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Mutations
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Frameshift mutations
A single base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
WT
Met Phe Ser Phe Ser Phe Ser
Delete C
AUG UUU AGU UUA GCU UUA GC
Met Phe Ser Leu Ala Leu
Insert C
AUG UUU AGC CUU UAG CUU UAG C
Met Phe Ser Leu STOP
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Sequencing the whole genomes of a family (2010 Science 328
636).
98 crossovers in maternal genome
57 crossovers in paternal genome
Mutation rate is 1x10-8 per position per haploid genome
~70 new mutations in each diploid human genome
CpG sites mutate at a rate 11 times higher than other sites
Exome sequencing of 2440 individuals (Science 2012 337 40)
Each person has ~100 loss of function mutations (~35
nonsense). 20 loss of function mutations are homozygous
Common SNP frequencies are the same in all geographic
populations
Rare SNPs are population specific and their frequencies vary
for each geographic population
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Frameshift mutation
Insertion/Deletion
A single base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Delete C
Delete GC
Delete AGC
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Frameshift mutations- Deletion
A single base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Delete C
AUG UUU AGU UUA GCU UUA GC
Met Phe Ser Leu Ala Leu
Delete GC
AUG UUU AUU UAG CUU UAG C
Met Phe Ile Stp
Delete AGC
AUG UUU UUU AGC UUU AGC
Met Phe Phe Ser Phe Ser
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Frameshift mutations-Insertion
A single base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Insert C
Insert CC
Insert CCC
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Frameshift mutations-Insertion
A single base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Insert C
AUG UUU AGC CUU UAG CUU UAG C
Met Phe Ser Leu STOP
Insert CC
AUG UUU AGC CCU UUA GCU UUA GC
Met Phe Ser Pro Leu Ala Leu
Insert CCC
AUG UUU AGC CCC UUU AGC UUU AGC
Met Phe Ser Pro Phe Ser Phe Ser
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Missense mutations
Missense mutations alters ONE codon so that it encodes
a different amino acid
UUU UUU UGC UUU UUU
Phe Phe Cys Phe Phe
WT
UUU UUU UGG UUU UUU
Phe Phe Trp Phe Phe
mut
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Consequences of Missense Mutations
Missense mutations alter one of the many amino acids
that make a protein
Its consequences depend on which amino acid is altered
Conservative mutations:
K to R
Nonconservative mutations:
K to E
Surface Vs buried
Mutations in globular domains Vs un structured tails
Silent mutations
Mutations in non-coding regions
Nonsense mutations
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Silent Mutations
Silent mutations do not alter the amino acid sequence!
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
WT
AUG UUC AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Mut
Mutations that occur in introns are also silent
Mutations that occur in non-genic regions are often silent
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Mutations in non-protein coding regions
Mutations in the promoter, splicing junction or ribosome
binding site are also mutagenic
Reduced expression of mRNA might result in reduced levels
of proteins
OR
Increased expression of mRNA might result in increased
levels of protein
Mutations in splicing junctions may also be mutagenic
improperly spliced mRNA will result in the intron being
translated
Mutations in tRNA or aminoacyl-tRNA synthase are
mutagenic
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Lactose intolerance in humans
Lactose========>Glucose + Galactose
Lactase
Human milk is 7% lactose. Lactose is not absorbed through the wall
of the digestive tract.
In human infants, lactase is secreted in intestine which breaks the
lactose into easily absorbed Glucose and Galactose.
Production of the lactase enzyme declines in adults.
The unabsorbed lactose creates cramps, diarrhea, and nausea.
In some humans, lactase continues to be produced throughout
adulthood. These individuals are called lactose absorbers (LA).
Adult lactose absorption is inherited as an autosomal dominant
trait.
Lactose persistence and non-persistence reflect inheritence of
different alleles of the lactase gene.
Lactose intolerance is the result of being homozygous for the
recessive lactase (WT) allele
Being homozygous or heterozygous for the mutant allele allows
lactase expression in adults when normally lactase expression is
turned off.
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lactose tolerance
There are no mutations in the coding region of the lactase gene.
A mutation is observed in the enhancer -13910 bp upstream of
the gene in an AP2 consensus sequence.
CCCCAGGC
•the polymorphism modifies a transcription factor binding site
(AP2)
•AP2 acts as a repressor but in the mutant it cannot bind and
cannot repress the gene- so adults keep producing lactase
C/C
T/
T
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Mutations in splicing of RNA
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Nonsense mutations
Nonsense mutations alter one codon so that it now encodes
for a STOP codon
UUU UUU UGC UUU UUU
Phe Phe Cys Phe Phe
UUU UUU UGA UUU UUU
Phe Phe STOP
Nonsense mutations insert a stop codon which results in
premature termination
Truncated polypeptide usually results in loss of function
for polypeptide
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There are NO tRNAs in cells with anti-codons that
recognize STOP codons in mRNA
What happens if there is a mutation in the anti-codon
loop of a specific tRNA Gene that allows a tRNA to
recognize a stop codon
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Nonsense suppressor mutations!
These are the result of a mutation in the anti-codon loop of
a specific tRNA Gene
It allows the tRNA to recognize a nonsense codon and base
pair with it.
DNA
Gene encoding tRNATRP
Point mutation occurs in the anticodon loop
This allows this tRNA to base pair with a stop codon and ?
Trp
Trp
AUG
---UAC---
AUC
---UAG---
Normal tRNA
Mutant tRNA
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Nonsense suppressor
--- UUU UUU UAG UUU UUU ------- Phe Phe STOP
Trp-tRNA has mutation
In anticodon
This allows it to pair
with a stop codon
MetAla
Phe
Phe
Trp
AAA AUC
5’--- UUU UUU UAG UUU UUU -----3’
--- Phe Phe Trp Phe Phe ---->
A mutant protein that is larger than normal will be synthesized!!
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Nonsense and Nonsense suppressor
--- UUU UUU CAG UUU UUU ------- Phe Phe Gln Phe Phe --Nonsense mutation
--- UUU UUU UAG UUU UUU ------- Phe Phe STOP
What will happen if an
individual carries both a
nonsense mutation in a gene
and a nonsense suppressor
mutation in the anticodon
loop of one of the trptRNA genes.
Trp
AUC
---UAG---
MetAla
Phe
Phe
Trp
Phe
Phe
AAA AUC AAA AAA
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5’--- UUU UUU UAG UUU UUU -----3’
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Generation of mutations
Spontaneous mutations
Replication induced mutations of DNA
Usually base substitutions (Most errors are corrected)
Meiotic crossing over can induce mutations
Small additions and deletions AND Large changes as well
Environment induced changes
Exposure to physical mutagens - radioactivity or chemicals
Depurination (removal of A or G)
Repair results in random substitution during replication
Deamination (removal of amino group of base) (nitrous acid)
Cytosine--uracil--bp adenine--replication-Oxidation (oxoG)
guanine--oxoguanine--bp adenine--replication -Base analog incorporation during replication BU-T
Intercalating agents
X-rays-
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Mutation rate
There are approximately 1013 cells in the human body
Each cell receives 10,000 DNA lesions per day (Lindahl and
Barnes 2000).
Most pervasive agent is UV. 100,000 lesions per exposed cell
per hour (Jackson and Bartek 2009)
Ionizing agents are more toxic because they generate double
strand breaks (Ward 1988)
Chromosome instability (gain or loss of entire segments) is
frequent in dividing cells- during manipulation like in vitro
fertilization. Occurs even in cleavage stage embryos. 40% of
imbalances are entire arm imbalance while 45% are terminal
segment imbalance (double strand break and nondysjunction)
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Methods used to study mutations
Gross chromosomal changesdeletions, insertions, inversions, translocations
Cytology- microscopy- karyotype
Point mutations
Small deletions, insertions
Recombinant DNA technologies
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Recombinant DNA
technology
When genes are mutated - proteins are mutatedDISEASE STATES OCCUR
Sickle cell Anemia
Globin
2 alpha globin chains
2 beta globin chains
Mol wt 16100 daltons xfour = 64650 daltons
Single point mutation in beta-globin
Converts Glu to Val at position 6
Need to know mutation
Need to look at genes of individuals
Genes lie buried in 6billion base pairs of DNA
(46 chromosomes).
Molecular analyses necessary
Take advantage of enzymes and reactions that naturally
occur in bacteria
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Why all the Hoopla?
Why all the excitement over recombinant DNA?
It provides a set of techniques that allows us to study
biological processes at the level of individual proteins
in individuals!
It plays an essential role in understanding the genetic basis
of cancer in humans
Recently found that mutations in a single gene called p53
are the most common Genetic lesion in cancers.
More than 50% of cancers contain a mutation in p53
Cells with mutant p53
Chromosomes fragment
Abnormal number of chromosomes
Abnormal cell proliferation!
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Alkaptonuria
Degenerative disease. Darkening of connective tissue, arthritis
Darkening of urine
1902
Garrod characterized the disorderusing Mendels rules- Autosomal recessive.
Affected individuals had normal parents and normal offspring.
1908
Garrod termed the defect- inborn error of metabolism
Homogentisic acid is secreted in urine of these patients.
This is an aromatic compound and so Garrod suggested that it
was an intermediate that was accumulating in mutant individuals
and was caused by lack of enzyme that splits aromatic rings of
amino Acids.
1958
La Du showed that accumulation of homogentistic acid
is due to absence of enzyme in liver extracts
1994
Seidman mapped gene to chromosome 3 in human
1996
Gene cloned and mutant identified P230S &V300G
2000
Enzyme principally expressed in liver and kidneys
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p53
To understand the complete biological role of p53 protein
and its mutant phenotype we need to study the gene at
multiple levels:
Genetics- mutant gene- mutant phenotype
Now what?
Genetics will relate specific mutation to specific phenotype
It usually provides No Information about how the protein
generates the phenotype
For p53
We would like to know
The nucleotide sequence of the gene and the mutation that
leads to cancer
When and in which cells the gene is normally expressed
(in which cells is it transcribed)
At the protein level--Amino acid sequence
Three-dimensional structure
Interactions with other proteins
Cellular information
Is the location in the cell affected
How does it influence the behavior of the cell during division
Organism phenotype
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