional needs for a dramatically increased worldwide population 28.
These issues have been deliberated in government policy hearings, panel discussions, newspapers, and television programs. It is of the utmost importance that both the scientific community and those with limited understandings about GMOs disseminate information and communicate correctly. The information available needs to be clear and specific and written in an everyday language that is accessible to the general public. It needs to include the benefits and rationale for implementing GMO in biotechnology, its evolution, protocols, risks, and levels of predictability 29. Additionally, risk assessment, management, and oversight of GMO practices and protocols should be publically available and openly discussed. Only then can a comprehensive perspective emerge to fuel public understanding and independent family decision making.
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