Molecular Marker

Molecular Marker 

A molecular marker is a DNA sequence in the genome which can be located and identified. As a result of genetic alterations (mutations, insertions, deletions), the base composition at a particular location of the genome may be different in different plants. These differences, collectively called as polymorphisms can be mapped and identified. Plant breeders always prefer to detect the gene as the molecular marker, although this is not always possible. The alternative is to have markers which are closely associated with genes and inherited together.

The molecular markers are highly reliable and advantageous in plant breeding programmes.

• Molecular markers provide a true representations of the genetic make up at the DNA level.
• They are consistent and not affected by environmental factors. 
• Molecular markers can be detected much before development of plants occur.
• A large number of markers can be generated as per the needs.

Basic principle of molecular marker detection

Let us assume that there are two plants of the same species-one with disease sensitivity and the other with disease resistance. If there is DNA marker that can identify these two alleles, then the genome can be extracted, digested by restriction enzymes, and separated by gel electrophoresis. The DNA fragments can be detected by their separation. For instance, the disease resistant plant may have a shorter DNA fragment while the disease-sensitive plant may have a longer DNA fragment . Molecular markers are of two types.

1. Based on nucleic acid (DNA) hybridization (non-PCR based approaches). 
2. Based on PCR amplification (PCR-based approaches).

Mapping of The Human Genome

 The most important objective of human genome project was to construct a series of maps for each chromosome.

1. Cytogenetic map: This is a map of the chromosome in which the active genes respond to a chemical dye and display themselves as bands on the chromosome.

2. Gene linkage map: A chromosome map in which the active genes are identified by locating closely associated marker genes. The most commonly used DNA markers are restriction fragment length polymorphism (RFLP), variable number tandems repeats (VNTRS) and short tandem repeats (STRS). VNTRs are also called as minisatellites while STRs are microsatellites.

3. Restriction fragment map: This consists of the random DNA fragments that have been sequenced.

4. Physical map: This is the ultimate map of the chromosome with highest resolution base sequence. The methods for DNA sequencing are given in next blog page. Physical map depicts the location of the active genes and the number of bases between the active genes.

Terms Used In Tissue Culture

 A selected list of the most commonly used terms in tissue culture are briefly explained

Explant: An excised piece of differentiated tissue or organ is regarded as an explant. The explant may be taken from any part of the plant body e.g., leaf, stem, root.

Callus: The unorganized and undifferentiated mass of plant cells is referred to as callus. Generally, when plant cells are cultured in a suitable medium, they divide to form callus i.e., a mass of parenchymatous cells.

Dedifferentiation: The phenomenon of mature cells reverting to meristematic state to produce callus is dedifferentiation. Dedifferentiation is possible since the non dividing quiescent cells of the explant, when grown in a suitable culture medium revert to meristematic state.

Redifferentiation: The ability of the callus cells to differentiate into a plant organ or a whole plant is regarded as redifferentiation.

Totipotency: The ability of an individual cell to develop into a whole plant is referred to as cellular totipotency. The inherent characteristic features of plant cells namely dedifferentiation and redifferentiation are phenomenon of totipotency.

Major highlights of Human Genome Project

 Major highlights of Human Genome Project:-

The most important feature of a DNA molecule are the nucleotide sequences, and the identification of genes and their activities. 

• Approximately 1.1% to 1.5% of the genome codes for proteins.
• The number of protein coding genes is in the range of 30,000-40,000.
• An average gene consists of 3000 bases, the sizes however vary greatly. Dystrophin gene is the larget known human gene with 2.4 million bases.
• Approximately 24% of the total genome is composed of introns that split the coding regions (exons), and appear as repeating sequences with no specific functions.
• The draft represents about 90% of the entire human genome. It is believed that most of the important parts have been identified.
• The remaining 10% of the genome sequences are at the very ends of chromosomes (i.e. telomeres) and around the centromeres.
• Chromosome 1 contains the highest number of genes (2968), while the Y chromosome has the lowest. Chromosomes also differ in their GC content and number of transposable elements.
• About 100 coding regions appear to have been copied and moved by RNA-based transposition (retro transposons).
• A vast majority of the genome (~97%) has known functions.
• More than 3 million single nucleotide polymorphisms (SNPs) have been identified.
• About 200 genes are close to that found in bacteria.
• Genes and DNA sequences associated with many diseases such as breast cancer, muscle diseases, deafness and blindness have been identified.
• Human genome is composed of 3200 Mb (or 3.2 Gb) i.e. 3.2 billion base pairs (3,200,000,000).
• Human DNA is about 98% identical to that of chimpanzees.
• Repeated sequences constitute about 50% of the human genome.
• Between the humans, the DNA differs only by 0.2% or one in 500 bases.

special properties of plant cells

 Special Properties of Plant Cells:-

Among eukaryotic cells the most striking difference is between those of animals and plants. Plants have evolved a sedentary lifestyle and a mode of nutrition that means

they must support a leaf canopy. Their cells are enclosed within a rigid cell wall that gives

shape to the cell and structural rigidity to the organism. This is in contrast to the flflexible boundaries of animal cells. Plant cells frequently contain one or more vacuoles

that can occupy up to 75% of the cell volume. Vacuoles accumulate a high concentration

of sugars and other soluble compounds. Water enters the vacuole to dilute these sugars,

generating hydrostatic pressure that is counterbalanced by the rigid wall. In this way the

cells of the plant become stiff or turgid, in the same way that when an inner tube is inflflated

inside a bicycle tire the combination becomes stiff. Vacuoles are often pigmented, and

the spectacular colors of petals and fruit reflflect the presence of compounds such as the

purple anthocyanins in the vacuole. Cells of photosynthetic plant tissues contain a special

organelle, the chloroplast, that houses the light-harvesting and carbohydrate-generating

systems of photosynthesis. Plant cells lack centrosomes although these are found in many algae.

mRNA processing in eukaryotes

Messenger RNA Processing:-

A newly synthesized eukaryotic mRNA undergoes several modifications before it leaves the nucleus(Fig.). The first is known as capping. Very early in transcription the 5' -terminal triphosphate group is modified by the addition of a guanosine via a 5' -5'-phosphodiester link. The guanosine is subsequently methylated to form the 7-methyl guanosine cap. The

3' ends of nearly all eukaryotic mRNAs are modified by the addition of a long stretch of adenosine residues, the poly-A tail (Fig.). A sequence AAUAAA is found in most eukaryotic mRNAs about 20 bases from where the poly-A tail is added and is probably a signal for the enzyme poly-A polymerase to bind and to begin the polyadenylation process. The length of the poly-A tail varies, it can be as long as 250 nucleotides. Unlike DNA, RNA is an unstable molecule, and the capping of eukaryotic mRNAs at their 5' ends and

the addition of a poly-A tail to their 3' end increases the lifetime of mRNA  molecules  by protecting them from digestion by nucleases.

Many eukaryotic protein-coding genes are split into exon and intron sequences. Both the exons and introns are transcribed into mRNA. The introns have to be removed and the exons joined together by a process known as RNA splicing before the mRNA can be used to make protein. Removal of introns takes place within the nucleus. Splicing is complex and not yet fully understood. It has, however, certain rules. Within an mRNA the first two bases following an exon are always GU and the last two bases of the intron are AG. Several small nuclear RNAs (snRNAs) are involved in splicing. These are complexes with a number of proteins to form a structure known as the spliceosome. One of the snRNAs is complementary in sequence to either end of the intron sequence. It is thought that binding of this snRNA to the intron, by complementary base pairing, brings the two exon sequences together, which causes the intron to loop out (Fig.). The proteins in the spliceosome remove the intron and join the exons together. Splicing is the final modification made to the mRNA in the nucleus. The mRNA is now transported to the cytoplasm for protein

synthesis.

As well as removing introns, splicing can sometimes remove exons in a process called alternative splicing. This allows the same gene to give rise to different proteins at different times or in different cells. For example, alternative splicing of the gene for the molecular motor dynein produces motors that transport different types of cargo.

Fig. mRNA processing in eukaryotes.

WHAT ARE ENZYMES

 WHAT ARE ENZYMES?

Enzymes are biological catalysts. They increase the rate of chemical reactions taking

place within living cells without themselves suffering any overall change. The

reactants of enzyme-catalysed reactions are termed substrates. Each enzyme is

quite specific in character, acting on a particular substrate or substrates to produce a

particular product or products.

All enzymes are proteins. However, without the presence of a non-protein

component called a cofactor, many enzyme proteins lack catalytic activity. When

this is the case, the inactive protein component of an enzyme is termed the

apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The

cofactor may be an organic molecule, when it is known as a coenzyme, or it may be

a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor

is bound so tightly that it is difficult to remove without damaging the enzyme, it is

sometimes called a prosthetic group.

To summarize diagrammatically:

CO-ENZYME < INACTIVE-PROTEIN+ACTIVE PROTEIN+COFACTOR < METAL ION+COENZYME 

As we shall see later, both the protein and cofactor components may be directly

involved in the catalytic processes taking place.

Elephantiasis/Filariasis

Elephantiasis/Filariasis 

Synopsis:-

• Introduction
• Types of Diseases
• Elephantiasis
• History
• Pathogen
• Symptoms
• Prevention And Treatment
• Conclusion
• Reference

                         

                                                   Introduction

Illness or disease is that condition. In which the organism becomes Structurally and Functionally deformed or irregular. When an organism has a disease. Then some symptoms appear in his body due to his structural or functional irregularity. These symptoms are called symptoms of that disease. When some organisms found in nature enter our body cause many disease.

Types of Disease:-

Diseases are divided into two categories on the basis of their nature and causes.

       A. Congenital Disease

       B. Acquired Disease

A. Congenital Disease:-

They are disease who lives in the organism from birth These disease dries due to developmental or metabolic disorders. Examples- hemophilia, diabetes,

B. Acquired Disease:-

They are disease which dries in living beings after birth due to various factors. It is of two types

              1. Communicable or infectious disease

              2. Non-communicable or Non infectious

        

Communicable or infectious disease:-

Those are disease which are caused by living factors such as Bacteria, Viruses, Protozoa, Fungi and Warms and spreads person to person.

These are of the following Types:-

Ø 1. Protozoan Diseases

Ø 2. Helminth Disease

Ø 3. Bacterial Disease

Ø 4. Viral Disease

Ø 5. Fungal Diseases

1. Protozoan Disease:-

These parasites spread due to infection of Protozoa Example- Malaria, dysentery,

2. Helminth Disease:-

These disease are caused by different types of warms, Examples- Filariasis, Ascariasis,

3. Bacterial Disease:-

These are caused by the infection of parasitic bacteria Example- Typhoid, Pneumonia,

4. Viral Disease:-

These are caused by due to infection of Viruses, Example- Dengue, Rabies,

5. Fungal Disease:-

These are caused by different types of fungi Example- Ringworm

2. Non-communicable or Non infectious    Diseases:-

They are disease which does not spread from person to person. The causative dgents of these disease are not organism rather, They happen for redsons other than organisms.

Example- Rickets, Cancer,

Elephantiasis:-

Elephantiasis is also known as lymphatic Filariasis. It’s caused by parasitic worms and can spread from person to person through mosquitoes. Elephantiasis causes swelling of the legs or breasts (chest).

Elephantiasis is considered a neglected tropical disease (NTD). It’s mare common in tropical and subtropical area of the world, including Africa and Southeast Area.

Which is commonly found in such areas in India. Where there is mare outbreak of mosquitoes and people use dirty water mare.  

History:-

• In 1876 Joseph Bancaroft discovered the adult form of the worm.
• Wuchereria bancrofti named in honor of the astralian physician Joseph Bancroft.
• 
  
  
  

Pathogen:-

Elephantiasis is caused by parasitic worms

The worms affect the lymphatic system in the body. There are three types of worms involved,

                  1. Wuchereria bancrofti

                  2. Brugia Malayi

                  3. Brugia timori

Ø That are spread by mosquitoes.

Ø Lymphatic Filariasis is mosquitoes for example by the culex mosquito widespread across urban and semi-urban areas.

Ø Anopheles mainly found in rural areas.

Aedes mainly in endemic island in the pacific.

Symptoms:-

Mild fever and body aches.

Hardening and thickening of the skin

Swelling of the legs, arms, (chest) breast and genitals.

Prevention:-

The best way to prevent elephantiasis is to avoid mosquito bites.

Sleep in an air conditioned room or under a mosquito net at night.

Dirty water should not be used.

Treatment:-

Gently washing the swollen and damaged skin every day with soap and water.

Moisturizing the skin.

Exercising regularly to support the lymphatic system. AS directed by a doctor.

Check for wounds and use medicated cream on any sore spots.

Conclusion:-

Elephantiasis is a highly uncommon phenomenon in western countries.

Wuchereria Bancrofti can cause elephantiasis and it left untreated. It can cause death.

Reference:-

Center for Disease control and prevention “Lymphatic Filariasis”

U.S centers for Disease control lymphatic Filariasis Treatment.

Sources of dietary fiber

Sources of dietary fiber

Fruits, leafy vegetables, vegetables, whole wheat legumes, rice bran etc. are rich

sources of fiber. The ideal way to increase fiber intake is to reduce intake ofrefined carbohydrates, besides eating vegetables, fresh fruits and whole grains.

In general, vegetarians consume more fiber than non-vegetarians. An average

daily intake of about 30g fiber is recommended.

Applications of DNA fingerprinting

Applications of DNA fingerprinting

The amount of DNA required for DNA fingerprint is remarkably small. The

minute quantities of DNA from blood strains, body fluids, hair fiber or skin

fragments are enough. Polymerase chain reaction is used to amplify this DNA

for use in fingerprinting. DNA profiling has wide range of applications—most of

them related to medical forensics. Some important ones are listed below.

• Identification of criminals, rapists, thieves etc.

• Settlement of paternity disputes.

• Use in immigration test cases and disputes.

In general, the fingerprinting technique is carried out by collecting the DNA

from a suspect (or a person in a paternity or immigration dispute) and matching

it with that of a reference sample (from the victim of a crime, or a close relative

in a civil case).

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Diagnosis of sicklecell anemia

 Diagnosis of sicklecell anemia:-

1. Sickling test : This is a simple microscopic examination of blood smear

prepared by adding reducing agents such as sodium dithionite. Sickled

erythrocytes can be detected under the microscope.

2. Electrophoresis : When subjected to electrophoresis in alkaline medium (pH

8.6), sicklecell hemoglobin (HbS) moves slowly towards anode (positive

electrode) than does adult hemoglobin (HbA). The slow mobility of HbS is

due to less negative charge, caused by the absence of glutamate residues that

carry negative charge. In case of sicklecell trait, the fast moving HbA and

slow moving HbS are observed. The electrophoresis of hemoglobin obtained

from lysed erythrocytes can be routinely used for the diagnosis of sicklecell

anemia and sicklecell trait.

FIG. - Electrophoresis of hemoglobins at pH 8.6 (HbA–Normal adult

hemoglobin; HbS–Sickle cell hemoglobin). 

Sicklecell trait provides resistance to malaria

Sicklecell trait provides resistance to malaria:-

The incidence of sicklecell disease coincides with the high incidence of malaria

in tropical areas of the world (particularly among the black Africans).

Sicklecell trait (heterozygous state with about 40% HbS) provides resistance

to malaria which is a major cause of death in tropical areas. This is explained as

follows

1. Malaria is a parasitic disease caused by Plasmodium falciparum in Africa. The

malarial parasite spends a part of its life cycle in erythrocytes. Increased lysis

of sickled cells (shorter life span of erythrocytes) interrupts the parasite cycle.

2. More recent studies indicate that malarial parasite increases the acidity of

erythrocytes (pH down by 0.4). The lowered pH increases the sickling of

erythrocytes to about 40% from the normally occurring 2%. Therefore, the

entry of malarial parasite promotes sickling leading to lysis of erythrocytes.

Furthermore, the concentration of K+ is low in sickled cells which is

unfavourable for the parasite to survive.

Sicklecell trait appears to be an adaptation for the survival of the individuals

in malaria-infested regions. Unfortunately, homozygous individuals, the

patients of sicklecell anemia (much less frequent than the trait), cannot live

beyond 20 years.

The future of gene therapy

 The future of gene therapy:-

Theoretically, gene therapy is the permanent solution for genetic diseases. But

it is not as simple as it appears since gene therapy has several inbuilt

complexicities. Gene therapy broadly involves isolation of a specific gene,

making its copies, inserting them into target tissue cells to make the desired

protein. The story does not end here. It is absolutely essential to ensure that the

gene is harmless to the patient and it is appropriately expressed (too much or too

little will be no good). Another concern in gene therapy is the body's immune

system which reacts to the foreign proteins produced by the new genes.

The public, in general, have exaggerated expectations on gene therapy. The

researchers, at least for the present, are unable to satisfy them. As per the

records, by 1999 about 1000 Americans had undergone clinical trails involving

various gene therapies. Unfortunately, the gene therapists are unable to

categorically claim that gene therapy has permanently cured any one of these

patients! Some people in the media (leading news papers and magazines) have

openly questioned whether it is worth to continue research on gene therapy!!

It may be true that as of now, gene therapy due to several limitations, has notprogressed the way it should, despite intensive research. But a breakthrough

may come anytime, and of course, this is only possible with persistent research.

And a day may come (it might take some years) when almost every disease will

have a gene therapy, as one of the treatment modalities. And gene therapy will

revolutionize the practice of medicine!

Gene replacement therapy

Gene replacement therapy:-

A gene named p53 codes for a protein with a molecular weight of 53 kilodaltons

(hence p53). p53 is considered to be a tumor-suppressor gene, since the protein it

encodes binds with DNA and inhibits replication. The tumor cells of several

tissues (breast, brain, lung, skin, bladder, colon, bone) were found to have

altered genes of p53 (mutated p53), synthesizing different proteins from the

original. These altered proteins cannot inhibit DNA replication. It is believed

that the damaged p53 gene may be a causative factor in tumor development.

Some workers have tried to replace the damaged p53 gene by a normal gene by

employing adenovirus vector systems. There are some encouraging results in the

patients with liver cancer.

The antisense therapy for cancer is discussed as a part of antigene and

antisense therapy

Structure of RNA

 Structure of RNA 

1. The acceptor arm : This arm is capped with a sequence CCA (5′to 3′). The

amino acid is attached to the acceptor arm.

2. The anticodon arm : This arm, with the three specific nucleotide bases

(anticodon), is responsible for the recognition of triplet codon of mRNA. The

codon and anticodon are complementary to each other.

3. The D arm : It is so named due to the presence of dihydrouridine.

4. The TψC arm : This arm contains a sequence of T, pseudouridine

(represented by psi, ψ) and C.

5. The variable arm : This arm is the most variable in tRNA. Based on this

variability, tRNAs are classified into 2 categories :

(a) Class I tRNAs : The most predominant (about 75%) form with 3–5 base

pairs length.

(b) Class II tRNAs : They contain 13–20 base pair long arm.

Classification of proteins on the basis of functional and chemical nature

Classification of Proteins

Proteins are complex macromolecules that perform a wide variety of biological functions. They can be classified based on function, chemical nature, and modern structural properties.

A. Functional Classification of Proteins

Proteins can be classified based on the role they play in organisms:

1.     Structural proteins – Provide mechanical support

o    Examples: Keratin (hair and nails), Collagen (bone)

2.     Enzymes (Catalytic proteins) – Speed up biochemical reactions

o    Examples: Hexokinase, Pepsin

3.     Transport proteins – Carry molecules across membranes or in blood

o    Examples: Hemoglobin, Serum albumin

4.     Hormonal proteins – Regulate physiological processes

o    Examples: Insulin, Growth hormone

5.     Contractile proteins – Facilitate movement

o    Examples: Actin, Myosin

6.     Storage proteins – Store nutrients and amino acids

o    Examples: Ovalbumin (egg), Glutelin (wheat)

7.     Genetic proteins – Associated with nucleic acids

o    Example: Nucleoproteins

8.     Defense proteins – Protect organisms from pathogens or toxins

o    Examples: Immunoglobulins, Snake venom proteins

9.     Receptor proteins – Bind hormones, viruses, or ligands

o    Example: Hormone receptors

B. Classification Based on Chemical Nature and Solubility

This system classifies proteins based on amino acid composition, structure, shape, and solubility.

1. Simple Proteins

Composed entirely of amino acids. They are further divided into:

(a) Globular Proteins – Spherical or oval, water-soluble, digestible

·         Albumins: Soluble in water and dilute salts, heat-coagulable (e.g., Serum albumin, Ovalbumin, Lactalbumin)

·         Globulins: Soluble in neutral or dilute salt solutions (e.g., Serum globulins, Vitelline)

·         Glutelins: Soluble in dilute acids/alkalis; mostly plant proteins (e.g., Glutelin, Oryzenin)

·         Prolamines: Soluble in 70% alcohol (e.g., Gliadin, Zein)

·         Histones: Strongly basic, soluble in water/dilute acids, insoluble in ammonium hydroxide (e.g., Thymus histones)

·         Globins: Not basic; soluble proteins associated with oxygen transport

·         Protamines: Strongly basic, smaller than histones, soluble in ammonium hydroxide (e.g., Sperm proteins)

·         Lectins: Carbohydrate-binding proteins; maintain tissue structure and used in affinity chromatography (e.g., Concanavalin A, Agglutinin)

(b) Fibrous Proteins – Insoluble, fiber-like, resistant to digestion

·         Collagens: Connective tissue proteins; gelatin obtained by boiling

·         Elastins: Found in elastic tissues like tendons and arteries

·         Keratins: Present in hair, nails, horns; high cysteine content (~14% in human hair)

2. Conjugated Proteins

Proteins containing a non-protein prosthetic group:

·         Nucleoproteins: DNA or RNA as prosthetic group (e.g., Nucleohistones, Nucleoprotamines)

·         Glycoproteins: Contain carbohydrates (<4%); mucoproteins if >4% (e.g., Mucin, Ovomucoid)

·         Lipoproteins: Proteins with lipid prosthetic groups (e.g., Serum lipoproteins)

·         Phosphoproteins: Contain phosphate groups (e.g., Casein, Vitelline)

·         Chromoproteins: Contain colored prosthetic groups (e.g., Hemoglobin, Cytochromes)

·         Metalloproteins: Contain metal ions (e.g., Ceruloplasmin-Cu, Carbonic anhydrase-Zn)

3. Derived Proteins

Derived proteins are denatured or hydrolyzed products of simple or conjugated proteins.

(a) Primary Derived Proteins – Initial denaturation or hydrolysis products

·         Coagulated Proteins: Denatured by heat, acids, or alkalies (e.g., Cooked proteins, Coagulated albumin)

·         Proteans: Early hydrolysis products, insoluble in water (e.g., Fibrin from fibrinogen)

·         Metaproteins: Second-stage hydrolysis products (e.g., Acid/alkali metaproteins)

(b) Secondary Derived Proteins – Progressive hydrolytic products

·         Include proteoses, peptones, polypeptides, and peptides formed during enzymatic or chemical breakdown of proteins

Summary Table (Optional for Quick Revision)

Classification	Subtype / Examples	Solubility / Property
Globular	Albumin, Globulin	Water / Salt soluble
Fibrous	Collagen, Keratin	Insoluble, fiber-like
Conjugated	Nucleoprotein, Glycoprotein, Metalloprotein	Depends on prosthetic group
Derived	Coagulated, Proteans, Peptones	Denatured or hydrolyzed