mutation chapter3

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.