ETHICAL ISSUES

ETHICAL ISSUES

The manipulation of living organisms by the human race cannot go on any further, without regulation. Some ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms.

Going beyond the morality of such issues, the biological significance of such things is also important.Genetic modification of organisms can have unpredictable result when such organisms are introduced into the ecosystem.

Therefore, the Indian Government has set up organisations such as GEAC (Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services.

The modification/usage of living organisms for public services (as food and medicine sources, For example) has also created problems with patents granted for the same.

GM crops are already in cultivation in U.S.A. Europe and several other countries. In India, some insect resistant cotton varieties expressing cry genes have reached the farmers, fields. It has been argued that transgenic crops may be harmful to the environment. The two points. Firstly, the transgene may be transferred through pollen from these crops to their wild relatives secondly GM crops may pollute the environment.

BIO-PATENT

A patent is a right granted by a government to an inventor to prevent others from commercial use of his invention A patent is granted for -

1. An invention [including product].

2. An improvement in an earlier invention. 

3. The process of generating products and

4. A concept or design.

There is growing public anger that certain 

 Companies are being granted patents for products and technologies that  make use of the genetic material, plants and other biological resources that have  long been identified, developed and used by farmers and indigenous people of specific region/country.

Rice is an important food grain, the presence of which goes back thousands of years in Asia's agricultural history. There are an estimated 200,000 varieties of tice in lndia alone. The diversity of rice in India is one of the richest in the world, Basmati Rice is distinct for its unique aroma atd flavour and 27 documented varieties of Basmati grown in India. There is reterence to Basmati in ancient texts,folklore and poetry, as it has been grown for centuries. In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed abroad This the company to sell a 'new' variety of Basmati in, the US and abroad. The 'New' variety of Basmati had actually been derived from Indian farmers varieties. Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty. The patent extends to functional equivalents, implying that other people selling Basmati rice could be restricted by the patent.

Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal e

medicines e.g. Turmeric neem. If we are not vigilant and we do not immediately counter these patent applications, other countries/individuals may encash on our rich legacy and we may not be able to do anything about it.

BIOPIRACY

Bio-piracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concemed without compensatory payment.

Most of the industrialised nations are rich financially but poor in biodiversity and traditional knowledge. In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Taditional knowledge related to bio-resources can be exploited to develop modern application and can also be used to save time, effort and expenditure during their commercialisation.

There has been growing realisation of the injustice, inadequate compensation and benefit sharing between developed and developing countries. Therefore, some nations are developing laws to prevent such unauthorised exploitation of their bio-resources and traditional knowledge. 

The Indian Parliament has recently cleared the second amendment the Indian Patents Bill, that takes issues  into consideration, including patent terms emergency provision and research and development initiatives.

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BIOTECHNOLOGY AND ITS APPLICATION

BIOTECHNOLOGY AND ITS APPLICATION

 

Biotechnology, as you would have learnt  essentially deals with industrial scale production of biopharmaceuticals and biological using genetically modified microbes, fungi, plants and animals. The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.

(A) BIOTECHNOLOGICAL APPLICATION IN AGRICULTURE :

Let us take a look at the three options that can be thought for increasing food production 

i. agro-chemical based agriculture

ii. organic agriculture and 

iii.  genetically engineered crop-based agriculture. 

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of batter management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive and further increases in yield with existing varieties are not possible using conventional breeding is there any alternative path that our undestanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Use of genetically modified crops is a possible solution.

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding. Is there any alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Uses genetically modified crops is possible solution.

Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).

GM has been used to create tailor made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.

Use of genetically modified (GM) plant:

1. To enhance nutritional quality of food

eg. Golden rice - Vitamin A enriched rice (In this rice gene of B-carotene is transfered).

2. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).

3. Helped to reduce post harvest losses eg. Flavr Savr Tomato : Transgenic variety of Tomato - Flavr Savr due to the inhibition of polygalacturonase enzyme which degrades pectin. So that tomato variety remains fresh and retain flavour much longer. Flavr Savr Tomato develop by anti-sense technology.

4. Increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).

5. To produce pharmaceutical product

eg. Production of Hirudin : Hirudin is a protein that prevents blood clotting. The gene incoding hirudin wes chemically synthesized and transfered into Brassica napus. Where hirudin accumulates in seeds. The hirudin is purified and used as medicine.

6. To produce herbicide resistant plant eg. First transgenic plant was tobacco. It contains resistant gene against weedicide (Glyphosate).

7. Pest-resistant crops reduced reliance on chemical pesticides.

i. Insect resistant plant 

eg. Bt cotton : Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B.thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.

The Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.

Bacillus thuringiensis, produces crystal [Cry] protein. This Cry protein is toxic to Larvae of certain insects. Each Cry protein is toxic to a different group of insects. The gene encoding cry protein is called "cry gene". This Cry protein isolated and transferred into several crops. A crop expressing a cry gene is usually resistant to the group of insects for which the concerned Cry protein is toxic. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.

However, gene symbol italics, e.g., cry. The first letter or the protein symbol, on the other hand is always capital and the symbol is always written in roman letters. e.g., Cry.

ii. Nematode resistant plant:

Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidogyne incognitia infects the roots of tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA Interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense.

This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing). The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and antisense RNA in the host cells. These two RNA's being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

A. Genetically Engineered Insulin

It is a proteinaceous hormone having 51 Amino acids arranged in two polypeptides A and B having 21 and 30 Amino Acids respectively and joined by S-S disulphide

bridges.

Sir Edward sharpy-Shafer (1916) was the first to note that diabetes of some persons was because of failure of some islands of pancreas to produce a substance which he called insulin (Derived from the latin, insula, meanning island).

Banting and best (1921) were the first to isolate insulin from dog's pancreas and used it to cure diabetes in man. The first genetically engineered insulin obtained by recombinant DNA technique with the help of E-Coli was produced by the American firms, Eli-Lilly on July 5, 1983. It has been given the trade name humulin and has been approved for clinical use.

Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein. Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges. In mammals, including humans, insulin is synthesised as a prohormone (like a pro-enzyme,the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone) which contains an extra stretch called the C peptide.

 This C peptide is not present in the mature insulin and is removed during maturation into insulin. The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E.coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

maturation of pro-insulin after removal of C-peptide (to be simplified)

B.Gene Therapy: 

A new system of medicine gene therapy, may develop to treat some hereditary diseases such as SCID, haemophilia etc.

Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/embryo. Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involved delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.

The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase. In some children ADA deficiency can be cured by bone marrow transplantation, in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. But the problem with both of these approaches that they are not completely curative. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture Outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes.

which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

 C. Medical Diagnosis of Disease (Molecular diagnosis) 

You know that for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno- sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.

Presence of a pathogen (bacteria, viruses, etc) is normally suspected only when the pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR. PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to detect mutations in genes in suspected cancer patients too. It is a powerful technique to identify any other genetic disorders.

A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complimentarity with the mutated gene.

ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.

TRANSGENIC ANIMALS

Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 percent of all existing transgenic animals are mice.

1. Normal physiology and development: Transgenic animals can be specifically designed to allow the study of how genes are regulated, and how they affect the normal functions of the body and its development e.g. study of complex factors involved in growth such as insulin-like growth factor. By intoducing genes from other species that alter the formation of this factor and studying the biological effects that result, information is obtained about the biological role of the factor in the body.

2. Study of disease: Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These are specially made to serve as models for humane diseases so that investigation of new treatments for diseases is made possible. Today transgenic models exist for many human diseases, such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.

3. Biological products : Medicines required to treat certain human diseases can contain biological products, but such products are often expensive to make. Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein ( α-1-antitrypsin) used to treat emphysema, Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre). The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies that natural cow-milk.

4. Vaccine safety : Transgenic mice are being developed for use in testing the safety of vaccines before they are used on humans. Transgenic mice are being used to test the safety of the polio vaccine. If Successful and found to be reliable, they could replace the use of monkeys to test the safety of batches of the vaccine.

5. Chemical safety testing: This is known as toxicity/safety testing. The procedure is the same as that used for testing toxicity of drugs. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are then exposed to the toxic substances and the effects studied. Toxicity testing in such animals will allow us to obtain results in less time.

Note:-

1. First transgenic animal was mouse contain gene for growth hormone. This enlarged mouse was known as supermouse.

2. First introduced transgenic crop in India (2002) is Bt-cotton.

3. Charles weismann of University of Zurich, obteined interferon through recombinant E. Coli (1980).

4. Microbe have been engineered to produce Human growth Hormone (HGH) for curing dwarfism.

5. Vaccines which are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus.

6. Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production without using expensive fertilizers.

7. Bioremediation: in pollution control, microbes have been engineered to break up the crude oil spills. Dr. Ananda Mohan chakarborthi introduced plasmids from different strains into a single cell of Pseudomonas putida. The  result was new genetically engineered bacterium which would clean the oil spills called "Superbug" (Oil eating bug). He transferred four types of genes/ plasmids in this bacteria. These are OCT, XYL, CAM and NAH.

8. Genetic modified food- the food is prepared from genetically modified crop (transgenic) is called genetically modified food or G.M. Food.

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BIOTECHNOLOGY- PRINCIPLES AND PROCESSES

BIOTECHNOLOGY - PRINCIPLES & PROCESSES

Biotechnology term given by Karl Ereky. Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and process useful to humans.

 Types of Biotechnology Two types- 

(1) Old/traditional- 

Old biotechnology are based on the natural capabilities of micro organisms.

e.g. formation of Citric acid, production of penicillin by Penicillium notatum, making curd bread or wine, which are all microbe mediated processes a form of biotechnology.

(2) New/modern-

Based on Recombinant DNA technology.

e.g Human gene producing Insulin has been transferred and expressed in bacteria like E.coli.

 According to the European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modem molecular biotechnology. The definition given by EFB is as  follows: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Paul berg (Father of genetic engineering)-  He transferred gene of SV-40 virus (simian virus) in to E.coli with the help of  -phage (Nobel prize 1980).

Stanley Cohen and Herbert Bover: First made recombinant DNA by linking an  antibiotic resistance gene with a native plasmid of Salmonella typhimurium.

Principles of Biotechnology 

Among many, the two core techniques that enabled birth of modern biotechnology are:-

1. Genetic Engineering/Recombinant DNA Technology:

 Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. (i.e. formation  of genetically modified organism).

2. Bioprocess Technology:

Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture biotechnological products like antibiotics, vaccines.enzymen, etc.

The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial research. These were-

1. presence of extrachromosomal DNA fragments called plasmids in the bacterial cell, which replicate along with chromosomal DNA of the bacterium.

2. Presence of enzymes restriction endonucleases which cut DNA at specific sites. These enzymes are, Therefore, called 'molecular scissors'.

The main basis of Recombinant DNA Technology is DNA cloning:- it is  making multiple identical copies of any template DNA.

There are three basic steps of DNA clonning-

i) identification of DNA with desirable genes.

ii) introduction of the identified DNA into the host.

iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY :

Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rDNA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, desired DNA and host cells.

1. Enzymes :- A number of specific kinds of enzymes are employed in genetic engineering.

These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.

Lysing enzymes - These enzymes are used for opening the cells to get DNA for genetic experiment.

Bacterial cell : is commonly digested with the help of lysozyme.

Plant cell : is commonly digested with the help of cellulase and pectinase

• Fungal cell : is commonly digested with the help of chitinase.

Cleaving enzymes - These enzymes are used for DNA molecules. Cleaving enzymes are of two types: exonucleases and endonucleases.

Exonuclease remove nucleotides from the ends of the DNA.

Endonucleases make cuts at specific positions within the DNA.

eg. Restriction endonucleases

Restriction Endonuclease Enzymes (Molecular scissors or molecular knife) Bestriction enzymes belong to a larger class of enzymes called endonucleases.

Restriction enzymes are used in recombinant DNA Technology because they can be used in vitro to recognize and cleave within specefic DNA sequence typically consisting of 4 to 8 nucleotides. This specie 4 to 8 nucleotide sequence  is called restriction site and is usually palindromic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.

Restriction enzymes are obtained from bacteria.

 What is function of restriction enzymes in bacteria?

They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome. This is an adaptation against bacteriophages. These enzymes exist in many bacteria beside cleavage some restriction on endonuclease, also have capability of modification.

Modification in the form of methylation, by methylation the bacterial DNA modifies and therefore protects its an own chromosomal DNA from cleavage by these restriction enzymes.

 The first restriction endonuclease-Hind II, whose functioning depended on a specific DNA nucleotide sequence was  isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.

Restriction enzyme (Eco R-I) was discovered by Arter Smith & Nathans (1978 Nobel prize).

Besides Hind II,  today we know more thán 900 restriction enzyme that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Nomenclature of enzyme -

The first letter- indicates bacterial genus (In italic).

 Second and third letter- indicate species of bacteria (in italics).

 Fourth letter- indicates strain of bacteria (optional).

 Roman numeraical- signifying the order in which the enzymes were Isolated from that strain of bacteria.

eg. EcoRI comes from Escherichia coli RY 13. In EcoRL the letter 'R' is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes forms two types of ends on the basis of mode of cutting.

Sticky end (Free end) : Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands.

This leaves single  stranded portion at the ends. There are overhanging stretches called sticky ends on each. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

eg. EcoRI, Hind III, Bam HI, Sal l.

Blunt end (non sticky end) : some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.

eg. Sma l, EcoRV, Hae III.

 Sma l (Serratia marcescens)

Synthesizing enzymes- These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.

Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates.

DNA polymerases- help in the synthesis of complementary DNA strands on DNA templates. 

Joining enzymes- These enzymes help in joining the DNA fragments. For example DNA ligase from Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.

2. Vehicle DNA or Vector DNA-  The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA.

You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

The following are the features that are required to facilitate cloning into a vector.

A. Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

B. Selectable marker: In addition to 'ori', the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

• Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E.coli

• Enzyme forming gene also act as selectable marker gene, eg Lac. Z gene Note: The normal E. coli cells do not carry resistance against any of these antibiotics.

C. Restriction sites/Cloning sites : In order to link the alien DNA, the vector needs, recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

A vector should have restriction site for many enzyme but only one restriction site for each enzyme, Otherwise vector will get fragmented.

Selection of vector depends on :-

1. Size of desired gene 

2. Type of host 

Some examples of vectors:-

a. Plasmids- They are double stranded, circular and extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzymes and DNA ligase. A plasmid carrying DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or Chimeric plasmid.

eg. pBR 322, PUC 18 (used for gene transfer in bacteria).

Ti plasmid, Ri Plasmid (used for gene transfer in dicot plant).

b. Viruses- The DNA of certain viruses is also suitable for use as a vehicle DNA.

Bacteriophage DNA also used as a vector DNA for gene transfer.

Lambda phage (λ phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.

Retro virus - useful for gene transfer in animal cell.

3. Desired DNA/ Alien DNA/ Foreign DNA/ Passenger DNA:-

It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. The passenger DNA can be complementary, synthetic or random.

i. Complementary DNA (cDNA)- It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides. cDNA formed through reverse transcription is shorter than the actual or in vivo gene because of the absence of introns or non-coding regions.

ii. Synthetic DNA (sDNA)- It is synthesized with the help of DNA polymerase on DNA template.

Kornberg (1961) synthesized first synthetic DNA.

Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene coding for yeast alanine t-RNA, which contained only 77 base pairs. However, it did not function in the living system. In 1979, Khorana was able to synthesis a functional tyrosine t-RNA gene of E. coli with 207 nucleotide pairs. Since then a number of genes have been synthesized artificially.

4. Host organism :- 

This organism is used for DNA cloning. Host may be plant, animal, bacteria (E.col. fungi (Yeast).

PROCESSES OF RECOMBINANT DNA TECHNOLOGY:

1. Isolation of DNA: The DNAs which are to be used as passenger DNA and the vehicle DNA are extracted out of their cells by lysing  the cells with the suitable enzyme.

The DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacterial), cellulose (plant cells), chitinase (fungus). You know that genes are located on long molecules of DNA intertwined with proteins such as histones.

The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed ty appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension DNA that separates out can be removed by spooling method.

2. Fragmentation of DNA by restriction endonucleases : Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.

Both the passenger and vehicle DNAs are then, cleaved by using the same restriction endonuclease so that they have complementary sticky ends.

Note : The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

3. Amplification of gene of interest using PCR

Polymerase chain reaction technology (PCR-technology)

This technique was invented by Kary mullis (1983).

In 1993 Kary Mullis got nobel prize for PCR (for chemistry).

PCR is a method for amplifying a specific region of DNA molecule without the requirement for time consuming cloning procedures.

PCR reaction takes place in Eppendorf tube.

Using PCR-technique very low content of DNA available from samples of blood or semen or any other tissue or hair cell can be amplified many times and analysed. In this technique Taq-Polymerase is used. Taq polymerase enzyme is used in PCR which is a special type of DNA polymerase enzyme which is resistant to high temperature.

Taq Polymerase is isolated from Thermus aquaticus bacteria.

Some other examples of polymerase which are used in PCR are-

Pflu Polymerase - Isolated from Pyrococcus furiosus bacteria.

Vent Polymerase - Isolated from Thermococcus litoralis bacterium

Main steps in PCR :-

Denaturation (94°) :- In this step a double stranded DNA molecule is placed at 94°C. So double stranded DNA becomes single stranded & each single stranded DNA functions as a template.

Annealing/Cooling (54°) - In this step two primer DNA are attached at 3' end of single stranded DNA.

Extension (72°):- In this process Taq polymerase enzyme synthesize DNA strain over template.

PCR is automatic process because Taq. polymerase enzyme is heat resistant.

4. Ligation of the DNA fragment into a vector- The complementary sticky ends of the passenger and vehicle DNAs are joined with ligase enzyme. This gives rise to a recombinant DNA.

5. Transferring the recombinant DNA into the host (Gene transfer):-

Transfer of desired genes from one organism into another is an important aspect of genetic engineering.

Gene transfer is achieved by two kinds of transfer methods:

1. Indirect method through vectors or carriers and

2.  Director vector less transfer method.

Indirect method :-

Gene Transfer in bacterial cell : Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by Incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

Note: The bacteria to be used as hosts should be without plasmids. The host cells are treated with calcium chloride or lysozyme.

Transformant E.coli with plasmid.

Non-transformant E.coli without plasmid.

Gene Transfer in plant cell 

1. A plant pathogenic bacterium-Agrobacterium tumefaciens produces crown galls or plant tumours in almost all dicotyledonous plants.

2. This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and cotton but not cereals.

3. Tumour formation is induced by it's plasmid which is therefore called Ti plasmid (TI - tumour inducing) Agrobacterium tumefaciens naturally transfers some part of Ti-Plasmid into host plant DNA without any human effort so it is called natural genetic engineer of plant.

In the transformation process two essential component in Ti-plasmid -

 (a) T-DNA - (Transferred DNA)

(b) Vir-region-(Virulence region)

Inside the host plant cell T-DNA is seperated from Ti-plasmid, and integrated into host plant DNA that causes crown gall tumour.

Vir-region contains genes which are essential for T-DNA transfer and integration to host plant DNA.

When we use Ti-plasmid as a vector, first we remove the tumour causing gene from T-DNA region. Then desired gene inserted inplace of it. Now, this plasmid is called disarmed plasmid.

Same as Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene transfer to plant cell.

Gene transfer in animal

Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

Direct method

Foreign genes can also be transferred directly by the following methods:-

a. Electroporation- It creates transient (temporary pores) in the plasma membrane to facilitate entry of foreign DNA.

b. Chemical mediated genetic transformation- It involves certain chemicals such as polyethylene gycol (PEG), that help in the uptake of foreign DNA into host cells.

c. Microinjection- it in the introduction of foreign genes mainly into animal cells using micropipette ar glass needles.

d. Particle gun/Biolistic method- It is a technique in which tungsten or gold particles coated with foreign DNA bombarded into target cells to facilitate entry of the foreign genes.

e. Liposome mediated gene transfer- In this method DNA encloses within lipid begs. Thest lipid begs funed with protoplast.

Selection of Transformed with recombinant cell:-

1. Selection by two antibiotic resistance gene 

You can ligate a foreign DNA at the Bam HI site of tetracycline resistance gene in the vector pBR322.

The recombinant plasmids will loss tetracycline resistance due to insertion of foreign DNA (insertional inactivation) now, it can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transferd) growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non recombinants wil grow on the medium containing both the antibiotics. In This case, one antibiotic resistance gene helps in selecting the transformants.

Note:- 

Insertional inactivation- Due to Insertion of desired gene within selectable marker gene of vector, selectable marker gene become inactive or loose their Function. This is called Insertional inactivation.

2. Selection by one Lac Z gene and one antibiotics resistant gene-   Selection of recombinants due to inactivation of antibiotics is a cumbersome (troublesome) procedure because it requires simultaneous plating on two plated having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate X-gal gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase (reporter enzyme) and the colonies do not produce any colour, these are identified as recombinant colonies.

Obtaining the Foreign Gene Product :

When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial plant or animal cell the alien DNA gets multiplied. In almost all recombinant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed. The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.

After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale. Can you think of any reason why there is a need for large-scale production? If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc. useing microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, Oxygen).

The most commonly used bioreactors are of stirring type -

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively, air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

Downstream Processing -

After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.

The processes include separation and purification, which are collectively referred to as downstream processing.

The product has to be formulated with suitable preservatives. Such formulation has to undergo through clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product.to product.

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HUMAN GENOME PROJECT

HUMAN GENOME PROJECT

Genetic make up of an organism or an individual lies in the DNA sequences. If two individuals differ, then their DNA sequences should also be different, at least at some places. These assumptions led to the quest of finding out the complete DNA sequence of human genome. With the establishment of genetic engineering techniques where it was possible to isolate and clone any piece of DNA and availability of simple and fast techniques for determining DNA sequences, a very ambitious project of sequencing human genome was launched in the year 1990.

Human Genome Project (HGP) was called a mega project. You can imagine the magnitude and the requirements for the project if we simply define the aims of the project as follows:

Human genome is said to have approximately 3 x 10⁹ bp, and if the cost of sequencing required is US $3 per bp (the estimated cost in the beginning), the total estimated cost of the project would be approximately 9 billion US dollars. Further, if the obtained sequences were to be stored in typed form in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to store the information of DNA sequence from a single human cell. HGP was closely associated with the rapid development of a new area in biology called as Bioinformatics.

Goals of HGP 

Some of the important goals of HGP are as follows :

1. Identify all the genes in human DNA.

2. Determine the sequences of the 3 billion chemical base pairs that make up human DNA.

3. Store this information in databases

4. Improve tools for data analysis.

5. Transfer related technologies to other sectors, such as industries

6. Address the ethical, legal and social issues (ELSI) that may arise from the project.

The project was completed in 2003. Knowledge about the effects of DNA variations among individuals can lead to revolutionary new ways to diagnose, treat and someday prevent the thousands of disorders that affect human beings. Besides providing clues to understanding human biology, learning about non-human organisms, DNA sequences can lead to an understanding of their natural capabilities that can be applied toward solving challenges in health care, agriculture energy production, environmental remediation. Many non-human model organisms, such as bacteria, yeast, Caenorhabditis elegans (a freeliving non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and Arabidopsis), etc., have also been sequenced.

Methodologies : The methods involved two major approaches (1) Expressed Sequence Tags (ESTS) - Identifying all the genes that expressed as RNA (2) Sequence Annotation - The blind approach of simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions. For sequencing, the total DNA from a cell is isolated and converted into random fragments of relatively smaller sizes (recall DNA is a very long polymer, and there are technical limitations in sequencing very long pieces of DNA) and cloned in suitable host using specialised vectors.

The cloning resulted into amplification of each piece of DNA fragment so, that is subsequently could be sequenced with ease. The commonly used hosts were bacteria and yeast, and the vectors were called as BAC (bacterial artificial chromosomes), and YAC (yeast artificial chromosomes).

The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger (Remember, Sanger is also credited for developing method for determination of amino acid sequences in proteins). These sequences were  then arranged based on some overlapping regions present in them. This required generation of overlapping fragments for sequencing. Alignment of these sequences was humanly not possible. Therefore, specialised computer based programmes were developed. These sequences were subsequently annotated and were assigned to each chromosome. The Sequence of chromosome I was completed only in May 2006 (this was the last of the 24 human chromosomes 22 autosomes and X and Y to be sequenced). Another challenging task was assigning the genetic and physical maps on the genome. This was generated using information on polymorphism of restriction endonuclease recognition sites, and some repetitive DNA sequences known as microsatellites.

Salient Features of Human Genome-

 Some of the salient observations drawn from human genome project are as follows :

1. The human genome contains 3164.7 million nucleotide bases.

2. The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases.

3. The total number of genes is estimated at 30,000 much lower than previous estimates of 80,000 to 1,40,000 genes. Almost all (99.9 per cent) nucleotide bases are exactly the same in all people.

5. The functions are unknown for over 50 percent of discovered genes.

6. Less than 2 percent of the genome codes for proteins.

7. Repeated sequences make up very large portion of the human genome.

8. Repetitive sequences are stretches of DNA sequences that are repeated many times, sometimes hundred to thousand times. They are thought to have no direct coding functions, but they shed light on chromosomes structure, dynamics and evolution.

9. Chromosome 1 has most genes (2968). and the Y has the fewest (231).

10. Scientists have identified about 1.4 million locations where single base DNA differences (SNPS- single nucleotide polymorphism, pronounced as 'snips) Occur in humans. This information promises to revolutionise the processes of finding chromosomal locations for disease-associated sequences and tracing human history.

(a) First prokaryotes in which complete genome was sequenced is Haemophilus influenzae.

(b) First Eukaryote in which complete genome was sequenced is Saccharomyces cerviceae (Yeast).

(c) First plant in which complete genome was sequenced is Arabidopsis thaliana (Small mustard plant).

(d) First animal in which complete genome was sequenced is Caenorhabditis elegans (Nematode).

  β-globin and insulin gene are less than 10 kilo base pair T.D.F. gene is the smallest gene (14 base pair) and Duchenne muscular Dystrophy gene is made up of 2400 kilo base pair.(Longest gene)

Thank you....🤞

DNA FINGERPRINTING

DNA fingerprinting/ DNA typing/ DNA profiling/ DNA test

It is technique to identify a person on the basis of his/her DNA specificity.

This technique was invented by sir Alec. Jeffery (1984).

In India DNA Finger printing has been started by Dr. V.K. Kashyap & Dr. Lal Ji Singh.

DNA of human is almost the same for all individuals but very small amount that differs from person to person that forensic scientists analyze to identify people.

These differences are called Polymorphism (many forms) and are the key of DNA typing. Polymorphisms are

most useful to forensic scientist. It is consist of variation in the length of DNA at specific loci is called Restricted fragment. It is most important segment for DNA test made up of short repetitive nucleotide sequences, these are called VNTRs (variable number of tandem repeat).

VNTRs also called minisatellites were discovered by Alec Jeffery. Restricted fragment consist of hypervariable repeat region of DNA having a basic repeat sequence of 11-60 bp and flanked on both sites by restriction site.

The number and position of minisatellites or VNTR in restriction fragment is different for each DNA and length of restriction fragment is depend on number of VNTR.

Therefore, when the genome of two people are cut using the same restriction enzyme the length of fragments obtained is different for both the people.

These variations in length of restricted fragment is called RFLP or Restriction fragment length polymorphism.

Restriction Fragment Length Polymorphism distributed throughout human genomes are useful for DNA Fingerprinting.

 DNA Fingerprint can be prepared from extremely minute amount of blood, semen, hair bulb or any other cell of the body.

DNA content of 1 - Microgram is sufficient.

Technique of DNA Fingerprinting involves the following major steps.

Extraction - DNA extracted from the cell by cell lysis. If the content of DNA is limited then DNA can be amplified by Polymerase chain reaction (PCR). This process is amplification.

Restriction Enzyme Digestion : Restriction enzyme cuts DNA at specific 4 or 6 base pair sequences called restriction site.

 Hae III (Haemophilus aegyptius) is most commonly used enzyme. It cuts the DNA, every where the bases are arranged in the sequence GGCC. These restriction fragment transferred to Agarose Polymer gel.

Gel Electrophoresis :

Gel electrophoresis is a method that separates macromolecules either nucleic acid or proteins-on the basis of size, electric charge.

Gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provides the driving force. A molecule's properties determine, how rapidly an electric field can move the molecule through a gelatinous medium.

Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids posses ionisable groups and therefore, at any given pH, exist in solution as electrically charged species either as cation (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

By the gel electrophoresis these restricted fragments move towards the positive electrode (anode) because DNA  has -ve electric charge (PO₄³⁻).

Smaller Fragment more move towards the positive pole due to less molecular weight. So after the gel electrophoresis DNA fragment arranged according to molecular weight.

These separated fragments can be visualized by staining them with a dye that fluoresces ultraviolet radiation.

Southern transfer / Southern blotting :

The gel is fragile. It is necessary to remove the DNA from the gel and permanently attaches it to a solid support. This is accomplished by the process of Southern blotting. The first step is to denature the DNA in the gel which means that the double-stranded restriction fragments are chemically separated into the single stranded form.

The DNA then is transferred by the process of blotting to a sheet of nylon. The nylon acts like an ink blotter and "blots" up the separated DNA fragments, the restriction fragments, invisible at this stage are irreversibly attached to the nylon membrane the "blot".

This process is called Southern blot by the name of Edward Southern (1970).

Hybridization: To detect VNTR locus on restricted fragment, we use single stranded Radioactive Phosphorus-32 (P32) DNA probe which have the base pair sequences complimentary to the DNA sequences at the VNTR locus. Commonly we use a combination of at least 4 to 6 separate DNA probes.

Labelled Probes are attached with the VNTR loci of restricted DNA Fragments, this process is called Hybridization.

Autoradiography: Nylon membrane containing radio active probe exposed to X-ray. Specific bands appear on X-ray film. These bands are the areas where the radioactive probe bind with the VNTR.

This appears the specific restriction fragment length pattern. This length pattern is different in different individual. This is called Restriction Fragment length Polymorphism (RFLP).

These allow analyzer to identify a particular person DNA, the occurrence and frequency of a particular genetic patter contained in this X-ray film. These x-ray film called DNA signature of a person which is specific for each individual.

The probability of two unrelated individual having same pattern of location and repeat number of minisatellite (VNTR) is one in ten billion (world population 6.1 billion).

In India the centre for DNA fingerprinting and diagnosis (CDFD - center for DNA finger printing & diagnosis) located at Hyderabad.

Application of DNA Fingerprinting

1. Paternity tests- The major application of DNA finger printing is in determining family relationships. For identifying the true (biological) father, DNA samples of Child, mother and possible fathers are taken and their DNA finger prints are obtained. The prints of child DNA match to the prints of biological parents.

2. Identification of the criminal- DNA fingerprinting has now become useful technique in forensic (crime detecting) science, specially when serious crimes such as murders and rapes are involved. For identifying a criminal, the DNA fingerprints of the suspects from blood or hair or semen picked up from the scene of crime are prepared and compared. The DNA fingerprint of the person matching the one obtained from sample collected from scene of crime can give a clue to the actual criminal.

schematic representation of DNA fingerprinting: few representative chromosomes have been shown to contain different copy number of VNTR. For the sake of understanding colour schemes have been used to trace the origin of each band in the gel. The two alleles (parental and maternal) of chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime sceme matches with individual B, and not with A.

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MUTATION

MUTATION

Sudden heritable change in genetic material of an organism is called as Mutation.

Mutation are source of discontinuous variation.

Only those mutation are heritable which occur in germinal cell of an organism. While somatic mutations are non heritable. Somatic mutations are also heritable in vegatative propagated plants.

Mutation word was given by Hugo De Vries.

 De Vries studied mutations in the plant Oenothera lamarckiana (evening primrose).

Mutation was first observed by Seth Wright. He observed some short legged sheep (Ancon) variety in a population of long legged sheep.

Beadle and Tatum induced mutations in Neurospora by the help of U. V. rays. or X-rays.

Wild neurospora (PROTOTROPH)----U.V. Rays----Mutant Neurospora (AUXOTROPH)

Normal Neurospora can be grown in minimal medium because Neurospora can make all essential nutrients required for it. This is known as Prototroph.

Mutant Neurospora doesn't has capability to grow in minimal medium because due to mutation It loses those genes which codes for the enzyme that helps to prepare some special nutrients for it. They gave "one gene one enzyme" concept. This form is known as Auxotroph.

M.S. Swaminathan induced mutations in wheat by the help of γ-rays to obtain good varieties tor eg. Sharbati Sonora, Pusa Lerma. Swaminathan established     γ (gamma) garden in IARI-New Delhi (Pusa Institute).

Types of mutation :

i. CHROMOSOMAL MUTATION 

ii. GENE MUTATIONS

1. Chromosomal Mutations- Each species of organisms has constant number of chromosomes. There are two sets of homologous chromosomes in the somatic cells of an organisms. The number, type and sequences of genes on chromosomes is fixed and all the genes are oriented at their definite loci. The gene loci retain the same sequence they have had for many generations past.

Sometimes, during cell division, as a results of certain abnormalities, changes in structure and number of chromosomes are produced. Such type of change are called chromosomal aberration. These chromosomal aberration can be classified into following two classes:-

Types of chromosomal mutation :

1.Heteroploidy/Genomatic mutation-change in chromosome number.

2. Chromosomal aberration-change in structure of chromosome.

1. Heteroploidy / Genomatic mutationor ploidy- In different species of all the organism, the number of chromosomes remains fixed and stable throughout their life, but sometimes change in the number of chromosomes occurs. The Change in number of one or few chromosomes in a set or number of entire set of chromosome.

It is of two types:

Euploidy-Change in number of chromosome sits

Aneuploidy - Change in number of chromosome in a set

EUPLOIDY- Change in number of sets of chromosome  i.e. either loss or addition of sets of chromosomes.

Monoploidy (x) - Presence of one set of chromosomes.

Diploidy (2x) - Presence of two sets of chromosomes.

Polyploidy - Presence of more than two sets of chromosomes.

It may be : Triploidy (3x) Tetraploidy (4x) Pentaploidy (5x) Hexaploid (6x) Heptaploid (7x) octaploidy  (8x)

Polyploid plants with even number of sets are always fertile, reproduce sexually and form seeds.

Polyploid plants with odd number of sets are always sterile don't reproduce by sexual reproduction. They don't produce seeds but they may produce seedless fruits by parthenocarpy. eg. Banana and seedless grapes.

ANEUPLOIDY- Loss or addition of chromosomes in a set of chromosomes.

Types of Aneuploidy:-

1. Hypoaneuploidy (loss) 

2n-1=Monosomy - (loss of one chromosome in one set) 

2n-1-1=Double monosomy (loss of one chromosome from each set, but these are non homologous).

2n -2=Nullisomy (loss of two homologous chromosome) 

2. Hyperaneuploidy (addition) 

2n +1=Trisomy : addition of one chromosome in one set.

2n +1 +1=Double Trisomy : addition of one chromosome in each set

2n +2=Tetrasomy: addition of two chromosome in one set.

• Cause of aneuploidy is chromosomal nondisjunction means chromosomes fail to separate during meiosis.
• Chances of aneuploidy are more in higher age female due to less activity of oocyte, so chances of syndrome increase in children who are bom from higher age female.

2. Chromosomal Aberrations : Change in structure of chromosome. The change in the number or orientation of genes located on chromosomes is called structural chromosomal abberation. Such types of changes are produced mainly as a results of some irregularities occured during meiotic divisons. Such irregularities include breaking, reattachment, and rearrangement etc. Structural changes in the chromosomes are of following four types:-

(i) Deletion : Loss of a part or segment of chromosome which leads to loss of some gene is called as deletion.

It is of 2 types :

(A) Terminal deletion -Loss of chromosomal segment from one or both ends.Eg. The cry-du-chat syndrome is an example of terminal deletion in short arm of 5th chromosome.

(B) Intercalary deletion - Loss of chromosomal part between the ends.

(ii) Inversion : Breakage of chromosomal segment but reunion on same chromosome in reverse orders. It leads to change in distance between genes on chromosome or sequence of genes on chromosome so crossing over is affected

It is of 2 types:-

1. Paracentric -If inversion occur only in one arm and inverted segment does not include centromere.

2. Pericentric - In this type of inversion inverted segment include centromere.

(iii) Duplication : Occurence of a chromosomal segment twice on a chromosome.

Example : In drosophila "Bar eye character" is observed due to duplication in X-chromosome. Bar eye is a character whose eyes are narrower as compared to normal eye shape.

(iv) Translocation : In this, a part of the chromosome is broken and may be joined with non homologous chromosome. This is also known as illegitimate crossing over (illegal crossing over).

Types of translocation

(A) Simple Translocation- When a chromosomal segment breaks and attached to the terminal end of a non homologous chromosome.

(B) Reciprocal Translocation- exchange of segments between two non homologous chromosome.

In man, STRUCTURAL CHROMOSOMAL ABERRATIONS.

In man, many examples of chromosomes breakage with resulting aberration have been found. 

For example, a terminal deletion of a part of long arm of chromosome 21 in man causes Chronic granulocytic leujemia. Since this shortened chromosome was discovered in Philadelphia, it is known as the Philadelphia chromosome. Similarly deletion of a part of short arm of chromosome 5 in man causes undeveloped larynx  in children as a result of which they produce mewing sound. Because of this it is called as Cri-du-chat or cat cry syndrome.

Chronic myeloid leukemia (C M L) is a type of blood cancer. This disease is a result of reciprocal transiocation between 22 and 9 chromosome.

Note: If exchange of segments take place in between homologous chromosomes then it is called crossing over.

(2) GENE MUTATION OR POINT MUTATION

Two types:-

1. Substitution 

2. Frameshift mutation.

A. Substitution:

Replacement of one nitrogenous base by another nitrogenous base is called as substitution.

 It causes change in one codon in  genetic code which leads to change in one amino acid in structure of protein. eg. Sickel cell anaemia.

Change may not occur some time because for one amino acid more than one type of codons are present

Substitution is of two types

1. Transition- Replacement of one purine by another  purine or replacement pyrimidine by another pyrimidines.

2. Transversion- Replacement of purine by pyrimidine or pyrimidine by purine is called transversion.

B. Frame shift mutation/Gibberish mutation:

Loss or addition of one or rarely more than one nitrogenous bases in structure of DNA.

Frame shift mutation is of two types.

1. Addition-Addition of one or rarely  more than one nitrogenous bases in structure of DNA.

2. Deletion- loss of ons or rarely more than one nitrogenous bases in structure of DNA.

 Due to frame shift mutation complete reading  of genetic code changed. It leads  to change in all amino acids in structure of proterin so a new protein is formed which is completely different from previous protein.

 So frameshift mutations are more harmful as compared to substitution.

 eg: Thalassemia (lethal genetic disorder)

MUTAGENS- 

Mutagens are those substances which cause mutations.

Non ionising:- U.V. Rays.

Thank you👍

CANOPY ARCHITECTURE

Canopy architecture

" The overall profile of the tree top is known as canopy architecture".

Vegetation canopy structure describes the three-dimensional arrangement of leaves and includes such canopy attributes as canopy architecture, leaf angle distribution, ground cover fraction, leaf morphology, vegetation spatial heterogeneity, and shadows.

Canopy architectures in plants:-

Canopy architecture is the overall profile of tree the  tops. The trees gets grouped together leading to the formation of canopy and reduces the amount of light beneath them.

Factors controlling the canopy architecture:-

1. Pattern of growth in plant axis.

2. Arrangements and distribution of branches on main shoot.

3. Angle of branches on shoot.

4. Direction of branches.

5. Size, arrangement, distribution and shapes of leaves.

6. Genetic, environmental and nutritional factors.

Heliophytes or shade-introlerant plants- those plants which grows in full sunlight. eg. Sunflower, poplars, pines etc.

Sciophytes or shade-tolerant plants- certain plants which survive only under shade. eg. Fir, spruce etc.

However some plants can be facultative sciophytes as well as facultative heliophytes.

fig : Heliophytes and Sciophytes

The forest is made up of some individual tall plants which protrudes above normal level of canopy and are called as emergents.

The process of stratification i.e. arrangements of different plants or animals in different layer of substratum takes place in canopy.

Eg. Stratification in a tropical rain forest.

Based upon branching a tree, it may classified as:-

1. Culm- The stem is unbranched showing distinct nodes and internodes imparting a jointed apperance. Eg. Bambusa arundinacea (Bamboo).

2. Columnar (caudex)- In such case trunk is unbranched and bears a crown of leaves at its apex. Eg. Fan plam, areca catechu (supari) , Cocos nucifera (coconut) etc.

3. Excurrent- In such case lateral branches do not complete with the growth of main stem. Branches appear in acropetal succession. Thus, tree gives cone-like apperance. Eg. Pinus, Eucalyptus etc.

4. Decurrent or Deliquescent- When some of the lateral branches grows vigorously, the trunk Disappears after some distance due to the suppression of growth of terminal bud, the form called Decurrent. Eg. Magnifera indica (mango), Dalbergia(Shisham), Ficus benghalensis etc.

SHAPE OF CANOPY ARCHITECTURE

a. Round canopy- Trees which shows Decurrent or Deliquescent branching shows round canopy. Branch appears in alternate pattern. Branches present on upper side grows towards upper and outer sides. However, lower branches spread  out. Minimum overlapping of leaves in such cases. Eg. Magnifera indica , lagerstromia, Dalbergia, Ficus etc.

b. Conical canopy- In such case lateral branches do not complete with the growth of main stem. Branches appears acropetally (Monopodial/Racemose).

Branches appear either horizontally (eg. Araucaria) or ascent at a particular angle (eg. Thuja). Shape of tree is conical.

Eg. Pinus, Eucalyptus etc.

c. Oblong or cylindrical canopy- Branching in these trees can be Racemose (Gnetum) or cymose type (peltophorum). Main trunk remains upright. Lateral branches are short and grows towards upper side providing cylindrical shape to trees. 

Eg. Artocorpus (jack fruit).

d. Flat topped canopy- Growth of apical bud on main trunk stops after attaining a certain height. It bends on upper side, a twig appears which further give rise to Obliquely rising branches. Thus, tree imparts umbrella shape. Eg. Cassia, Terminalia (imli), Thespesia actifolia, Enterolobium saman (rain tree) etc.

e. Weeping tree- Due to weak branching or due to drooping branching pattern, trees impact weeping appearance.

Stem is Monopodial (Polyalthia) or stem is sympodial (Zizyphus jujuba).

Eg. Salix (weeping willow), Acacia arabica (Kikar).

f. Pagoda trees- Trees bear spirally arranged compacts, simple or palmate type of leaves. The branches on trunk arises in wholed fashion. The pagoda shape is very distinct when the tree is young. As the whorls ascent from the base to tip, length internode shortens and gives a typical pagoda shape to trees. Eg. Salmalia, Achras sapota (Chiku), Terminalis etc.

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