DIABETES

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

Diabetes is a metabolic disorder characterized by hyperglycaemia which results in a lack of insulin secretion, insulin action, or both the conditions. Metabolic abnormalities are caused due to a low level of resistance to insulin. The effect of symptoms can be classified based on the type and duration of diabetes. Diabetes has also been associated with many metabolic disorders such as acromegaly and hypercortisolism for example insulin resistance has been observed in patients with acromegaly in the liver. Hypercortisolism (Cushing syndrome) produces visceral obesity, insulin resistance, dyslipidaemia which leads to hyperglycaemia and reduces glucose tolerance. Besides, diabetes been associated with metabolic disorders, clinical convergence between type 1 diabetes (T1D), and type 2 diabetes(T2D) is also observed. T2D patients develop a progressive decline in total beta-cell mass. Thus there are many interlinked complications due to diabetes.

According to the report by WHO 2019, 10 main issues demand attention one of them is noncommunicable diseases such as diabetes, cancer, and heart disease. These are collectively responsible for 70% of deaths worldwide. According to the National Health Portal, the Government of India, nearly 5.8 million deaths occur due to noncommunicable diseases in India (WHO 2015). As per data provided by Directorate General of Health Services Ministry of Health & Family Welfare, Government of India (MoHFW) 2016-2017, 2.24 core persons were screened for Common noncommunicable diseases like diabetes, hypertension, cardiovascular disorders, and common cancers. From this, 9.7 % was diagnosed to be diabetes, 12.09% was diagnosed to be hypertension, 0.55% was diagnosed to be cardiovascular disease and 0.17% was with common cancers.

Events occurred from discovery of Diabetes to development of various drugs 

YEAR	EVENTS
1552 BC	HESY-RA documented urination as symptom of mysterious disease
133 AD	Araetus of Cappodocia coined the word diabetes
1675	Thomas Willis coined the word mellitus
1776	Dobson confirmed presence of excess sugar in patients
1800	Discovered chemical test for presence of sugar in urine
1700’s and 1800’s	Physician began to realize dietary changes help manage diabetes
1857	Claude Bernard confirmed that the diabetes occur due to excess glucose production
1870’s	During Franco Prussian war French physician Apollinaire Bouchardat proved that the diabetes patients symptoms improved due to war related food rationing
1889	Oskar Minkowski and Joseph Von Mering extract obtained from dogs pancreas
Early 1900	Development of oat cure, potato therapy, starvation diet.George Zuelzar injected pancreatic extract to control diabetes
1916	Boston scientist Elliott Joslin wrote book “ The Treatment Of Diabetes Mellitus “
1922	Frederick Banting discovered insulin to treat diabetes and won Nobel Prize in medicine 1923
1978	Production of recombinant human DNA insulin
1996	For the treatment of type 22 diabetes Thiazolidinediones (TZDs) were introduced.
2005	The  amylin analogue known as pramlintide, which was approved by the FDA
2008	Colesevelam approved for type 2 diabetes by FDA
2009	Bromocriptine approved for diabetes
2013	Canagliflozin  is the first SGLT- 2 inhibitor  approved by FDA  [Sodium Glucose Co-Transporter 2 Inhibitors], Dapagliflozin approved in 2014 by FDA

(Source: Saudi Med et al., 2002, John et al., 2014)

Diagnosis of Diabetes: 

There are several methods used for the diagnosis of Diabetes Mellitus. According to American Diabetes Association (ADA) the most standard diagnostic criteria is as follows 

1. Hemoglobin A1c (HbA1c)
2. Fasting Plasma Glucose (FPG)
3. Oral Glucose Tolerance Test (OGTT)

 Hemoglobin A1c (HbA1c):

The average level of blood sugar over past two to three months can be diagnosed using hemoglobin A1c test. The main advantage of this type of diagnosis is that there is no need of fasting. A1c is measured using percentage The standard referred by ADA for normal person is less than 5.7%.

 Diagnosis of Diabetes by checking Hemoglobin A1c (HbA1c)

	Hemoglobin A1c
Normal	Less than 5.7%
Prediabetes	5.7% to 6.4%
Diabetes	6.5% higher

Fasting Plasma Glucose (FPG):

It is used to check fasting blood sugar levels. The patient should fast for 8 hours before the test. It is mainly done during morning. For normal person the FPG is lower than 100mg/dl.

Diagnosis of Diabetes by checking Fasting Plasma Glucose (FPG)

	FPG
Normal	100mg/dl or less
Pre diabetes	100 mg/dl to 125 mg/dl
Diabetes	126 mg/dl or high

Oral Glucose Tolerance Test (OGTT)

This method is used to diagnose blood sugar level before and after 2 hours of a sweet drink. For normal person the OGTT is less than 140mg/dl

 Diagnosis of Diabetes by checking Oral Glucose Tolerance (OGTT)

	OGTT
Normal	140mg/dl or less
Pre diabetes	149 to 199mg/dl
Diabetes	200 mg/dl or high

Human Mitochondrial DNA

BY: Ezhuthachan Mithu Mohanan (MIWM043)

DNA within mitochondria was first detected in 1963. Human mitochondria represent the mammalian mtDNA. Human mtDNA is 16,569 bp, double stranded and circular. It codes for 13 polypeptides, belonging to OXPHOS family. mtDNA also codes for 22 tRNA,2 rRNA. It also has control noncoding regions. Various nuclear coded factors also known as precursor polypeptides are essential for the expression and maintenance of mtDNA. It was Sanger who found that circular mtDNA in vertebrates, has both Light Strand and Heavy strand. There are many differences considering nuclear DNA and mitochondrial DNA, which are as follows 

MITOCHONDRIAL DNA	NUCLEAR DNA
Present in mitochondria	Present in nucleous
One cell contains 0.25% mtDNA	One cell has 99.75% n DNA
Mutation rate of mtDNA is 20 times faster than nDNA	Slow mutation rate
circular	Linear
1000’s of mtDNA copies/ cell	2 Copies / cell
Haploid	Diploid
Maternally inherited	Biparental inheritance
Replication repair mechanism is absent	Replication repair mechanism is present
Reference sequence by Anderson  and Colleagues in 1981	Reference sequence in Human Genome Project in 2001
Size of genome is 16,569 bp	Size of genome is 3.2 billion base pair

The genetic code of nuclear DNA differ from mtDNA as such ‘TGA’ codes for tryptophan in vertebrate mitochondria, while it is a stop codon for nuclear DNA. ‘ATA’ codes for Isoleucine in cytosol, while it codes for methionine in mitochondria.

Mitochondrial Inheritance: 

With most of the evidence provided, mostly there is maternal inheritance of mitochondria. Due to nucleotide imbalance and reduction in fidelity of polymerase γ, it causes higher mutation rate. This can be used as approach for human identity test, studying evolutionary and migration pattern.

mtDNA replication:  

Factors for mtDNA  replication: 

DNA Polymerase γ is the polymerase enzyme, it is a heterotrimer with one catalytic subunit ( POLγA). POLγA has 3’-5’ exonuclease activity for proofreading. TWINKLE is DNA Helicase which unwinds double stranded DNA. mtSSB is binds with single stranded DNA to protect from nucleases. Vinograd and coworkers proposed the strand displacement theory, which emphasize continuous DNA synthesis on H and O strand. The replication initiation begins from OH Strand, which proceeds unidirectional. During OL replication stem loop structure is formed which block mtSSB from binding, initiating primer synthesis. Thus the two strand synthesis occur in a continuous manner , until two complete double stranded DNA is formed.A triple-stranded displacement loop structure also known as D Loop is formed, When 7S DNA remains bound to parental L strand, while parental H-Strand is displaced.  The role of mtDNA D loop is not completely understood. 

Mitochondrial Diseases:

A dysfunction in mitochondria leads to mitochondrial disorder. Heteroplasmy  is condition due to presence of mutant mitochondrial DNA .  Various Mitochondrial disorders are as follows:

1. Mitochondrial Myopathy:  Presence of ragged red muscle fibres is due to accumulation glycogen and neutral lipids which leads to decreased reactivity of cytochrome c oxidase
2. Leber’s hereditary optic neuropathy : This is maternally inherited, which causes degeneration of retinal ganglion cells
3. Leigh syndrome: It is a neurometabolic disorder affecting CNS( central nervous system)
4. Myoneurogenic gastrointestinal encephalopathy : Autosomal recessive disorder. It is due to mutation of TYMP gene
5. Mitochondrial DNA depletion syndrome: It is also known as Aplers disease. This is caused my mutation inTK2 gene.

TRANSGENIC ANIMALS AND DISEASE RESISTANCE

BY: Reddy Sailaja M (MSIWM030)

TRANSGENIC ANIMALS

‘Transgenesis’ is a molecular method of introducing a foreign gene (of interest) into the genome of an organism to express the desired trait or characteristic and further pass the trait to the progeny successfully. The gene that is being introduced is called a ‘transgene’.

A transgenic animal or genetically modified animal is the one that is being introduced with a desired foreign gene into its genetic material through recombinant DNA technology, a molecular biology technique.

Transgenesis has been widely applied in most of the domestic animals, aquaculture and agriculture that aids in human welfare and development.

Ralph Brinster and Richard Palmiter were the pioneers in creating first transgenic animal – “Super mouse” in 1982 by introducing human growth hormone in the mouse genome. The offspring produced were larger in size than the parent.

Figure 1: Transgenic super mouse (right) produced by recombinant DNA technology

Pig, goat, sheep, fish, cattle and insects like Drosophila melanogaster (fruit fly) are the most common transgenic animals that are being used in basic and applied research for human welfare.

PRODUCTION OF TRANSGENIC ANIMALS

Two methods are principally followed to generate transgenic animals

1. Embryonic stem cell method
2. Pronucleus method by microinjection

1. Embryonic stem cell method for Transgenesis:
2. Inner cell mass of mammalian blastocysts contain embryonic stem cells (ESCs). ESCs have the ability to produce all kinds of organisms’ cells, including gametes.
3. Desired gene is selected from the donor organism.
4. Vector DNA is chosen that carries the desired DNA to the host cell.
5. Vector contains promoter and other regulatory sequences that are crucial for transgene transfer, selection and expression in the host organism.
6. ESCs were cultured along with the vector containing desired DNA
7. Successfully transformed cells will be selected based on the selection methods like antibiotic resistance.
8. The transformed cells are injected into inner cell mass of embryonic blastocysts of the mouse for further propagation.
9. A pseudo pregnant mouse (stimulus of mating results in making mouse uterus receptive for the blastocysts due to hormonal changes) was prepared and the transformed blastocyst stage embryo was introduced into the uterus.
10. Blastocyst would implant successfully and the mouse gives birth to pups. 10-20% pups will be having the transgene and is heterozygous in nature (only one copy of the gene was transformed and the other was wild).
11. Heterozygous mice are allowed to mate to get homozygous offspring (1 in 4, Mendelian ratio) was selected and propagated further to generate transgenic trait.

Figure 2: Embryonic stem cell method for transgenic animal generation

• Microinjection method:
• Manipulation of the pronucleus is the most common method to create a transgenic animal and is first described by Gordon et al.
• Superovulating female is induced with specific hormones and the eggs are harvested.
• The male and female pronuclei are visible under microscope several hours after the sperm is allowed to enter into the oocyte. As male pronucleus is larger in size, the transgene is microinjected easily into it.
• Pronucleus stage is advantageous as it allows early incorporation of the transgene into the host DNA and the entire host cells could express it.
• Once the transgene is introduced, male and female pronuclei are allowed to fuse to form a fertilized egg.
• Once the blastocyst stage is reached, it was implanted into the pseudo pregnant mother and the progeny was checked for the transgene expression as in the ESCs method. 

Figure 3: Generation of transgenic animal by microinjection method

Other techniques followed to generate transgenic animals are listed in table 1.

TRANSGENIC TECHNIQUES	INTERPRETATION
Cre-lox technique	Ideal technique with more control over resulting phenotype; time-consuming
Viral vectors	difficult; largely restricted to avian species
Cytoplasmic injection	Less efficient than direct pronuclear microinjection
Primordial germ cells	Chimeric animals result
Nuclear transfer	Large potential for genetically modifying livestock
Spermatogonial manipulation	Transplantation into recipient testes

Table 1: Other techniques used to generate transgenic animals

APPLICATION OF TRANSGENESIS

1. Disease resistant transgenic animals:

Selection and cross breeding of animals is a natural way of producing superior quality livestock animals with respect to disease resistance, more milk production, larger size etc. However, maintaining these qualities to be passed to the generations is unpredictable.

1. Neurodegenerative disease resistant animals: Spongiform encephalopathy (Scrapie disease) in sheep, bovine spongiform encephalopathy (BSE) (Mad cow disease) in cattle, Creutzfeldt Jacob disease in humans are some of the major neurodegenerative diseases. These diseases occur because of the expression and misfolding of “prion” protein. ‘Gene knock out’ of ‘prion protein through rDNA technology helps in generating prion protein free livestock and resistant to neurodegenerative disorders. RNA interference (RNAi) is the new rDNA technique that helps in knocking down of the desired gene by forming a double stranded DNA construct and suppressing its expression.

RNAi method has extensive applicability, one of which is to generate knock down transgenic animals that can survive RNA based viral infections like foot and mouth disease, classic swine fever and the most resent SARS-CoV-2 disease.

Figure 4: Prion free sheep (Denning et al 2001)

• Bacterial disease resistant cattle: Mastitis is a bacterial infection of the mammary gland in the cows that affects quality and quantity of the milk being produced. Scientists have developed transgenic cattle that express lysostaphin protein, which kills mastitis causing bacteria by cleaving their cell wall.

Similarly, lysozyme producing transgenic goats are also generated that prevents mastitis causing bacterial lysis and healthier mammary glands.

• Disease resistance in fishes: Catfish often prone to microbial infection and death. Cecropin B is a small protein expressed in Hyalophora cecropia moth that has anti-microbial protperties. Scientists have generated Cecropin gene expressing transgenic catfish that confers resistance against microbial infections.
• Disease resistant cattle against Brucellosis: Brucellosis is a deadly zoonotic disease, that can spread across animals without limit and even to humans. A large number of animals in American Bison area have been affected badly and the grazing cattle used to acquire the infection that lead to abortions, low fertility rates, reduced milk production etc. In humans, the disease is called undulant fever and its effects are severe. Recently, it was discovered that bovineNRAMP1 gene is efficient in providing resistance to brucellosis. Transgenic cattle with this gene offer protection against the disease.

Mastitis resistant transgenic cow (Agricultural research service, US)

Identification and integration of genes through transgenesis is the need of the hour that provides disease resistance and improved immune response in livestock and poultry. Scientists are focusing on genetic engineering based disease resistant animal models that would help livestock show resistance to diseases as shown in the table 2.

Table 2: Diverse applications of disease-resistant genetically engineered animals.

• Medical applications:
• Disease models: Understanding the disease and its effects are crucial for effective drug development and vaccine creation. Transgenic method is widely applied to generate disease models to understand causes and effects of human diseases. For example, mouse with various cancers or cystic fibrosis were produced through rDNA technology. These models give insights into the disease and further effective drug development and treatment.
• Understanding gene functions: Mouse/rat genetic composition is closely related to humans. Hence, mice or rat models are chosen to produce genetically modified organism with alteration of gene like, gene knock-out (removal of a particular gene) or gene knock-in (insertion of a gene) or damaging the gene.

This category of transgenic models helps to understand the crucial functions of the gene and its role in human development and disease. This method is widely used to produce transgenic animals with superior quality. For example, transgenic cow – with disease resistance and improved milk production.

• Production of therapeutic proteins and antibodies: Animals like horse, goat and cows were genetically modified to develop and secrete useful chemical substances like antibodies, and therapeutic proteins that help treat the human diseases efficiently. For example, transgenic cow that secretes egg proteins into its milk. These transgenic animals with therapeutic reagents production are also called ‘walking pharmacies’.
•  Production of xenotransplants: Scientists have developed transgenic farm animals by ‘knocking out’ the gene that is responsible for eliciting immune response and rejection of an organ when introduced into the human body. For example, knock out transgenic pig organs can be now used for organ transplantation in humans. This method solves the issue of organ donor shortage and saves many critical lives.
• Transgenic fishes: Transgenic fishes are produced by introducing genes responsible for disease resistant/temperature tolerant/ better growth etc. For example, a company called Aqua Bounty Farms has requested United States Food and Drug Administration (USFDA) to approve its genetically modified salmon that has the ability to grow three times bigger size than normal within a year of its growth.

DISADVANTAGES OF TRANSGENIC ANIMALS

• Genetically modified animals (like disease laboratory study models) will show negative impact on ecosystem if they escape and released into the environment.
• May act as human disease reservoirs for critical pathogens like virus, prions etc.
• May cause severe allergies in humans as it is not a natural product.
• Genetically modified animals may show alterations in its behavior if the foreign gene undergoes any changes like mutations, leading to any unexpected harm to the mankind.

Even though, the chance of adverse effects is minimal, one can’t rule out completely.

ETHICAL CONCERNS OF TRANSGENIC ANIMALS

It is considered as unethical to produce transgenic animals because it is kind of violating animal rights and disrespect to animals.

Unless there is a balance maintained between need and the production of transgenic animals and effective application in human welfare like medical purpose, agriculture and scientific understanding and application.

CONCLUSION

From its origin, transgenic method has been creating revolutionary output for human well being by producing therapeutic proteins, superior quality breeds of animals and plants and xenografts. A transgenic animal has full potential to play a significant role in biomedical field. However, it is important to maintain ethical standards in effective usage of transgenic method for human welfare. As there is an equal chance of enormous harm that may cause to humans and the environment with the misuse of the technique.

MOLECULAR MARKERS

BY: Reddy Sailaja M (MSIWM030)

A molecular marker is a specific gene fragment present at a specific position called ‘locus’ (pleural loci) in the genome of a cell. These molecular markers are ‘phenotypically neutral’ i.e., they won’t exhibit any genotypic or phenotypic properties. Rather, they ‘flag’ or give ‘sign’ of a particular gene, its functions, variations and inheritance. They act as ‘tags’ if they are present in close proximity of a gene of interest.

These markers help to detect a particular character/trait by analyzing the variations that occur in a particular gene fragment over a period of time.

Ideal characteristics of a molecular marker:

• Polymorphic
• Co-dominant inheritance
• Frequent and even incidence across the genome
• Easy, cost and time effective to use
• Consistency in results
• Reliable data across globe
• Reproducibility

There are three types of molecular markers as follows:

1. Morphological markers: These markers are widely used in animal breeding and selection of superior quality farm animals. Morphological characters like skin color, body structure, coat color etc. were considered on visual observation and classify superior quality breeds. However, this technique is not always accurate.
2. Cytological markers: These are used to identify proper location of a gene, its genetic diversity with respect to chromosome number and structure in the domesticated animals in comparison to their wild ancestors. Karyotypes, translocations, insertions, deletions, repeats etc. are the characteristics of these cytological markers to be investigated to understand the function of the gene of interest in inheritance, principally in the origin and phylogenetic classification of a species.
3. Biochemical markers: Blood type and isozymes are the major biochemical markers that are being investigated at protein level with respect to amino acids composition. These are helpful in understanding phylogenetic relationships at intra and inter-species level. But these are not widely used as proteins are not genetic entities, but an end product of the expressed gene after many modifications.

Figure 1: Types of molecular markers

In short, molecular markers at gene level are the more reliable markers to understand variations and inheritance of a particular gene.  

In this session, four major types of molecular markers are discussed as follows:

1. Restriction Fragment Length Polymorphism (RFLP): This is one of the early and widely used techniques for DNA analysis. Main principle of the technique is to generate restriction fragments with different sizes that were formed because of nucleotide base insertions, deletions, substitutions, inversions, and duplications etc. in the gene of interest that belongs to the same species. This technique helps scientists to generate gene map/ profile/ finger printing of a particular disease. This technique helps to understand the genetic disease inheritance within the family like hemophilia, a rare blood disorder. Autoradiography (using radioactive probes) or Chemiluminiscence (enzyme linked probe labeling) are the common methods used to visualize the RFLP results.

Figure 2: RFLP process illustration

Advantages:

• Simple
• Co-dominant markers expression
• No PCR is requirement
• Distinguish homozygous or heterozygous condition

Disadvantages:

• Needs large amount of pure DNA
• Identification of suitable markers is laborious
• Time consuming
• Requires trained technician to operate

• Random Amplified Polymorphic DNA (RAPD):

RAPD is the most widely used technique to develop DNA markers. It uses short, random oligonucleotide primers (10 – mers) that amplify random sequences at various loci and the PCR to detect variations in the genome. This is a dominant marker selection system and detects polymorphism by analyzing difference in the primer binding site in the DNA sequence between closely arranged sequences of less than 2kb (kilobases).

Figure 3: RAPD process illustration

Advantages:

• Requires small amount of DNA
• Doesn’t require specific primers
• Detects polymorphism effectively

Disadvantages:

• It’s a dominant trait
• Sensitive to PCR conditions
• Generation of non-parental bands in the progeny of known pedigree warns its use
• Not reproducible

• Amplified Fragment Length Polymorphism:

AFLP is a combination of RFLP and polymerase chain reaction (PCR) techniques that detects variation in the DNA sequence from two individuals of a species. Whole genome is digested with the known and rare restriction endonuclease to generate DNA fragments. Adapters are linked to these DNA fragments and primers that are complimentary to these adapters are used to amplify the DNA fragments.

Fragments that are generated by PCR are analyzed using agarose gel electrophoresis. AFLP technique was majorly used in determining genetic variation in the population and has applicability in phenotyping, population genetics, DNA finger printing and quantitative trait loci (QTL) mapping.

Figure 4: AFLP process illustration

Advantages:

• Sensitive – can distinguish homo and heterozygotes
• Wide range of applicability
• Gene mapping

Disadvantages:

• Expensive
• Require large amount of DNA
• Needed trained personnel for sequencing the gels.
• Simple Sequence Repeats (SSR)/Microsatellites:

SSRs are 1 – 6 nucleotides in length and are present throughout the genome as repeats in most of the eukaryotes and a few prokaryotes. They occur as di-, tri-, tetra nucleotide repeats that occur 5-20 times in the genome. The number of repeats varies among different alleles of a gene among population. SSR uses unique sequences as primers that act as flanking regions of a specific DNA fragment. These DNA fragments are further amplified by PCR to generate enough DNA for visualization on agarose or polyacrylamide gels.

Figure 5: SSR process illustration

Advantages:

• Simple to use
• Co-dominant marker
• Map based cloning
• Used to identify genetic distances between population, inbreeds and breeding material during evolution.

Disadvantages:

• Development of proper primers for the satellite region is time consuming and costly.
• Require DNA sequencing

Major applications of molecular markers:

• DNA fingerprinting
• Measure of genetic diversity among the species
• Selection of a Genotype
• Marker assisted selection of a particular trait

HUMAN INSULIN PRODUCTION

BY: Reddy Sailaja M (MSIWM030)

INSULIN

Insulin is a peptide hormone that plays a critical role in human metabolism. It is synthesized and secreted by beta cells of Islets of Langerhans in the pancreas. It is the first peptide hormone to be discovered (by Frederick Banting and Charles Herbert Best, 1921). It is the first protein to be sequenced in 1951 by Frederick Sanger. Dorothy Hodgkin has determined the crystal structure of insulin in 1969. Nevertheless, it is the first hormone to be synthesized by recombinant DNA technology.

Figure 1: Structure of insulin

STRUCTURE AND FUNCTION

 Human insulin is made up two polypeptide chains of 51 amino acids (A-chain- 21 amino acids and B-chain, 30 amino acids) with a molecular mass of 5808 Daltons.  Insulin is an anabolic hormone that plays a crucial role in the metabolism of carbohydrates and fats by converting the free glucose available in the blood into glycogen that can be stored in the muscles.

Figure 2: Functions of insulin

SYNTHESIS

 Insulin is synthesized as a single polypeptide called ‘preproinsulin’ along with a 24 residue signal peptide in the pancreatic beta cells. The signal peptide guides preproinsulin to endoplasmic reticulum (ER), where the signal peptide gets separated, resulting in ‘proinsulin’ formation. In the ER, the proinsulin is further processed and folded with the formation of three disulphide bonds and gets transported to golgi complex. In golgi, the folded proinsulin is converted to ‘active insulin’ by cellular endopeptidases, namely prohormone convertases 1 & 2 and exoprotease carboxypeptidase E. These endonucleases cleave at two positions in the proinsulin, resulting in the separation of a fragment called C-peptide. The active and mature insulin now consists of two chains: A-chain (21 amino acids) and B-chain (30 amino acids), both liked to each other by two disulphide bonds.

Figure 3:  Synthesis of active insulin from precursor

INSULIN MALFUNCTION AND THE ASSOCIATED DISEASES

Insulin helps maintain blood sugar level normal at all the times. When the blood sugar level is high, insulin directs liver to store glucose in the form of glycogen. In need, insulin directs the liver and muscles to release the stored glycogen in the form of glucose to boost energy to the body.

When the insulin production is less or uncontrollable, malfunction of the hormone results in the development of a condition called as diabetes mellitus (DM), where the body is unable to maintain balance between normal blood sugar levels and sysnthesis or breakdown of glycogen. Malfunction of Insulin hormone leads to two major types of diabetes milletus: Type 1 and Type 2.

Type 1 DM: It is an autoimmune disease, where one’s own immune system attacks the pancreatic cells and results in low or no insulin production. Environmental factors, genes and certain viruses trigger the immune system to damage the pancreatic cells.

Type 2 DM: The condition develops either by low insulin production by pancreatic cells or inability of the body to utilize the released insulin for glycogen synthesis. Insulin resistance is the condition developed when the major organs like muscles, body fat and liver starts ignoring the signals form insulin and fail in converting free glucose into glycogen. As more insulin is being produced, pancreatic cells get damaged and the free glucose (that was not being stored) affects the body with surge of energy. This, type 2 DM is a lifestyle disease that results majorly of  over body weight, lack of exercise, smoking, lower belly fat etc.

HUMAN INSULIN PRODUCTION BY RECOMBINANT DNA TECHNOLOGY

 Recombinant DNA technology is a revolutionary technique, where DNA molecules from two different organisms are joined together and inserted into a host organism in order to generate new genetic combination that adds value to varied fields like science, health care, agriculture, poultry and industry.

Human insulin is being produced by recombinant DNA technology using E.coli or Saccharomyces cerevisiae as host organisms in many ways. The popular one is the production of insulin A-chain and B-chain separately in two E.coli strains and then joined together by disulphide bonds to produce active insulin.

The mRNA sequence of A-chain (basically, mRNA is a blue print of functional protein after modifications) is fused with ß-galactosidase gene (lac Z) present in the pBR322 plasmid (now called recombinant plasmid) and inserted into E.coli by transformation process. The recombinant bacteria is allowed to grow in the presence of an antibiotic, so that only transformed E.coli with A-chain will be selected. The whole lacZ gene and the fused A-chain will synthesize ß-galactosidase enzyme and A-chain. Similarly the B-chain was also synthesized separately.

Both the chains are purified from bacteria, combined, oxidized and reduced to form disulphide bridges to produce active insulin.

Figure 4: Human insulin production using recombinant DNA technology

Recombinant human insulin was first approved in 1982 for human administration. Humulin, is the first human insulin that was released into market in 1986.

Major insulin manufacturers include: Novo Nordisk A/S (Denmark), Sanofi S.A. (France), Eli Lilly and Company (U.S.), Bioton S.A. (Poland), Wockhardt Ltd. (India) and Julphar (UAE).

SECONDARY METABOLITE

 BY: SHREE LAKSHMI (MSIWM012)

Second metabolites are natural compounds that are not directly involved in normal growth, growth or metabolism. The second metabolism of the plant produces products that help the growth and development of the plant but are not necessary for the plant to survive. A common role of secondary metabolites in plants is to protect the egg that is used to fight weeds, insects and germs. In humans they plant the second most important metabolite as it is used in many fields such as medicine, flavoring agent, dye etc. The main contributors are the specific aroma, color and taste of the plant parts. The secondary metabolites play a role and play an important role in potential immune systems, especially in the chemical warfare between plants and their bacteria. Some of these compounds have also been shown to play a role in the fight against pests and pollinators, allopathic agents, and toxins, UV light protection, and signal transmission. Food, cosmetics, drugs and their important role in plant protection.

 The general role of secondary metabolites

 Plants to protect plants such as weed control, weed control and insecticides. For humans, plant second most important metabolites as they are used in many fields such as medicines, flavoring agents, dyes etc.

 Functions of secondary plant metabolites

• Major activities include

• Competitive weapons against biological agents such as animals, plants, insects and micro-organisms.

• Metal transport agents

• Agents for compatibility with other organisms

• Reproductive agent

• They make a difference

• Biodiversity coordinators

CONSTRUCTION OF SECONDARY PLANT METABOLITE

Alkaloids

 An alkaloid is defined as a natural or synthetic product, which contains one or more nitrogen atoms, usually heterocyclic, and contains certain substances in the human or animal body, if used in small amounts. Most are based on a few amino acids. Chemicals have a ring structure and nitrogen residues. Indole alkaloids are the largest group in the family, based on tryptophan. They are widely used as medicine.

 Structure

 It is hard, crystalline and has a sharp melting point. Some alkaloids are amorphous compounds and some like coniine, nicotine are naturally liquid. Some alkaloids are naturally colored e.g. Betanidine shows red, Berberine shows yellow Most of the alkaloid salts are dissolved in water. Water bases only melt in water. Pseudo and proto alkaloids provide high solubility in water. Melting and alkaloid salts help the pharmaceutical industry to extract They provide the color edge with a halogenated compound

Chemical properties

• Basic in response

• It turns neutral or acidic when active groups are associated with the release of electrons such as amide grp.

• Formation of salt by inorganic Acid – their decomposition during storage

• It contains one or more nitrogen

• Natural – can be in the form of free salt such as amine or as a salty acid or alk. N-oxide

TERPENES

 Various kinds of fruits and flowers and spices. The word is derived from the word turpentine. Turpentine, also known as “pine resin”, is viscous, fragrant, and does not melt in flowing water when cutting or carving new bark and wood of pine species (Pinaceae). Turpentine contains “resin acids” and other hydrocarbons, formerly called terpenes. They are used in cloves, essential oils and medicines. Terpenes are mainly composed of Fusion of 5 Carbon Isoprene Units The basic elements of the structure of terpenes are sometimes called units of isoprene because terpenes can decompose at high temperatures to provide isoprene. All terpenes are sometimes called isoprenoids. Therefore, Terpenes are unused compounds made by joining together isoprene units.

Terpenes are divided into five carbon components.

• Ten terpenes, containing two units of C5, are called monoterpenes

• Three carbon terpenes (three C5 units) are sesquiterpenes

• Two carbon terpenes (four C5 units) are diterpenes.

• Large terpenes include triterpenes (30 carbons), tetraterpenes (40 carbons), and polyterpenoids

TERPENOIDS

Are hydrocarbons of plant origin of the common formula (C5H8) n their oxygen, which comes from hydrogenated and water.

Body structures

 • Colorless liquid, soluble in natural solvents and soluble in water, active, environmentally friendly, boiling area: 150-180 ° C.

Chemical properties

Impure Chemicals, They deal with additional reactions with hydrogen, halogen acid to form additional products such as NOCl, NOBr and hydrates. They can undergo polymerization and dehydrogenation in the ring In hot rot, terpenoids release isoprene as early as production.

Terpenoids are classified based on the number of rings present in terpenoids

• Acyclic terpenoids (Eg Citronellal, Citral)

• Monocyclic terpenoids (Ex. Menthol, alpha-terpineol)

• Bicyclic terpenoids (The size of the first ring is the same in all, and the size of the second ring varies). Depending on the size of the second ring, the other is divided –

1. From Bornane (Eg Camphane)

2. Based on Norbornane

• I-terriceno terriceno

• Tetracyclic terpenoids

 Phenolics

 The phenolic compounds of benzene have one or more hydroxyl groups.The plants are produced mainly to protect against stress.They are widely distributed in the plant kingdom.Many second plant metabolites. Forms 40% plant soluble protein. More than 8000 phenolics properties are known. Lignin (polyphenol) is the second most abundant compound in the plant

 Sources of phenolics

• Fruits

• Vegetables

• Books

• Olive

• Legumes

HUMAN GENOME PROJECT

BY: SREE LAKSHMI (MSIWM012)

The Human Genome Project was an international research effort to determine the genetic makeup of humans and to identify the genes that it contains. The Human Genome Project was launched in 1984 but officially launched in October 1990. It is a major international co-operation program, with the ultimate goal of achieving a “nucleotide sequence across nuclear genetics”. The Global Research Group consisted of six different countries namely USA, UK, France, Germany, Japan and China as well as several laboratories, a large number of scientists and experts from various fields.

Objectives of the Human Genome Project:

1. Establish a complete genetic sequence and make it easy to access.

2. Improve sequencing technology by developing new and more efficient methods.

3. Analyze genetic variation in the human genome, such as single nucleotide polymorphisms (SNPs) and other DNA sequence variants.

4. Improving the performance of genomics technology. Includes the creation of additional cDNA sources and detailed genetic analysis technologies; a complete study of non-protein coding sequence activities; and to promote the development of global protein analysis technologies.

5. Learn comparative genomics by completing a sequence of a particular type of model (e.g. mouse etc.) that can enhance our understanding of human genes.

6. Consider the moral, legal and social consequences of a growing knowledge base. It is expected that the conflict between this new and advanced knowledge and existing philosophical ideas could have undesirable consequences, which need to be addressed.

7. Develop bioinformatics and computational biology to transfer advanced training to young scientists and to promote the development of academic fields in genetic research.

 Most important features of HGP:

• The human genome contains the details of 23 chromosomes

• Itcontains more than three billion nucleotides.

• The human genome is estimated to have over 30,000 genes. The average gene has 3000 bases. But gene sizes vary, and the gene dystrophin gene has 2.5 million bases.

• Only about 3% of the genome contains amino acid sequences of polypeptides and the rest is in the trash (DNA duplicated).

• Jobs are known to more than 50% of the genes found.

• Repeated sequences make up the largest part of the human genome. Repeated sequences do not use direct coding but illuminate chromosome formation, dynamics and evolution.

• Chromosome 1 has many genes (2968) and Y chromosome is very small (231).

• Almost all nucleotide bases are exactly the same for all humans. The genome sequence of different populations varies less than 0.2% of base pairs.

• Most of the differences come in the form of variations from one foundation to another. One primary variation which is called single nucleotide polymorphisms (SNPs) is derived from all ~ 1,000 bp of the human genome. About 85% of all differences in human DNA are due to SNPs.

Main Objectives:

• Find the complete sequence of DNA extracted from cells donated by several unknown donors, to determine the sequence of DNA in each chromosome.

• Genetic mapping to simplify genetic linking studies.

• Obtaining all human genes to allow for the continuation of human genetic studies.

• Develop simple and automated DNA sequencing technologies.

Updated objectives:

• Determining the sequence of human DNA

• Identify all the genes in a person’s DNA

• Store this information in archives

• Improving data analysis tools

• Transfer of technology related to the private sector

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

Project result:

• Has a base base pair of 3164.7

• Genetics have 3000 bases, but sizes vary widely. The gene known as dystrophin has 2.4 million bases.

• The total number of genes is estimated was 30,000.

• Jobs are known to more than 50% of the genes found

• Less than 2% of genome protein codes.

• Dense men with “urban middle-class genomes” are made up of DNA blocks of G and C.

• In contrast, genetically engineered “deserts” are rich in DNA A and T genes.

Chromosome 1 has many genes (2968), and Y chromosome has very few (231). Scientists have identified about 1.4 million places where single DNA (SNPs) mutations occur in humans, and these findings will help to link disease sequences to chromosomes. Cancer with the help of human maps (SNPs) produced in the Human Genome Project.

Applications:

• Identification of human genes and their functions.

• Understand polygenic disturbances e.g. cancer, high blood pressure, diabetes

• Advances in gene therapy

• Advanced diagnosis

• Development of pharmacogenomics

• Genetic basis for mental disorders

• Understanding the norms of the general public

• Advanced knowledge of genetic modification

DNA SEQUENCING

BY: SREE LAKSHMI (MSIWM012)

Maxam-Gilbert sequence:

In 1976-1977, Allan Maxam and Walter Gilbert developed DNA sequences based on chemical reactions and subsequent purification. It depends on the associated chemical bond of different nucleotide bonds. Chemical degradation is also known. Chain jumping method is the most widely used method due to its speed and ease. This process requires the installation of radiation labels on one side and the cleaning of the DNA fragment to be followed. The following chemical treatments are:

• G reaction: Dimethyl Sulfate + Piperidine treatment

• A + G Response: Dimethyl Sulfate + Piperidine + Formic Acid Treatment

• T + C reaction: Hydrazine + Piperidine

• C reaction: Hydrazine + Piperidine + 1.5M Sodium Chloride

• Dimethyl sulfate attacks the purine ring (A & G)

• Attack of Hydrazine ring pyrimidine (C&T)

• Piperidine restores the binding capacity of the phosphodiester where the base is removed

 Chemical therapy creates breaks with a small amount of one or two four nucleotides based on each of the four reactions (G, A + G, C, C + T). So a series of labelled pieces were made, from the end of the radio beams to the first ‘cut’ site in each molecule. The four reaction fragments were arranged separately on the gel electrophoresis for size separation. These fragments are shown using a gel shown on an X-ray film of autoradiography showing a series of black bands attached to each radiolabelled DNA fragment, respectively.

Benefits

• Pure DNA can be read directly
• Homopolymeric DNA run is best followed as a unique DNA sequence
• It can be used to analyze the interaction of DNA proteins
• Can be used to analyze the formation of nucleic acids and epigenetic changes in DNA

Disadvantages:

• Requires extensive use of hazardous chemicals.
• He has advanced technology.
• It is difficult to “grow” and cannot be used to analyze more than 500 basic pairs.
• Learn reading length decreases from incomplete response to completion.
• It is difficult to make Maxam-Gilbert’s DNA kits based on sequence.

Sanger sequence

Also known as chain termination method or video sequencing method. The terminator process or video sequencing process of DNA sequences in two layers of DNA polymerases:

• Their ability to faithfully integrate a complimentary copy of a single-tailed DNA template.
• Their ability to use 2 ‘, 3’-dideoxynucleotide as substrates

 When the analog is embedded in a growing DNA sequence, the 3 ‘end has no hydroxyl group and is no longer a substrate for chain expansion. Thus, the growing DNA sequence is broken, meaning that dideoxynucleotide acts as terminators. In practice, the Klenow piece of DNA polymerase is used because this does not have 5 ‘→ 3’ remission functions associated with an incomplete enzyme. DNA synthesis regeneration needs to be done first and this is usually the chemical oligonucleotide bound to the sequence of analyzes. dideoxynucleoside triphosphate. Thus, in each reaction there are fragments of active DNA fragments that are slightly divided, each with the same end of 5, but each varies in length to the end of a particular 3. After a good incubation period, the DNA in each structure was shown to be electronically converted into a successive gel. The terminator chain method works very well and uses fewer toxic chemicals and a lower amount of radioactivity than the Maxam and Gilbert method. The main purpose of the Sanger method was to use dideoxynucleotide triphosphates (ddNTPs) as breakers in DNA chains.

WORKING

This chain-breaking process requires a single-stranded DNA template, DNA primer, DNA polymerase, radiotide or shiny nucleotides, and modified nucleotides that complete the DNA strand expansion. The DNA samples was divided into four sequences, which included all four deoxynucleotides dATP, dGTP, dCTP, dTTP and also DNA polymerase. In each reaction only dideoxynucleotide (ddATP, ddGTP, ddCTP, and ddTTP) is added to the nucleotide terminals, lacking the 3′-OH group needed to form a phosphodiester bond between the two nucleotides, thus ending DNA expansion strand and lead to DNA fragments of various lengths. Freshly formed and transcribed DNA fragments are burned, and measured with gel electrophoresis in a gel that explains the polyacrylamide-urea gel for each of these four processes is performed in one of four ways (lines A, T, G, C). DNA strands are then detected by autoradiography or UV light, and DNA sequences can be read directly from an X-ray film or gel image. The black band on the track shows a DNA strip that leads to the removal of chains after the introduction of dideoxynucleotide (ddATP, ddGP, ddCTP, or ddTTP). chain fragmentation involves the marking of nucleotides containing radios with a phosphorus label, or the use of a labeled primer at the end of 5 in a fluorescent color.Dye-primer sequences help optical system study to speed up analysis and cost.

NUCLEIC ACIDS

BY: SREELAKSHMI S (MSIWM011)

Nucleic acid molecules maintain details of cell growth and reproduction. These are polymers that contain long chains of monomers called nucleotides. Nucleotide has a base of nitrogenous, pentose sugar and phosphate group .There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

• Purines: Adenine (A) and Guanine (G)

• Pyrimidine: Cytosine (C), Thymine (T) and Uracil (U)

• Pentose Sugars: There are two related pentose sugars:

• RNA contains ribose

• DNA contains deoxyribose

Sugar contains its carbon atoms composed of primes to separate them from nitrogen bases

Nucleosides

The nucleoside contains a nitrogen base attached to the glycosides bond to C1 ’of ribose or deoxyribose.Nucleosides are named for the substitution of the nitrogen base ending in -osine purines and -idsine pyrimidine

Nucleotides

Nucleotide is a nucleoside that forms a phosphate ester with the C5 ‘OH group of ribose or deoxyribose.Nucleotide is named after the nucleoside term followed by 5’-monophosphate. Additional phosphate groups can be added to nucleoside 5’-monophosphates to form triphosphates and triphosphates. ATP is a major source of energy for cellular activity

The main structure of Nucleic Acids:

The main structure of nucleic acid is the nucleotide sequence. Nucleotides in nucleic acids are associated with phosphodiester bonds. Group 3’-OH sugar in one nucleotide forms an ester bond in the phosphate group to 5’-carbon sugar for the next nucleotide. The nucleic acid polymer has a free 5′-phosphate group at one end and a free 3′-OH group on the other. Sequences are learned from 5′-end free using base characters.

 In RNA, A, C, G, and U a 3′-5 ‘ester bond is connected between ribose and phosphate

In DNA, A, C, G, and T are linked by 3’-5’ ester bonds between deoxyribose and phosphate

 

DNA Double Helix

In 1953 Watson and Crick wrote a three-dimensional model of the DNA structure (The Double Helix) In DNA there are two strands of polynucleotides that combine around the same axis to form a double right helix. The hydrophilic nuclei of deoxyribose groups interacting with phosphates are outside the double helix, facing the surrounding water. The strands work on opposite sides of the bases arranged in pairs such as steps .two bases are held together by binding hydrogen. The pairing of the bases from both ropes is very clear

Two basic compliments are A-T and G-C

The two forms of hydrogen bond are between A and T

The three forms of hydrogen bond are between G and C

Each pair contains purine and pyrimidine, so they have the same width, keeping the two strands the same distance from each other. In the first type proposed by Watson and Crick, the adjacent bases are separated by 3.4Å.

In eukaryotic cells (animals, plants, fungi) DNA is stored in the nucleus, which is separated from the cell by an entire insignificant membrane. DNA is organized only by chromosomes during cell duplication. During reproduction, DNA is stored in a compound ball called chromatin, and it is wrapped in a protein called histones to form nucleosomes

DNA types:

The Watson-Crick structure is also called B form DNA, or B-DNA. Form B is the most stable structure. The two structural variations that are best represented in crystal structures are types A and Z. Form A is popular in many waterless solutions. DNA is still arranged with a double helix of the right hand, but the helix is ​​wider and the number of base pairs per helical turn is 11, rather than 10.5 as in B-DNA. The base pair of A-DNA pairs is approximately 200 in relation to the helix axis. These structural changes deepen the large groove while making the small groove less shallow. The Z-form DNA difference is within the left helical exchange. There are 12 basic pairs per helical curve, and the structure appears very small and compact. The DNA sequence assumes a zigzag pattern. The large groove is hardly visible in Z-DNA, while the small groove is small and deep. Whether A-DNA is derived from cells is uncertain, but there is evidence of some simple Z-DNA strands in both prokaryotes and eukaryotes.

Differences between RNA and DNA:

o pentose sugar in RNA is ribose, in DNA is deoxyribose

o In RNA, uracil replaces basic thymine (U pairs in A)

o RNA is left alone while DNA is doubled

RNA molecules are much smaller than DNA molecules

Three main types of RNA:

Ribosomal (rRNA), messenger (mRNA) and transmission (tRNA)

Ribosomal RNA

It is part of the RNA of the ribosome

It is a large part of the ribosomes. Protein synthesis is important. Ribosomes are areas of protein synthesis. They contain ribosomal rRNA (65%) and protein (35%).Ribosomal RNAs make up two subunits, a large subunit (LSU) and a small subunit (SSU).

Messenger RNA

      They are RNA strands that attach to the DNA of a component so that the protein is synthesized .They carry details (genetic code) of protein synthesis from DNA in a fraction of the nucleus to ribosomes.

Transfer RNA

RNA transfer translates genetic code from the RNA messenger and delivers certain amino acids to the ribosome for protein synthesis. Each amino acid known as one or more tRNA.tRNA has an L-shaped higher education structure. The structure is compacted and reinforced with foundation bonding and foundation installation. One end binds to amino acids and the other binds to mRNA in a favorable 3-base sequence.

MITOSIS

                                       BY: ABHISHEKA (MSIWM013)

INTRODUCTION:

1. Mitosis is a type of cell division that takes place in living organisms and it is commonly defined as the process of duplication of chromosomes in eukaryotic cells and distributed during cell division.
2.  The process where a single cell divides resulting in two identical cells, each resulted cell contains the same number of chromosomes and same genetic composition similar to the parent cell.
3. Mitosis was first discovered in plant cells by Strasburger in 1875. In 1879, mitosis is also discovered in animal cells by W. Flemming. Flemming in 1882 gave the term Mitosis.
4. The term mitosis is derived from the Greek word such as ‘Mitos’ means thread.

OCCURRENCE OF MITOSIS:

1. The mitosis takes place in somatic cells. The cells which undergo mitosis are called  Mitocytes.
2. In plants, the mitocytes are called meristematic cells. Some of the major sites of Mitosis in plants are root apex, shoot apex, intercalary meristem, lateral meristem, leaves, embryo, and seeds.
3. In animals, the mitocytes are stem cells, germinal epithelium, and embryonic cells. In animals, it mainly takes place in Embryo, skin, and bone marrow.
4. Mitosis also occurs during the regeneration of the cells. The mitosis takes place for three main reasons such as growth, repair, and asexual reproduction.

NUCLEUS:

It is a membrane-bound cell organelle present in both animal and plant cells. It is the center of the cell where genetic material is stored in the form of DNA. The DNA is arranged into a group of proteins into thin fibers. During the Interphase of the cell division, the fibers are uncoiled and dispersed into the chromatin. During mitosis, these chromatin condenses to become chromosome.

 CHROMOSOME:

The chromosomes carry genetic material and they are made of DNA. The mitotic chromosomes possess two sister chromatids, they are narrow at the centromere. They also contain identical copies of original DNA. These Mitotic chromosomes are homologous, they are similar in shape, size, and location of centromere.

STAGES OF MITOSIS CELL DIVISION:

The mitosis cell division is broadly explained in two stages such

1. Karyokinesis: Division of Nucleus. Greek ‘karyon’ means the nucleus, whereas ‘kinesis’ means movement.
2. Cytokinesis: Division of Cytoplasm.

KARYOKINESIS:

4 different stages that take place in Karyokinesis.

1.Prophase

2.Metaphase

3.Anaphase

4.Telophase

PROPHASE:

1. The nucleus becomes spherical and the cytoplasm becomes more sticky.
2. The chromatin slowly condenses into well-defined chromosomes.
3. During Prophase, the chromosomes appear as a ball of wool. The chromosomes consists of two threads which are longitudinal known as chromatids.
4. The chromosomes appear as two sister chromatids joined at the centromere.
5. The microtubules are formed outside the nucleus.
6. In plant cells, the spindle apparatus is formed without centriole. In animal cells, the centriole is divided into two moves towards opposite poles.

METAPHASE:

1. Nuclear envelop breaks down into membrane vesicles and the chromosomes are set free into the cytoplasm.
2. Chromosomes are attached to spindle microtubules through kinetochore.
3. Nucleolus disappears.
4. Kinetochore microtubules arrange the chromosomes in one plane to form a central equatorial plate.
5. Centromeres lie on the equatorial plane while the chromosome arms are directed away from the equator called auto orientation.
6. Smaller chromosomes remain towards the center while larger ones arrange at the periphery.
7. Metaphase is the longest stage of Mitosis and takes place for about 20 minutes. It is the best stage to study the structure of chromosomes.

ANAPHASE:

1. Chromosomes split simultaneously at the centromeres so that the sister chromatids separate.
2. The separated sister chromatids move towards the opposite poles.
3. The daughter chromosomes appear in different shapes such as V-shaped(metacentric), L-shaped(sub-metacentric), J-shaped(acrocentric), rod-shaped(telocentric).
4. The spindle fibers are attached to the centromere and pull the chromosomes to the poles.
5. Anaphase is the shortest stage of Mitosis.

TELOPHASE:

1. Daughter chromosomes arrive at the poles. Kinetochore microtubules disappear.
2. Chromosomes uncoil into chromatin.
3. Nucleolus reappears. The formation of nuclear envelope occurs around each pair of chromosomes.
4. The Viscous nature of the cytoplasm decreases.
5. Telophase is called a reverse stage of the prophase.

CYTOKINESIS:

Cytokinesis is defined as the division of cytoplasm.

1. It starts during the anaphase and is completed by the end of the telophase.
2. It takes place in 2 different methods.

a) Cell plate method: It takes place in plant cells. The vesicles of Golgi fuse at the center to form a barrel-shaped phragmoplast. The contents of the phragmoplast solidify to become a cell plate, this cell plate separates the two daughter cells.

b) Cleavage or cell furrowing method: It takes place in Animal cells. In this method, a Cleavage furrow appears in the middle, which gradually deepens and breaks the parent cell into two daughter cells.

SIGNIFICANCE OF MITOSIS:

1. Mitosis is called as an equational division in which daughter cells produced are identical.
2. It maintains the constant number of chromosomes and genetic stability in somatic and vegetative cells of the living organisms.
3. It helps to increase the cell number so that zygote transforms into a multicellular adult.
4. Healing wounds takes place by Mitosis.
5. It helps in asexual reproduction.
6. Mitosis is necessary for growth, maturity and to repair damaged cells.