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Genetically Modified Organisms

Assignment
Topic: Genetically Modified Organisms (GMOs)
Department: Submitted To: Submitted By: 1) Abdul Rehman Rashid (101) Biochemistry Dr. Awais Asif

Submitted on:
Monday, 23rd January 2012

GENETICALLY MODIFIED ORGANISMS (GMOs)


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Genetically Modified Organisms

Definition:

GMO is an organism whose genetic makeup is modified or changed, not by mutation or accidental cross-breeding, but by using latest bio-techniques like Recombinant DNA technique, in which different genetic materials are combined. This process of modification is called Genetic Modification OR Genetic engineering. This step is taken to make organisms more useful and adaptable.

Description:
Genetic Engineering of organisms involved in it, by changing the genome of organisms using mostly, recombinant DNA technique. In which desired traits and characteristics are taken by removing and inserting genes of interests. Genes of interest are taken from any other species of organism having that desired characteristics.

History:
Generally, GMOs are under the results of genetic engineering. It has been going on for very long time by the name of Selective Breeding Artificial Selection and other common methods results from hairless cats to sweet corn and human like versions of mice gene FOXP2. The alternation of genetic material by insertion or removal was made possible after the discovery of Recombinant DNA Bactrian in 1973. E-coli were this bacterium first ever genetically modified by great scientist HERB BOYER and STANELY COHEN. In 1978, Genentech (Biotechnology Corporation founded by venture capitalist Robert A. Swanson and biochemist Dr. Herbert Boyer.in 1976) announced that E.colis one strain can produce human insulin. Glofish was first GMO animal sold as pet. Since 1996, Genetically Modified foods and crops are being cultivating rapidly. Now-a-days, there is a huge diversity in G.M.Os as food, animals, agriculture and medicine.

What are not Genetically Modified Organisms?


These do not include organisms whose modification is due to: Fusion of animal cells unless the product can form an animal. Plants formed X-protoplast fusion. Plants formed by embryo rescue or in vitro fertilization. Formed by zygote implantation. Organisms formed by natural DNA transfer.

Techniques Used For Genetic Modification:


There are many techniques used in genetic modification or engineering Following are the most commonly used techniques in Genetic Modification. Page 2 of 17

Genetically Modified Organisms 1. Recombinant DNA Technology 2. Bioballistics Method 3. Electro and Chemical proration 4. Microinjection

1.Recombinant DNA Technology:


To cut a gene of interest from one organism and insert to other organism to get the benefit of that gene of interest. Following are the steps involved in Recombinant DNA techniques: 1. Isolation of Gene of interest. 2. Piece of DNA is cut with a restriction enzyme and then ligate the DNA insert into the vector(on which the gene of interest is placed) with DNA Ligase enzyme. 3. Now, the gene or DNA along with the vector is called recombinant DNA is produced, and is introduced into an expression system as a result of which specific result or product is obtained. Explanation of the Complete Process: Following are the steps in rDNA process.

1.Isolation of gene of interest:


In rDNA is the isolation or getting of gene of interest .Three possible ways to get the gene of interest

A. To isolate it from the Chromosomes:


Gene can be isolated from the chromosomes by cutting the chromosomes and getting genes using special enzymes Known as restriction Endonucleases.

Restriction Endonuclease: An enzyme which cuts the double stranded DNA is known as restriction endonuclease or restriction enzyme.

Structure of Restriction Endonuclease Page 3 of 17

Genetically Modified Organisms Such enzymes, found in archaea and bacteria, are thought to have evolved to provide a defense mechanism against invading viruses.Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA but does not harm to bacterial chromosome.In 1970,Hamilton O.smith,at Johns Hopkins University isolated the first restriction enzyme. These enzymes cut the DNA at very specific sites(known as restriction sites) characterized by specific sequence of four or six nucleotides symmetrically in the reverse order.Thes sequences are called Palindromic sequences. In fig. below AATT (5'-3' ) and TTAA (3'-5' ) are palindromic sequences.

Palindromic sequence When it cuts DNA sticky ends are created from palindromic sequences.

Restriction enzyme EcoR1 ,cuts specific sequence of nucleotides in such a way that sticky ends are produced

There are more than 400 such enzymes have been isolated and out of these 20 are frequently used in recombinant technology.Most commonly used restriction enzyme is EcoR1(isolated from strains of E. coli).

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Genetically Modified Organisms

EcoRI crystal structure

EcoRI recognition site with cutting pattern indicated by a line Some other restriction enzymes are: EcoRII (isolated from Escherichia coli) , BamHI (isolated from Bacillus amyloliquefaciens), TaqI(isolated from Thermus aquaticus).

B.To synthesize it chemically If genes are small they can be synthesized in the laboratory
C.To make it from mRNA: This is another method used for getting gene of interest. Using messenger RNA by reverse transcriptase. This DNA molecule is called complementary DNA.

2.Selection of Vector:
A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. Selection of a vector is also important step to make the recombinant DNA.Means by which rDNA is introduced into host cell. Two types of vectors are commonly used in recombinant DNA technology , 1. Plasmid 2. Viruses

3. Recombinant DNA:
Recombinaanat DNA is DNA which have foreign piece of DNA is present.For the preparation r DNA ,the plasmid is cut with the same restriction enzyme which was used for isolation of the gene of interest the of interest is then joined with sticky ends produced after the cutting of the plasmid with the help of an enzyme DNA ligase.This enzyme seals the foreign piece of DNA into vector Now the two different pieces of DNA have been joined together ,which is known as recombinant DNA or chimaeric DNA. Page 5 of 17

Genetically Modified Organisms Types Of Recombinant DNA Technique On the basis of the usage of vector there are two types of rDNA technology are:

1. Vector method:
In this method viral DNA is used as vector,its product is directly inserted into the genome via a viral chromosome.

2. Plasmid method:
Plasmid is a DNA molecule that and can replicate independently.These are doublestranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, Plasmids were discovered by investigators studying the sex life of the intestinal bacterium Escherichia coli. Plasmid are natural extra chromosomal circular DNA.

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Genetically Modified Organisms

Bacterial cell plasmid inside the cell

In this method plasmid is used as vector to carry gene of interest.and this can be implemented the gene into plants and animals. Schematic Diagram of plasmid method is shown below

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Genetically Modified Organisms

Applications of Recombinant DNA technology Method


Recombinant DNA is widely used in biotechnology, medicine and research.The most common application of recombinant DNA is in basic research, where it is important to most current work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine their function. Many additional practical applications of recombinant DNA are as: 1.Recombinant human insulin. Recombinant insulin has almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. 2.Recombinant blood clotting factor VIII. Recombinant factor VIII is a blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation 3.Golden rice: golden rice is a recombinant variety of rice that has been engineered to express enzymes responsible for -carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in world's population. 4.Herbicide Resistant Crops: Varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed which incorporate a recombinant gene that results in resistance to the herbicide glyphosate and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries. 5.Insect-resistant crops. Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties.The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed which express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved

2. Bioballistic Method:
This is also called micro-projectile bombardment or gene gun or biolistics. This technique is used for in vivo, within a living organism, transformation and has been especially useful in transforming monocot species like corn and rice. This approach literally shoots genes into plant cells and plant cell chloroplasts. DNA is coated onto small Page 8 of 17

Genetically Modified Organisms particles of gold or tungsten approximately two microns in diameter. This technique can also be used for RNA, if desired. The particles are placed in a vacuum chamber and the plant tissue to be The Helios Gene Gun by BioRad engineered is placed below the chamber. The particles are propelled at high velocity using a short pulse of high pressure Helium gas into any target cell or tissue.

Gene Gun used for Bioballistics The biolistic method is most useful for inserting genes into plant cells such as pesticide or herbicide resistance. Different methods have been used to accelerate the particles: these include pneumatic devices; instruments utilizing a mechanical impulse or macroprojectile; centripetal, magnetic or electrostatic forces; spray or vaccination guns; and apparatus based on acceleration by shock wave, such as electric discharge .

Helios Gene Gun schematic

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Genetically Modified Organisms

3.Electro and Chemical proration:


In this method,the targt cell and selected DNA are placed in a special solution and electric shock is passed through solution which causes pores in target cells and DNA goes inside the cell.This is done in an instrument called electroporator. Special chemicals, such as polyethylene glycol, are also used to make pores in cell surface. Then cells are placed in another solution & allow to repair their cell walls .thus locking new DNA. Selected DNA incorporates into host chromosomes.

Diagram of the major components of an electroporator

4.Microinjection:
The process in which a glass micropipette is used to insert substances at a microscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and the nuclear envelope. The desired genes are then injected into the desired sub-cellular compartment and the needle is removed. The injected genes are automatically enter into the nucleus where they incorporate with the host cells genetic material and replicate with it.

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Genetically Modified Organisms

Introduction of Foreign DNA in a Host Cell with a Microinjection Needle

List of Genetically Modified organisms


Foods:
Food Soybeans Properties variety of the genetically modified Modification Herbicide resistant gene taken from bacteria inserted into soybean New genes, some from the bacterium Bacillus thuringiensis, added/transferred into plant genome. Bt crystal protein added/transferred plant genome New added/transferred plant genome. gene into genes into gene

Resistant to glyphosate or glufosinate herbicides

Resistant to glyphosate or glufosinate herbicides. Insect resistance via producing Bt proteins, some Corn, field previously used as pesticides in organic crop (Maize) production. Vitamin-enriched corn derived from South African white corn variety M37W has bright orange kernels, with 169x increase in beta carotene, 6x the vitamin C and 2x folate. Cotton (cottonseed Pest-resistant cotton oil) Alfalfa Hawaiian Resistant to glyphosate or glufosinate herbicides

Variety is resistant to the papaya ringspot New Page 11 of 17

Genetically Modified Organisms papaya virus. added/transferred plant genome into

Tomatoes

A reverse copy Variety in which the production of the enzyme (an antisense gene) of the polygalacturonase (PG) is suppressed, gene responsible for the retarding fruit softening after harvesting. production of PG enzyme added into plant genome Resistance to herbicides (glyphosate glufosinate), high laurate canola or New added/transferred plant genome genes into genes into genes into

Canola

New Resistance to certain pesticides, high sucrose Sugar cane added/transferred content. plant genome Sugar beet Resistance herbicides to glyphosate, glufosinate New added/transferred plant genome

Rice

Current version of Golden Rice under development contains genes from maize and a common soil Golden Rice: genetically modified to microorganism. Previous contain beta-carotene(a source of vitamin A) prototype version contained three new genes: two from daffodils and the third from a bacterium Resistance to watermelon, cucumber and Contains coat zucchini yellow mosaic viruses genes of viruses. Resistance to virus protein

Squash (Zucchini) Sweet Peppers

Contains coat protein genes of the virus.

Bactaria:
Bacteria were the first organisms to be modified in the laboratory, due to their simple genetics. These organisms are now used for Page 12 of 17

Genetically Modified Organisms several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine. Genetically modified bacteria are used to produce the protein insulin to treat diabetes. Similar bacteria have been used to produce clotting factors to treat haemophilia and human growth hormone to treat various forms of dwarfism.

Animals:
Some chimeras, like the blotched mouse shown, are created through genetic modification techniques like gene targeting. Transgenic animals are used as experimental models to perform phenotypic and for testing in biomedical research. Genetically-Modified (Genetically Engineered) animals are becoming more vital to the discovery and development of cures and treatments for many serious diseases. By altering the DNA or transferring DNA to an animal, we can develop certain proteins that may be used in medical treatment. Stable expressions of human proteins have been developed in many animals, including sheep, pigs, and rats. Some examples are: Human-alpha-1-antitrypsin, which has been developed in sheep and is used in treating humans with this deficency and transgenic pigs with human-histocompatibility have been studied in the hopes that the organs will be suitable for transplant with less chances of rejection. Transgenic livestock have been used as bioreactors since the 1990s. Many medicines, including insulin and many immunizations are developed in transgenic animals. In March 2011, the bioactive recombinant Human Lysozyme was expressed in the milk of cloned transgenic cattle. This field is growing rapidly and new pharming uses are being discovered and developed. The extent that trangenic animals will be useful in the medical field as well as other fields is very promising based on results thus far.

Fruit flies
In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms used to study the effects of genetic changes on development. Fruit flies are often preferred over other animals due to their short life cycle, low maintenance requirements, and relatively simple genome compared to many vertebrates. Mosquitoes Page 13 of 17

Genetically Modified Organisms Scientists created "malaria-resistant mosquitoes" in the laboratory.The World Health Organisation estimated that Malaria killed almost one million people in 2008. Genetically modified male mosquitoes containing a lethal gene have been developed in order to combat the spread of Dengue fever. Aedes aegypti mosquitoes, the single most important carrier of dengue fever, were reduced by 80% in a 2010 trial of these GM mosquitoes in the Cayman Islands.Between 50 - 100 million people are affected by Dengue fever every year and 40,000 people die from it. Bollworms A strain of Pectinophora gossypiella (Pink bollworm) has been developed that contains a fluorescent marker in their DNA. This allows researchers to monitor bollworms that have been sterilized by radiation and released in order to reduce bollworm infestation. Mammals Genetically modified mammals are an important category of genetically modified organisms.

Mice:
Genetically Modified mice are commonly used for research or as animal models of human diseases,often used to study cellular and tissue-specific responses to disease. They have been also used to study and model obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease

Enviropig:
Enviropig have capability to digest plant phosphorus more efficiently than ordinary unmodified pigs The Enviropig excretes from 30 to 70.7% less phosphorus in manure depending upon the age and diet. The benefits of the enviropig if commercialized include reduced feed cost and reduced phosphorus pollution as compared to the raising of ordinary pigs. In 2009, scientists in Japan announced that they had successfully transferred a gene into a primate species (marmosets) and produced a stable line of breeding transgenic primates for the first time.Their first research target for these marmosets was Parkinson's disease, but they were also considering Amyotrophic lateral sclerosis and Huntington's disease. In 2011, scientists in China released news that they have introduced human genes into 300 dairy cows to produce milk with the same properties as human breast Page 14 of 17

Genetically Modified Organisms milk. Aside from milk production, the researchers claim these transgenic cows to be identical to regular cows. Cnidarians Cnidarians such as Hydra and the sea anemone Nematostella vectensis have become attractive model organisms to study the evolution of immunity and certain developmental processes. An important technical breakthrough was the development of procedures for generation of stably transgenic hydras and sea anemones by embryo microinjection.

Fish Genetically modified fish have promoters driving an over-production of "all fish" growth hormone. This resulted in dramatic growth enhancement in several species, including salmonids, and tilapias.

Advantage and Disadvantage


Advantages of GMO: 1. GMO have more resistance for diseases and the widespread plant diseases cannot spread in GMO widely 2. Unexpected frost usually destroys sensitive seedlings. To make plants tolerant from unexpected frost, an anti-freeze gene from cold water fishes has been introduced into plants such as tobacco and potato 3. GMO are more droughts tolerant. Theses can withstand long periods of drought or high salt content in soil, and water. Farmers can grow crops in unsuited places, due to GMO. 4. GMO foods have high nutrition. The nutrients that are not naturally present in some foods are inoculated in them using Genetic Modification .E.g. rice 5. Scientists are working to develop edible and less costly vaccines for third world countries. They are working on tomatoes and potatoes. 6. Plants are Genetically Modified to have greater tolerance against the herbicidic sprays. So that the herbs will die but plants will withstand against the spray of herbicides to remove herbs. This improves the quality of plant products. Page 15 of 17

Genetically Modified Organisms 7. Plants are genetically modified to be resist against the harms of different kinds of pests.

Disadvantages of GMOs:
1. Harm to other organisms. For example genes and their effect included in a crop may turn out to be poisonous to insects (monarch butterfly poisoned by GMO corns). 2. Taste of GMOs are not as good or "natural".. 3. Cross-pollination with traditional, organic plants. Cross pollination can occur at quite large distances. New genes may also be included in the offspring of the traditional, organic crops miles away. This makes it difficult to distinguish which crop field is organic, and which is not, posing a problem to the proper labeling of non-GMO food products. 4. Spread of new, more resistant "super weeds 5. Spread of new, more resistant "super pests". 6. Major trading countries that obtain most of the benefit from the production and trade of genetically modified crops. This might cause more geopolitical conflicts. 7. New trade, tariff and quota issues may arise between countries, regions. 8. Critics say GMOs may cause health problems. 9. As the USA is the biggest producer of GMO crops, their exports may rouse more anti-American feeling, due to Americanization worldwide. 10. 11. Possible damages to the environment. Possible greed of GMO manufacturing firms. Unharmonized test-and safety standards around the world.

12.

13. GMOs are made because it is possible to make them, not because consumers feel their need. 14. Possible creation of new kinds of weapons; genetic food and beverage weapons. 15. Additional costs of labeling whether products are GMOs or not. This might increase costs of foods. 16. Widening corporate size gaps between food producing giants and smaller ones. This might cause a consolidation in the market: fewer competitors increase the risk of oligopolies, which might increase food prices..

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Genetically Modified Organisms 17. Activists increased ability to boycott and influence food market, food retailing, and food prices. 18. Allergies may become more intense, and also, new allergy types may develop. 19. Discrepancies in information flow. GMO producers stress the benefits, but are reluctant to talk about risks and dangers.

Refrences : 1. http://en.wikipedia.org 2. http://library.thinkquest.org/C004367/be9.shtml 3. http://www.ucsusa.org/food_and_agriculture/science_and_impacts/scienc

e/genetic-engineering-techniques.html 4. http://csanad.hubpages.com/ 5. http://www.biotechnologyforums.com/thread-43.html 6. Intermedite book part 2 7. http://courses.washington.edu/z490/gmo/natural.html 8. http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Mc Donald/Gene_gun.html

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