bacterial mutation

1. Mutation
• Inheritance based on transfer of genes & transfer
characters from one to another gen. Gene
duplicate before sexual rep.
• Genes are DNA seq. encode information Duplicate
accurately during sexual reproduction DNA
Polymerase copy exactly to existing DNA & Have
proof reading ability
• In spite defects occur
• These changes whether expressible or non
expressible in the Gene are called as Mutations

2. Mutation
• Sudden, random change in genetic material of a
cell
• cause the cell and all cells derived from it differ
in appearance or behavior from normal type.
• Cells with mutation–Mutant cell
• The changes may cause change in
chromosome no./ structure or change in
individual gene. May involve a change of single
base pair or substitution of one base pair to
another

3. • Mutation is ultimate source of all genetic variations provides
raw material for evolution
• Recombination merely rearranges the genetic variability to play
new combinations & natural selection preserves the
combination best adapted to existing Env. Cond.
• Adaptation cause temp changes from specified gene expression
Changes in heritable traits is due to mutation rather than
adaptation towards environmental influences
Transcription & Translation
Normal phenotype
Normal Gene
abnormal/partially def. product
Mutated Gene

4. • First report of Mutation dates back to 1791 English farmer Seth
Write found a male lamb with unusual short legs
• Hugo De Vries 1901 Oenothera lamarkiana gave the term
mutation (Latin Mutare means Change) Book The Mutation
Theory
• 1910 TH Morgan analysed white eyed mutants of Drosophila
Chromosomal basis of inheritance
• Search for inducing agent started
• 1927 HJ Mullar discovered mutagenic effect of X ray on
Drosophila
• 1929 Stadler mutagenic effect of X ray on Barley
• 1946 C Auerbach & Robson mutagenic effect of Mustard gas &
some other chemical compounds
• 1950 B McClintock Transposon Biological Mutagens

5. Types of Mutations
(A)On basis of types of cells
i) Somatic in the somatic cells & not pass to next
generation Naval oranges, golden delicious
apples, seedless emperor grapes propagated
vegetatively
ii) Germinal Potential to express in all cell of a
mutant mutation in germinal cells

6. (B) On the basis of type of chromosome
Sex chromosome or Autosomes
(C) On the basis of site of mutation
Chromosomal mutation at chromosomes
Structural
Numerical
Gene Mutation at the level of genes may be of
two types
Point Mutation gene mutation involving a single
base May be of three types
Substitution when a single base is replaced by
another AT GC may be of 2 types

7. Transition one purine replaced by another
purine & vice versa A=G; C=T
Transversion one purine replaced by
pyrimidine & vice versa A=T; G=C; A=C;
G=T
Substitution may change the composition of a
triplet code so that it codes for a different a
acid & in turn may change the protein

8. Types of Substitution
Missence Mutation involves alteration of a base changing a
codon in such a way that encoded a acid is altered usually in
one of first two bases (3rd likely to cause a mutation). The
change of a acid is such that it alters the st & Function of entire
protein
TTC GAT GAG CCC TTG TGC ACG DNA
UUC GAU GAG CCC UUG UGC ACG RNA
Phe Asp Glu Pro Leu Cys Thr Protein
TTC GAT AAG CCC TTG TGC ACG DNA
UUC GAU AAG CCC UUG UGC ACG RNA
Phe Asp Lys Pro Leu Cys Thr Protein

9. Non sense Mutation If by substitution a nonsense codon
comes in mRNA prior to onset of terminator codon. Thus
polypeptide formed with less no. of a acids
AUG GCC AAA GGC UUU CCC
Met Ala Lys Gly Phe Pro
A = U substitution
AUG GCC UAA GGC UUU CCC
Met Ala Ochre Terminator
Silent Mutation some times a change is in such a way that any
change is not noticed due to degeneracy of genetic code same
a acid is encoded
AUG CCC AGG AAA GGC UUU
Met Ala Arg Lys Gly Phe
AUG CCC AGA AAA GGC UUU
Met Ala Arg Lys Gly Phe

10. Neutral Some times it may go unnoticed if a substitution
of nt codes a similar type of a acid & the function of
protein is not altered
AGG AAG; Arg Lys
Missence Mutation
Ala Mutation GCA to GGA GLY
Amino acid change
Ala Gly
Original Protein Mutated Protein
This may have a major or minor change when change of one base
alters a codon severity depends on location & nature of a.acid
substituted Genx123 mutation at 123rd position of gene. eg Arg 185
Leu or R185L This indicates arginine at 185th position has been
replaced by Leucine. Main effect is observed if substitution occurs
for conserved aacids (Active site or proteins involved in folding

11. Chemical nature of original a acid & replacing one is also imp.
Codon UCU (Serine) change to ACU (Threonine) both are smaller
hydrophilic with hydroxyl groups. This is called as conservative
substitution
Ala (-) Glu
When one a acid replaced with other having different
physical & chemical properties is known as radical
replacement. Codon GUA valine changed to GAA
glutamic acid Replacing bulky hydrophobic residues
smaller hydrophobic residue carrying a negative charge.
Thus crippling most of proteins. If located on surface
much effect not observed if this residue is on active site

12. An interesting mutant is temperature sensitive mutant (Ts). Protein
folds properly at low temp. but folds properly at low temp but unstable
at high temp.& unfolds
UGU Cys UUU Phe UCU Ser
CAU His UAU UAC Tyr
Tyr
AAU Asn
GAU Asp UAG stop
UAA stop

13. Addition & Deletion addition/deletion of a
single base and causes
Frame Shift Mutation Going to alter entire
reading frame
AAG AGU CCA UCA CUU AAU GCC
Lys Ser Pro Ser Leu Asn Ala
AAG GUC CAU CAC UUA AUG CCC
Lys Val His His Leu Met Ala

14. On basis of origin
Spontaneous & Induced
Spontaneous mutations
naturally occurring mutations, by chance no specific
agent for their origin, arise due to inherent errors of DNA
replications, Sometimes cell environment is also
responsible
Induced mutations
• occur when an organism is exposed to a physical or
chemical agent, known as mutagen, that interacts with
DNA to cause a mutation Biological agents also
• Induced mutations typically occur at a much higher
frequency than spontaneous mutations

15. On basis of activity
Leaky Mutant expressing very low level of activity
Null Mutant When the function of a gene becomes zero
On basis of Direction
Forward mutation in wild type leading to production of
mutant is called as forward mutant
Back or suppressor Mutation if a mutant allele is
converted to wild type is known as back or suppressor
Other types 1. suppressor Mutation if mutation occurs at
a second site which completely or partially restores a
function lost at another site because of earlier primary
mutation. The mutation does not reverse original site but
compensates for its effect & infact the organism becomes
double mutant

16. Polar Mutation If a mutation occurs which not
only results in defective gene product but also
interferes with expression of one or more
adjacent gens
Conditional mutation when mutant phenotype
expressed under specific condition eg chain
termination & temperature sensitive mutant
Auxotrophic Mutation Nutritionally defective
mutation

17. Spontaneous Mutation
G CA G G C TTTTTTTTT C G A
G CA G G C TTTTTTTTT C G A
AAAAGCT
slippage TT
G CA G G C TTTTT TT C G A
C GT C CG AAAAA AA G C T
Spontaneous Mutation can occur by mutagens arise
spontaneously eg DNA replication errors, tautomeric shifts
DNA Replication Errors
Enzymes replicating DNA are not perfect and also cause errors
although DNA Polymeras has proof reading ability

18. In some cases it is part of DNA poly. Itself but in some cases
accessory protein such as DNAQ associated with E coli DNA Poly
III Cells having mutation in proof reading ability shows high rate of
mutation
Drake isolated mutator mutants (high Spon. Mut. than normal) &
anti mutator (lower rate than normal) it was found that former have
high Polymerization in comparison to Exonuclease and later have
lower
Genes give rise to altered mutation rates when they themselves are
mutated are commonly called as mutator genes(E coli dna Q
mutant originally called as mutator D)
Error rate is low 1 in 10 million & 20 times high in lagging than
leading
DNA Poly I less effective than DNA Poly III in proof reading (gaps in
lagging filled by DNA Poly I)

19. In addition to putting a wrong base polymerase may or rarely omit
bases or insert extra bases due to strand slippage if a run of
several identical bases occur in case template & newly formed
strands may become misaligned
As in Fig. template strand of DNA contains numerous T in row
during replication T pairs with A & long A track may cause
confusion here T may slip & pair out of register This does not pair
and bulge out
Mutation can result from mispairing & recombinations occur
between closely related Seq of DNA eg 2 alleles of a gene & many
rearrangements include deletion, inversion, translocation,
duplication may result from mistaken pairing followed by
recombination

20. DNA
Copy 1 Gene1 Gene 2 copy 2
Copy 1 Two molecules of DNA pairs up
Copy 2 Copy1 Gene1 Gene 2 copy 2
Copy1 Gene1 Gene2 copy2
Result of Recombination
Gene 2
Similar Seq on same orientation mispairing
Gene 1 mispairing followed by crossing over generate a
duplication on one molecule & corresponding deletion on other

21. 2 copies of seq on same DNA but facing each other e.g. opposite
orientation mispairing followed by crossing over will generate an
inversion eg E. coli contains 7 copies of genes for rRNA. Some
strains of E. coli where rRNA operons are inverted.. Such strains
grow slightly slower but unstable.
copy 1 Gene1 Gene2 Gene 3 copy 2
Inverted repeat Inverted repeat
Gene1 gene2
Gene 3
Copy 1 copy2 Recombination
copy 1 Gene3 Gene2 Gene 1 copy 2

22. DNA containing 2 copies of seq. inverted relative to each other.
Three intervening genes 123 with their direction of
transcription (arrow). Duplicate seq may pair up & forms stem
loop and under go recombination. Result is inversion of region
between the duplicate seq. This reverses the direction of
transcription of 3 enclosed genes with respect to DNA molecule
Spontaneous mutants can be result of Tautomerism
Bases of DNA are subject to spontaneous alteration called as
Tautomerism They are capable to exist in two forms between
which they interconvert G exist in both keto & enol forms
produced by shifting of electrons & protons. The forms are
known as tautomers or structural isomers. tautomers are
capable of hydrogen bond with non-complementary base T can
have enol form while A & C can exit in amino & imino forms.
During DNA replication G is in enol form polmerase will add T
across it instead of C. This result in GC to AT transition.

23. Spontaneous Mutation Tautomer of Thymine

24. Tautomer of Cytosine

26. Tautomer

27. Spontaneous mutation can also be caused by inherent chemical instability
DNA relatively stable but some components show level of spontaneous
chemical reaction, Some times deamination (Cytosine provide Uracil)
methyl cytosine forms methyl uracil means T.
Deamination of A (to hypoxanthin) & G (to xanthin) occasionally & both
pairs with C & sometimes mutations occurs.
Oxidative damage of DNA also occurs, hydroxyl & super oxide radicals
derived from molecular oxygen attack several bases. Most common target
is G oxidised to hydroxyguanine which pairs with A & GC Mutates to TA
Often bonds linking Nitrogenous bases to Sugar spontaneously hydrolyse
most often purine than pyr generating apurinated sites
Insertional mutations are due to transposon
Most insertional mutations are due to mobile Genetic elements inserting
themselves into DNA e.g. IS elements, transposons, retroposon, certain
viruses integrate their DNA into host chromosome. Insertion are indicated
by symbol :: between target gene and inserted element
LacZ::Tn10 Insertion of transposon Tn10 into LacZ gene. Presence of
transposons increase the freq. of various other rearrangements eg deletion
& inversion

28. Transposons & viruses usually contains multiple transcription terminators.
RNA Poly can not transcribe through them. So presence of transposon block
transcription. So any other gene downstream sharing same promoter as the
gene suffering the insertional events. This effect is known as polarity (in
prokaryotes the genes are in clusters & transcribed together, shows more
likely the polarity effect in eukaryotes to insertion.
DNA Promoter Gene1 Gene2 Gene2 Gene3 Terminator
Insertion of Transposon
Transposon
Transcription
transposon promoter

29. Small amount of streptomycin when added to bacterial cultures all
bacteria killed within few minutes
Bacteria exposure to phages lysis & death of host cell
Small fraction 1 in million survive when propagated progeny become
resistant
Resistant population of bacteria owe their survival to mutation
Changes in a gene which encode a ribosomal protein results in the ability
of the streptomycin resistant bacteria to synthesize protein even in
presence of streptomycin
Similarly resistant bacteria isolated from a population of cells killed by
phages have altered the genes for synthesis of cell wall components which
do not allow bacterial viruses to attach or enter the cell
These mutations in bacteria arise only following exposure to the
appropriate agent debated for many years whether mutations are
adaptive changes where a sp. agent would induce the required alteration
in the genetic material
Alternative hypothesis supported by Darwinian theory of evolution
proposed changes to occur spontaneously & continuously in a population

30. It is selective process which allows us merely to
identify such bacteria which carries a specific
mutation
Controversy resolved in early 1940’s when M
Delbruck & S Luria demonstrated Fluctuation test
which demonstrated that mutations are specific &
independent of selective environment
Original test performed was based on the ability of E
coli to mutate into a form which is resistant to
phageT1. When exposed to T1 a population of T1
resistant colonies obtained.

31. Fluctuation Test
Cultures of E coli propagated in
flasks & divided in two
Tubes
Content of
One tube sub
Divided into
50 tubes
All cultures grown to stationary phage (from single to 50 tubes)
Small aliquot was spread on agar densely seeded with phage
Which will kill susceptible but not to resistant
Incubated overnight & phage resistant colonies were observed

32. Cultures incubated in single tube yielded 3-7 resistant colonies.
The range of such colonies ranged from none to 100 or more.
What was the reason for this variation in no. of resistant
colonies ?
If mutation occurred from result of phage contact aliquots from
large & small cultures must produce equal no of colonies
If phage resistant mutants arising all the time individual culture
should have great variations in no. depending upon time
mutation occurred during growth
Mutation occurred immediately after subdivision as many as
100 of resistant bacteria will be identifiedin tube following
plating on phage containing media. If mutation occurred long
after subdivision or not at all small no of resistant colonies will
be identified.
Results favor a next hypothesis

33. Replica plating
J Lederberg 1952 demonstrated Replica Plating technique
Reported E coli cells mutate spontaneously & become resistant to T1
E coli cells grown on master plate on nutrient agar media
Sample of cells transferred by pressing it on a sterile velvet surface
supported on a circular block to a fresh agar medium coated with T1
phage. Identical pattern of colonies observed. Question arises about the
origin of these mutant colonies. If mutation had already occurred on
master plate. Same colony may appear on same location on new plate. He
repeated the experiment several times. Phage resistant colony was
obtained on same location & true breeding resistant strain could develop.
Result suggest that resistant cells arose by mutation on master plate
& not induced by the phage to become resistant.
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