Category: Biology

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Transfer of Genetic Material

Normally, genes and the characteristics they code for are passed down from parent to progeny. This is called vertical gene transfer. Bacteria and some lower eukaryotes are unique in that they can pass DNA from one cell of the same generation to another.

The exchange of genes between two cells of the same generation is referred to as horizontal gene transfer. Mechanisms like transformation, transduction and conjugation takes place naturally and may bring about genetic variation and genetic recombination.

These gene transfer mechanisms are also employed in genetic engineering to introduce desired gene into the cells. Introducing a foreign gene or recombinant DNA into the cells is one of the techniques used in genetic engineering. The success of cloning depends on the efficiency of gene transfer process.

The most commonly employed gene transfer methods are transformation, conjugation, transduction, electroporation, lipofection and direct transfer of DNA. The choice of the method depends on the type of host cell (bacteria, fungi, plant, animal). Figure 12.15 shows methods of DNA transfer.

Note: The term Transfection is used for the transfer of DNA into eukaryotic cells by various physical or chemical means.

Transformation

Transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings. Transformation occurs naturally in some species of bacteria, but it can also take place by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence.

Competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial. Transformation works best with DNA from closely-related species. The naturally-competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).

There are some differences in the mechanisms of DNA uptake by gram positive and gram negative cells. However, they share some common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA
translocase.

Only single stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes. Figure 12.16 shows mechanism of transformation.

Artificial competence can be induced in laboratory by procedures that involve making the cell passively permeable to DNA. Typically, the cells are incubated in a solution containing divalent cations; most commonly, calcium chloride solution under cold condition, which is then exposed to a pulse of heat shock.

Electroporation is another method of promoting competence. Using this method, the cells are briefly shocked with an electric field of 10-20 kV / cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.

Conjugation

The initial evidence for bacterial conjugation, came from an experiment performed by Joshua Lederberg and Edward L Tatum in 1946. Later in 1950, Bernard Davis gave evidence that physical contact of the cells was necessary for conjugation. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges (e.g. pilus) and transfer single stranded DNA (from donor to recipient).

Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids). Conjugation can occur among the cells from different genera of bacteria, while transformation takes place among the cells of a bacterial genus.

A plasmid called the fertility or F factor plays a major role in conjugation. The F factor is about 100 kilobases long and bears genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation. F factor is made up of:-

• tra region (tra operon / transfer genes): genes coding the F pilus and DNA transfer,
• Insertion sequence: genes assisting plasmid integration into host cell chromosome.

Thus, the F factor is an episome – a genetic material that can exist outside the bacterial chromosome or be integrated into it.

During F⁺ × F^– mating or conjugation (Figure 12.17 a) the F factor replicates by the rolling circle mechanism and a copy moves to the recipient. The channel for DNA transfer could be either the hollow F pilus or a special conjugation bridge formed upon contact. The entering strand is copied to produce double – stranded DNA.

F factor can integrate into the bacterial chromosome at several different locations by recombination between homologous insertion sequences present on both the plasmid and host chromosomes. The integration of F factor into bacterial chromosome results in formation of HFR (High Frequency Recombination) cell.

When integrated, the Fplasmid’s tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling circle replication, and transfer genetic material to an F- recipient cell.

An HFR cell is so called because it exhibits a very high efficiency of chromosomal gene transfer in comparison with F⁺ cells. In F⁺ cells the independent F factor rarely transfer chromosomal genes hence the recombination frequency is low. Figure 12.17 b shows formation of HFR cell.

When an HFR cell is mated with F^– cell the F^– recipient does not become F⁺ unless the whole chromosome is transferred as explained in Figure 12.17 c. The connection usually breaks before this process is finished. Thus, complete F factor usually is not transferred, and the recipient remains F^–.

Because the F plasmid is an episome, it can leave (deintegrate) the bacterial chromosome. Sometimes during this process, the plasmid makes an error in excision and picks up a portion of the chromosomal material to form an F′ plasmid. Figure 12.17 d shows formation of F′.

During F′XF^– conjugation (Figure 12.17 e) the recipient becomes F′ and is a partially diploid since it has two set of the genes carried by the plasmid.

The natural phenomenon of conjugation is now exploited for gene transfer and Recombinant DNA technology. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

Transduction

Transduction is the transfer of bacterial genes from one bacteria to other by viruses. Example: Bacteriophage (Bacterial viruses). To understand the role of bacteriophage in gene transfer, the lifecycle of bacteriophage is described below briefly.

After infecting the host cell, a bacteriophage (phage for short) often takes control and forces the host to make many copies of the virus. Eventually the host bacterium bursts or lyses and releases new phages. This reproductive cycle is called a lytic cycle because it ends in lysis of the host.

The lytic cycle (Figure 12.18) has four phases.

1. Attachment – Virus particle attaches to a specific receptor site on the bacterial surface.
2. Penetration – the genetic material, which is often double stranded DNA, then enters the cell.
3. Biosynthesis – After adsorption and penetration, the virus chromosome forces the bacterium to make viral componentsviral nucleic acids and proteins.
4. Assembly – Phages are assembled from the virus components. Phage nucleic acid is packed within the virus’s protein coat.
5. Release – mature viruses are released by cell lysis.
   

Bacterial viruses that reproduce using a lytic cycle often are called virulent bacteriophages (e.g. T phages) because they destroy the host cell. The genome of many DNA phages such as the lambda phage, after adsorption and penetration do not take control of its host and does not destroy the host.

Instead the viral genome remains within the host cell and is reproduced along with the bacterial chromosome. The infected bacteria may multiply for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyses under appropriate environmental conditions. This relationship between phage and its host is called lysogeny (Figure 12.19).

Bacteria that can produce phage particles under some conditions are said to be lysogens or lysogenic bacteria. Phages which are able to establish lysogeny are called temperate phages. The latent form of virus genome that remains within the host without destroying the host is called the prophage.

The prophage usually is integrated into the bacterial genome. Sometimes phage reproduction is triggered in a lysogenized culture by exposure to UV radiation or other factors. The lysosomes are then destroyed and new phages released – This phenomenon is called induction (Figure 12.20)

Sometimes, bacterial genes are incorporated into a phage capsid because of errors made during the virus life cycle. The virus containing these genes then infects them into another bacterium, resulting in the transfer of genes from one bacterium to the other. Transduction may be the most common mechanism for gene exchange and recombination in bacteria.

There are two very different kinds of transduction.

1. Generalized transduction
2. Specialized transduction

Generalized transduction (Figure 12.21 a) occurs during the lytic cycle of virulent and temperate phages. During the assembly stage, when the viral chromosomes are packaged into protein capsids, random fragments of the partially degraded bacterial chromosome also may be packaged by mistake. The resulting virus particles often injects the DNA into another bacterial cell but does not initiate a lytic cycle.

Thus in generalized transduction any part of the bacterial chromosome can be transferred. Once the DNA has been injected it may integrate into the recipient cell’s chromosome to preserve the transferred genes. About 70 to 90% of the transferred DNA is not integrated but is often able to survive and express itself. However, if the transferred DNA is degraded gene transfer is unsuccessful.

Specialized Transduction (Figure 12.21 b) is also called restricted transduction in which only specific portions of the bacterial genome is carried by the phage. When a prophage is induced to leave the host chromosome, exicision is sometimes carried out improperly.

The resulting phage genome contains portions of the bacterial chromosome next to the integration site. When this phage infects another bacterium, it transfers the bacterial genes from the donor bacterium along with phage DNA. Here only the bacterial genes that are close to the site of prophage are transferred. So, this transduction is called specialized.

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Formation of Mutants

The term mutant refers to an organism in which either the base sequence of DNA or the phenotype has been changed. A mutant is an organism whose genotype differs from that found in nature. The process of formation of mutant organism is called mutagenesis.

In nature and in the laboratory, mutations sometimes arise spontaneously without any help from the experimenter. This is called spontaneous mutagenesis. The two mechanisms that are most important for spontaneous mutagenesis are

1. Errors occurring during replication and
2. Spontaneous alteration of bases.

Mutations can also be induced experimentally by application of mutagens. Mutagens are agents that cause mutations.

Mutagens and their Mode of Action

Physical Mutagens

UV radiation:

UV light causes mutations because the purine and pyrimidine bases in DNA absorb light strongly in the ultraviolet range (254 to 260 nm). At this wavelength, UV light induces point mutations primarily by causing photochemical changes in the DNA.

One of the effects of UV radiation on DNA is the formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand, or between pyrimidines on the opposite strands, of the double helix.

This bonding is induced mostly between adjacent thymines, forming what are called thymine dimers (Figure 12.10), usually designated TT. This unusual pairing produces a bulge in the DNA strand and disrupts the normal pairing of T’s (thymines) with corresponding A’s(adenines) on the opposite strand. If UV induced genetic damage is not repaired, mutations or cell death may result.

Chemical Mutagens

Chemical mutagens include both naturally occurring chemicals and synthetic substances. These mutagens can be grouped into different classes on the basis of their mechanism of action. They are

(i) Base analogs are bases that are similar to the bases normally found in DNA.
E.g. 5 – bromouracil (5-BU). TA to CG (Figure 12.11).

(ii) Base Modifying Agents are chemical that act as mutagens by modifying the chemical structure and properties of bases. The three types of mutagens that work in this way are

• A deaminating agent e.g: Nitrous acid removes amino groups (- NH₂) from the bases guanine, cytosine, and adenine.
• Hydroxylamine (NH₂ OH) is a hydroxylating mutagen that react specifically with cytosine, modifying it by adding a hydroxyl group (OH) so that it can pair solely with adenine instead of with guanine.
• Alkylating agents like methymethane sulfonate (MMS) introduces alkyl groups onto the bases at a number of location.

(iii) Intercalating agents

Acridine, proflavin, ethidium bromide are a few examples of intercalating agents. These insert (intercalate) themselves between adjacent bases in one or both strands of the DNA double helix. Intercalating agents can cause either additions or deletions.

The Ames Test: A Screen for Potential Carcinogens

Everyday we are exposed to a wide variety of chemicals in our environment, such as drugs, cosmetics, food additives, pesticides, and industrial compounds. Many of these chemicals can have mutagenic effects, including genetic diseases and cancer. Some banned chemical warfare agents (e.g. mustard gas) also are mutagens.

A number of chemicals (subclass of mutagens) induce mutations that result in tumorous or cancerous growth. These chemical agents are called chemical carcinogens. Directly testing the chemicals for their ability to cause tumors in animals is time consuming and expensive. However, the fact that most chemical carcinogens are mutagens led Bruce Ames to develop a simple, inexpensive, indirect assay for mutagens.

In general Ames test is an indicator of whether the chemical is a mutagen. The Ames test assays the ability of chemicals to revert mutant strains of the bacterium Salmonella typhimurium to wild type. The mutant strain of S.typhimurium is auxotrophic to histidine (histidine), that is it requires histidine for its growth and
cannot grow in the absence of histidine. The mutant strain is grown in a histidine deficient medium containing the chemical to be tested.

A control plate is also set up which does not contain the chemical. After incubation the control plates may have few colonies resulting from spontaneous reversion of the his – strain. Compared to the control plates if there are increased number of colonies on test plate, it indicates that the chemical has reverted the mutant strain back to wild type. This chemical is likely to be a carcinogen. Figure 12.14 shows steps in Ames test.

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Types of Mutation

The base sequence of DNA determines the amino acid sequence of a protein. The chemical and physical properties of each protein are determined by its amino acid sequence, so a single amino acid change is capable of altering the activity of, or even completely inactivating, a protein.

Genotype refers to the genetic composition of an organism. Phenotype is an observable property of organism. The functional form of a gene is called Wildtype because presumably this is the form found in nature.

Mutation is the process by which the sequence of base pairs in a DNA molecule is altered.The alteration can be a single base pair substitution, insertion or deletion. Mutations can be divided into two general categories:

1. Base – pair substitution

Base – pair substitution mutation involves a change in the DNA such that one base pair is replaced by another.

• A mutation from one purine – pyrimidine base pair to the other purine – pyrimidine base pair is a transition mutation (Figure 12.7 a). E.g. AT to GC, CG to TA.
• A mutation from a purine pyrimdine base pair to a pyrimidine – purine base pair is a transversion mutation (Figure 12.7 b). E.g. AT to TA, CG to GC.
  

2. Base pair insertion or deletions

Involves the addition or deletion of one base pair. If one or more base pairs are added to or deleted from a protein coding gene, the reading frame of an mRNA can change downstream of the mutation. An addition or deletion of one base pair, for example, shifts the mRNA’s downstream reading frame by one base, so that incorrect amino acids are added to the polypeptide chain after the mutation site.

This type of mutation, called a frame shift mutation (Figure 12.8) usually results in a nonfunctional protein.

Frame shift mutations:

• May generate new stop codons, resulting in a shortened protein.
• May result in a read through of the normal stop codon, resulting in longer than normal proteins
• Or may result in a complete alteration of the amino acid sequence of a protein.
  

Point mutations are single base changes, that do not affect the reading frame, that is, the mutation only makes a single change in a single codon, and everything else is undisturbed. Mutations can also be defined according to their effects on amino acid sequences in proteins. They are:-

1. A missense mutation (Figure 12.9 a) is a gene mutation in which a base – pair change in the DNA changes a codon in an mRNA so that a different amino acid is inserted into the polypeptide.

2. A neutral mutation (Figure 12.9 b) is a subset of missense mutations in which the new codon codes for a different amino acid that is chemically equivalent to the original and therefore does not affect the proteins function. Consequently, the phenotype does not change.

3. A silent mutation (Figure 12.9 c) is also a subset of missense mutations that occurs when a base – pair change in a gene alters a codon in the mRNA such that the same amino acid is inserted in the protein. In this case, the protein obviously has a wild type function.

4. A nonsense mutation (Figure 12.9 d) is a gene mutation in which a base – pair change in the DNA, changes a codon in an mRNA to a stop (nonsense) codon (UAG, UAA or UGA). Nonsense mutation cause premature chain termination so instead of complete polypeptides, shorter than normal polypeptide fragments (often nonfunctional) are formed.

Forward mutations change the genotype from wild type to mutant and reverse mutations (or reversions or back mutations) change the genotype from mutant to wild type or to partially wild type. An organism which has reverted is a Revertant. The effects of mutation may be diminished or abolished by a suppress or mutation.

Suppressor mutation is a mutation at a different site from that of the original mutation. A suppressor mutation masks or compensates for the effects of the initial mutation, but it does not reverse the original mutation.

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Microbial Genetic Code and its Features

A tRNA molecule “reads” the base sequence of mRNA. The language read by the tRNA molecules is called the genetic code, which is a set of relations between sequences of three adjacent bases on an mRNA molecule and particular amino acids. (A RNA base sequence (a set of 3 bases) corresponding to a particular amino acid is called a codon).

The genetic code is the set of all codons. Only four bases in DNA serve to specify 20 amino acids in proteins, so some combination of bases is needed for each amino acid. Before the genetic code was elucidated, it was reasoned that if all codons were assumed to have the same number of bases, then each codon would have to contain at least three bases.

Codons consisting of pairs of bases would be insufficient because four bases can form only 42 = 16 pairs, and there are 20 amino acid. Triplets of bases would suffice because, these can form 43 = 64 triplets. In fact, the genetic code is a triplet code, and all 64 possible codons carry information of some sort.

Several different codons designate the same amino acid. Furthermore, in translating mRNA molecules the codons do not overlap but are used sequentially. The same genetic code is used by almost all biological systems and hence is said to be universal (exceptions are mitochondria and a few unusual microorganisms). The codons are by convention written with the 5′ end at the left. The complete code is shown in Table 12.1.

Features of the Code:

Sixtyone codons correspond to amino acids. Four codons are signals. These are the three stop codons – UAA, UAG, UGA – and the one start codons, AUG.

The start codons (initiation codon) also specifies the amino acid methionine. In rare cases, certain other codon (E.g. GUG) initiate translation. No normal tRNA molecule has an anticodon (a sequence of three bases on tRNA that can base – pair with a codon sequence in the mRNA) complementary to any of the stop codons UAG, UAA or UGA, which is why these codons are stop signals.

The code is highly redundant i.e. more than one codons code for an amino acid. Only tryptophan and methionine are specified by one codon. The synonymous codons usually differ only in third base (except for serine, leucine and arginine).

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Microbial Genetics Transcription

An important feature of RNA synthesis is that even though the DNA molecule being copied is double stranded, in any particular region of DNA only one strand serves as a template. The DNA strand copied into RNA molecule is called CODING OR SENSE STRAND. The synthesis of RNA consists of five discrete stage (Figure 12.2):

1. Promoter recognition:

RNA polymerase binds to DNA within a specific base sequence (20-200 bases long) called a promoter. The sequence TATAAT (or a nearly identical sequence) often called a pribnow box or – 10 region is found as part of all prokaryotic promoters.

The RNA polymerase of the bacterium E.coli consists of five protein subunits. Four of the subunits comprise the core enzyme (catalyzes the joining of the nucleoside triphosphates to the RNA) and fifth subunit, the σ subunit (required for promoter binding).

2. Local unwinding

Local unwinding of DNA occurs and RNA polymerase forms an open promoter complex.

3. Nucleoside Triphosphate

The first nucleoside triphosphate is placed at polymerization start site (near to the initial binding site) and synthesis begins.

4. RNA

RNA polymerase then moves along the DNA, adding ribonucleotides, to the growing RNA chain.

5. RNA polymerase

RNA polymerase reaches chain termination sequence and both the newly synthesized RNA and the polymerase are released. Two kinds of termination events are known those that are self – terminating (dependent on the base sequence only) and those that require the presence of the termination protein Rho.

Initiation of a second round of transcription need not await completion of the first, for the promoter becomes available once RNA polymerase has polymerized 50-60 nucleotides. In bacteria most mRNA molecules are degraded within a few minutes after synthesis. This degradation enables cells to dispense with molecules
that are no longer needed.

In prokaryotes mRNA molecules commonly contain information for the amino acid sequences of several different polypeptide chains. In this case, such a molecule is called polycistronic mRNA. Cistron is a term used to mean a base sequence encoding a single polypeptide chain.

The genes contained in polycistronic mRNA molecule (Figure 12.3) often encode the different portions of a metabolic pathway. For example, in E. coli the ten enzymes needed to synthesize histidine are encoded in one mRNA molecule.

In prokaryotes the immediate product of transcription (called the primary transcript) is mRNA, in contrast in eukaryotes the primary transcript must be converted to mRNA. This conversion called RNA processing consists of two types of events – modification of termini and excision of untranslated sequences (noncoding sequence or introns) embedded within coding sequences (exons).

Introns excision and the joining of exons to form an mRNA molecule is called RNA splicing. The introns are present in almost all eukaryotic transcripts but are rare in the free – living unicellular eukaryotes such as yeast. Some bacterial genes do contain introns.

Synthesis of rRNA and tRNA Ribosomal RNA and tRNA are also transcribed from genes. The production of these molecules is not as direct as synthesis of bacterial mRNA. The main difference is that these RNA molecules are excised from large primary transcripts. Highly specific RNA excise rRNA and tRNA from these large transcripts, and other enzymes produce the modified bases in tRNA.

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Concept of Gene Microbial

The fundamental unit of information in living systems is the gene. Genome is the set of all genes and genetic signals of a cell. The information contained in genes is converted to molecules that determine the metabolism, structure and form of microorganisms.

Gene is expressed through a sequence of events. A gene can be defined biochemically as a segment of DNA (or, in a few cases, RNA) that encodes the information required to produce a functional biological product.

The final product is usually a protein. Not all genes are involved in protein synthesis; some code instead for rRNA and tRNA. The central dogma of molecular biology, comprises the three major processes (Figure 12.1). The first is replication, the copying of parental DNA to form daughter DNA molecules with identical nucleotide sequences. The information contained in the base sequence of DNA is copied into protein molecule through an RNA molecule.

The second is transcription, production of mRNA from DNA. It is the process by which the segment corresponding to a particular gene is selected and an RNA molecule is synthesized. The third is translation, The production of an amino acid sequence from an RNA base sequence. The genetic message encoded in messenger RNA (mRNA) is translated on the ribosomes into a polypeptide with a particular sequence of amino acids. The order of amino acid in a polypeptide chain is determined by DNA base sequence.

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Updated National Immunization Schedule Chart

Immunization/vaccination produce a response in the body that is similar to the body’s response to a natural infection (Table 11.4). Immunization or vaccines can therefore protect the body from a disease before the disease has a chance to cause illness. Immunization has helped to reduce the impact of communicable disease on health and well being.

Some diseases have been well controlled and other has been eliminated from some parts of the world because of vaccination. Stopping vaccination may lead to epidemic.

Table 11.4: National immunization schedule

Vaccine	Due age	Route
BCG	At birth	Intra dermal
Hepatitis B-Birth dose	At birth	Intra muscular
OPV-O	At birth	Oral
OPV 1, 2 & 3	At 6 weeks, 10 weeks & 14 weeks	Oral
Pentavalent 1, 2 & 3 (Diphtheria + Pertuss is + Tetanus + Hepatitis B + Hib)	At 6 weeks, 10 weeks & 14 weeks	Intra muscular
Inactivated polio vaccine	At 6 & 14 weeks	Intra muscular
Rotavirus (where applicable)	At 6 weeks, 10 weeks & 14 weeks	Oral
Pneumococcal conjugate vaccine (where applicable)	At 6 weeks & 14 weeks. At 9 completed months -booster	Intra muscular
Measles/Rubella 1st dose	At 9 completed months – 12 months	Subcutaneous
DPT Booster-1	16–24 months	Intra muscular
Measles/Rubella 2nd dose	16–29 months	Subcutaneous
OPV Booster	16–24 months	Oral
DPT Booster – 2	5–6 years	Intra muscular
TT	10 years & 16 years	Intra muscular

proteins, antibodies and hormones. There are four kinds of ELISA assay tests. They are: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA. Western blotting technique is used for the identification of particular protein from the mixture of proteins.

The most common protein sample used for Western blotting is cell lysate. Blotting refers to the transfer of the protein from the gel to the nitrocellulose paper by capillary action.

The substances causing allergic/hypersensitivity is known as allergens. Allergic rhinitis develops when the body’s immune system becomes sensitized and overreacts to something in the environment like pollen grains, strong odour of perfumes, dust etc.

Certain drugs such as penicillin, cephalosporin and streptomycin can absorb non-specifically to protein on surface of RBC forming complex similar to hapten-carrier complex.

Transfer of living cells, tissues or organs from one part of the body to another or from one individual to another is known as transplantation. The graft tissue antigens induce an immune response in the host. This type of immune response is called host versus graft reaction. The ultimate goal of any immunization program is the eradication of the disease.

Active natural immunization involves activation of immune system in the body to produce antibodies. It is achieved in both clinical and subclinical infections Immunization has helped to reduce the impact of communicable disease on health and well being.

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Types of Immunization/Vaccination

Father of Immunology is Edward Jenner. He produced the vaccine for small pox from cow pox virus. Vaccine is a substance that is introduced into the body to prevent the disease produced by certain pathogens. Vaccines consist of dead pathogens or live but attenuated (artificially weakened) organisms.

Immunization programmes and the development of new vaccines play an important role in protecting individuals against illness. Vaccination works by safely exposing individuals to a specific pathogenic microbe, artificially increasing their immunity to it.

Vaccines are made from

• Live micro-organisms that have been ‘treated’ so that they are weakened (attenuated) and are unable to cause disease.
• Dead micro-organisms.
• Some part or product of the microorganism that can produce an immune response.

Vaccine Types

Live attenuated vaccines:
These vaccines contain modified strains of a pathogen that have been weakened but are able to multiply within the body and remain antigenic enough to induce a strong immune response. Example: Oral Polio vaccine

Heterologous vaccine:
These are a group of live attenuated vaccines produced from the strains that are pathogenic in animals and not in humans. It is a vaccine that confers protective immunity against a pathogen that shares cross-reacting antigens with the microorganisms in the vaccine. Example: Cow pox virus that protects against small pox in humans.

Killed inactivated vaccines:
These groups of vaccine are produced either by killing or inactivating the bacteria or virus by chemical treatment or heat. Example; Polio virus.

Sub unit vaccine:
The antigenic determinant / epitope (the very specific part of the microbe) is used to prepare the vaccine.

DNA Vaccines:
When the genes for microbe’s antigens are introduced into the body some cells will take up the DNA. The DNA then instructs those cells to make the antigen molecules. The cells secrete the antigens and display them on their surfaces. The body’s own cells become vaccine generating factories.

Routes of Administration

• Deep subcutaneous or intramuscular route – most vaccines
• Oral route – Oral BCG vaccine
• Intradermal route – BCG vaccine
• Scarification – Small pox vaccine
• Intranasal route – Live attenuated influenza virus

Types of Immunization

Immunization is of two types:

1. Passive Immunization
2. Active Immunization

1. Passive Immunization

• Passive immunization is produced without challenging the immune system of the body. It is done by administration of serum or gamma globulins from a person who is already immunized to a non-immune person.
• Passive immunization is the administration of preformed antibodies either intravenously or intramuscularly.
• It is used to provide rapid protection in certain infections such as diphtheria or tetanus or in the event of accidental exposure to certain pathogens such as hepatitis B.
• It is also used to provide protection in immune compromised individuals.

Passive natural immunization:

Acquired from the mother before and after birth. Before birth, immunity is transferred from mother to the fetus in the form of maternal antibodies through placenta. After birth, the antibodies (Ig A) are transferred through breast milk (Table 11.2).

Table 11.2: Passive Immunization

Infection	Source of Antiserum	Indications
Tetanus	Immune human; horse	Post exposure (Plus vaccine)
Diptheria	Horse	Post-exposure
Gas gangrene	Horse	Psot-exposure
Botulism	Horse	Post-exposure
Varicella-Zoster	Immune human	Post-exposure in immunodeficiency
Rabies	Immune human	Post exposure (Plus vaccine)
Hepatitis B	Immune human	Post-exposure prophylaxis
Hepatisis A	Pooled human Ig	Prophylaxis
Measles	Immune human	Prophylaxis
Snakebite	Horse	Post-bite
Some autoimmune disease	Pooled human ig	Acute thrombocytopenia and neutropenia

Passive artificial immunization:

Developed by injecting previously prepared antibodies using serum from humans or animals. This type of immunity is useful for providing immediate protection against acute infections like tetanus, measles etc.

2. Active Immunization

Active immunization is the administration of vaccines containing microbial products with or without adjuvants in order to obtain long term immunological protection against the offending microbe.

At present the normal route of vaccination in most instances is either intramuscular or subcutaneous. Oral immunization is the method of choice for polio and Salmonella typhi vaccines. However, there is an increasing awareness that this route of immunization may be the best for most immunizations since nearly all infectious agents gain entrance through the mucosal surfaces.

Active natural immunization involves activation of immune system in the body to produce antibodies. It is achieved in both clinical and subclinical infections. Active artificial immunization is achieved by the administration of vaccines or toxoids.

Antigen preparations

Most vaccines consist of attenuated organisms, killed organisms, inactivated toxins, or sub cellular fragments and more recently genes for antigens in viral ‘vectors’, and DNA itself. Thus, vaccines must be capable of targeting the immune system appropriately i.e. cellular/or humoral mechanisms (Table 11.3).
Table 11.3: Antigen Preparation Used in Vaccines.

Adjuvants

Nonliving vaccines, especially those consisting of small molecules require the inclusion of agents to enhance their effectiveness.

These adjuvants include microbial, synthetic and endogenous preparations having adjuvant activity, but at present only aluminium or calcium salts are generally used in humans.

Adjuvants should enable antigens to be slowly released, preserve antigen integrity, target antigen presenting cells and induce cytotoxic lymphocytes.

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Overview of an Transplantation

Transfer of living cells, tissues or organs from one part of the body to another or from one individual to another is known as transplantation. A tissue or organ that is removed from one site and placed to another site usually in a same or different individual is called graft. The individual who provides the graft is called donor and the individual who receives the graft is called host or recipient.

If the graft is placed into its normal anatomic location, the procedure is called orthotopic transplantation. If the graft is placed in a different site it is called heterotopic transplantation. Transplantation is the only form of treatment for most end-stage organ failure.

In clinical practice, transplantation is used to overcome a functional and anatomic deficit in the recipient. Transplantation of kidneys, hearts, livers, lungs, pancreas and bone marrow are widely done today.

Methods of Transplantation

Auto grafting:
The transfer of self tissue from one body site to another in the same individual

Allografting:
The transfer of organs or tissues from human to human

Xenografting:
The transfer of tissue from one species to another (Figure 11.11).

Graft Acceptance

When transplantation is made between genetically identical individuals the graft survives and lives as healthy as it is in the original places. When the graft tissue remains alive, it is said to be accepted and the process is called graft acceptance.

Graft Rejection

When transplantation is made between genetically distinct individual the graft tissue dies and decays. When the graft tissue dies, the graft is said to be rejected and the process is called graft rejection. It is of two types. They are:-

1. Host Verses Graft Reaction
2. Graft Verses Host Rejection.

Host Verses Graft Reaction (HVG)

The graft tissue antigens induce an immune response in the host. This type of immune response is called host versus graft reaction.

Allograft Rejection

Types of allograft rejection

• Acute rejection-Quick graft rejection. It is due to stimulation of thymocytes and B lymphocytes
• Hyperacute rejection-It is a very quick rejection. It is due to pre-existing humoral antibodies in the serum of the host as a result of presensitization with previous grafts.
• Insidious rejection-It is a secret rejection due to deposition of immune complex on the tissues like glomerulus membrane that can be demonstrated in kidney by immune fluorescence.

Mechanism of Allograft Rejection

Immunological contact

When tissue is implanted as graft, its antigen can pass into local lymph nodes of the host. The graft antigens then make contact with the lymphocytes of the host. Production of sensitized T cells and cytotoxic antibodies are produced in the host. This brings about graft rejection.

First set rejection

When the graft is made between genetically different individuals, the graft gets blood supply from the host and it appears to be normal for the first 3 days. But on the 5th day, sensitized T cells, macrophages and a few plasma cells invade the graft. Inflammation starts in the graft. This leads to necrosis. It is similar to the primary immune response to an antigen.

Second set rejection

When a graft is implanted in an individual who has already rejected a graft is second set rejection. This is similar to the secondary immune response of our body.

Cell mediated cytotoxic reaction

The 1st set of rejection of allograft is brought about mainly by CMI response. In this process the cells involved in the cytotoxic mediated immunity involves. On stimulation of these cells interferon causes the lysis of the graft.

Antibody mediated cytotoxic reaction

The 2^2nd set rejection of graft is brought about mainly by HMI response. This is one of the hyperacute rejection brought about by the antibodies. Complement, macrophages, mast cells, platelets, B cells bring about this reaction.

Graft versus Host Rejection (GVH)

Sometimes the graft tissue elicits an immune response against the host antigens. This immune response is called graft versus host reaction. It occurs when:

• Graft remains inside the host and the host should not reject the graft.
• The graft should have immune competent T cells.
• The transplantation antigens of the host should be different from that of the graft.

Mechanism of the graft rejection

The graft lymphocytes aggregate in the host lymphoid organs and are stimulated by the lymphocytes of the host. The stimulated lymphocytes produce lymphokines. Lymphocytes in turn activate the host T cell. Activated T cell further activates the B cells. The stimulated B cell reacts with the self antigen and causes the damage.

How to prevent graft rejection?

Before transplantation the following things should be done to avoid graft rejection.

• Perform blood grouping and Rh grouping
• HLA typing should be done
• Immuno suppressive drugs should be administered
• Suitable donor should be chosen

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Hypersensitivity Types and its Classification

Hypersensitivity is defined as the exaggerated immunological response leading to severe symptoms and even death in a sensitized individual when exposed for the second time. It is commonly termed as allergy. The substances causing allergic/hypersensitivity is known as allergens. Example: Drugs, food stuffs, infectious microorganisms, blood transfusion and contact chemicals.

Classification of Hypersensitivity (Coombs and Gell Classification)

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

Type II:
Antibody-dependent Hypersensitivity

Type III:
Immune complex mediated Hypersensitivity

Type IV:
Cell mediated or delayed Hypersensitivity

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

This type of hypersensitivity is an allergic reaction provoked by the re-exposure to a specific antigen. The antigen can make its entry through ingestion, inhalation, injection or direct contact. The reaction may involve skin, eyes, nasopharynx and gastrointestinal tract. The reaction is mediated by IgE antibodies (Figure 11.7).

IgE has very high affinity for its receptor on mast cells and basophils. Cross linking of IgE receptor is important in mast cell trigerring. Mast cell degranulation is preceded by increased Ca⁺⁺ influx.

Basophils and mast cells release pharmacologically active substances such as histamines and tryptase. This causes inflammatory response. The response is immediate (within seconds to minutes). Hence, it is termed as immediate hypersensitivity. The reaction is either local or systemic.

Hay Fever

Allergic rhinitis is commonly known as hay fever. Allergic rhinitis develops when the body’s immune system becomes sensitized and overreacts to something in the environment like pollen grains, strong odour of perfumes, dust etc that typically causes no problem in most people. When a sensitive person inhales an allergen the body’s immune system may react with the symptoms such as sneezing, cough and
puffy swollen eyelids.

Type II Hypersensitivity: Antibody dependent hypersensitivity

In this type of hypersensitivity reactions the antibodies produced by the immune response binds to antigens on the patient’s own cell surfaces. It is also known as cytotoxic hypersensitivity and may affect variety of organs or tissues. Ig G and Ig M antibodies bind to these antigens and form complexes. This inturn activates the classical complement pathway and eliminates the cells presenting the foreign antigen. The reaction takes hours to day (Figure 11.8).

Drug induced haemolytic anaemia Certain drugs such as penicillin, cephalosporin and streptomycin can absorb non-specifically to protein on surface of RBC forming complex similar to hapten-carrier complex. In some patients these complex induce formation of antibodies, which binds to drugs on RBC and induce complement mediated lysis of RBC and thus produce progressive anaemia. This drug induced haemolytic anaemia is an example of Type II hypersensitivity reaction.

Type III Hypersensitivity: Immune complex mediated hypersensitivity

When a huge amount of antigen enters into the body, the body produces higher concentrations of antibodies. These antigens and antibodies combine together to form insoluble complex called immune complex. These complexes are not completely removed by macrophages.

These get attached to minute capillaries of tissues and organs such as kidneys, lung and skin (Figure 11.9). These antigen-antibody complexes activate the classical complement pathway leading to vasodilation. The complement proteins and antigen-antibody complexes attract leucocytes to the area. The leukocytes discharge their killing agents and promote massive inflammation. This can lead to tissue death and haemorrhage.

Arthus reaction

It was first observed by Arthus. It is a local immune complex reaction occurring in the skin. Horse serum and egg albumin are the antigens that induce the arthus reaction. It is characterized by erythema, induration, oedema, haemorrhage and necrosis. This reaction occurs when antibody is found in excess. It appears in 2-8 hours after injection and persists for about 12-24 hours (Table 11.1).

Table 11.1: Difference between Immediate Hypersensitivity and Delayed Hypersensitivity

Immediate Hypersensitivity	Delayed Hypersenstivity
1. It appears and disappers rapidly	1. It appears slowly and last longer.
2. It is induced by antigens or haptens by any route	2. Induced by infection, injection of antigen intra dermally or with adjuvants of by skin contact.
3. The reaction is antibody mediated B-cell response	3. The reaction is T-cell mediated response.
4. Passive transfer is possible with serum	4. Cannot be transferred with serum but can be transferred by lymphocytes
5. Desensitization is easy, but does not last long	5. Desensitization is difficult but long lasting.

It is often called as delayed hypersensitivity reaction as the reaction takes two to three days to develop. Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious diseases such as tuberculosis and leprosy. T lymphocytes, monocytes and macrophages are involved in the reaction. Cytotoxic T Cells cause direct damage whereas the T helper cells secrete cytokines and activate monocytes and macrophages and cause the bulk damage (Figure 11.10).

Type IV hypersensitivity: Cell Mediated Delayed Hypersensitivity

Tuberculin reaction (Mantoux Reaction)

When a small dose of tuberculin is injected intra dermally in an individual already having tubercle bacilli, the reaction occurs. It is due to the interaction of sensitized T cell and tubercle bacterium. The reaction is manifested on the skin very late only after 48-72 hours.

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Immunology of Western Blot Techniques

Macromolecules immobilized or fixed on nitrocellulose membrane i.e., blotted can be subjected to a variety of analytical techniques more easily. Southern blotting was the first blotting technique developed which made the analysis and recording of DNA easy.

Later the technique was extended for analysis of RNA and proteins and they have acquired the jargon terms Northern and Western Blotting respectively.

Western blotting is also known as immunoblotting because it uses antibodies to detect the protein. Western blotting is a quantitative test to determine the amount of protein in sample.

Principle

Western blotting technique is used for the identification of a particular protein from the mixture of a proteins. In this method, the proteins are first extracted from the sample. Extracted proteins are subjected to Poly Acryl – amide Gel Electrophoresis (PAGE).

Transfer of proteins from poly acryl amide to the nitrocellulose paper is achieved by applying electric field. When radio labelled specific antibody is added on such membrane it binds to the specific complementary protein. Finally the proteins on the membrane can be detected by staining or through ELISA technique.

Steps

Step I:
Extraction of Protein

The most common protein sample used for Western blotting is cell lysate. The protein from the cell is generally extracted by mechanical means or by adding chemicals which can lyse the cell. The extraction step is termed as tissue preparation.

Protease inhibitor is used to prevent the denaturing of proteins. Using spectroscopy the concentration of the protein sample is analysed and diluted in loading buffer containing glycerol. This will help the sample to sink in the well. Bromothymol blue is used as tracking dye and is used to monitor the movement of the sample.

Step II:
Gel electrophoresis

The protein sample is loaded in well of SDS-PAGE (Sodium dodecyl sulfatepoly-acryl amide gel electrophoresis). The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of all these. Proteins are negatively charged, so they move toward positive (anode) pole as electric current is applied. Smaller proteins move faster than the larger proteins.

Step III:
Blotting

Blotting refers to the transfer of the protein from the gel to the nitrocellulose paper by capillary action. Electro blotting is done nowadays to speed up the process. In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.

Step IV:
Blocking

The nitrocellulose membrane is nonspecifically saturated or masked by using casein or Bovine serum albumin (BSA) before adding the primary antibody. This blocking step is very important in western blotting as antibodies are also proteins and they are likely to bind to the nitrocellulose paper.

Step V:
Treatment with primary and secondary antibody

The primary antibody is specific to desired protein so it forms Ag-Ab complex. The secondary antibody is enzyme labelled and is against primary antibody (antiantibody) so it can bind with Ag-Ab complex. Alkaline phosphatase or Horseradish peroxidase (HRP) is labelled
with secondary antibody.

Step VI:
Treatment with suitable substrate

Finally, the reaction mixture is incubated with specific substrate. The enzyme convert the substrate to give visible coloured product, so band of colour can be visualized in the membrane (Figure 11.6).

Application

1. The size and concentration of protein in given sample is determined by western blotting.
2. It is used in the detection of antibody against virus or bacteria in serum and helps in the disease diagnosis.
3. Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
4. Useful to detect defective proteins.