Analytical/Preparative Centrifugation

 Analytical/Preparative Centrifugation:-

The 2 most common types of centrifugation are analytical and preparative; the distinction is between the 2 is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles, such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by optical system during centrifugation, to allow observation of macromolecules in solution as they move in the gravitational field. The samples are centrifuged in cells with windows that lie parallel to the plane of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system onto film or a computer. The concentration of the solution at various points in the cell is determined by absorption of a light of the appropriate wavelength. This can be accomplished either by measuring the degree of blackening of a photographic film or by the deflection of the recorder of the scanning system and fed into a computer. The other type of centrifugation is called preparative and the objective is to isolate specific particles that can be reused. There are many type of preparative centrifugation such as rate zonal, differential, and isopycnic centrifugation.

The major categories of innate lymphoid cells (ILCs) and their properties

 

Innate immune recognition by Toll-like receptors.

Properties of Vectors in RDT

 Vectors

The term vector refers to the DNA molecules that act as transporting vehicle which carries target DNA into a host cell for the purpose of cloning and expression. Cloning vectors are used to clone target DNA whereas expression vectors are engineered so that desirable target DNA can be transcribed in RNA and translated into protein, A viral DNA or plasmid is generally used as a vector. The important features of a cloning vector are as follows:

1. Ability to replicate in host cells. All cloning vectors have origin of replication for autonomous replication within the host cell. The origin of replication is a specific sequence in DNA from where replication starts. When target DNA is linked to vector containing origin of replication then along with vector replication, desirable target DNA also starts replicating within the host cell.

2. Unique restriction sites for insertional cloning, All cloning vectors have features that allow a target DNA to be conveniently inserted into the vector. This may be a multiple cloning site (also called polylinker site), which contains many unique restriction sites. The restriction site(s) in the polylinker site are first cleaved by specific restriction enzyme(s), and a target gene is then ligated into the vector using DNA ligase.

3. Genetic marker to select for host cells containing the vector Genetic marker is a gene that allow the selection of transformed cell from non trans formed cells and recombinant containing transformed cells from non-recombinant containing transformed cells. Marker genes belong to two broad categories: selectable markers and screen able markers. A selectable marker gene encodes a product that allows the growth of one type of cells under specific conditions that kill or restrict the growth of other types of cells. A screenable marker gene, also called reporter gene, gives a product that can be detected using a simple and often quantitative assay. 

4. Low molecular weight (minimum amount of non-essential DNA). The advantages of a low molecular weight are several. First, the plasmid is more resistant to damage by shearing and is readily isolated from host cells. Secondly, low molecular weight plasmids are usually present as multiple copies. Finally, with a low molecular weight there is less chance that the vector will have multiple sites for any restriction endonuclease.

CHEMICAL COMPOSITION OF LIVING ORGANISMS

Synopsis:-

• Introduction
• Composition of elements in living system
• Biogenic elements
• Water and Mineral substances
• Organic Matters
• Conclusion
• Reference 

        1.      Introduction to the topic:-                                                                                                                               The life on Earth can be understood as a form of existence of matter. All the living matter, i.e. living organisms, is composed of the same particles (atoms, ions, molecules) as the non-living organisms and chemical laws and laws of physics are applicable to both of them. There is a close connection between living and non-living nature, however they differ in chemical composition, structure, complexity and organization. While the chemical composition of the non-living nature is varied, the existence of living organisms is based on the presence of a few chemical elements, especially carbon, oxygen, nitrogen and hydrogen.

       All the chemical compounds in living organisms are composed of chemical elements. In these days almost 120 chemical elements are known. Out of this number 92 elements are naturally present in the nature (the rest were made in laboratories). Out of the 92 elements only 30 elements create the living matter and they are called biogenic elements.

There are 92 elements in the Earth´s crust. Oxygen and silicon represent the highest percentage - 75% of all elements. Both these elements of the Earth´s crust, as well as the other elements, are bonded especially in minerals (e.g. oxides, silicates) and rocks. The remaining 90 elements represent about 25 % of all elements.

Fig.: Representation of chemical elements in the Earth´s crust

2.     Composition of elements in living systems:-

                                     In all living systems we can always find 4 basic elements: carbon, oxygen, nitrogen and hydrogen. Carbon is the basic building unit contained in living matter. The percentage of carbon in the mass of living matter is 19.4 %. Oxygen and hydrogen are present in almost all organic compounds which create living organisms. The percentage of oxygen in the mass of living systems is 62.8 %, the percentage of hydrogen is 9.3 %. The source of hydrogen for organisms is water, the source of oxygen is water and the atmosphere. Nitrogen is bonded mainly in amino acids, proteins and nucleic acids. Its percentage is 5.1 %.

Chemical element	Average representation in living matter (%)	Average representation in non-living matter (%)
Carbon	19.37 %	0.18 %
Oxygen	62.80 %	49.40 %
Hydrogen	9.31 %	0.95 %
Nitrogen	5.14 %	0.63 %

2.1  Biogenic elements

 

       All elements contained in living matter are called biogenic elements. They are present in compounds, in the form of ions and in some special cases they are unbound (e.g. oxygen). According to their representation in organisms, the biogenic elements are divided into 3 groups: macrobiogenic, microbiogenic and trace elements. Trace and microbiogenic elements are sometimes also called oligobiogenic elements.

 

       I. Macrobiogenic elements – C, O, H, N, S, P, Na, K, Ca, Mg, Cl, Fe.  Four of these elements – O, C, H, N – represent up to 95 % of living matter. The rest of the elements mentioned above represent up to 4.9 %. Macrobiogenic elements have a building function.

      Carbon is the basis for all living matter. The typical feature of carbon atoms is the ability to bond to each other or to atoms of other elements. That is why there are many organic compounds of carbon. Carbon is also present in carbon dioxide and carbonates.

      Oxygen and hydrogen in organisms they are present both in the form of organic and inorganic compounds and they are a part of the basic micromolecule – water. Oxygen is produced by autotroph organisms (especially by plants and cyanobacteria) during the process of photosynthesis.

Nitrogen is a component of proteins and nucleic acids. It is also a part of nitrates and ammonium carbonate, which are necessary for the mineral nutrition of plants and also the synthesis of plant proteins.

      II. Microbiogenic elements – Cu, I, Mo, Mn, Zn, Co. The average content of these elements in living organisms is less than 0.1%. Microbiogenic elements have catalytic function, i.e. they are part of enzymes.

      III. Trace elements – e.g. Al, As, B, Br, F, Li, Ni, Se, Si, Ti, V. Their content in organisms is less than 0.001 %. As well as microbiogenic elements, trace elements are parts of enzymes and their function is catalytic.

 

3.  Chemical composition of living systems

 Living organisms are composed of several types of substances called biomolecules. According to their molecular weight, substances in living organisms are divided into two groups:

 1. Low molecular substances (M_r < 10 000)

•         water

•         inorganic (mineral) substances

•         intermediates of metabolic pathways (carboxylic acids etc.)

• final products of metabolic pathways (amino acids, monosaccharides, lipids, nucleotides)

 2. High molecular substances (M_r > 10 000)

•         proteins

•         polysaccharides

•         nucleic acids

      High molecular substances, which are present in living organisms, are also named as biological macromolecules or biopolymers. The building units of proteins are amino acids, the building units of polysaccharides are monosaccharides, and the building units of nucleic acids are nucleotides.

      According to their origin, the substances included in the living organisms are divided into inorganic substances (water, carbon dioxide, mineral substances) and organic substances (the most important are nucleic acids, proteins, saccharides, lipids).

  

Fig.: Average representation of the main groups of substances in organisms

3.1.  Water and mineral substances

The most frequent and the simplest biomolecule in living systems is water. Water  is basic and the most spread inorganic compound contained in living organisms. The average content of water in organisms is 60-70 %. The amount of water depends on the surroundings  in which the organism lives, on a kind of organism, on its age. The amount of water also depends on specific parts of body, e.g. the highest percentage of water in human body is in body fluids and the lowest in fatty, dental or bone tissue. Water in organisms helps to create their inner environment and keep their stability. Water is a dissolving agent, transporting medium and a thermoregulator. Bochemical reactions in living systems happen in water environment.

      

Inorganic salts can be either water-soluble, i.e. dissociated into ions, or insoluble. Insoluble salts are present in hard connective tissue such as teeth, bones or shells. Examples: Ca₃(PO₄)₂ (bones, teeth), CaCO₃ (bones, shells of invertebrates), CaF₂ (teeth). Soluble salts in the form of ions are mainly in body fluids. The main extracellular ions are cation Na⁺ and anion Cl^-.  The main intracellular ions are cation K⁺ and cation  Mg²⁺ .

     Very important compound is carbon dioxide, which is necessary for photosynthesis. It is produced in metabolic (catabolic) processes, e.g. when breathing.

 

3.2.  Organic matters                  

 

The most important organic matters necessary for organism structure and function are:

ü  proteins

ü  nucleic acids

ü  saccharides

ü  lipids

       Organic matters represent more than 30% of organism mass. The rest is represented by water and mineral matters.

Characteristics of biosensor

Biosensor characteristics:-

1. Sensitivity is the response of the sensor to per unit change in analyte concentration.

2. Selectivity is the ability of the sensor to respond only to the target analyte. That is, lack of   response to other interfering chemicals is the desired feature.

3. Range is the concentration, range over which the sensitivity of the sensor is good.

4. Response time is the time required by the sensor to produce r responses.

5. Reproducibility is the accuracy with which the sensor's output can be obtained.

6. Detection limit is the lowest concentration of the analyte to which there is a measurable,

    response.

7. Life time is the time period over which the sensor can be used without significant deterioration in performance characteristics.

8. Stability characterizes the change in its baseline or sensitivity over a fixed period of time.

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.