Biology

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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

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

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.