Molecuar Genetics 8.2012

8/2/2019 Molecuar Genetics 8.2012

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Mutations


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Mutagen:Any environmental agent that significantly increases the rate

of mutation above the spontaneous rate is called a mutagen.

Causes of Mutations

Chemical treatment

Exposure to X-ray, UV light (Radiations)

Transposons that insert into a gene and disrupt the normal reading

frame


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Mutagens
http://www.ncbi.nlm.nih.gov/books/NBK21114/figure/A8368/?report=objectonly


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Induced mutations: chemicalmutagens

Base analogs

Similar to normal bases,incorporated into DNA duringreplication.

Some cause mis-pairing (e.g.,5-bromouracil).

Not all are mutagenic.


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5-Bromouracil (a base analog) resembles thymine, except that it has a bromineatom in place of a methyl group on the 5-carbon atom. Because of the similarity intheir structures, 5-bromouracil may be incorporated into DNA in place of thymine.Like thymine, 5-bromouracil normally pairs with adenine but, when ionized, it may pairwith guanine.


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Mutagenic efffects of 5-bromouracil


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Induced mutations: Chemical mutagens

Base modifying agents, act at any stage of the cell cycle:

Deaminating agents

Hydroxylating agents

Alkylating agents


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Base-modifying agents.


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Base-modifying agents


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Induced mutations: chemicalmutagens

Intercalating agents:

Thin, plate-like hydrophobicmolecules insert themselvesbetween adjacent base-pairs,

Mutagenic intercalating agentscause insertions during DNAreplication.

Loss of intercalating agent can

result in deletion.

Examples: proflavin, ethidiumbromide Fig. 7.13


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Induced mutations

Radiation (e.g., X-rays, UV)

Ionizing radiation breaks covalent bonds including those in DNA and is theleading cause of chromosome mutations.

Ionizing radiation has a cumulative effect and kills cells at high doses.

UV (254-260 nm) causes purines and pyrimidines to form abnormal dimerbonds and bulges in the DNA strands.


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B. UV radiation

260-280 nm is wavelength at which maximum absorptionoccurs for DNA.

Ultraviolet light causes mutations primarily by producing pyrimidinedimers that disrupt replication and transcription.

Non-ionizing radiation (Ultraviolet radiation) excites electrons to a higher

energy level.

Two nucleotide bases in DNA - cytosine and thymine-are most vulnerable to

excitation that can change base-pairing properties.

UV light can induce adjacent thymine bases in a DNA strand to pair with each

other, as a bulky dimer.

DNA has so-called hotspots, where mutations occur up to 100 times more

frequently than the normal mutation rate. A hotspot can be at an unusual base,

e.g., 5-methylcytosine.

Radiation


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Radiations

Ionizing radiation such as X-rays and gamma rays damage DNA bydislodging electrons from atoms; these electrons then break

phosphodiester bonds and alter the structure of bases.

Ionizing radiation causes three types of damage to DNA

Single-strand breaks - mostly sealed by DNA ligase

Double-strand breaks - often lethal because can't be resealed by ligase so

degraded by nucleases. Alteration of bases - this type of oxidative damage is usually lethal because

forms a replication barrier at that site.


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A major problem with chemically-induced and irradiation-induced

mutations, however, is that they are generated essentially at

random. In order to identify a mutant phenotype of interest, a

laborious screen for mutants needs to be conducted by close

examination of the phenotypes following mutagenesis.


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The Ames test is based on the principle that both cancer and

mutations result from damage to DNA.

Ames test


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Detecting environmental mutations: Ames Test (after Bruce Ames)

Ames Test is an inexpensive method used to screen possible carcinogens and

mutagens.

Histidine auxotroph Salmonella typhimurium (requires Histidine to grow) are

mixed with rat liver enzymes and plated on media lacking histidine.

Liver enzymes are required to detect mutagens that are converted to

carcinogenic forms by the liver (e.g., procarcinogens).

Test chemical is then added to medium.

Control plates show only a small # of revertants (bacteria cells growingwithout histidine).

Plates innoculated with mutagens or procarcinogens show a larger # of

revertants.

Auxotroph will not grow without Histidine unless a mutation has occurred.


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Mutagenesis

Mutagenesis is a term that refers to the deliberate production ofgenetic variability through the use of various forms of energy

(neutrons, gamma rays, X-rays) or various chemical treatments/

molecular methods.

A fundamentally important DNA technology which seeks to change

the base sequence of DNA and test its effect on gene or DNA

function

Either of these treatments, at appropriate levels, will cause

changes in the DNA of an organism.

As these changes are random, any gene (and any number of genes)

could be disrupted with consequences that will depend on their

function.


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What is the distinction between

mutagenesis and genetic engineering?

Mutagenesis Genetic engineering

Mutation is a random event. To isolate, clone and incorporate genes.

It requires the production of very large

numbers of individuals that one or more

organism/plants will carry the desired

mutation. The products of mutagenesis

probably carry other changes in their DNA.

Genetic engineering is a more precise

technique than mutation in that the basis

for the change is understood at both the

DNA and the protein level.

It is not possible to direct this process and

the changes induced in the DNA are not

known.

This is probably less the case with genetic

engineering.

Mutagenesis can only modify the genes of an

organism / produce mutations.

whereas genetic engineering can add a new

gene/genes. In genetic engineering, control

sequences for the gene also have to be

inserted.


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The mutagenesis can be conducted;

In vivo (in studies of model organisms, or cultured cells).

In vitro mutagenesis can be directed to a specific site in a pre-

determined way (site-directed mutagenesis), or can be random.

MUTAGENESIS


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In vivo mutagenesis; Gene targeting

Gene targeting involves engineering a mutation in a predetermined

gene within an intact cell.

Form of artificial site directed in vivo mutagenesis.

Useful for studying gene function

Result in inactivation of gene expression (a knock-out' mutation), or

altered gene expression,

Therapeutic potential; Same method can be used to correct' a

pathogenic mutation by restoring the normal phenotype.


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Gene targeting typically involves several steps;

1. Introducing a mutation by homologous recombination.

2. A cloned gene (or gene segment) closely related in sequence to

endogenous target gene is transfected into the appropriate cells.3. In some of the cells, homologous recombination occurs between the

introduced gene and its chromosomal homolog.

4. Once a mutation has been engineered into a specific mouse gene within

the ES cells, the modified ES cells can then be injected into the

blastocyst of a foster mother and eventually a mouse can be produced

with the mutation in the desired gene in all nucleated cells.


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The production of either random or specific mutations in a piece

of cloned DNA.

DNA will then be reintroduced into a cell or an organism to

assess the results of the mutagenesis.

Gene targeting


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Involve essentially random approaches to mutagenesis, which may bevaluable in producing libraries of new mutants.

If a gene is cloned and a functional assay of the product is available, it is

also very useful to be able to employ a form of in vitro mutagenesis which

results in alteration of a specific amino acid or small component of the gene

product in a predetermined way.

In vitro mutagenesis

Methods for making a precise alteration in a gene sequencein order to change the structure and possibly the activity

of a protein


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Site Directed Mutagenesis

Oligonucleotide-directed mutagenesis

Site-directed mutagenesis by PCR

5 Add-on mutagenesis

In vitro Mutagenesis

Method by which mutant alleles can be synthesized in thelab and transformed into cell culture and animals.

Commonly used to study mutations of human genes in miceor other model organisms.


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site-directed Mutagenesis

Create mutations at specific locations in a process called site-directed

mutagenesis and then to study the effects of these mutations on the

organism.

One strategy is to cut out a short sequence of nucleotides with restriction

enzymes and replace it with a short, synthetic oligonulceotide that contains the

desired mutated sequence.

The success of this method depends on the availability of restriction sites

flanking the sequence to be altered.


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An in vitro mutagenesis technique in which a synthetic

oligonucleotide is used to introduce a predetermined

nucleotide alteration into the gene to be mutated. A short oligonucleotide is synthesized, complementary to the

relevant region of the gene but containing the desired

nucleotide alteration.

This oligonucleotide is hybridized to the DNA and used as the

primer for a strand synthesis reaction that is allowed to

continue all the way around the circular template molecule.


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A single-stranded oligonucleotide is produced that differs from the targetsequence by one or a few bases. Because they differ in only a few bases, the

target DNA and the oligonucleotide will pair under the appropriate

conditions.

When successfully paired with the target DNA, the oligonucleotide can actas a primer to initiate DNA synthesis, which produces a double-stranded

molecule with a mismatch in the primer region.

When this DNA is transferred to bacterial cells, the mismatched bases will

be repaired by bacterial enzymes.

About half of the time the normal bases will be changed into mutant bases,

and about half of the time the mutant bases will be changed into normal

bases.

The bacteria are then screened for the presence of the mutant gene.


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Concept:

Oligonucleotide-directed mutagenesis is used tostudy gene function when appropriate restrictionsites are not available.


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Mutations can also be introduced in addition to single nucleotidesubstitutions.

For example, it is possible to introduce a three-nucleotide deletion that willresult in removal of a single amino acid from the encoded polypeptide, or an

insertion that adds a new amino acid. Provided the mutagenic oligonucleotide is long enough, it will be able to bind

specifically to the gene template even if there is a considerable centralmismatch.

Larger mutations can be introduced by using cassette mutagenesis in whichcase a specific region of the original sequence of the original gene isdeleted and replaced by oligonucleotide cassettes.

In vitro mutagenesis



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Site-directed mutagenesis by PCR

PCR can be used to couple desired sequences or chemical groups to a

target sequence and to produce specific pre-determined mutations in

DNA sequences

A form of mutagenesis known as 5 add-on mutagenesis permits addition

of a desired sequence or chemical group.


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5 Add-on mutagenesis by PCR

This is a commonly used practice in which a new sequence or chemical

group is added to the 5 end of a PCR product by designing primers

which have the desired specific sequence for the 3 part of the primer

while the 5 part of the primer contains the novel sequence or a

sequence with an attached chemical group.


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5 Add-on mutagenesis

The extra 5 sequence does not participate in the first annealing step of the

PCR reaction (only the 3 part of the primer is specific for the target

sequence), but it subsequently becomes incorporated into the amplified

product, thereby generating a recombinant product.

Alternatives for the extra 5 sequence include:

(i) a suitable restriction site which may facilitate subsequent cell-based

DNA cloning;

(ii) a functional component, e.g. a promoter sequence for driving expression,

(iii)a modified nucleotide containing a reporter group or labeled group, such

as a biotinylated nucleotide or fluorophore.


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Mismatched primer mutagenesis

The primer is designed to be only partially complementary to thetarget site but in such a way that it will still bind specifically to thetarget. Mutation is introduced close to the extreme end of the PCR product.

This approach may be exploited to introduce an artificial diagnostic

restriction site that permits screening for a known mutation.

Mutations can also be introduced at any point within a chosensequence using mismatched primers. Two mutagenic reactions are designed in which the two separate PCR

products have partially overlapping sequences containing the mutation. The denatured products are combined to generate a larger product with the

mutation in a more central location.


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Molecuar Genetics 8.2012

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