Mutation3
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1. Chemical mutagens include alkylating agents, base analogues, intercalating agents, and deaminating agents. Alkylating agents can add alkyl groups to molecules and cause mutations by altering DNA bases. 2. Intercalating agents like proflavin insert between DNA base pairs, increasing rigidity and altering DNA confirmation which can cause frameshift mutations. 3. Base analogues like 5-bromouracil are structurally similar to normal bases but have slightly altered base-pairing properties, inducing nonsense or missense mutations.
Mutation3
- 1. Chemical mutagens Chemical name Common name structure 1 Alkylating agents Di-(2chloroethyl)sulfide Mustard gas or sulfur Cl-CH2-CH2-S-CH3-CH2-Cl mustard Di-(2chloroethyl) methyl Nitrogen Mustard Cl-CH2-CH2-N-CH3-CH2-Cl Amine CH3 Ethyl ethane sulfonate EES CH3-CH2-O-SO2-CH2-CH3 Ethyl methane sulfonate EMS CH3-CH2-O-SO2-CH3 N-Methyl-N-Nitro-N NTG NH=C-NH-NO2 nitrosoguanidine O=N-N-CH3 2 Base Analogue 5 bromouracil 5BU 2aminopurine 2AP
- 2. Intercalating Agents 2,8 –Diamino acridin Proflavin CH CH CH H HC C C C C C C C NH2 CH +NH N NH3+ Acridine orange Deaminating Agent Nitrous Acid HNO2 Miscellaneous NH2OH
- 3. Alkylating agents Agents that can add alkyl groups to another molecule. They usually include a large number of molecules that carry one (monofuctional) or more (multifunctional) of the alkylating groups (-CH3) Electrophilic reactants Alkylated nitrogen bases are altered in base pairing abilities eg Alkylated Guanine pairs with T in place of C EMS Guanine
- 4. Alkylation of purines causes hydrolysis of sugar base linkage &purines are lost from DNA backbone forming gaps Alkylating agents are said to be electrophilic (e- deficient) reactant because they combines with nucleophiles which have e- rich centres. Hence they are known as Radiomimetic. As their effect resembles ionizing radiations However on other hand alkylating agents also induce somehow to activate repair process similar to that of Thymine dimer repair mechanism & during repair sometimes occasional errors are made which leads to point mutations. Some alkylating agents (those having 2 alkyl groups) crosslink two DNA strands &or molecules & induce chromosomal breaks as well as aberrations
- 5. Intercalating Agents Substances like acridine orange, proflavin, acriflavin. Ethidium bromide, ICR-170, ICR191 are potent mutagens and induce frame shift mutation. These are planer structures and have dimension almost same as those of Pu-py base pairs. On the other hand they have various side chains alkyl groups. Positively charged acridines intercalates or sndwitch themselves between stacked base pairs. Thus, increase rigidity & alter confirmation of DNA possibly by producing kinks in DNA molecule. The thickness is almost equal to one base pair. So intercalation results in pushing away adjacent bp by a distance equivalent to 1 bp. The gap probably results in the insertion of randomly chosen NT during replication & produce frame shifts
- 6. Frame shift due to intercalation intercalation replication (1 strand shown) next round replication Frame shift due to addition of A:T
- 7. Base Analogues nucleoside analog: molecules which are structurally similar to normal nitrogenous bases but have slightly altered base-pairing properties Normal nitrogenous base Analog Adenine 2-Aminopurine Thymine 5-Bromouracil pair up with pair up with adenine and cytosine thymine and guanine induce nonsense or missense mutation
- 9. GC AT G 5BU G C A 5BU A T GC A 5BU AT G5BU AT A 5BU GC A5BU GC AT AT GC Total AT GC
- 10. 5BU has same structure to thart of thymine except for bromine at 5th position. Bromine & methyl group has same vander waals radii. This feature makes BU a virtual chemical twin of thymine and it can make H bond with Adenine just like thymine. So, it can fool replication machinery and get incorporated in growing DNA chain at place of T pairs with A.
- 11. Deaminating Agent Nitrous Acid (HNO2) • Source of Nitrous Acid Nitrite –a common food preservative used to keep meat “red” Ingested almost daily Eventually form nitrous acid at low pH in stomach Oxidative deamination of base • Amino group (-NH3) to keto (=O) group, once the mutated gene is expressed without correction What mutation it causes ? Base-pair mutation by altering its structure 1. Cytosine to Uracil Now the base pairs with A, instead of G, in DNA replication Correctable, because Uracil is foreign
- 12. 2. Adenine to Hypoxanthine Now A pairs with the C, instead of T • In correctable
- 13. 3. Guanine to Xanthine Still pair with Cytosine Less harmful
- 15. Hydroxylating agent hydroxylamine: • amino group of cytosine is hydroxylated • hydroxylaminocytosine base pairs with adenine • G : C →A : T NNNH2oh
- 16. Hydrogen peroxide H2O2 Fe2+ + H+ + H2O2→Fe3+ + .OH + H2O • Because of the above reaction, free radical (.OH) is produced • Oxidized nitrogenous bases which cause DNA damage • Guanine is the most vulnerable on because of its lowest ionization potential -among the nucleic acid component • Induced frame shift mutation
- 17. Ames Test Which chemical can cause cancer in human beings? Difficult to determine may be administered in mice/rat Many substances which can cause cancer can also cause mutation both of them effect DNA Bruce Ames- Univ of California developed a routine test for mutagenicity. He worked with a strain of Salmonella typhimurium requiring histidine for growth (auxotroph not capable to grow on minimal media). Strain was grown after treatment with compound in question if it grows on minimal media means it is revert back to prototype. Few cells revert back normally but if the reversion frequency is high this indicates that chemical has mutagenic potential. To improve test ability rat liver extract is added to the medium. This improves the ability of test because some compounds are as such not carcinogenic but sometimes their breakdown in liver produces carcinogenic substances. Rat liver acts in same way as that of human. Liver enzymes also convert some mutagen to non mutagenic form. Once identified these substances are immediately removed from work place/lab environment. Commonly used are hair dyes, food preservatives, coloring agents and even artificial sweetners
- 18. 1996 International agency for researches on cancer classified bacterium Helicobacter pylori as carcinogens. Any specific mechanism was not proposed to explain relationship between H. pylori & gastric cancer There must be some relation- diet and normal microbiota Importance of microbes in cancer induction Mice feed on cyacasin (compound in nut of Cycas) bacterial enzyme Bglucosidase convert cyacasin to Methylazoxymethanol Caused cancer in experimental rats & rats not infected with H. pylori unaffected by cyacasin Rats with usual microflora developed colon cancer This determines the role of intestinal components in convert chemicals to carcinogens
- 19. Elena McCoy & her coworkers at NY Medical College used Ames test to compare mutagenic ability of 2 amino flourene when activated by liver cell enzymes, intestinal cell enzymes alone, Bacteroides fragalis enzyme alone & bacterial enzyme plus intestinal cell enzymes Found that liver cell enzymes produced highest conversion of this chemical to mutagenic form Alone bacterial & intestinal produced conversion Combination of both had as great effect as that produced by liver enzymes Role of bacteria in bladder cancer (Researches at Kyushu Medical Centre, Japan) Used Ames test to evaluate mutagenic abilities of human urine activated by bacteria Bacteria from UTI infection tested for their ability to reduce nitrate to nitrite Mutagenic activity seen with addition of nitrite ion indicates that nitrite is carcingenic Results indicated that the chemical reaching the bladdercan be activated to a carcinogen by bacterial enzymes
- 20. Hot spots of Mutation Certain DNA seq are called as mutational hot spots because more liable to under go mutation than other sequences. These are Monotonous runs of single NT Tendem repeats of short seq. gain or loss of copies by variety of mechanisms & are found at many sites through out the genome & within genes, means a small no. of sites for account for a disproportionately large fraction of all mutations. Sites with cytosine methylation are usually highly mutable GC AT Transitions (Dam & DCm methylases) 1% of C residue are methylated at C5 forming 5methyl cytosine in place of Cytosine
- 22. Mutation Rate & Frequency Mutation rate can be defined as probability of mutation at a locus (or in genome) in a specific unit of time measured as organism generation or cell generation or cell division Or Frequency with which a gene changes from wt to specific mutant, expressed as no. of mutations per biological unit Frequency refers to the number of cells mutaed within a known population. Suppose 1millon cells used for nutation and 14 mutant cplpnies obtained then mutation freq is 1.4X103
- 23. Penicillin enrichment Technique is used to isolate mutants eg auxotroph. Original strain is prototroph can grow on minimal media use single carbon compound as source of C & energy. Suppose for isolating mutants for Arg – one has to expose prototype to mutagens for increasing freq. of mutations. Cells grown on minimal media with C source as well as enriched with Arg. When pop reached to max density Cells harvested & washed and transferred to media but lacking Arg. When penicillin added to cultures only auxotrophs will survive. Penicillin kills growing bacteria because it inhibits crosslinking of newly synthesized cell wall & eventually cause lysis of cell. While non growing do not possess new peptidoglycan material. Prototrophs killed because capable to grow on minimal media. Mutants incapable to grow survive.