Document mutation.docx
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Mutagens are agents that cause genetic mutations by altering gene expression and can be physical, chemical, or biological in nature. The document outlines various types of mutagens, their effects on DNA, and methods for isolating mutants, including the Ames test for evaluating potential carcinogens. It also describes specific examples of mutagens and their mechanisms, including the roles of radiation and chemical compounds in inducing mutations.
Document mutation.docx
- 1. INTRODUCTION – mutagen and isolation of mutant ⚫ Mutagens are the known agents either physical, chemical or biological cause mutations by altering the genotype or gene expression which results in genetic abnormality. Not all mutations are caused by mutagens only Induced mutations were caused by mutagens. Spontaneous mutations are naturally occurring mutations. Mutagens causing cancer, are likely to be known as Carcinogens. Discovery - The first mutagens to be identified were Carcinogens. The mutagenic property of mutagens was first demonstrated in 1927 by Muller, he discovered that X- rays can cause genetic mutations in fruit flies.Edger Altenburg also demonstrated theMutational effect of UV radiation in 1928. Charlotte Auerbach and J.M Robson found that mustard gas can cause mutations in fruit flies. EFFECTS- At chromosomal level mutagens alter the structure or number of chromosomes,
- 2. as deletion, duplication, insertion, translocation, monosomy and nondisjunction are some of the chromosomal abnormalities caused by mutagens. Teratogens- are class of the mutagens which causes congenital malformations. Eg: X-rays, valporate, toxoplasma. Carcinognes -are the class of mutagens which Induces tumour formation and thus cause cancer.Carcinogenic agents include X-rays/ UV rays,Aflatoxins, retrovirus etc. Clastogens -are the class of mutagens responsible for chromosomal-breakage, deletion, duplication and rearrangements. UV rays, bleomycins, HIV virus are common type of clastogens. Mutagens also alters the codon, deletes bases, alters bases, breaks hydrogen bonds, phosphodiester bonds or changes gene expression. TYPES OF MUTAGENS Three different types of common mutations are observed in nature physical, chemical & biological agents. Many mutagens are not mutagenic by themselves. But can form mutagenic metabolites through cellular
- 3. Process. Such mutagens are called promutagens. Physical agents: Heat and radiation Chemical agents: Base anlogues Intercalating agents Alkylating agents Deaminating agents Metal ions Biological agents: Viruses Bacteria Transposons. PHYSICAL MUTAGENS RADIATION: The first mutagenic agent reported in 1920. ii. Radiations directly damage the DNA or nucleotide structure which might be either lethal or sub lethal Radiation causes cross linking of DNA or protein, chromosomal break, stand break or loss of chromosomes.
- 4. Iv. At the molecular level it causes deletion of bases or DNA strand bases. X-RAY RADIATION: X-rays are one of the most common types of ionizing radiation used in many of the medical practices. At molecular level the lethal dose of X-ray (350 - 500rems)breaks phosphodiester bonds between DNA and thus results in strands breakage. It causes multiple strand breakage. If strand breakage occurs in both the strands, it becomes lethal for cell. UV-rays: It is non ionizing type of radiation having less energyIn it. The major causes of UV radiation are base deletion Strand breakage, cross linking and generation of nucleotide dimers. UV induced mutations are dimer formation, thymine Thymine dimer & thymine-cytosine dimers are Commonly formed as the lesions block replication as Well as transcription. Formation of pyrimidine dimerization cause distortion in structure of DNA and prevents Formation of replication fork. CHEMICAL MUTAGENS
- 5. BASE ANALOGS: They are similar to the bases of DNA-purine and pyrimidines or structurally resemble the DNA bases. Bromouracil and aminopurine are two common base analogs incorporated in DNA instead of normal Bases. Bromouracil pairs with adenine as like the thymine And causes mutation. Aminopurines cause AT to GC or GC to AT transition During replication. ALKYLATING AGENTS: Mutagens, types of mutations AnuKiruthika Ethylnitrosourea, mustard gas and vinyl chloride are common alkylating agents, add alkyl group to the DNA and damages it. The agents cause base pairing errors by increasing Ionization and produces gaps in DNA strand. Alkylated purine bases are removed by the Phenomenon called depurination. Common alkylating agents are: Methylhydrazine, Temozolomide, Busulfan, Thio-TEPA, Dimethyl sulphate, Ethyl ethane sulphate. INTERCALATING AGENTS:
- 6. Ethidium bromide, proflavine, acridine orange are Few intercalating agents. The molecules intercalate between the bases od DNA & disrupt its structure. If incorporated during replication, it can cause Frameshift mutation. It may also block transcription. METAL IONS: Nickel, chromium, cobalt, cadmium are common metal ions that cause mutations. The metal ions work by producing ROS(reactive oxygen species), hindering DNA repair pathway. Cause DNA hypermethylation or may directly damage DNA. OTHER CHEMICAL MUTAGENS: ROS, benzene, synthetic rubber and rubber products, sodium azide, aromatic amines, deaminating agents etc are other mutagens which create different mutations BIOLOGICAL MUTAGENS Viruses, bacteria and transposon are biological mutagens. VIRUS: Virus insert their DNA into our genome and disrupt the normal function of DNA or a gene. Once it inserts DNA, the DNA is replicated transcribed and translated viral protein instead of our own protein. Eg: HIV.
- 7. BACTERIA: Some bacteria can cause inflammation. It provokes DNA damage and DNA breakage. TRANSPOSONS: They are non coding DNA sequences, jumps from one place to another in a genome and influence function of genes, ISOLATION OF MUTANTS: Mutation occurring in microorganism can be Detected and efficiently isolated from the parent organism of other mutants. While studying we must be aware of wild type characters of an organism,so the mutants can Easily detected. In bacteria and other haploid microorganism, the detection system are straight forward because any new allele should be observed immediately. In albino mutation, the detection is very simple. It requires only change in colour of bacterial colony. The other detection systems are rather complex. SOME DETECTION METHODS Replica plating technique. Resistance selection method. Substrate utilization method. Ames method REPLICA PLATINGTECHNIQUE:
- 8. Joshua and Esther Ledgerberg (1952) developed a New technique called replica plating. This technique is used to detect auxotrophic mutants and wild type strains on the basis of ability to grow in the absence of amino acids. Also this test is used to demonstrate the presence of antibiotic resistance in bacterial cultures prior to exposure of antibiotic STEPS INVOLVED Generate the mutants by treating a culture with a mutagen e.g.nitrosoguanidine. Inoculate a plate containing complete growth medium and incubate it at proper temperature. Both Wild type and mutant survivors will from complete medium This plate containing complete medium is called master plate. Prepare a piece of sterile velvet and gently on the upper surface of the master plate to pick up bacterial cell from each colony. As pressed the master plate, again gently press the velvet on the replica plates containing complete medium in one set and lacking cine in only leucine in the other set. Thus, the bacterial cells are transferred in replica plates in the same position as in master plate. Incubate the plates and compare the replica
- 9. plate with master plate for bacterial colony not growing on replica plate.. 2.RESISTANCE SELECTION METHOD This is another method used for isolation of Mutants. Generally the wild the wild type cells not resistant either to antibiotics or bacteriophage. • Therefore, it is possible to grow the bacterium in the Presence of agent. This method is applied for isolation of mutants resistant to chemical compounds that can be amended in agar, phage resistant mutants. 3.SUBSTRATE UTILIZATION METHOD: This method is employed in the selection of bacteria. Several bacteria utilize only a few carbon sources. The cultures are plated on to medium containingAlternate carbon sources. Any colony that grows on medium can use the Substrate and are possibly mutants. These can be Isolated. Sugar utilization mutants are also isolated by Means of color indicator plates . EMB medium is used for this purpose.This medium contain lactose sugar as carbonSource and complete mixture of amino acids.
- 10. Therefore both lactose wild type and lactose mutant cells can grow and form colonies on EMB agar plates. The lac+ cells catabolize lactose and secrete acids, therefore the pH of the medium decreases. This will result in staining of colony to dark purple. On the other hand, Lac- cells are unable to utilize Lactose and use some of the amino acids as carbon Source. After utilization of amino acid, ammonia is produced that increases the pH and de colorize the dye resulting in white colony. Ames test In 1974 Bruce Ames developed a method for evaluating the potential of chemical to cause cancer, known as Ames test. Ames test is based on the principle that both Cancer and mutations results from the damage of DNA, and results of experiments have Demonstrated that 90% of known carcinogen areAlso mutagens. Several species of salmonella typhimurium are employed. Each strain contains a different mutation in the operon histidine biosynthesis. STEPS OF AMES TEST:
- 11. Prepare the culture of Salmonella histidine Auxotrophs (His-). Mix the bacterial cells and test substance( mutagen) in dilute molten top agar with a small amount of histidine in one set, and control with cmplete medium plus large amount of histidine. Pour the molten mix on the top of minimal agar plates and incubate at 37°C for 2-3 days. Until histidine is depleted all the His- cells will grow In the presence of test mutagen. When the histidine is completely exhausted only the revertants will grow on the plate. 。 The number of spontaneous revertants is low, Whereas the number of revertant induced by Carcinogen is quite high. High number of colonies represent the greater Mutagenicity. A mammalian liver extract is added to the above Molten top agar before plating. The extract converts the carcinogen in to electrophilic derivatives which will soon react with DNA molecule. In natural way it is occurs in mammalian system When foreign particle are metabolized in the liver. Bacteria does not have metabolizing capacity,
- 12. therefore, the liver extract is added to this test, to promote transformation.