Genetics: Principles and Applications

In this article we will discuss about Genetics:- 1. Meaning of Genetics 2. Nucleic Acid 3. Structure of Chromosomes 4. Replication of DNA 5. RNA 6. Transcription and Translation 7. Plasmids 8. Genetic Engineering 9. Separation of DNA Fragments 10. Application of Gene Cloning 11. Principle of Genetics 12. Application of Genetic.

Contents:

1. Meaning of Genetics
2. Nucleic Acid
3. Structure of Chromosomes
4. Replication of DNA
5. RNA
6. Transcription and Translation
7. Plasmids
8. Genetic Engineering
9. Separation of DNA Fragments
10. Application of Gene Cloning
11. Principle of Genetics
12. Application of Genetics

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1. Meaning of Genetics:

Genetics is the study of inheritance, except for RNA viruses, all hereditary characteristics are encoded in DNA. Each bacterial colony consists of descendants of a single cell. The progeny thus formed maintained their character with the parent cell from generation to generation.

The chromosomal DNA plays a pivotal role in the maintenance of genetic stability within the organism and the species by providing genetic information to the progeny possessed by the parent. This is accomplished by most accurate replication of its DNA. Occasional inaccuracies result in heritable variations in a small proportion of daughter cells which is about one per 10⁴ to 10¹⁰ cell division.

2. Nucleic Acid:

Nucleic acid is a polynucleotide and nucleotides are linked by means of 3,5 phosphodiester bonds. They are of two types-DNA (deoxyribonucleic acid), and RNA (ribonucleic acid). RNA is structurally similar to DNA except for two major differences The sugar in RNA is ribose instead of deoxyribose in DNA and the base uracil (instead of thymine present in DNA).

The following products are obtained on hydrolysis of the nucleic acids:

3. Structure of Chromosomes:

The structure of DNA molecule is composed of two strands of complementary polynucleotide chains wound together in the form of double helix. Bacteria, like other prokaryotic cells, are haploid (with one copy of each gene) and its nucleus consists of a circular chromosome of DNA molecules of approximately 1 mm long when straightened.

Unlike eukaryotic cell, it is not associated with histone or non-histone proteins and lies free in the cytoplasm of bacteria. Each strand has a backbone of deoxyribose and phosphate groups. One of the four nitrogenous bases are purines; adenine (A) and guanine (G) and the pyrimidine’s: Thymine (T) and Cytosine (C) is attached to each deoxyribose.

The Two strands:

These two strands are held together by hydrogen bonding between the bases on the opposite strands in such a specific manner that hydrogen bonds can only be formed between adenine and thymine (A-T), between guanine and cytosine (G-C).

4. Replication of DNA:

During replication of the DNA molecule, the two strands separate at one end, each then acts as a template for the synthesis of a complementary strand. The newly formed double stranded DNA molecule from the parent cell DNA are sorted between the two daughter cells. DNA replication is, therefore, semi-conservative as each strand is conserved intact during replication.

DNA dependent DNA polymerase is the main replicase enzyme in replication of complementary strand of DNA. It is believed that the enzyme of DNA replications is fixed of the specific site of cell membrane, possibly of mesosome, where the replication of DNA begins.

Synthesis of DNA occurs in short segments which are late joined together by ligases. There are three DNA polymerase, polymerase III is thought to be responsible for replication of DNA and polymerase I for repair work. Polymerase II is of minor importance.

Repair mechanisms:

The incorrect nucleotide sequences are excised by the help of nuclease and replacement is done by the appropriate nucleotides with relegation of the sequence.

Restriction enzymes (restriction endonucleases):

These enzymes provide bacteria with a mechanism to distinguish between their own DNA and DNA from other biological sources, and thereby depend many bacteria against incoming foreign nucleic acids.

5. RNA:

The DNA gene represents a code which is transcribed on m-RNA and then translated as the particular polypeptide. RNA mostly occurs in a single stranded form. There are three distinct types of RNA in a cell — messenger RNA, (m-RNA), ribosomal RNA (r-RNA), and transfer RNA (t-RNA),

6. Transcription and Translation:

RNA polymerase attaches itself to the beginning of a gene on the DNA and synthesizes single complementary polyribonucleotide strand, called m-RNA using one of the strand in the DNA-double helix as a template. This process is known as transcription. After transcription, m-RNA passes into cytoplasm and the m-RNA and t-RNA comes together on the surface ribosome which contains r-RNA.

The triplet base sequence on m-RNA is known as codon. The t-RNA molecule has got a triplet at one end the amino acid at the other end. The ribosome moves along the m-RNA carrying the amino acid from t-RNA, the polypeptide growing sequentially until the entire m-RNA molecules has been translated into corresponding sequences of amino acid. This process is called as translation.

7. Plasmids:

Plasmids are extra chromosomal DNA molecules and consist of circular double stranded DNA of 1-2000 mega Daltons which replicate autonomously in the host cell. Besides the chromosomal DNA molecule some bacteria such as Escherichia, Klebsiella, Salmonella, Shigella and Proteus possess extra-chromosomal DNA which is about 1-3% of the weight of bacterial chromosome.

These have been described by two different terms. Those situated in the cytoplasm in free state and replicate autonomously (independent replicons) are known as Plasmids.

DNA molecules that remain attached to the bacterial chromosome are called Episomes. It is not possible to differentiate between plasmids and episomes and the two terms are synonymously used. Like chromosome, plasmids contain their own genetic information which control their replication and ensure segregation of one copy into each daughter cell at cell division.

Plasmids are not essential for normal function of host bacterium but their presence in bacteria confers properties of drug resistance, toxigenicity, conjugative plasmid and others.

Plasmid types:

Plasmids are three main types based on their function:

1, R-plasmid

2, Col-factors

3, For Fertility factor.

1. R. plasmid:

R-plasmid (R-factor) consists of two components, One component is resistance transfer factor (RTF) which carries the genes that govern the process of intercellular transfer and other component is called resistant determinant (R. determinant) carrying resistant genes for each of the several drugs.

2. Col-factors:

Colcino genie (Col) factors are found in several species of coliforms which produce extracellular colicines. These bactericidal substances are lethal toxins for other strains of the same or closely related species of bacteria. Since similar antibacterial substances are also produce by other bacteria other than coliforms, this group of substances has been named as bactericides.

Colicins are produced by Esch. coli, pycocin by Pseudomonas, diphthericin by Corynebacterium diphtheriae. Composition of bacteriocin varies from protein to lipoprotein-polysaccharide complex. Bacterial strains producing bacteriocid in are resistant to their own bacteriocin which helps inter species typing of organisms.

3. For fertility factor:

The fertility plasmid, F-factor is transfer factor, that contains the basic genetic information necessary for extra-chromosomal existence, self-transfer and for the synthesis of sex pilus, but it is devoid of other identifiable genetic marker such as drug resistance genes.

Though F plasmid is inherited as a stable trait it is not required for normal cell functioning and is lost spontaneously on occasion. Cells carrying F plasmid free in the cytoplasm (F+ cells) have no unusual characters apart from their ability to mate with F cells and renders them F+ by conjugation.

Cell carrying F factor (F+ cell) possess sex pilus and extrude an extra-cellular protein that attaches donor cell (F+) to recipient cell lacking fertility factor (F-). The sex pilus of male bacteria (F+ cell) is probably responsible for forming conjugation-tube with female (F-) bacteria, through which genes are transferred from F+ to F- Cells.

Cell properties encoded by plasmids:

1. Drug resistance.

2. Virulence-toxigenicity and invasiveness.

3. Antimicrobial agents-antibiotics and bacteriocin production.

4. Genes determining metabolic pathways.

5. Specialised recombination system e.g. RTF R determinant association and dissociation.

6. Conjugation foundations (transfer gene).

8. Genetic Engineering:

Genetic engineering is the recombinant DNA technology by which any genetic system can be analysed. By genetic engineering, it is now possible to separate and isolate gene virtually from any cell type (bacteria, yeasts and higher forms of life including man) and to insert into bacterial cell in such a way so that it can be expressed in the formation of desired protein.

Specified gene can be identified by chemical or radioactive probes containing complementary sequences of DNA so that the DNA sequence can be used as the probe in hybridization reaction. Hybridization is the technique in which two single strands of nucleic acid come together forming a stable double stranded molecule.

9. Separation of DNA Fragments:

Desired genes or DNA fragments are separated by several techniques:

Gel electrophoresis:

The DNA fragments are separated by gel electrophoresis based on their size, the smaller the fragments, the more rapid the rate of migration.

DNA probe:

Bacteria possess a variety of restriction endonucleases, commonly called restriction enzymes. Certain restriction enzymes protect their host by cleavage of foreign (external) DNA that might be introduced by a virus and therapy protects the host. This property is utilized in gene separation.

The enzyme recognizes a specific oligonucleotide sequence in the double stranded DNA and breaks the DNA molecule at this site and produces discrete fragments that can be separated by gel electrophoresis. This technique is generally applied for analysis or manipulation of genes in bacteria. This method is not applicable with DNA of higher organisms, as they contain introns.

In higher organisms, messenger RNA, synthesizing desired protein, is isolated from the cell— with the help of reverse transcriptase a DNA copy is prepared from the RNA.

Thus a double stranded DNA is prepared using DNA polymerase separated DNA fragments can be used for gene cloning and also can be identified by gene probing by labelled DNA probe. These nucleic acid probes (DNA and RNA probes) can be utilized for various investigation (Table 96.2).

10. Application of Gene Cloning:

(1) Genetic cloning in bacteria is now widely used in developed countries for preparation of many mammalian proteins, e.g., somatostatin, human growth hormone, insulin, several interferon and tissue plasminogen activators.

(2) Preparations of some vaccines are done by making only the antigen against which the antibody is required, e.g. Hepatitis B virus, rabies, and malaria, B. pertussis.

Polymerase chain reaction (PGR for DNA amplification.

The development of PCR has made a revolutionary impact in molecular biology from 1983.

11. Principle of Genetics:

The technique is based on the nucleic acid sequence in a region which, for a diagnostic application, is produced in large amounts in vitro from small amounts of complex template.

Procedure:

The PCR involves three main stages.

The reaction is subjected to a series of temperate variations:

(1) Melting of DNA:

The double stranded DNA is dissociated to a single stranded DNA at a denaturing temperature of 94°C.

(2) Hybridisation of primers to target DNA:

In an annealing temperature of 50°-70°C, oligonucleotide primers hybridize to target DNA.

(3) DNA synthesis:

The synthesis of DNA is carried out at a polymerisation temperature using heat resistant DNA polymerase derived from thermus aquaticus (Taq) in the presence of free deoxynucleotide triphosphate — resulting in duplication of starting material. Melting the product of DNA duplexes and repeating the cycles, typically to 20 to 30 or more, an exponentia increase in the amount of target DNA occurs.

12. Application of Genetics:

The PCR provides extremely rapid analysis (one day), ease of automatic relative economy and 100 per cent efficiency.

There are many variations and modifications of PCR concept which include:

(a) Synthesis of C DNA with reverse transcriptase followed by the amplification of cDNA.

(b) Single stranded DNA can be synthesised by later alteration in the ratio of oligonucleotide primers.

(c) Detection of nucleotide sequences of infectious agents (Table 96.3).

(d) Research application: Recombinant DNA construction, mutagenesis of cloned DNA and detection of rare nucleotide sequences.