Category: Biology

Learninsta presents the core concepts of Biology with high-quality research papers and topical review articles.

Major Abiotic Components or Factors

The abiotic factors include the chemical and physical factors which influence or affect organisms and their functioning in their environment. The common abiotic factors are:

Temperature

Temperature or degree of hotness and coldness is an essential and variable factor in any environment. It influences all forms of life by affecting many vital activities of organisms like metabolism, behaviour, reproduction, development and even death in the Biosphere. The minimum and maximum temperature of an environment regulates the survival of a cell.

The metabolism of organisms is regulated by enzymes which are temperature sensitive. In many organisms, determination of sex and sex ratio, maturation of gonads, gametogenesis and reproduction is influenced by temperature. In certain environments, the size and colouration of animals are inflenced by temperature. Birds and mammals attain greater body size in colder regions than warmer regions (Bergmann’s rule).

Warm blooded animals, living in colder climates, tend to have shorter limbs, ears and their appendages when compared to the members of the same species in warmer climates (Allen’s rule). In some aquatic environments, an inverse relationship between water temperature and fish meristic characters is observed lower the temperature, more the vertebrae (Jordon’s rule).

Temperature influences the distribution of organisms. The tropics have higher diversity and density of populations, when compared to temperate and polar regions.

Adaptations to temperature

Adaptation to temperature is essential for the survival of the species/organisms. Organisms which can survive a wide range of temperature are referred to as Eurytherms (cat, dog, tiger, human). Eurythermy can be an evolutionary advantage: adaptations to cold temperatures (cold-eurythemy) are seen as essential for the survival of species during ice ages.

In addition, the ability to survive in a wide range of temperatures increases a species ability to inhabit other areas, an advantage for natural selection. Eurythermy is an aspect of thermoregulation in organisms. These organisms which can tolerate only a narrow range of temperature are Stenotherms (Fish, Frogs, Lizards and Snakes).

Over the course of time, by evolution, animals of different ecological habitats have developed different variations and adaptations to temperature changes. It enabled them to survive in different habitats and develop niches. In case of extreme temperatures, organisms have adapted by forming heat resistant spores, cysts (Entamoeba), antifreeze proteins (Arctic fihes).

Hibernation (winter sleep) and Aestivation (Summer sleep) are useful adaptations to overcome extreme winters and summers. In certain conditions, migration is an appropriate adaptation to overcome extreme temperatures and resultant water and food scarcity. (Fig 10.2).

Light

It is an important and essential abiotic factor. Ecologically, the quality (wavelength or colour), the intensity (actual energy in gram calories) and duration (length of day) of light are considered signifiant for organisms.

Light influences growth, pigmentation, migration and reproduction. The intensity and frequency of light influences metabolic activity, induce gene mutations (UV, X – rays). Light is essential for vision. This is proved by the poorly developed or absence of eyes in cave dwelling organisms. Diapause is also influenced by light in animals. Gonads of birds become more active with increasing light in summer. Light inflences the locomotion and movement of lower animals.

WATER

Life on earth began in the seas and water is essential for the survival of all forms of life. About three-fourth of the earth’s surface is covered with water (hydrosphere). Water is found in three states: gaseous, liquid, and solid.

There are two types of water on Earth. They are the Fresh water (rivers, lakes, ponds) and the Salt water (seas and oceans). Based on the dissolved salts, water can be hard water (sulphates/nitrates of Calcium/Magnesium) or soft water. If hardness can be removed by boiling, it is temporary hard water, and if boiling does not help, it is permanent hard water.

Essential properties of water

• Water is one of the main agents in Pedogenesis (soil formation).
• It is the medium for several different ecosystems.
• It is present as moisture in the atmosphere and the outer layers of the lithosphere and is uneven in distribution on the earth.
• Water is heavier than air and imparts greater buoyancy to the aquatic medium. This enables organism to flat at variable levels.
• Water has high heat capacity and latent heat, due to which it can withhold large amounts of heat. This, oceans and lakes tend to maintain a relatively constant temperature, and the biosphere is relatively thermostable.
• Water is physically unique because it is less dense as a solid (ice) than as a liquid.
• When water freezes (0oC), it contracts. The maximum density of liquid water occurs at 4oC. Below that, it expands markedly.
• This enables ice to flat on the top of water bodies. Hence, only the surface of water bodies will freeze, while below the surface, water will be in liquid form, sustaining life (Fig. 10.3).
• Water is considered as the Universal solvent. It is the main medium by which chemical constituents are transported from abiotic components to the living components of an ecosystem.
• Water has high surface tension. This allows pollen, dust, and even water striders to remain at the surface of a water body even though they are denser than the water.

Soil

It is a mixture of organic matter, minerals, gases, liquids and organisms that together support life. The soil zone is known as Pedosphere. Soil is formed from rocks which are the parent materials of soil, by weathering and is called embryonic soil (Pedogenesis).

It has four major functions:-

• Medium for plant growth
• Means for water storage and purification
• Modifier of earth’s atmosphere
• Habitat for many organisms, which in turn modify the soil.

Soil is formed of many horizontal layers called as Soil Profile.

Properties of Soil

1. Texture of soil:

The texture of soil is determined by the size of the soil particles. The types of soil include sand, silt and clay on the basis of their size differences.

2. Porosity:

The space present between soil particles in a given volume of soil are called pore spaces. The percentage of soil volume occupied by pore space or by the interstitial spaces is called porosity of the soil.

3. Permeability of soil:

The characteristic of soil that determines the movement of water through pore spaces is known as soil permeability. Soil permeability is directly dependent on the pore size. Water holding capacity of the soil is inversely dependent on soil porosity.

4. Soil Temperature:

Soil gets its heat energy from solar radiation, decomposing organic matter, and heat from the interior of earth. Soil temperature effects the germination of seeds, growth of roots and biological activity of soil-inhabiting micro-and macroorganisms.

5. Soil water:

In soil, water is not only important as a solvent and transporting agent, but also maintains soil texture, arrangement and compactness of soil particles, making soil habitable for plants and animals.

Wind

Wind is the natural movement of air of any velocity from a particular direction. The two main causes are differential heating between the equator and the poles and the rotation of the planet (Coriolis effect). Wind helps to transport pollen grains, seeds, and even flight of birds. While it is the source of wind energy, it also causes erosion. Wind speed is measured with an Anemometer.

Humidity

Moisture in the form of invisible vapor in the atmosphere is called humidity. which is generally expressed in terms of absolute humidity, relative humidity or specific humidity. Absolute humidity is the total mass of water vapour present in a given volume or mass of air. It does not take temperature into consideration.

Relative humidity is the amount of water vapour present in air and is expressed as a percentage of the amount needed for saturation at the same temperature Relative humidity is expressed as a percentage; a high percentage means that the air-water mixture is more humid at a given temperature. Humidity is measured with a Hygrometer.

Altitude

This factor is mainly the elevation or gradient and it affects temperature and precipitation in an ecosystem or biome. As altitude increases, temperature and density of oxygen decreases.

Higher altitudes usually receive snow instead of rain because of low temperature. Animals are known to modify their response to environmental changes (stress) in reasonably short time spans. This is known as Acclimatization. This is observed when people who have moved from the plains to higher altitudes show enhanced RBC count within a few days of settling in their new habitat. This helps them cope with
lower atmospheric oxygen and higher oxygen demand.

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Habitat Definition and Examples

Habitat refers to the place where an organism or a community of organisms live, including all biotic and abiotic factors or conditions of the surrounding environment.

The collection of all the habitat areas of a species constitutes its geographical range. Organisms in a habitat interact with each other and can be part of trophic levels to form food chains and food webs.

Examples: In a xerophytic habitat, the camel is able to use water efficiently and effectively for evaporative cooling through their skin and respiratory system. They excrete highly concentrated urine and can also withstand dehydration upto 25% of the body weight. The hoofs and hump are also suitable adaptations for survival in this dry sandy environment.

In an aquatic media, maintaining homeostasis and osmotic balance is a challenge. So, marine animals have appropriate adaptations to prevent cell shrinkage. While freshwater organisms have suitable adaptations to withstand bursting of their cells.

Apart from this, organisms such as fish have a wide range of adaptations like fins (locomotion), streamlined body (aerodynamic), lateral line system (sensory), gills (respiration), air sacs (flatation) and kidneys (excretion).

Niche (or) Ecological Niche

As every organism has its unique habitat, so also it has an ecological niche which includes the physical space occupied by an organism and its functional role in the community. The ecological niche of an organism not only depends on where it lives but also includes the sum total of its environmental requirements.

Charles Elton (1927) was the first to use the term ‘niche’ as the functional status of an organism in its community. Groups of species with comparable role and niche dimensions within a community are termed ‘guilds’. Species that occupy the same niche in different geographical regions, are termed ‘ecological equivalents’.

Many animals share the same general habitat. But their niches are well defined. The life style of an individual population in the habitat is known as its niche. For example, crickets and grasshoppers are closely related insects that live in the same habitat, yet they occupy different ecological niches. The grasshopper is very active during daylight. It can usually be found on a plant, feeding on the plant parts.

Although the cricket lives in the same field, it is quite different. During the day, the cricket hides under leaves or plant debris and is usually inactive. It is active at night time (nocturnal). The cricket and the grasshopper do not interfere with each other’s activities in the same habitat. Thus, niche of an organism can be defined as the total position and function of an individual in its environment.

In a pond ecosystem, where Catla, Rohu and Mrigal are present, the ecological niche of the Catla is a surface feeder, Rohu is a column feeder and Mrigal is a bottom feeder. Their mouths are designed to suit their niche and hence have different positions and functions in their habitat (Fig.10.1).

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Organism and Its Environment

Every living organism has its own specific surrounding, medium or environment with which it continuously interacts and develops suitable adaptations for survival there. Environment is a collective term which includes the different conditions in which an organism lives or is present. The common and infulencing factors in any environment are light, temperature, pressure, water, salinity. These are collectively referred to as Abiotic components.

Environments are variable and dynamic, in which temperature changes and light changes are diurnal and seasonal. These inflence the organisms inhabiting them. An organism’s growth, distribution, number, behaviour and reproduction is determined by the different factors present in the environment.

Ecology is the study of how living organisms interact with each other and with their environment. Abiotic factors are the parts of the environment that have never been alive, while biotic factors are the parts of the environment that are alive, or were alive and then died.

Ecology is the study of the interaction of organisms in an area with the surrounding environment. This interaction constitutes an overall adaptation of the organisms to their environment which also includes the continuity of species.

Ecology is the study of organisms and how they interact with the environment around them. An ecologist studies the relationship between living things and their habitats.

Environment is the living and non living things surrounding the living organism. An organism’s habitat refers to an ecological or environmental area inhabited by particular species of plants, animals, fungi, etc. It refers to an organism’s natural environment. Life has to adapt to specific environmental conditions.

Ecology is the study of how organisms interact with one another and with their physical environment. The distribution and abundance of organisms on Earth is shaped by both biotic, living-organism-related, and abiotic, nonliving or physical, factors.

7 Ecological Principles

The seven principles are:-

1. Maintain diversity and Redundancy
2. Manage connectivity
3. Manage slow variables and feedbacks
4. Foster complex adaptive systems thinking
5. Encourage learning
6. Broaden participation and
7. Promote polycentric governance systems.

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Biotechnology of Ethical Issues

Biotechnology has given to the society cheap drugs, better friuts and vegetables, pest resistant crops, indigenious cure to diseases and lot of controversy. This is mainly because the major part of the modern biotechnology deals with genetic manipulations. People fear that these genetic manipulations may lead to unknown consequences.

The major apprehension of recombinant DNA technology is that unique microorganisms either inadvertently or deliberately for the purpose of war may be developed that could cause epidemics or environmental catastrophies. Although many are concerned about the possible risk of genetic engineering, the risks are in fact slight and the potential benefits are substantial.

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Biotechnology Of Animal Cloning

Cloning is the process of producing genetically identical individuals of an organism either naturally or artificially. In nature many organisms produce clones through asexual reproduction.

Cloning in biotechnology refers to the process of creating copies of organisms or copies of cells or DNA fragments (molecular cloning). Dolly was the first mammal (Sheep) clone developed by Ian Wilmut and Campbell in 1997. Dolly, the transgenic clone was developed by the nuclear transfer technique and the phenomenon of totipotency. Totipotency refers to the potential of a cell to develop different cells, tissues, organs and finally an organism.

The mammary gland udder cells (somatic cells) from a donor sheep (ewe) were isolated and subjected to starvation for 5 days. The udder cells could not undergo normal growth cycle, entered a dormant stage and became totipotent. An ovum (egg cell) was taken from another sheep (ewe) and its nucleus was removed to form an enucleated ovum.

The dormant mammary gland cell/udder cell and the enucleated ovum were fused. The outer membrane of the mammary cell was ruptured allowing the ovum to envelope the nucleus. The fused cell was implanted into another ewe which served as a surrogate mother. Five months later dolly was born. Dolly was the first animal to be cloned from a diffrentiated somatic cell taken from an adult animal without the process of fertilization (Fig. 9.8).

Advantages and Disadvantages Of Cloning Animals

• Offers benefis for clinical trials and medical research. It can help in the production of proteins and drugs in the field of medicine.
• Aids stem cell research.
• Animal cloning could help to save endangered species.
• Animal and human activists see it as a threat to biodiversity saying that this alters evolution which will have an impact on populations and the ecosystem.
• The process is tedious and very expensive.
• It can cause animals to suffer.
• Reports show that animal surrogates were manifesting adverse outcomes and cloned animals were affected with disease and have high mortality rate.
• It might compromise human health through consumption of cloned animal meat.
• Cloned animals age faster than normal animals and are less healthy than the parent organism as discovered in Dolly.
• Cloning can lead to occurrence of genetic disorders in animals.
• More than 90% of cloning attempts fail to produce a viable offspring.

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Biological Products And Their Uses

A biological product is a substance derived from a living organism and used for the prevention or treatment of disease. These products include antitoxins, bacterial and viral vaccines, blood products and hormone extracts. These products may be produced through biotechnology in a living system, such as a microorganism, plant cell or animal cell, and are often more difficult to characterize than small molecule drugs.

Though recombinant DNA technology it is possible to produce these biological products on demand. There are many types of biological products approved for use – they are, therapeutic proteins, monoclonal antibodies and vaccines. Health care and pharmaceutical industries have been revolutionised by biotechnological proteins.

Hormones and antibodies are produced commercially, primarily for the medical industry. Recombinant hormones like Insulin, Human growth hormone, Recombinant vaccines and recombinant proteins like human alpha lactalbumin are available today.

Animals are used as bioreactors to produce desirable proteins. Antibodies are substances that react against the disease causing antigens and these can be produced using transgenic animals as bioreactors. Monoclonal antibodies, which are used to treat cancer, heart disease and transplant rejection are produced by this technology. Natural protein adhesives are non toxic, biodegradable and rarely trigger an immune response, hence could be used to reattach tendons and tissues, fill cavities in teeth, and repair broken bones.

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Transgenic Animals

In early days selective breeding methods were carried out to improve the genetic characteristics of live stock and other domestic animals. With the advent of modern biotechnology it is possible to carry out manipulations at the genetic level to get the desired traits in animals. Transgenesis is the process of introduction of extra (foreign/exogenous) DNA into the genome of the animals to create and maintain stable heritable characters.

The foreign DNA that is introduced is called the transgene and the animals that are produced by DNA manipulations are called transgenic animals or the genetically engineered or genetically modified organisms.

The various steps involved in the production of transgenic organisms are

• Identification and separation of desired gene.
• Selection of a vector (generally a virus) or direct transmission.
• Combining the desired gene with the vector.
• Introduction of transferred vector into cells, tissues, embryo or mature individual.
• Demonstration of integration and expression of foreign gene in transgenic tissue or animals.
  Transgenic animals such as mice, rat, rabbit, pig, cow, goat, sheep and fish have been produced (Fig. 9.7).

Uses Of Transgenesis

• Transgenesis is a powerful tool to study gene expression and developmental processes in higher organisms.
• Transgenesis helps in the improvement of genetic characters in animals.
• Transgenic animals serve as good models for understanding human diseases which help in the investigation of new treatments for diseases.
• Transgenic models exist for many human diseases such as cancer, Alzheimer’s, cystic fibrosis, rheumatoid arthritis and sickle cell anemia.
• Transgenic animals are used to produce proteins which are important for medical and pharmaceutical applications.
• Transgenic mice are used for testing the safety of vaccines.
• Transgenic animals are used for testing toxicity in animals that carry genes which make them sensitive to toxic substances than non-transgenic animals exposed to toxic substances and their effects are studied.
• Transgenesis is important for improving the quality and quantity of milk, meat, eggs and wool production in addition to testing drug resistance.

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Molecular Diagnostics

Early diagnosis of infectious diseases or inherent genetic defects is essential for appropriate treatment. Early detection of the disease is not possible using conventional diagnostic methods like microscopic examinations, serum analysis and urine analysis. These laboratory techniques are indirect and not always specific. Scientists are continuously searching for specific, sensitive and simple diagnostic techniques for diagnosis of diseases.

Recombinant DNA technology, Polymerase Chain Reactions (PCR) and Enzyme Linked Immunosorbent Assay (ELISA) are some of the techniques that are reliable and help in early diagnosis. Presence of pathogens like virus, bacteria, etc., is detected only when the pathogen produces symptoms in the patient. By the time the symptoms appear concentration of pathogen becomes very high in the body. However very low concentration of a bacteria or a virus, even when the symptoms of the disease does not appear, can be detected by amplification of their nucleic acid.

ELISA [Enzyme Linked Immunosorbent Assay]

ELISA is a biochemical procedure discovered by Eva Engvall and Peter Perlmanin (1971) to detect the presence of specific antibodies or antigens in a sample of serum, urine, etc., It is a very important diagnostic tool to determine if a person is HIV positive or negative.

ELISA is a tool for determining serum antibody concentrations (such as the antibodies produced in a person infected by pathogens such as HIV) and also for detecting the presence of specific antigens and hormones such as human chorionic gonadotropins.

During diagnosis the sample suspected to contain the antigen is immobilized on the surface of an ELISA plate (Fig. 9.5). The antibody specific to this antigen is added and allowed to react with the immobilized antigen. The anti-antibody is linked to an appropriate enzyme like peroxidase.

The unreacted anti-antbody is washed away and the substrate of the enzyme (hydrogen peroxidase) is added with certain reagents such as 4-chloronaphthol. The activity of the enzyme yields a coloured product indicating the presence of the antigen. The intensity of the colour is directly proportional to the amount of the antigen. ELISA is highly sensitive and can detect antigens in the range of a nanogram.

There are four kinds of ELISA namely, Direct ELISA, Indirect ELISA, sandwich ELISA and competitive ELISA. It is a highly sensitive and specific method used for diagnosis. ELISA possesses the added advantages of not requiring radioisotopes or a radiation counting apparatus. PCR (Polymerase Chain Reaction) The polymerase chain reaction (PCR) is an invitro amplification technique used for synthesising multiple identical copies (billions) of DNA of interest. The technique was developed by Kary Mullis (Nobel laureate, 1993) in the year 1983.

Denaturation, renaturation or primer annealing and synthesis or primer extension, are the three steps involved in PCR (Fig. 9.6). The double stranded DNA of interest is denatured to separate into two individual strands by high temperature .

This is called denaturation. Each strand is allowed to hybridize with a primer (renaturation or primer annealing). The primer template is used to synthesize DNA by using Taq – DNA polymerase.

During denaturation the reaction mixture is heated to 95° C for a short time to denature the target DNA into single strands that will act as a template for DNA synthesis. Annealing is done by rapid cooling of the mixture, allowing the primers to bind to the sequences on each of the two strands flanking the target DNA. During primer extension or synthesis the temperature of the mixture is increased to 75° C for a sufficient
period of time to allow Taq DNA polymerase to extend each primer by copying the single stranded template.

At the end of incubation both single template strands will be made partially double stranded. The new strand of each double stranded DNA extends to a variable distance downstream. These steps are repeated again and again to generate multiple forms of the desired DNA. This process is also called DNA amplification.

The PCR technique can also be used for amplifications of RNA in which case it is referred to as reverse transcription PCR (RT-PCR). In this process the RNA molecules (mRNA) must be converted to complementary DNA by the enzyme reverse transcriptase. The CDNA then serves as the template for PCR.

PCR In Clinical Diagnosis

The specificity and sensitivity of PCR is useful for the diagnosis of inherited disorders (genetic diseases), viral diseases, bacterial diseases, etc., The diagnosis and treatment of a particular disease often requires identifying a particular pathogen. Traditional methods of identification involve culturing these organisms from clinical specimens and performing metabolic and other tests to identify them.

The concept behind PCR based diagnosis of infectious diseases is simple – if the pathogen is present in a clinical specimen its DNA will be present.

Its DNA has unique sequences that can be detected by PCR, often using the clinical specimen (for example, blood, stool, spinal fluid, or sputum) in the PCR mixture. PCR is also employed in the prenatal diagnosis of inherited diseases by using chorionic villi samples or cells from amniocentesis. Diseases like sickle cell anemia, β-thalassemia and phenylketonuria can be detected by PCR in these samples.

CDNA from PCR is a valuable tool for diagnosis and monitoring retroviral infections e.g., Tuberculosis by Mycobacterium tuberculosis. Several virally induced cancers, like cervical cancer caused by Papilloma virus can be detected by PCR. Sex of human beings and live stocks, embryos fertilized invitro can be determined by PCR by using primers and DNA probes specific for sex chromosomes. PCR technique is also used to detect sex-linked disorders in fertilized embryos.

Applications of PCR

The differences in the genomes of two different organisms can be studied by PCR. PCR is very important in the study of evolutions, more specifically phylogenetics. As a technique which can amplify even minute quantities of DNA from any source, like hair, mummified tissues, bones or any fossilized materials.

PCR technique can also be used in the field of forensic medicine. A single molecule of DNA from blood stains, hair, semen of an individual is adequate for amplification by PCR. The amplified DNA is used to develop DNA fingerprint which is used as an important tool in forensic science. Thus, PCR is very useful for identification of criminals. PCR is also used in amplification of specific DNA segment to be used in gene therapy.

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Stem Cell Therapy

Stem cells are undifferentiated cells found in most of the multi cellular animals. These cells maintain their undifferentiated state even after undergoing numerous mitotic divisions.

Stem cell research has the potential to revolutionize the future of medicine with the ability to regenerate damaged and diseased organs. Stem cells are capable of self renewal and exhibit ‘cellular potency’. Stem cells can differentiate into all types of cells that are derived from any of the three germ layers ectoderm, endoderm and mesoderm.

In mammals there are two main types of stem cells – embryonic stem cells (ES cells) and adult stem cells. ES cells are pluripotent and can produce the three primary germ layers ectoderm, mesoderm and endoderm. Embryonic stem cells are multipotent stem cells that can differentiate into a number of types of cells (Fig. 9.4). ES cells are isolated from the epiblast tissue of the inner cell mass of a blastocyst.

When stimulated ES can develop into more than 200 cells types of the adult body. ES cells are immortal i.e., they can proliferate in a sterile culture medium and maintain their undifferentiated state. Adult stem cells are found in various tissues of children as well as adults. An adult stem cell or somatic stem cell can divide and create another cell similar to it. Most of the adult stem cells are multipotent and can act as a repair system of the body, replenishing adult tissues. The red bone marrow is a rich source of adult stem cells.

The most important and potential application of human stem cells is the generation of cells and tissues that could be used for cell based therapies. Human stem cells could be used to test new drugs.

Stem Cell Banks

Stem cell banking is the extraction, processing and storage of stem cells, so that they may be used for treatment in the future, when required. Amniotic cell bank is a facility that stores stem cells derived from amniotic fluid for future use.

Stem cells are stored in banks specifically for use by the individual from whom such cells have been collected and the banking costs are paid. Cord Blood Banking is the extraction of stem cells from the umbilical cord during childbirth. While the umbilical cord and cord blood are the most popular sources of stem cells, the placenta, amniotic sac and amniotic fluid are also rich sources in terms of both quantity and quality.

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Biotechnology Of Gene Therapy

If a person is born with a hereditary disease, can a corrective therapy be given for such disease? Yes, this can be done by a process known as gene therapy. This process involves the transfer of a normal gene into a person’s cells that carries one or more mutant alleles. Expression of normal gene in the person results in a functional gene product whose action produces a normal phenotype.

Delivery of the normal gene is accomplished by using a vector. The main thrust of gene therapy has been directed at correcting single gene mutations as in cystic firosis and haemophilia. At present most genetic diseases have no effective treatment and so gene therapy could offer hope for many people.

There are two strategies involved in gene therapy namely; Gene augmentation therapy which involves insertion of DNA into the genome to replace the missing gene product and Gene inhibition therapy which involves insertion of the anti sense gene which inhibits the expression of the dominant gene (Fig. 9.3). The two approaches to achieve gene therapy are somatic cell and germ line gene therapy.

Somatic cell therapy involves the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently whereas Germline gene therapy involves the introduction of DNA into germ cells which is passed on to the successive generations. Gene therapy involves isolation of a specific gene and making its copies and inserting them into target cells to make the desired proteins.

It is absolutely essential for gene therapists to ensure that the gene is harmless to the patient and it is appropriately expressed and that they body’s immune system does not react to the foreign proteins produced by the new genes.

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Applications In Medicine

Recombinant Human Insulin

The Human insulin is synthesized by the β cells of Islets of Langerhans in the pancreas. It is formed of 51 aminoacids which are arranged in two polypeptide chains, A and B. The polypeptide chain A has 21 amino acids while the polypeptide chain B has 30 amino acids. Both A and B chains are attached together by disulphide bonds.

Insulin controls the levels of glucose in blood. It facilitates the cellular uptake and utilization of glucose for the release of energy. Deficiency of insulin leads to diabetes mellitus which is characterized by increased blood glucose concentration and a complex of symptoms which may lead to death, if untreated. A continuous program of insulin dependence is required to treat this deficiency.

In the early years, insulin isolated and purified from the pancreas of pigs and cows was used to treat diabetic patients. Due to minor differences in the structure of the animal insulin as compared to human insulin, it resulted in the occurrence of allergic reactions in some diabetic patients.

Production of insulin by recombinant DNA technology started in the late 1970s. This technique involved the insertion of human insulin gene on the plasmids of E.coli. The polypeptide chains are synthesized as a precursor called pre-pro insulin, which contains A and B segments linked by a third chain (C) and preceded by a leader sequence.

The leader sequence is removed after translation and the C chain is excised, leaving the A and B polypeptide chains (Fig. 9.1). Insulin was the first ever pharmaceutical product of recombinant DNA technology administered to humans. The approval to use recombinant insulin for diabetes mellitus was given in 1982. In 1986 human insulin was marketed under the trade name Humulin.

Interferons

Interferons are proteinaceous, antiviral, species specific substances produced by mammalian cells when infected with viruses. Interferons were discovered by Alick Isaacs and Jean Lindemann in 1957. Based on the structure of interferons they are classifid as α, β and γ interferons.

They stimulate the cellular DNA to produce antiviral enzymes which inhibit viral replication and protect the cells. Interferons could be isolated from blood, but the amount of blood required for isolation of interferons is enormous and not practical. To overcome this issue interferons could be produced by rDNA technology.

The yeast Saccharomyces cerevisiae is more suitable for production of recombinant interferons than E.coli, since E.coli does not possess the machinery for glycosylation of proteins. Interferons are used for the treatment of various diseases like cancer, AIDS, multiple sclerosis, hepatitis C and herpes zoster. In spite of the therapeutic applications interferons are not within the reach of the common man due to high cost for its production.

Recombinant Vaccines

Recombinant DNA technology has been used to produce new generation vaccines. The limitations of traditional vaccine production could be overcome by this approach. The recombinant vaccines are generally of uniform quality and produce less side effects as compared to the vaccines produced by conventional methods. Different types of recombinant vaccines include subunit recombinant vaccines, attenuated recombinant vaccines and DNA vaccines.

Subunit recombinant vaccines

Vaccines that use components of a pathogenic organism rather than the whole organism are called subunit vaccines; recombinant DNA technology is very suited for developing new subunit vaccines. It includes components like proteins, peptides and DNAs of pathogenic organisms. The advantages of these vaccines include their purity in preparation, stability and safe use.

Attenuated recombinant vaccines

This includes genetically modified pathogenic organisms (bacteria or viruses) that are made nonpathogenic and are used as vaccines. It is now possible to genetically engineer the organisms (bacteria or viruses) and use them as live vaccines and such vaccines are referred to as attenuated recombinant vaccines.

DNA Vaccines

Genetic immunisation by using DNA vaccines is a novel approach that came into being in 1990. The immune response of the body is stimulated by a DNA molecule. A DNA vaccine consists of a gene encoding an antigenic protein, inserted onto a plasmid, and then incorporated into the cells in a target animal. DNA instructs the cells to make antigenic molecules which are displayed on its surfaces.

This would evoke an antibody response to the free floating antigen secreted by the cells. The DNA vaccine cannot cause the disease as it contains only copies of a few of its genes. DNA vaccines are relatively easy and inexpensive to design and produce. Vaccines produced by these new techniques have definite advantages like producing target proteins, long lasting immunity and trigger immune response only against specific pathogens with less toxic effects.

Recombinant hepatitis B vaccine as a subunit vaccine is produced by cloning hepatitis B surface antigen (HbsAg) gene in the yeast, Saccharomyces cerevisiae (Fig. 9.2).