Mutations and the code frameshift mutations
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Mutations and the code Frameshift mutations. A single base-pair deletion or insertion results in a change in the reading frame. AUG UUU AG C UUU AGC UUU AGCWT Met Phe Ser Phe Ser Phe Ser Delete C AUG UUU AGU UUA GCU UUA GC Met Phe Ser Leu Ala Leu Insert C

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Mutations and the code Frameshift mutations

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Mutations and the code frameshift mutations

Mutations and the code Frameshift mutations

A single base-pair deletion or insertion results in a change

in the reading frame

AUG UUU AGC UUU AGC UUU AGCWT

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

Delete GC

Delete AGC


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

Insert CC

Insert CCC


Missense mutations

Missense mutations

Missense mutations alters ONE codon so that it encodes

a different amino acid

UUU UUU UGC UUU UUUWT

UUU UUU UGG UUU UUUmut


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!

AUG UUU AGC UUU AGC UUU AGCWT

AUG UUC AGC UUU AGC UUU AGCMut

Mutations that occur in introns are also silent

Mutations that occur in non-genic regions are silent


Mutations in non protein coding regions

Mutations in non-protein coding regions

Mutations in the promoter or ribosome binding site are

also mutagenic

Reduced expression of mRNA might result in reduced levels

of proteins

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


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


Nonsense suppressor mutations

Nonsense suppressor mutations!

These are the result of a mutation in the anti-codon loop of

a specific tRNA

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 ?


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.


Generation of mutations

Spontaneous mutations

Replication induced mutations of DNA

Usually base substitutions

Most spontaneous errors are corrected

Mutations during meiotic pairing

Small additions and deletions

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-

Generation of mutations


Methods used to study mutations

Methods used to study mutations

Gross chromosomal changes-

deletions, insertions, inversions, translocations

Cytology- microscopy- karyotype

Point mutations

Small deletions, insertions

Recombinant DNA technologies


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 position6

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!


Mutations and the code frameshift mutations

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


Alkaptonuria

Alkaptonuria

Degenerative disease. Darkening of connective tissue, arthritis

Darkening of urine

Garrod characterized the disorder-

using Mendels rules- Autosomal recessive.

Affected individuals had normal parents and normal offspring.

1908Garrod 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.

1958La Du showed that accumulation of homogentistic acid

is due to absence of enzyme in liver extracts

1994Seidman mapped gene to chromosome 3 in human

1996Gene cloned and mutant identified P230S &V300G

2000Enzyme principally expressed in liver and kidneys


Basic techniques

Basic techniques

---Nucleic acid hybridization

complementary strands will associate and form double

stranded molecules

---Restriction Enzymes

These enzymes recognize and cleave DNA at specific

sequences

---Blotting

Allows analysis of a single sequence in a mixture

---DNA cloning

This allows the isolation and generation of a large number

of copies of a given DNA sequence

---DNA sequencing

Determining the array of nucleotides in a DNA molecule

---PCR

---Transformation

Stably integrating a piece of DNA into the genome of an

organism

---Genetic engineering

Altering the DNA sequence of a given piece of DNA

---Genomics

Analyzing changes in an entire genome


Nucleic acid hybridization

Nucleic acid hybridization

Complementary strands of DNA or RNA will specifically associate

DNA is heated to 100C, the hydrogen bonds linking the two

strands are broken

The double helix dissociates into single strands.

As the solution is allowed to cool, strands with complementary

sequences readily re-form double helixes.

This is called Nucleic acid hybridization.

AAAAAAAATTTTAAAAAAA

Will associate with

TTTTTTTTAAAATTTTTTT

This occurs with complementary

DNA/DNA, DNA/RNA, RNA/RNA


Li fraumeni syndrome

Li-Fraumeni syndrome

This technique is very sensitive and specific.

A single 200 nucleotide sequence when added to a solution

of a million sequences will specifically hybridize with the

ONE complementary sequence

Usefulness

Li-Fraumeni syndrome

Individuals in a family have a propensity to develop tumors

at an early age

Often these families have a deletion in the p53 gene

When this family has a child, they might want to know

if their child has normal p53 or not

Nucleic acid hybridization provides a means to rapidly

determine whether the sequence is present or not


Restriction enzymes

Restriction Enzymes

Enzymes which cut DNA at specific sequences

SmaI

Analysis revealed that the enzyme recognized and cut the

following sequence

|

5’ CCCGGG3’

3’ GGGCCC5’

|

This sequence is symmetrical. If one rotates it about the axis

It reads the same


Linear circular dna

Linear/Circular DNA

A linear DNA molecule with ONE HindII site will be cut into

two fragments

A circular DNA molecule with ONE HindII site will generate

one DNA fragment


Restriction sites

Restriction sites

SmaI

5’ CCCGGG3’

3’ GGGCCC5’

5’CCC3’ 5’GGG3’

3’GGG5’ 3’CCC5’

EcoRI is another commonly used restriction enzyme

5’G3’ 5’AATTC3’

3’CTTAA5’ 3’G5’

5’GAATTC3’

3’CTTAAG5’

Unlike SmaI which produces a blunt end,

EcoRI produces sticky or cohesive ends

These cohesive ends facilitate formation of recombinant

DNA molecules


Restriction maps

Restriction maps

Restriction maps are descriptions of the number, type and

distances between Restriction sites on a piece of DNA.

Very useful for molecular biologists.

Restriction sites serve as landmarks in the DNA with which a

physical map of a specific DNA sequence can be created.


Sequence divergence

Sequence Divergence

The restriction map is also a reflection of the nucleotide sequence

arrangement of a gene

By comparing maps we can surmise differences in the sequence

between species


Deletions and additions

Deletions and additions

Normal Globin gene

3

5

8

4

EcoRI

EcoRI

EcoRI

HindIII

HindIII

Globin gene from a patient

3

5

3

4

EcoRI

EcoRI

EcoRI

HindIII

HindIII

With restriction maps, the relationship between genes can be

determined without having to actually sequence the genes.


Gel electrophoresis

1

3

5

2

EcoRI

EcoRI

EcoRI

HindIII

EcoRI

HindIII

HindIII

Gel electrophoresis

Agarose gel electrophoresis

The length of the DNA can

be accurately determined by

allowing the charged DNA to

run through an agarose gel.

DNA moves towards the

Positive electrode.

The rate of migration of a

DNA fragment is inversely

proportional to its size.

Larger the size, slower its

movement.


Mapping

HindIII

Marker

EcoRI

14

12

6

4

2

1

Mapping

You are given a 20 kb fragment of DNA

After trying many enzymes you find

That EcoRI and HindIII cut the fragment

HindIII 14kb and 6kb

EcoRI 12kb 6kb and 2kb

Solve the map


Mapping1

Mapping

Since HindIII cut the 20kb fragment once, in which of the

three EcoRI fragments. Does it cut?

A double digest with both enzymes will provide the answer

Fragments of 8kb, 6kb, 4kb and 2kb

The double digest does not alter the

size of the 6kb and 2kb fragments

The 12kb fragment is lost. Also 8+4=12

EcoRI+HindIII

HindIII

Marker

EcoRI

14

12

8

6

4

4

2

1


Mapping2

Mapping

How are these fragments ordered?

The HindIII single digest tells us that they must be ordered so

that One side adds up to 6kb and the other side adds up to 14kb

EcoRI+ HindIII

HindIII

Marker

EcoRI

14

12

6

4

2

1


Mapping3

HindIIIEcoRIHindIII/EcoRI

14128

666

4

22

Mapping


Mapping example

HiEcHi/Ec

12128

866

4

22

PsPs/Ec

1312

75

2

1

Mapping example

Three different enzymes

Hi

Ec

Ps


Mapping4

HindIIIEcoRIHindIII/EcoRI

12128

866

4

22

Mapping


Mapping5

EcoRI PstIPstI/EcoRI

121312

675

22

1

Mapping

4

8


Mapping deletions

Mapping deletions

Say you isolated this DNA from a region coding for the globin

gene, from a normal Patient and one suffering from thalassemia.

The fragment was 17kb rather than 20kb in the patient with

Thalassemia!

The restriction patterns were as following:

HindIIIEcoRIDouble

1498

366

22

1

With similar reasoning as described above, the following

map is produced:


Mapping6

Mapping

Often maps are more complex and difficult to analyze using single and double digests alone.

To simplify the analyses, you can isolate each EcoRI band From the gel and then digest with HindIII

2kb+HindIII

12kb+HindIII

6kb+HindIII

EcoRI+

HindIII

Marker

Marker

HindIII

Marker

Marker

2kb

12kb

EcoRI

6kb

14

14

14

14

12

12

12

12

6

6

6

6

4

4

4

4

2

2

2

2

1

1

1

1


Recombinant dna

Recombinant DNA

A reasonable question is how did we get the 20kb fragment in

the first place?

Also how did we obtain the p53 probe

To understand the origin of the fragment we must address

the issue of:

The construction of Recombinant DNA molecules

Recombinant DNA is generated through cutting and pasting of

DNA to produce novel sequence arrangements

Restriction enzymes such as EcoRI produce staggered cuts

leaving short single-stranded tails at the ends of the fragment.

These “cohesive or sticky” ends allow joining of different DNA

fragments

|

GAATTC

CTTAAG

|

When a piece of DNA is cut with EcoRI,

you get


Plasmids

AATT---------------------

---------------------TTAA

AATT---------------------

---------------------TTAA

Plasmids

Plasmids are naturally occurring circular pieces of DNA in E. coli

The plasmid DNA is circular and usually has one EcoRI site.

It is cut with EcoRI to give a linear plasmid DNA molecule


Ligation

AATT

TTAA

AATT

TTAA

Ligation

PLASMID

GENOMIC DNA

The EcoRI linearized plasmid DNA is mixed with human EcoRI

digested DNA

The sticky ends hybridize and anneal and a recombinant

plasmid is generated


Plasmid propagation

Plasmid propagation

The plasmid DNA can replicate in bacteria

and therefore many copies of the

plasmid will be made. The human DNA

fragment in the plasmid will also multiply

along with the plasmid DNA.

Normally a gene is present as 2 copies

in a cell. If the gene is 3000bp long

there are 6x103 bp in a total of 6x109 bp

of the human genome

Once cloned into a plasmid, unlimited copies

of a single gene can be produced.The

process of amplifying and isolating the

human DNA fragment is called cloning.


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