The following points highlight the top four applications of gene technology. The applications are: 1. Production of Pharmaceuticals 2. Diagnosis of Diseases 3. Insect Pest Control 4. Use of Genetically Engineered Microorganisms for Control of Pollution.
Contents
Application # 1. Production of Pharmaceuticals:
The production of medically useful human peptides and proteins (e.g. human growth hormones, insulin, somatostatin and interferon) are of much importance (Table 11.4).
Table 11.4 : Some human peptides and proteins synthesized by gene technology.
i. Recombinant Human Growth Hormone (hGH):
The pituitary gland of humans produces growth hormones that regulate the growth and development. However, in children stunted growth occurs due to deficiency of the hormone which is called pituitary dwarfism. Such children are regularly treated with growth hormone which is procured from the pituitary glands of deceased persons. The injections of hGH has been found effective in children.
Now, the hGH is available as recombinant protein. The hGH-coding DNA sequence is linked with the bacterial signal sequence of E. coli. The hGH is secreted into the periplasmic space of bacterial cell by the signal peptides where from the protein is purified.
The hGH lacks terminal methionine, hence it is called met-less hGH. In the USA, the recombinant growth hormone is extensively used for farm animals for increased milk production and leaner meat. But for the safety of food produced such methods may lead ethical problems.
ii. Recombinant Insulin:
Insulin is a peptide hormone secreted by the sets of Langerhans of pancreas. It catabolizes glucose in blood. Insulin is a boon for the diabetics whose normal function for sugar metabolism generally fails. However, diabetes affects a significant percentage of world population.
The diabetics take daily injection of insulin for its control. Previously insulin for injection had been isolated from the pancreas of cows, pigs, etc. It was quite effective for diabetics but some patients developed antibodies against insulin as it was antigen because insulin of human and animals has antigenic differences.
Insulin consists of two short polypeptide chains: A (21 amino acid long) and B (30 amino acid long). These two chains are linked by two sulfide bridges. These two peptides are connected by a third peptide chain-C (35 amino acid long). The precursor of insulin is pre- pro-insulin which is about 109 amino acids long.
The structure of pre-pro-insulin is as below:
NH2 – (peptide) β-chain-(peptide C)-A chain-COOH
For the production of mature insulin molecule, post translational proteolysis of pre hormone is essential. Itakura (1977) chemically synthesized DNA sequences for two chains (A and B) of insulin and separately inserted into the plasmid pBR322 by the side of β-galactosidase gene of E. coli.
The recombinant plasmids were separately transferred into E. coli cells which secreted fused β-galactosidase-A chain and β-galactosidase-B-chain separately. These two chains were isolated by detaching from β-galactosidase through CNBr (cyanogen bromide). It was obtained in pure form to about 10 mg/24 g of healthy and transformed cells.
Production of recombinant insulin by E. coli is given in Fig. 11.11. However, after addition of extra methionine codon to the N-terminus of each gene A and B, detachment of pro-insulin could be possible. The chains A and B are joined together in vitro to constitute a native insulin by sulphonating the two peptides with sodium dis-sulphonate and sodium sulphite.
The human insulin (humulin) is the first therapeutic product by means of recombinant DNA technology by Eli Lilly & Co. (USA).
iii. Recombinant Vaccines:
For the production of recombinant vaccines, genes for desired antigens are identified and cloned into suitable vectors. The vectors are introduced into suitable hosts for expression.
Production of recombinant vaccine through this method has several advantages. However, the major problem associated with them is the low level of immunogenicity (of recombinant proteins). Some of the recombinant vaccines are described in this section.
(i) Vaccine for Hepatitis B virus:
The characteristics of Hepatitis B virus (HBV) (Virus– I), after infection, HBV fails to grow and even in cultured cells it does not grow. This property has been explained to be due to inhibition of its molecular expression and development of vaccines. Plasma of human contained varying amount of antigens.
Three types of viral proteins are recognised to be antigenic:
(i) Viral surface antigen (HBsAg),
(ii) Viral core antigen (HBcAg), and
(iii) The e-antigen (HBeAg).
Recombinant vaccine for HBV was produced by cloning HBsAg gene of the virus in yeast cells. The yeast system has its complex membrane and ability of secreting glycosylate protein. This has made it possible to build an autonomously replicating plasmid containing HBsAg gene near the yeast alcohol dehydrogenase (ADH) I promoter (Fig. 11.12).
The HbsAg gene contains 6 bp long sequence preceding the AUG that synthesizes N-terminal methionine. This is joined to ADH promoter cloned in the yeast vector PMA-56. The recombinant plasmid is inserted into yeast cells. The transformed yeast cells are multiplied in tryptophan-free medium. The transformed cells are selected. The cloned yeast cells are cultured for expression of HBsAg gene.
This inserted gene sequence expresses and produces particles similar to the 22 µm particle of HBV as these particles are produced in serum of HBV patients. The expressed HBsAg particles have similarity in structure and immunogenicity with those isolated from HB V-infected cells of patients. Its high immunogenicity has made it possible to market the recombinant product as vaccine against HBV infection.
Indigenous Hepatitis-B Vaccine:
India’s first genetically engineered vaccine (Guni) against HBV developed by a Hyderabad based laboratory (Shantha Bio-technics Pvt.. Ltd.) was launched on August 18, 1997. India is the fourth country (after the U.S.A., France and Belgium) to develop this highly advanced vaccine. The indigenous yeast-desired HBV-vaccine is one third the cost of the imported vaccine.
This new vaccine had undergone human clinical trials at Nizam’s Institute of Medical Sciences, Hyderabad and K.E.M. Hospital, Mumbai. The clinical trials clearly proved that the seroprotection is about 98%. It was found more effective than the imported vaccine. The Drug Controller General of India has permitted it for commercial manufacture.
(ii) Vaccine for Foot and Mouth Disease (FMD) Virus:
FMD is a very serious disease of animals caused by an RNA virus belonging to the picorna virus group. It consists of ssRNA molecule of 8,000 nucleotides surrounded by a capsid. The capsid is made up of 60 copies of four proteins: VP1, VP2, VP3 and VP4. Only VP1 has a little immunogenic activity.
The gene coding for VP1 has been identified and cloned on pBR322. The recombinant plasmid was introduced in E. coli. About 1,000 molecules of VP1 per bacterial cell were synthesized.
An outline of making vaccine for FMD virus is as below:
Application # 2. Diagnosis of Diseases:
There are many microbial diseases.
These are also diagnosed through molecular techniques:
i. Use of DNA Probe in Diagnosis:
Recently, much work is being done on use of probe in disease diagnosis. DNA probes are single stranded oligonucleotide sequences, complementary to a particular DNA sequence of desired function which are labelled with radionuclide, enzyme or fluorescent molecule. Very specific DNA probes are constructed.
The specificity lies in such a way that the other related species or strains do not contain those sequences. The unrelated specific sequences of known parasite are recognised by using DNA hybridization technique. Then a DNA sequence, not found in any species is identified, cleaved by restriction enzyme and inserted into a cloning vector (plasmid). The bacterial cells are transformed by the recombinant vector.
The transformants are multiplied. Finally, the foreign DNA fragment is retrieved from the host cells. The DNA sequences of the parasite, thus obtained are labelled with radioisotope and used as a probe. The probe can also be chemically synthesized.
Following are the steps for diagnosis of a particular disease:
(a) Isolate the parasite from the infected tissue of the patient. Extract the DNA from the parasite and purify it.
(b) Break the DNA by using restriction enzyme.
(c) Electrophorise the DNA solution containing DNA fragment of different lengths by using agarose gel to get a smear of DNA.
(d) Attach DNA to more firmer support by Southern blotting technique. Thus, the filter paper carries the exact replica of the DNA adhered to it.
(e) Hybridize the immobilized DNA on filter paper by incubating it with radiolabelled probe. Probe DNA complementary to certain DNA sequence of parasite DNA sticks to it and forms the hybrid.
(f) Wash filter paper to remove unbound probe; pass filter paper through X-ray film. The hybridized specific sequences appear as dark bands. Thus a parasitic disease is diagnosed positive. If dark bands do not appear the parasite is reported to be absent.
This diagnosis system is very effective for viruses, bacteria and protozoa. Tuberculosis caused by Mycobacterium tuberculosis is diagnosed by this method. Gen-probe Inc. California has marketed a complete testing system of tuberculosis. Similar effort has also been made for diagnosis of leprosy, Kala Azar, malaria, etc.
ii. Use of PCR in Disease Diagnosis:
The PCR can detect even a single organism that has infected the humans which is present even in low number. From the suspected patients, sample of sera is taken and the region from DNA samples.
The PCR as diagnostic tool may be used in some diseases as given in Table 11.5:
Table 11.5 : Use of the PCR as diagnostic tool.
Application # 3. Insect Pest Control:
Mosquitoes are a menace and vector for transmission of several human diseases such as malaria, filaria, encephalitis, dengue, plague, etc. The increase in their population is augmented by environmental pollution particularly polluted water outlets from houses, huts, water in coolers of any site where stagnant water is found. In addition, they cause serious diseases to crop plants with great loss in yield.
To kill these insect pests, insecticides are being produced worldwide that in turn pose environmental pollution leading to health hazards in animals and humans. Who can forget en masse killing of more than 8,000 people, many animals, birds and plants, when methylisocyanate (MIC) gas leaked out in night of 2/3 December, 1984 from the underground reservoir of Union Carbide Factory of Bhopal (M.P.) Many gas affected people are suffering even today.
Small incidences of gas leakage and human death are many and occurring day-by-day. In this grim scenario, total ban on insecticide chemicals, and formulation and production of microbial bio pesticides are urgently required.
In recent years, attempts have been made to produce microbial insecticides i.e. bio-pesticides Bio-pesticides are the preparations of chemicals/microbial cells basically from bacteria, fungi and viruses for killing of insect pests. The examples are baculoviruses, iridovims, entomopox virus. Bacillus thuringiensis, B.popilliae, B. sphaericus, B. moritai, and species of Aspergillus, Coelomomyces, Entomophthora, Fusarium, Paecilomyces.
i. Bacterial Bio-Pesticides (Bioinsecticides):
Bacillus thuringiensis is a wide spread spore forming bacterium which is found in soil, litter and dead insects. It produces toxins viz., α-, β- and δ-exotoxins, and 5-endotoxin which can be obtained in crystalline form. The β-endotoxin is composed of a glycoprotein subunit.
These toxins have insecticidal properties. B. thuringiensis has been found as a strong antagonist against larvae of lapidoptera. After ingestion of spores larvae are damaged as the rod shaped bacterial cell secretes at the opposite end a single large crystal in the cell. This toxic crystal is proteinaceous in nature. It gets dissolved in alkaUne juice of caterpillar’s digestive cavity.
This toxin crystal is secreted by a plasmid present in bacterial cell. The plasmid has been transferred in cells of B.subtilis and E. coli where it successfully expressed the toxin.
Microbial bio-pesticides have been produced by many companies by using genetically engineered microbial cells of preparation of B. thuringiensis. In the U.S.A. commercial formulation of β-endotoxin from B.thuringiensis (Bt) has been banned. In other countries like France, formulations of Bt in the form of wettable powder and water suspension have been recommended for use.
ii. Production of Transgenic Plants:
Now, scientists have started producing transgenic plants instead of producing Bt preparations. Transgenic plants are those plants in which a gene of foreign origin i.e. other organisms has been introduced.
Recently, the scientists at the US Multinational Monsanto Co., Australia, Cotton Seed distributors and the Council of Scientific and Industrial Research Organisation (CSIRO) of Australia, have produced genetically engineered species of cotton known as Killer cotton.
It kills the predators especially the bullworm (Heliothis sp.). The leaves of transgenic plants, produced through cell culture, secrete lethal toxins. When the bullworm eats upon cotton leaves, toxin is taken up by them. Toxin is activated in their guts and results in death of bullworm. Toxin does not harm the spiders, humans and other mammals.
B. thuringiensis (Bt) β-endotoxin genes also called cry genes have been cloned in E. coli. Such transformed E. coli produced larvicidal proteins in large amount which accumulate in cells to form large crystals. Similarly, transgenic tomato plants have been produced through tissue culture/cell culture. Introduction of Bt gene in a plant is shown in Fig. 11.14.
Today bio-pesticide sales have attained 3% of the global chemical pesticide market and expected to reach 10% in ten years time. The bacteria based bio-pesticides have extremely popular in view of their efficiency and from the knowledge of genetic basis of their inseticidal activity. B. thuringiensis var. israelensis and B. sphaericus emerged as the two major microbes for mosquitoes and black flies.
When these ingest the bacilli which dissolve in alkaline pH of the gut, release the endotoxin contained in the inclusion bodies. The multiple toxic peptides bind specifically to the larval gut cells and dissolve the membrane by causing leakage of Ca++ resulting in paralysis of gut and death of larvae.
The bio-pesticides are preferred as biological alternative to environmentally toxic chemicals.
The bio-pesticides offer many advantages such as:
(i) Their nontoxic effects on non-target pathogen/microorganisms/pests (unlike chemicals),
(ii) They are biodegradable, and
(iii) Development of resistance by the insects to a low extent against the bi-pyramidal crystal shaped proteins.
The target pests of B. thuringiensis are given in Table 11.6:
Table 11.6 : Some important pests of Bacillus thuringinesis.
Additional types of transgenic plants have been produced in the last few years by using modern molecular biology. A synthetic toxin gene was created in which natural A-T-rich gene regions were replaced with G-C content sequences. This was done to minimize degradation of mRNA in plant.
After successful insertion of modified artificial gene into plant, over 0.2% of transgenic plants have been produced. Attempts are being made to commercialize the crops. The research has been carried out with soya bean, oilseed rape, sugar beet and sunflower, and also in monocots such as maize, rice and wheat.
iii. Viral Pesticides:
A number of viruses have been discovered which belong to the groups Baculovirases and Cytoplasmic Polypeptides Viruses (CPV). Use of viral preparations in disease control is done in the field of agriculture, horticulture and forestry.
They are free from pollution, toxicity of any hazardous chemicals related to plant or animal health. These viruses are specific and do not damage the useful pollinator insects yielding useful products, warm blooded animals and even man. These enter in digestive tract of insect pest and kill them.
Nuclear polyhedrosis viruses (NPV’s) have been used for preparation of potential pesticides. Heliothis sp. is a cosmopolitan insect that attacks 30 plant species. It is controlled by application of NPV’s of Baculovirus heliothis: In 1975, Environmental Protection Agency (U.S.A.) registered B. heliothis preparations.
At present, it is marketed under the name Elcar (Sandoz Inc.), Biotrol VHZ (Nutrihte products Int) and Virom/H (International Minerals Chemical Corp.). In 1974, Japan produced a commercial preparation of CPV under the name Matsukemin for the control of a pine caterpillar (Dendrolimus spectabilis). This insect was controlled fully after application of 1011 polyhedral inclusion bodies per hectare.
Application # 4. Use of Genetically Engineered Microorganisms for Control of Pollution:
In 1979, India-born American scientist, Anand Mohan Chakrabarty and his coworkers isolated the microbial cultures that utilized a number of toxic chemicals such as salicylate, 2.4-D, 3 chloro-benzene, ethylene, biphenyls, 1,2,4-tri-methylbenzene, 2-4-5-trichlorophenoxyacetic acid, etc.
Genes responsible for degradation of environmental pollutants, for example toluene, chloro-benzene, acids and other pesticides and toxic wastes have been isolated. For the degradation of every toxic compound one gene is required. However, it is not like that one plasmid can degrade all the toxic compounds of different groups.
A.M.Chakrabarty categorized the plasmids into four groups as below:
(i) OCT plasmid that degrades octane, hexane and decane.
(ii) XYL plasmid that degrades xylene and toluene.
(iii) CAM plasmid that decomposes camphor and,
(iv) NAH plasmid which degrades naphthalene.
Chakrabarty produced a new genetically engineered microbial strain called superbug (oil eating bug) after introducing all the four plasmids from different strains into one single cell of Pseudomonasputida. This superbug is such that degrades all the four types of substrates for which four separate plasmids were required (Fig. 11.15).
In addition, attempts have been made to render plants resistant to environmental stresses, and have shown much success. The genes for detoxification of glyophosphate herbicides were isolated from Salmonella. These genes were successfully cloned and interred into tobacco cells through Ti-plasmid of A. tumifaciens. The plantlets regenerated from transformed cells showed resistance against the herbicide.
Thus, herbicide-resistant tobacco and com plants have been developed. It is now known that many plants suffer from stress when treated with herbicides. Therefore, such type of plants seems to have much importance. The herbicide resistant plants will not suffer from stress when a herbicide is used to control weeds in a crop field.