Biology

Key Unit Competence
Evaluate how gene technology is applied in areas of medicine, forensic science and agriculture

Learning objectives
By the end of this unit, I should be able to:
–– Define the term bioinformatics.

–– Outline the role of bioinformatics following the sequencing of genomes, such as those of humans and parasites, e.g. Plasmodium. (Details of the methods of DNA sequencing are not required).

–– Explain the advantages of producing human proteins by recombinant DNA techniques. (Reference should be made to some suitable examples, such as insulin, factor VIII for the treatment of haemophilia and adenosine deaminase for treating severe combined immunodeficiency (SCID).

–– Outline the advantages of screening for genetic conditions. (Reference may be made to tests for specific genes such as those for breast cancer, BRCA1 and BRCA2, and genes for haemophilia, sickle cell anaemia, Huntington’s disease and cystic fibrosis).

–– Outline how genetic diseases can be treated with gene therapy and discuss the challenges in choosing appropriate vectors, such as: viruses, liposomes and naked DNA, (Reference may be made to SCID, inherited eye diseases and cystic fibrosis).

–– Explain the significance of genetic engineering in improving the quality and yield of crop plants and livestock in solving the demand for food in the world
e.g. Bt maize, vitamin A enhanced rice (Golden rice TM) and GM salmon.

–– Outline the way in which the production of crops such as maize, cotton, tobacco and rape seed oil may be increased by using varieties that are genetically modified for herbicide resistance and insect resistance.

–– Explain the ethical and social implications of using genetically modified organisms (GMOs) in food production.

–– Interpret a chart on the stages involved in the production of insulin by bacteria.

–– Analyse the application of gene technology in agricultural modernisation.

–– Research the benefits, hazards and implications of gene technology.

–– Appreciate the application of gene technology in medicine, and forensic science such as the detection of crimes e.g. rape, murder, and paternity disputes.

–– Appreciate the application of gene technology in agriculture through the improvement of crop varieties and animal breeds.

Techniques used by genetic engineers have been seen in unit 13. What can be done with these techniques? By far most numerous applications are still as research tools, and those techniques are helping geneticists to understand complex genetic systems. Despite all of those types, genetic engineering still has very few successful commercial applications, although these are increasing each year. The applications so far can usefully be considered in three groups.

–– Gene products using genetically modified organisms (usually microbes) to produce chemicals, usually for medical or industrial applications.
–– New phenotypes using gene technology to alter the characteristics of organisms (usually farm animals or crops).
–– Gene therapy using gene technology on humans to treat a disease.

The biggest and most successful kind of genetic engineering is the production of gene products. These products are of; medical, agricultural or commercial value. The table below shows some examples of genetically engineered products that are already available.

14.1 Bioinformatics

Bioinformatics is the collection, processing and analysis of biological information and data using computer software. In other words, it is the branch of biology that is concerned with the acquisition, storage, and analysis of the information found in nucleic acid and protein sequence data. Bioinformatics combines biological data with computer technology and statistics. It builds up databases and allows links to be made between them. The databases hold gene sequences of complete genomes, amino acid sequences of proteins and protein structures.

UniProt (universal protein resource) holds information on the primary sequences of proteins and the functions of many proteins, such as enzymes. The search tool BLAST (basic local alignment search tool) is an algorithm for comparing primary biological sequence information, such as the primary sequences of different proteins

or the nucleotide sequences of genes. Researchers use BLAST to find similarities between sequences that they are studying and those already saved in databases. When a genome has been sequenced, comparisons can be made with other known genomes. For example, the human genome can be compared to the genomes of the fruit fly, Drosophila, the nematode worm, or the malarial parasite, Plasmodium. All the information about the genome of Plasmodium is now available in databases. This information is being used to find new methods to control the parasite. For example, being able to read gene sequences is providing valuable information in the development of vaccines for malaria.

14.2 Production of human proteins by recombinant DNA technology

Recombinant DNA technology brought about a complete revolution in the way living organisms are exploited. By transferring new DNA sequences into microbes, plants, and animals, or by removing or altering DNA sequences in the endogenous genome, completely new strains or varieties can be created to perform specific tasks. One of the earliest commercial applications of gene manipulation was the production of human therapeutic proteins in bacteria. Not surprisingly, the first such products were recombinant versions of proteins already used as therapeutics: human growth hormone and insulin. Prior to the arrival of genetic engineering, human growth hormone was obtained from pituitary glands removed from cadavers and the insulin was extracted from the pancreas of pigs or cattle.

Production of Insulin

This hormone can be produced by genetically modified bacteria and has been in use since 1982. The human insulin gene is inserted into bacteria, which then secrete human insulin. The human insulin produced in this way is purer than insulin prepared from pigs or cattle that was used before, which sometimes provokes allergic reactions owing to traces of ‘foreign’ protein. The Genetically Modified(GM) insulin is acceptable to people with a range of religious beliefs who may not be allowed to use insulin from cows or pigs. The main advantage of this form of insulin is that there is now a reliable supply available to meet the increasing demand. The chart below summarises stages involved in the production of insulin by bacteria

There were problems in locating and isolating the gene coding for human insulin from all of the rest of the DNA in a human cell. Instead of cutting out the gene from the DNA in the relevant chromosome, these are steps involved in human insulin production:

– Researchers extracted mRNA for insulin from pancreatic β cells, which are the only cells to express the insulin gene. These cells contain large quantities of mRNA for insulin as they are its only source in the body.

– The mRNA was then incubated with the enzyme reverse transcriptase which comes from the group of viruses called retroviruses. As its name suggests, this enzyme reverses transcription, using mRNA as a template to make singlestranded DNA.

– These single-stranded DNA molecules were then converted to doublestranded DNA molecules using DNA polymerase to assemble nucleotides to make the complementary strand.

– The genetic engineers now had insulin genes that they could insert into plasmids to transform the bacterium Escherichia coli.

  – When the bacterial cells copy their own DNA, they also copy the plasmids and the donor genes that plasmids carry. After the cells have grown into colonies, on an industrial scale in large fermenters insulin is extracted from the bacteria.

14.3 Genetic technology applied to medicine and forensic science

14.3.1 Genetic screening

Genetic screening is the detection of mutations known to be associated with genetic disorders before they manifest themselves in an individual. This can be done in adults, in a foetus or embryo in the uterus, or in a newly formed embryo produced by in-vitro fertilization. For example, an adult woman with a family history of breast cancer may choose to be screened for the faulty alleles of the genes Brca-1 and Brca2, which considerably increase an individual’s chance of developing breast cancer. If the results are to be positive; the woman may choose to have her breasts removed (elective mastectomy) before such cancer appears.

Genetic disorders in the human foetus can also be detected using genetic screening of embryonic cells found in the amniotic fluid during gestation. Such prenatal screens are available for haemophilia, phenylketonuria, cystic fibrosis, and Duchenne’s muscular dystrophy. Couples with a family history of genetic disorders who are at risk of passing mutations on to their offspring are offered genetic counselling to better prepare for the birth of a child. The most common vectors that are used to carry the normal alleles into host cells are viruses (often retroviruses) or small spheres of phospholipid called liposomes.

14.3.2 The ethics of genetic screening

Many people believe that the law is allowing too much, while others think that it should allow more. For instance, in some countries, the law allowed an embryo screening for a genetic disease; also some countries allow a successful transplant of tissue from one person to another. But the law does not allow the addition of an allele to an egg, sperm or zygote. Other countries have different attitudes and regulations. For example, a foetus can now be screened for a genetic disease while in the uterus, using amniocentesis or chorionic villus sampling. From this screening parent can decide to terminate her pregnancy if the embryo is found to have a genetic disease.

Some parents have decided to terminate pregnancies simply because the child is not the sex that they want. Pre-implantation genetic diagnosis (PGD) is the technique that involves mixing the father’s sperm with the mother’s eggs in a dish (In vitro procedure). The PGD has been also used to select the sex of the embryo that is chosen to be implanted. Many people think that this sex pre-selection, as it is called, is totally unethical.

14.3.3 Treatment of genetic diseases by gene therapy

Gene therapy is the introduction of genes into suffering individual for therapeutic purposes. It holds great potential for treating disorders noticeable to a single defective gene. The first successful gene therapy performed was about the rare genetic disorder known as severe combined immunodeficiency (SCID). The defect in SCID involves the inability to make an enzyme, adenosine deaminase (ADA) which is vital for the functioning of the immune system. These enzymes are made by a genetically modified; insect larva, the cabbage looper moth caterpillar. This enzyme is administered to patients while they are waiting for gene therapy or when gene therapy is not possible. The work on SCID has led to increasingly successful gene therapies in the last few years, including the followings:

a. Inherited eye diseases

Inherited eye diseases called Leber congenital amaurosis is a form of hereditary blindness that primarily affects the retina, which is a specialised tissue at the back of the eye that detects light and colour. People with this disorder typically have severe vision impairment beginning at infancy. By gene therapy this condition has been improved.

b. Haemophilia

Haemophilia is an inherited bleeding disorder where the blood does not clot properly. It is caused when blood does not have enough clotting factor. Genetically modified hamster (small furry animal which is similar to a mouse) cells are used by several companies to produce factor VIII. This protein is essential for blood clotting, and people who cannot make it are said to have haemophilia. The human gene for making factor VIII has been inserted into hamster kidney and ovary cells which are then cultured in fermenters. The cells constantly produce factor VIII which is extracted and purified before being used to treat people with haemophilia. These people need regular injections of factor VIII which, before the availability of recombinant factor VIII, came from donated blood.

c. Cystic fibrosis

Cystic fibrosis which is a genetic disorder in which abnormally thick mucus is produced in the lungs and other parts of the body, is also treated using gene therapy. Cystic fibrosis is caused by a recessive allele of the gene that codes for a transporter protein called CFTR (cystic fibrosis transmembrane conductance regulator). This protein is found in the cell surface membranes of cells in the alveoli and allow chloride ions (Cl-) to pass out of the cells. The recessive allele codes for a faulty version of this protein that does not act properly as a chloride ion transporter.

If the normal dominant allele could be inserted into cells in the lungs, the correct CFTR should be made. In theory this should happen but in practice, there have been problem of getting the allele into the cell

Note that:

There were different trials of gene therapy using different vectors like liposomes and viruses which were not successful. DNA also has been inserted directly into tissues without the use of any vector. This so called naked DNA has been used in trials of gene therapy for skin, muscular and heart disorders. The advantages of using this method is that, it removes the problems associated with using vectors. Some proteins are even produced by transgenic animals. Sheep and goats have been genetically modified to produce human proteins in their milk: human antithrombin is produced by goats, this protein is used to stop blood clotting human alpha. Antitrypsin is produced by sheep, this is used to treat people with emphysema.

14.3.4 Application of gene technology in forensic science.

Forensic science deals with the application of scientific methods and techniques to matters under investigation by a court of law. For most people, forensic science is synonymous with criminal investigations, but it is also used to resolve civil disputes such as parenthood disputes.

DNA can be extracted from small sample of the cells found at the scene of the crime, for example in traces of blood, hair or saliva. In cases of rape, semen may be used.

a. Detection of crimes (Rape or murder)

b. In forensic science,

DNA fingerprinting is used to match material collected at the scene of crime to that of suspects. This diagram above( Figure 14.4 ) of the genetic fingerprints shows semen or blood (specimen from crime scene) found on the victim and blood samples taken from the suspect rapists or murder. The fingerprint results show an exact match between semen or blood sample obtained from the victim and the blood sample of suspect 2. As a result suspect 2 is confirmed to be the rapist or a murderer.

c. Paternity test

In perternity tests, DNA of suspected fathers are analysed together with the one of the child and the mother in order to find out the potential father among the suspect fathers that has the most DNA common with the child in question. Figure 14.5 shows an example of a Restriction Fragment Length Polymorphism (RFLP) used to determine which potential father between father 1 and 2 who is the real father of the child (C). As it is seen on the above figure, the second father tested (F2) seems to have more DNA in common with the child than of the first farther tested (F1).

14.4 Significance of genetic engineering in improving the quality and yield of crop plants and livestock

Scientists are working to learn more about the genomes of agriculturally important plants and animals. For a number of years, they have been using DNA technology in an effort to improve agricultural productivity. The selective breeding of both livestock (animal husbandry) and crops has exploited naturally occurring mutations and genetic recombination for thousands of years. As we described earlier, DNA technology enables scientists to produce transgenic animals, which speeds up the selective breeding process

14.4.1 Gene technology and agriculture

Many new products have been developed using this technology. Crops have been genetically engineered to increase yield, hardiness, uniformity, insect and virus resistance, and herbicide tolerance. The vast bulk of genetically modified plants grown around the world are crop plants modified to be resistant to herbicides or crops that are resistant to insect pests. These modifications increase crop yield. A few crops, such as vitamin A, enhanced rice, provide improved nutrition

a. Golden rice

Golden rice is a staple food in many parts of the world, where people are poor and rice forms the major part of their diet. Deficiency of vitamin A is a common and serious problem; its deficiency can cause blindness. In the 1990s, a project was undertaken to produce a variety of rice that contained carotene in its endosperm. Genes for the production of carotene were extracted from maize and the bacterium Pantonoea ananatis. These genes, together with promoters, were inserted into plasmids. The plasmids were inserted into bacteria called Agrobacterium tumefaciens. These bacteria naturally infect plants and so could introduce the genetically modified plasmid into rice cells. The rice embryos, now containing the carotene genes, were grown into adult plants.

This genetically modified rice is called golden rice, because it contains a lot of yellow pigment carotene. The genetically modified rice is being bred into other varieties of rice to produce varieties that grow well in the conditions in different parts of the world, with the same yield, pest resistance and eating qualities as the original varieties.

b. Herbicide-resistant crops: Oil seed rape

Herbicide-resistant crops called oil seed rape or Brassica napus, is grown in many parts of the world as a source of vegetable oil which is used as biodiesel fuel, as a lubricant and in human and animal foods. Natural rape seed oil contains substances that are undesirable in oil that is to be used in human or animal food. A hybrid, was made to produce low concentrations of these undesirable substances, called canola (Canadian oilseed low acid), and this name is now often used to mean any variety of oil seed rape. Gene technology has been used to produce herbicide-resistant strains. Growing an herbicide-resistant crop allows fields to be sprayed with herbicide after the crop has germinated, killing any weeds that would otherwise compete with the crop for space, light, water or ions. This increases the yield of the crop.

c. Insect pests-resistant plants

Another important agricultural development is that of genetically modified plants protected against attack by insect pests. Bt maize is genetically engineered (GE)  plant that produces crystal (Cry) proteins or toxins derived from the soil bacterium, Bacillus thuringiensis (Bt), hence the common name “Bt maize”. Bt maize plant has revolutionized pest control in a number of countries, but there still are questions about its use and impact.

14.5.2 Transgenic animals.

DNA technology enables scientists to produce transgenic animals, which speeds up the selective breeding process. Creating transgenic animals is aimed at improving quality and productivity.  For instance, to make a sheep with better quality wool, a pig with leaner meat, or a cow that will mature in a shorter time. Scientists might, for example, identify and clone a gene that causes the development of larger muscles (muscles make up most of the meat) in one breed of cattle and transfer it to other cattle or even to sheep.

Genetically modified animals for food production are much rarer than crop plants. An example is the genetically modified (GM) Atlantic salmon, developed in the USA and Canada. A growth-hormone regulating gene from a Pacific Chinook salmon and a promoter from another species of fish (an ocean pout), were injected into a fertilised egg of an Atlantic salmon. By producing growth hormone throughout the year, the salmon are able to grow all year, instead of just in spring and summer. As a result, fish reach market size in about eighteen months, compared with the three years needed by an unmodified fish. It is proposed to rear only sterile females and to farm them in land-based tanks. The characteristics of the GM salmon reduce their ability to compete with wild salmon in a natural environment. Below figure compares GM salmon the big one, and farm salmon the small; both fish are 18 months.

14.5 Ethical and social implications of using genetically modified organisms (GMOs).

Ethics includes moral principles that control or influence a person’s behaviour. It includes a set of standards by which a community regulates its behaviour and decides as to which activity is legitimate and which is not. Bioethics may be viewed as a set of standards that may be used to regulate our activities in relation to the biological world. Biotechnology, particularly recombinant DNA technology, is used for exploitation of the biological world by various ways.

Some genetically modified plants are grown in strict containment of glasshouses, but a totally different set of problems emerges when genetically engineered organisms such as crop plants and organisms for the biological control of pests are intended for use in the general environment. Few countries would object to the growth of genetically modified crops that produce vaccines for human or animal use, yet there are people who object to the growth of pro-vitamin A enhanced rice. The major bioethical concerns pertaining to biotechnology are summarized below:

–– When animals are used for production of certain pharmaceutical proteins, they are treated as factory machines.
–– Introduction of a transgene from one species into another species violates the integrity of species.
–– The transfer of human genes into animals or vice-versa is great ethic threat to humanity.
–– Biotechnology is disrespectful to living beings, and only exploits them for the benefit of humans.
–– Genetic modification of organism can have unpredictable/ undesirable effects when such organisms are introduced into the ecosystem.

Moreover, most objections are raised against the growth of herbicide-resistant or insect-resistant crops as follow:

–– The modified crop plants may become agricultural weeds or invade natural habitats.
–– The introduced gene may be transferred by pollen to wild relatives whose hybrid offspring may become more invasive.
–– The introduced gene may be transferred by pollen to unmodified plants growing on a farm with organic certification.
–– The modified plants may be a direct hazard to humans, domestic animals or other beneficial animals, by being toxic or producing allergies.
–– The herbicide that can now be used on the crop will leave toxic residues in the crop.
–– Genetically modified seeds are expensive, as is herbicide, and their cost may remove any advantage of growing a resistant crop.
–– Growers mostly need to buy seed each season, keeping costs high, unlike for traditional varieties, where the grower kept seed from one crop to sow for the next
–– In parts of the world where a lot of genetically modified crops are grown, there is a danger of losing traditional varieties with their desirable background genes for particular localities This requires a programme of growing and harvesting traditional varieties and setting up a seed bank to preserve them.