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Sunday, September 27, 2009

Mutation, Gene Mutation, Chemical Mutagen and Recombination

Sunday, September 27, 2009 - 0 Comments

MUTATION

Genes along with their chromosomes are fairly stable and transmit heredity more or less uncharged. They sometimes undergo changes called ‘Mutation’. Mutation is any change in the amount, organization or content of genetic material. It does not include exchange as a result of recombination between homologous chromosomes. Mutations may be obvious and observable by cytological techniques. Some changes in chromosomes are called chromosomal aberrations. They may be however invisible when they are in genes. Both types of mutations when passed from adult to the offspring alter the hereditary instructions.

GENE MUTATION

A change in genetic massage of a cell is called Mutation. Mutational changes that affect the message itself producing alterations in the sequence are called ‘point mutations’; since they usually involve only one or a few nucleotide. In both bacteria and eukaryotic individual genes may move from one place on the chromosome to another by a process called ‘transposition’. When a particular gene moves to different location there is often alternation in its expression or in that of neighbouring genes. In eukaryote large segments of chromosomes may change their relative location or undergo duplication. Such chromosomal rearrangement often has drastic effects on the expression of the genetic message.

Point mutations involving only one or few nucleotide, result either from chemical or physical damage to the DNA or from spontaneous pairing errors that occur during DNA replication. First class of mutation is of particular practical importance because modern industrial societies produce and release into the environment many chemicals capable of damaging DNA. These chemicals are called Mutagens.

CHEMICAL MUTAGENS

Many mutations result from the direct chemical modification of DNA bases. The chemicals that act on DNA fall into three classes (i) Chemicals that look like DNA nucleotides but pair incorrectly when they are incorporated into DNA (ii) chemicals that remove the amino group from adenine or cytosine, causing them to mispair and (iii) chemicals that add hydrocarbon groups to nucleotide bases, also causing them to mispair.

RECOMBINATION

Scientists J. Lederberg and E. L. Talum discovered specific type of sexual reproduction in E. coli bacteria. Two mutant kinds were found in E. coli bacteria. One mutant strain was named as Y10 and other mutant strain was known as Y24. Scientists prepared mixed cultures of Y10 and Y24 by providing all six additional substances which were necessary for their growth. They also discovered various recombinations of six mutations. Because the rate of back mutation was already determined in these bacteria which were very low rate therefore it was explained that the discovery of wild type bacteria and other recombinations of mutations could be the explanation that the exchange of hereditary material might have occurred during a process that was named as conjugation.

Genetic Engineering and Biotechnology and Basic Genetic- Engineering techniques

GENETIC ENGINEERING:

It is the manipulation of genetic material of any organism. Genetic engineering usually utilized bacterial cells and their plasmids, which are small circular DNA molecule. They can replicate freely within bacterial cells. Genetic engineering can produces cells that contain a foreign gene. These cells are capable of producing new and different protein. As a result of growth of these cells so many identical copies of plasmid with a foreign gene are produced. Basic steps in recombination of DNA technology are:

(1) Preparation of recombinant DNA (r DNA) molecule.

(2) Insertion of r DNA into host cell.

(3) Multiplication and production of numerous copies of host with r DNA in it.

(4) Selection of bacteria with required gene.

TRANSGENIC PLANTS:

Free living plants in the environment that have a foreign gene inserted into than are said to be transgenic organisms or genetically engineered organisms. Plants in particular are easy to genetic manipulation because they can grow by tissue culture, where entire plant can be grow from single cell. Some techniques have been developed to produce transgenic plants. r DNA can be introduced into embryo or must be in cell wall-removed cell called protoplast. The only plasmid for transgenic plants cell is Ti Plasmid, transferred by its host Agro bacterium to many but not all plants.

Main aims of developing transgenic plants are:

(i) To cultivate more nutritious plants, seeds of these plants contain all amino acids required by human. Protein enhanced beans, soybeans, corn and what are now developed.

(ii) Plants require less fertilizer and are able to make rise of nitrogen from atmosphere.

(iii) Plants grow under harsh and unfavourable condition.

GENE SEQUENCING:

Gene sequencing is a method of determining mucleotide sequence of a gene (DAA molecules) developed in late 1970s by Fredrick Singer. This gene sequence can provide great deal of information. It is often the fastest way to determine the amino acid sequences of its polypeptide, more over; it provides the location of restriction site within a gene which can be manipulated further later on.

The main phases of gene sequencing are firstly to cut genes into specific small pieces, then each fragment is individually sequenced.

Most sequences that are determined are being collected in computer data bank that are valuable for biotechnology.

Genomic Library: It consist of genetic information of a species in a preferred environment. This library provides easy access to a preferred gene for its further copying or manipulation. In order to establish a genomic library of particular species, its DNA is fragmented by means of restriction enzymes and then the fragments are inserted into plasmids and bacteriophages which are introduced into bacteria. Such bacteria are cultured at controlled conditions so that they can be used later on.

Role of BioteChNology in diagnosis of diseases:

Biotechnology is now playing very important role in the diagnosis of infections as well as genetic diseases. The use of PCR and DNA propes in providing an excellent tool for the diagnosis of such diseases even before the onset of sumptoms. Medical scientists can now diagnose more than 200 genetic diseases using such technologies. Though hybridization alalysis, now it is possibleto detect abnormal alletic forms of genes present in DNA samples.

Genetherapy

One of the potential benefits of genetic engineering is to treat genetic diseases in individuals. Theoretically it could be possible to replace or supplement the defective allele with a functional, normal allele. This could be inserted into somatic cells of the child or adult or into the germ cells or embryonic cells.

First illness likely to be treated by this techniques is called sever combined immuno deficiency disease (5CID) which is characterized by a very poor immune system. In this case the cells of bone marrow can not produce an enzyme called adenosine deaminase (ADA).

Gene therapy of germ live cells is another matter which has raised complex issues of safety and ethics. In this type recombinant DNA would be inserted into human sex cells so not only the treated individual be affected but so would all the individuals descendents.

GENETIC COUNSELLING

In most cases genetic counselling entails predicting the chances of recurrence of condition that has already affected one or more members of a family. Such counselling depends first on precise diagnosis of disorder; the counsellor must be able to explain why the disorder occurred and how it is inherited. Counselling is important for parents of a child with genetic disorder such as cystic fibrosis, hemophilia, Down’s syndrome.

Some uses and applications of biotechnology in agriculture and medicine: Biotechnology brings revolution in the field of agriculture, medicine and other fields of biological sciences. One of the most important advantage of biotechnology in that it allows mass production of proteins which were difficult to obtain in past. Another field of biotechnology is the production of hormone. Human growth hormone produced by biotechnology is used to treat dwarfs. Insulin also produced by biotechnology is being used to treat diabetics. Biotechnology also contributes to diagnose by making DNA probes available. DNA probe is a specific sequence of single stranded DNA often radio active which binds by complimentary base pairing to a gene of interest.

Cotton, corn, potato and Soyabean plants have been engineered to be resistant to either insect predation or herbicides which are also environmentally safe. In 1999 transgenic crops were grown on more than 70 million acres around the world.

Biotechnology made progress in enhancing food quality of crops like soyabeans which produced oleic acid unsaturated fatty acid.

Conjugation and Gene Recombination in E. Coli also Transduction and Transformation

Conjugation and Recombination:

J. Lederberg and E. L. Tatum discovered specific type of sexual reproduction E. coli bacteria which was named as Conjugation. Wild type E. coli usually grow on a minimal medium containgin inorganic salts and glucose. Two mutant kinds were found in E. coli bacteria which could not grow on the minimal medium. One mutant strain was named as Y10 and the other mutant strain was known as Y24.

Y24 strain was unable to synthesize three chemical substances i.e. threonine, Leucine (both amino acids) and a vitamin biotin. Various experiments revealed that both the strains can give rise to wild type back mutants but at very low rate i.e. 1/1000000 (one per million) or even 1/10000000 (one per hundred million). The scientists prepared mixed cultures of Y10 and Y24 by providing all six additional substances which were necessary for their growth. They discovered wild type bacteria from the mixed culture. They also discovered various recombinants of six mutations. Because the rate of back mutation was already determined in these bacteria which was very low rate, therefore it was explained that the discovers of wild type bacteria and other recombination of mutations could not have possibly originated due to back mutation.

This only was to explain the origin of coild type bacteria and recombinants could be the explanation that the exchange of hereditary material might have occurred during a process that was named as Conjugation.

SEXUAL MATING TYPE IN BACTERIA:

In E. coli the bacterial strains between which the exchange of hereditary material occur by Conjugation are known as mating types one mating type which donates its hereditary material to the other is usually known as F+; other bacterial strain which receives the hereditary material is called as F-. F+ cannot mate with F+ type; similarly F cannot mate with F- type. Frequency of recombination of hereditary material is relatively low. Period of 430minutes is normal time of Conjugation.

TRANSDUCTION:

When a phase virus is allowed to cause infection in bacteria, a few bacteria survive and are not destroyed by phage, although the phages chromosome had entered the bacteria. Such resistant bacteria are supposed to carry phage chromosome in inactive form known as prophage of temperate phage; however such passive phage itself is known as temperate phage. Resistant strains of bacteria which escape from Lysis are known as Lysogenic. The presence of phages chromosome in Lysogenic bacteria in an inactive form is useful to bacteria because it enables them to with stand infection and prevents the reproduction of phage particles. In 1952 Zinger and Lederberg discovered that such inactive phage in the bacteria might act as a carrier for the transfer of genes from one bacterium to another bacterium, the process is known as transduction.

Example: In Salmonella (bacteria) there are two mutant strains, one of which is tryptoplan (amino acid) dependent and the other is cysteine dependent. When these two mutant strains were grown together and were allowed to be infected with T4-phage virus, three types of bacteria were discovered from the culture i.e. wild type bacteria, tryplophan dependent bacteria and cysteine dependent bacteria. The rate of occurance of wild type bacteria in this case is much greater than can be expected by back mutation, therefore, it was concluded that the wild type bacteria in this case might have been produced by Transduction i.e. some of the wild type T4-phage virus which were containing some of the genes of the other bacteria would have been responsible to induce the property of synthesizing typtophan and cysteine.

TRANSFORMATION:

If some living strain of bacteria is exposed to purified DNA extract of some of other strain of bacteria, recombinant type bacteria arise which may show recombination for one gene that are known as single gene transformation or they may show recombination for two genes which are known as double transformants or may show recombination for three genes that are known as triple transformants. Various experiments of transformation on all Mutants have shown that many genes are involved to synthesize and enzyme amylomaltase which is actually responsible to metabolize maltose.

Example: There are two strains of bacteria one of which is resistant to sulpha drug and amethopterin (a toxic substance), the other strain is resistant to streptomycin and micrococin. When mixed culture of these two strains were grown in Laboratory or the DNA extract of one strain is added to the living culture of the other strain osme bacteria resistant to all the four substances were produced. Formation of such recombinant type bacteria by adding DNA extract of the other strain was indicative of genetic transformation.

Regulation of Gene Expression

The hereditary apparatus of the body works in much the same way as that of most primitive bacteria; all organisms use the same mechanism. RNA copy of each active gene is made and at a ribosome RNA copy directs the sequential assembly of a chain of amino acids. There are many minor differences in the details of gene expression between bacteria and eukaryote and a single major difference. The basic apparatus used in gene expressions appear to be the same in all organisms. It apparently has persisted virtually unchanged since early in the history of life. The process of gene expression occurs in two phases which are called transcription and translation.

(1) TRANSCRIPTION: First stage of gene expression is the production of RNA copy of the gene called messenger RNA or mRNA. Like all classes or RNA that occur in cells mRNA is formed on DNA template. The production of RNA is called transcription; messenger RNA molecule (as well as other t RNA and r RNA) is said to have been transcribed from DNA.

Transcription is initiated when a special enzyme called RNA Polymerase binds to a particular sequence of nucleotides on one of DNA strands. This sequence is located at the edge of a gene.

(97) FIG 6.10 PAGE 157 BIOLOGY XII SINDH TEXT BOOK BOARD

Starting at that end of the gene, RNA polymerase proceed to assemble a single strand of RNA with a nucleotide sequence Complementary to that of the DNA strand it has bound. Complementarity refers to the way in which two single strands of DNA that form a double helix relate to one another, with A (ademine) pairing with T (thymine) and G (guanine) pairing with C (cytosine). RNA strand complementary to thymine is uracilc.

As the RNA polymerase moves along the strand into gene, encountering each DNA nucleotide in turn. It adds the Corresponding Complementary RNA nucleotide to the growing RNA strand. When the enzyme arrives at a special stop signal at the for edge of the gene, it disengages from the DNA and releases the newly assembled RNA chain. This chain is complementary to DNA strand from which the polymerase assembled it; thus it is RNA transcript (copy) called primary RNA transcript, of the DNA nucleotide sequence of gene.

TRANSLATION:

The second stage of gene expression is the synthesis of a Polypeptide by ribosomes, which use the information contained in and mRNA molecule to direct the choice of amino acids. This process of mRNA directed Polypeptide synthesis by ribosomes is called ‘Translation’ because nucleotide sequence information is translated into amino acid sequence information.

Translation begins when r RNA molecule within the ribosome binds to one end of mRNA transcript. Once it has bound to mRNA molecule, a ribosome proceeds to move along mRNA molecules in increments of three nucleotides. At each step it adds amino acid to a growing polypeptide chain. It continues to do this until it eucounters a ‘stop’ signal that indicates the end of Polypeptide. It then disengages from mRNA end releases newly assembled Polypeptide.

Genetic Code: There is sued the word code several times to refer to the information stored in DNA and ultimately translated into amino acid sequence of Proteins. This genetic code is conceptually similar to Morse Code. One set of symbols (bases in nucleic acids, dots and dashes in Morse Code) can be translated into another set of symbols (amino acids in Proteins, letters of the alphabet). The question is what combinations of bases stand for which amino acids?

Translation in Protein synthesis

Translation:

The Mechanism of Protein synthesis: Translation is the final stage of gene expression during which mRNA molecules are translated into Polypeptides. The process is complex and occurs only in association with ribosomes. Three major types of RNA molecules produced during transcription, messenger, ribosomal and transfer RNA work together to synthesize proteins by the process of translation. However the main fact about translation is that sequence of amino acids in the polypeptides being synthesized in specified by the sequence of nucleotides in mRNA molecule being translated. The genetic code determines the sequence of nucleotides in mRNA molecule that specified the sequence of amino acids in a polypeptide. The process of translation can be subdivided into the following four phases.

(1) t RNA charging (2) Chain initiation

(3) Chain Elongation (4) Chain termination

(1) Charging t RNA:

Role of t RNA: Each cell contains a number of different kinds of t RNAs. Each t RNA is distinguished in functional terms by its specificity for one of 20 amino acids involved in Protein synthesis for example t RNA tyr is specific tyrosine and t RNA 2Ly for glycerine.

A t RNA forms a covalent linkage with its amino acid and can recognize and attach to a Codon specifying that amino acid. There may be more than one type of t RNA molecule for single amino acid as most amino acids are Coded by more than one Codon.

CHARGING OF AMINO ACYLATION:

Before translation starts t RNA molecules must be chemically linked to their respective amino acids. This is called charging or amino acylation. The charging is controlled by a group enzymes called amino acyl synthetases. There is specific amino acyl synthetase enzyme for each amino acid.

(2) Initiation of Polypeptide chain:

Initiation of translation involves ribosomal subunits, mRNA molecule, a specific initiater t RNA, GTP, Mg++ and at least three non ribosomal protein initiation factors which enhance binding between various components. Ribosomes do not exist free in the cytoplasm, rather they are dissociated into their large and small subunits. Small ribosomal subunit binds to mRNA molecule. The attachment takes place at specific point just upstream of the initiation Condon of the gene. The translation process itself starts when a charge t RNA base pair with small subunit. The resultant structure comprising of mRNA, small ribosomal subunit and charged t RNA is called initiation complex.

(3) Elongation of Polypeptide chain:

Once the initiation Complex has been formed, the large subunit of the ribosome attaches to the complex. The energy is provided by hydrolysis of GTP molecule. Large subunit contains two binding sites for charge t RNA molecules, P-site or Peptidyl and A site or amino acyl sites. The initiation t RNA binds to P-site provided AUG triplet is in corresponding position of the small subunit. The sequence of second triplet (anticodon) in m RNA dictates which charge t RNA molecule will become attached to A-site. If correct t RNA molecule is present, Peptidyl transferase catalyze the formation of Peptide bond which links two amino acid together. This enzyme is present in large ribosomal subunit. At the same time the covalent bond between amino acid and t RNA occupying P-site is broken. The product of this reaction is dipeptide which is attached to t RNA at A-site. This phase during which growing polypeptide chain increase in length by one amino acid is called elongation.

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