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Mutation 2014

The Problem Watson and Crick took on


What are the requirements for genetic material explaining the inheritance of molecules,
physiology, morphology, behaviour etc.
A code that is: (1) stable (2) replicable (3) expressible (4) can evolve
In April 1953, Watson and Crick proposed a structural model in Nature:
We propose
the double helix, (stable structure)
complementary base pairing (expressible) and
a mechanism for DNA replication by unwinding and separating
Followed by a scale model (1954 Proc. R. Soc. (A): 80 - 96) they also proposed a mechanism
for mutation (can evolve)
Several years later, Crick et al. (1961) provided evidence that the code involved triplet base
sequences.

L14 Mutation


Forward Genetics depends on phenotypic variation as an index of
genetic variation: its inheritance or transmission-genetic pattern; how
transmission is related to chromosome variation; how character and
phenotype differences depend on metabolic pathways involving several
genes that may or may not be functional; how they replicate, express
peptides and how this expression is regulated, but not source(s) of mutation .

Scale of mutation: this can range from a change in a single base (substitution),
through several bases (insertiondeletion), to a chromosome fragment.



Mutation rate is low What really governs mutation rate is the efficiency of
repair mechanisms. Regardless, the rate is never 0, some base changes (1) go
un-repaired, or, (2) repair mechanisms introduce a mutant variant.

If one of these (1 or 2) occurs in a germ cell (sperm,eggs) which happens to
segregate and produce an embryo, it will be passed on to descendent
generations.

Viruses and Prokaryotes the average


base mutation rate decreased
isometrically (slope ~-1) with
increasing genome size -

Multicellular eukaryotes- the base


mutation rate increases with
genome size - a tradeoff with speed
and the efficiency of repair
mechanisms or drift ?

M. Lynch.
2012.
Evolution of
the mutation
rate.

Trends in
Genetics. V
26 pp 345-362

Mutations are:
(1) potentially permanent (mutations in somatic cells), and



(2) possibly inherited changes in DNA sequence (mutations in germ cells).







Classify:


(A) spontaneous - occurring in the absence of known mutagens

or


(B) induced mutations - require an known agent that increases the
mutation rate significantly above the spontaneous rate.

Spontaneous mutations

Rare tautomers

Spontaneous mutations



Tautomeric or keto-enol,
amino - imino shifts
suggested by Watson &
Crick (1953), involve:

(1) the migration of a Hbond ,

(2) the switch of adjacent
single and double bonds



Depurination -covalent
bond between sugar and
purine is less stable than
sugar-pyrimidine.

= rare single base loss

Note this tautomeric shift must happen during the process, just
before/as DNA replicates.

An apurinic site - any complementary base could be substituted
during replication.

Spontaneous (single strand) mutations in germ


cells may be inherited, if a mutant gamete
forms a viable zygote that survives to
reproduce.

Faithful, but not perfect



spontaneous mutations during replication

Copy errors in replication could cause heritable changes to a DNA
sequence.

There are 3 error - checking steps in DNA replication:
(2) proof reading (3) mismatch repair

(1) base selection

(1) Base selection if the correct base is not chosen, the polymerase site in the
holoenzyme will not be activated.

(2) Proof reading: 3-5 exonuclease activity (DNA pol III)

(3) Mismatch repair after replication, the wrong nucleotide will not generally
pair correctly, it causes a bulge in the helix, mismatch repair enzymes (Mut S, L
and MutH) scan for bulges, if found soon after replication, non-methylated
strands are repaired based on its methylated complement. See fig 15-26

If both strands are methylated, the mutation may be inherited (pp 536-538).

Mutations and their molecular basis (15.3)





High energy radiation cause transitions and transversions.


Transitions - replacement of a base by the other base in the same


chemical category (purine/purine A-G, pyrimidine/ pyrimidine (C-T). e.g.
tautomeric shift (pp 520)

Transversions - replacement of a base of one category with a base


from a different category









Base analogues (pp 526) cause transitions or transversions in the
first replication. E.g. 5 bromouracil



Intercalating agents (pp 528) cause indels -










insertions and deletions.



?conservative chemically similar neutral mutation

?non-conservative - chemically different


5 bromouracil is an analogue of

thymine

It forces a tautomeric

shift

Rare, but frequent


compared to the enol
form of thymine.

Intercalating agents mimic base pairs and insert


themselves between them (intercalate) usually
causing an indel mutation - an insertion or
deletion of one or more bases

Classify by scale, whether spontaneous or induced


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Point Mutations - summary Fig 15-2


1. Point mutations base substitutions,
- synonymous or silent
- missense mutations,
- nonsense mutations
- sense mutations
2. Point mutations -Frame shift mutation -indel mutants
- insertion or deletion of nucleotides,
-insertion or loss of one or two nucleotides

Point mutations : Synonymous mutations are changes or

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substitutions in a nucleotide sequence that do not cause a change in
amino acid sequence.

DNA: 3TAC GCT CCT CTT GGT GCT


Protein Met- Arg- Gly- Glu- Pro- ArgMutant
DNA: 3TAC GCT CCT CTT GGT GCG
Protein Met- Arg- Gly- Glu- Pro- ArgSynonymous mutations have no effect on the protein function

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Point mutations: Missense mutations change a codon, and it

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mutates to a different amino acid, it is potentially non-silent, it may


be chemically similar, it may be chemically different , thus having a
non neutral effect on a character expression. The effect of the amino
acid substitution may be conservative (similar chemical properties) or
non conservative.
Protein Met- Arg- Gly- Glu- Pro- Arg- AspWild type
DNA: 3 TAC GCT CCT CTT GGT GCT CTA
Mutant
DNA:
Protein

TAC GCT CCT CTT GGT CCT CTA


Met- Arg- Gly- Glu- Pro- GLY- Asp-

Some point mutations are very deleterious those that change


critical amino acids in a protein.

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Point mutations: Nonsense mutations change a codon to a 15



STOP codon
Sense mutations - change a protein coding or
a stop codon to a START codon.
WT
DNA: 3TAC GCT CCT CTT GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Arg- AspMutant
DNA: TAC GCT ACT CTT GGT GCT CTA
Protein

Met- Arg STOP

Non sense mutations produce truncated proteins and are nearly


always deleterious except those at the extreme carboxyl end.

Nonsense mutations change a codon to a STOP


codon.


Sense mutations change a stop codon to a protein
coding codon.



Same - sense mutation may be a synonomous
mutation which may be silent or noisy
synonomous mutation affecting a splice site, an
RNAi site or a binding site ( no longer a
palindrome)



Missense mutation- change protein coding

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Mutation,
1. Point mutations substitutions,
- synonymous
- missense mutations,
- nonsense mutations
- sense mutations
2. Point mutations -Frame shift mutation - indels
- insertion or deletion of nucleotides,
-insertion or loss of one or two nucleotides

Frame shift mutation - the addition or loss of 1 or 2


nucleotides:
Frame shift mutations change all the amino acid sequence
after the mutation and often introduce stop codons
C

WT
DNA: 5ATG CGA CCT GAA GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Arg- AspMutant
DNA:
Protein

ATG CGA CCT CGA AGG


Met- Arg- Gly- Arg - Arg .

Frame shift mutations are deleterious

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A 3 base pair addition or deletion mutation
will add or delete one amino acid. In this case the effect
depends on the chemical similarity of the protein with or
without one of hundreds of amino acids. Any addition or
deletion not dividable by 3 will cause a frame shift.

Frame shift mutations can be corrected


by a second mutation up or downstream and
nearby. The negative effect increases with the
size of the frame-shifted sequence.
All amino acids encoded between the two mutations
will be changed. Sometimes short stretches of changed
amino acids will not inactivate a protein.

Frame shift correction by 2nd indel mutation:


WT
DNA: 5ATG CGA CCT GAA GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Ala- LeuC addition

Mutant
DNA:
ATG CGA CCT CGA AGG..
Protein
Met- Arg- Gly- Arg - Arg.
2nd

G deletion

Mutation
DNA:
ATG CGA CCT CGA AGT GCT CTA
Protein

Met- Arg- Gly- Arg - Arg Ala- Leu-

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Forward Genetic Analysis is only possible with genetic


differences: mutants, variants, polymorphisms etc..

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The most useful mutations for research are conditional


(lethal) mutations:
(a) Auxotrophic mutants - unable to synthesize essential
metabolites- e.g Beadle & Tathum
(b) Temperature sensitive mutations- grow or express at one
temperature .
(c) Suppressor-sensitive mutants are viable when a second
genetic factor a suppressor is present , inviable in the
absence of the suppressor

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Large Sequence Alterations


Large -scale changes in chromosome


structure. See fig 16.19

1. Gene Duplications

2. Deletions

3. Inversions

4. Translocations

Human
Normal
Karyotype

Karyotype
from
cancer cell.
Note extra copies
lost copies
translocations

Duplications
A B

A B

C C

A B

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E
D

F
E

G
F

E C D

H
G
E F

I
H

J
I

J
H

Duplications could create an imbalance in the number


of genes.
Evolutionarily, it is clear that duplications give rise to
extra copies that can evolve to produce novel genes
(paralogs Haemoglobin , ).

Duplication of globin molecule


Human chromosome 11, 5 - epsilon gammaG gammaA delta


beta - 3.

Human chromosome 16 : 5- zeta - pseudozeta - mu - pseudoalpha-1 alpha-2 - alpha-1 - theta - 3

, Molecular
order (5- 3)
reflects their order
of expression.


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Same gene, different functions, probably


evolved through an inactive duplication

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1.Duplications
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2. Deletions of genes or a large portion of its sequence
3. Inversions
4. Translocations
Many mutations occur in meiosis

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Deletions:
A B

A B

Deletions are usually deleterious they are the loss


of genes. They may exist in a heterozygous state. But, (1)imbalance in gene
number can be deleterious. (2) they can uncover deleterious recessive
alleles that may not otherwise be expressed (3) probably due to slippage during
replication, mispairing in meiosis breakage, deletion & duplication.

1. Duplications
2. Deletions
3. Sequence inversions of many bases to several genes.
4. Translocations

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Inversions either include the centromere or not


A B

Paracentric inversion:- no recombinant offspring (normal & inverted)

A B

F E

Pericentric inversion no recombinant offspring, includes the


centromere (normal & inverted)

A B

G F E

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In both, recombinants are not viable

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Recombinants are inviable, thus

inversion heterozygotes are never recovered


If inversions lead to reduction of numbers of
surviving offspring from crossovers in the inversion
region , what effect do they have on map distance of genes
in the region of the inversion ?

1. Duplications
2. Deletions
3. Inversions
4. Translocations of chromosome segments

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Reciprocal translocationsbreak and reciprocal fusion
of non-homologous
chromosomes.
Meiosis I:
Alternate segregation - both
pairs have a complete gene
set - fine
Adjacent segregation pattern
produces inviable gametes
Thus translocation
heterozygotes are semisterile
(fig. 16.30)

Mutjac deer are found in China


SE asia and India. While these
species are morphologically
similar, they are cytogenetically
distinct.



The Chinese muntjac
(Muntiacus reevesi) has a 2n
number of 46 in both

Sexes.



The Indian muntjac, Muntiacus

muntjak, possesses the lowest
diploid chromosomal

number in mammals (2n = 6 for
females and 7 for males).



However, these two species,

can produce viable F1 hybrids
(2n = 27) in captivity, and
partial spermatogenesis was
observed in hybrids .

Figure 4-14 Molecular Biology of the Cell ( Garland Science 2008)