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Methods used to study mutations. Gross chromosomal changes - deletions, insertions, inversions, translocations Cytology- microscopy- karyotype Small mutations Small deletions, insertions and point mutations Recombinant DNA technologies. Frameshift mutations.

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methods used to study mutations
Methods used to study mutations

Gross chromosomal changes-

deletions, insertions, inversions, translocations

Cytology- microscopy- karyotype

Small mutations

Small deletions, insertions and point mutations

Recombinant DNA technologies

frameshift mutations
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

frameshift mutations deletion
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

frameshift mutations insertion
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

missense mutations
Missense mutations

Missense mutations alters ONE codon so that it encodes

a different amino acid

UUU UUU UGC UUU UUU WT

Phe Phe Cys Phe Phe

UUU UUU UGG UUU UUU mut

Phe Phe Trp Phe Phe

consequences of missense mutations
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

silent mutations
Silent Mutations

Silent mutations do not alter the amino acid sequence!

The Genetic code is degenerate!

AUG UUU AGC UUU AGC UUU AGC WT

Met Phe Ser Phe Ser Phe Ser

AUG UUC AGC UUU AGC UUU AGC Mut

Met Phe Ser Phe Ser Phe Ser

Mutations that occur in introns are also silent

Mutations that occur in non-genic regions are often silent

mutations in non protein coding regions
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

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.

Lactose intolerance in humans
lactose tolerance
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

nonsense mutations
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

slide13

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

nonsense suppressor mutations

Trp

Trp

AUG

AUC

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.

Nonsense suppressor mutations!

DNA

Gene encoding tRNATRP

Point mutation occurs in the anticodon loop OF THE tRNA

This allows this tRNA to base pair with a stop codon and ?

---UAC---UAG

---UAG---UAG

Normal tRNA

Mutant tRNA

nonsense suppressor

Met

Ala

Phe

Phe

Trp

AUC

AAA

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

5’--- UUU UUU UAG UUU UUU -----3’

--- Phe Phe Trp Phe Phe ---->

A mutant protein that is larger than normal will be synthesized!!

nonsense and nonsense suppressor

Met

Ala

Phe

Phe

Phe

Phe

Trp

Trp

AUC

AUC

AAA

AAA

AAA

---UAG---

5’--- UUU UUU UAG UUU UUU -----3’

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 trp-tRNA genes.

recombinant dna technology
Recombinant DNA technology

When genes are mutated - proteins are mutated-

DISEASE 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

why all the hoopla
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!

slide20

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

p53
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