Mutations

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

Mutagens

Induced mutations: chemical mutagens Base analogs Similar to normal bases, incorporated into DNA during replication. Some cause mis-pairing (e.g., 5-bromouracil). Not all are mutagenic.

5-Bromouracil (a base analog) resembles thymine, except that it has a bromine atom in place of a methyl group on the 5-carbon atom. Because of the similarity in their 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 pair with guanine.

Mutagenic efffects of 5bromouracil

Induced mutations: Chemical mutagens

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

Base-modifying agents.

Base-modifying agents

Induced mutations: chemical mutagens Intercalating agents: Thin, plate-like hydrophobic molecules insert themselves between adjacent base-pairs, Mutagenic intercalating agents cause insertions during DNA replication. Loss of intercalating agent can result in deletion. Examples: proflavin, ethidium Fig. 7.13 bromide

Induced mutations

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

Ionizing radiation breaks covalent bonds including those in DNA and is the leading 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 dimer bonds and bulges in the DNA strands.

Radiation
B. UV radiation 260-280 nm is wavelength at which maximum absorption occurs for DNA. Ultraviolet light causes mutations primarily by producing pyrimidine dimers 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.

Radiations
Ionizing radiation such as X-rays and gamma rays damage DNA by dislodging 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.

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.

Ames test
The Ames test is based on the principle that both cancer and mutations result from damage to DNA.

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 growing without histidine). Plates innoculated with mutagens or procarcinogens show a larger # of revertants. Auxotroph will not grow without Histidine unless a mutation has occurred.

Mutagenesis
Mutagenesis is a term that refers to the deliberate production of genetic 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.

mutagenesis and genetic engineering?

Mutagenesis Mutation is a random event. 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. It is not possible to direct this process and the changes induced in the DNA are not known. Mutagenesis can only modify the genes of an whereas genetic engineering can add a new This is probably less the case with genetic engineering. Genetic engineering To isolate, clone and incorporate genes. 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.

What is the distinction between

organism / produce mutations.

gene/genes. In genetic engineering, control

sequences for the gene also have to be inserted.

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

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.

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.

Gene targeting
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.

In vitro mutagenesis
Methods for making a precise alteration in a gene sequence in order to change the structure and possibly the activity

of a protein
Involve essentially random approaches to mutagenesis, which may be valuable 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
Method by which mutant alleles can be synthesized in the lab and transformed into cell culture and animals. Commonly used to study mutations of human genes in mice or other model organisms.

Site Directed Mutagenesis

Oligonucleotide-directed mutagenesis Site-directed mutagenesis by PCR

5 Add-on mutagenesis

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.

Oligonucleotide-directed mutagenesis

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.

Oligonucleotide-directed mutagenesis

A single-stranded oligonucleotide is produced that differs from the target sequence 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 act as 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.

Concept: Oligonucleotide-directed mutagenesis is used to study gene function when appropriate restriction sites are not available.

In vitro mutagenesis

Oligonucleotide-directed mutagenesis

Mutations can also be introduced in addition to single nucleotide substitutions. For example, it is possible to introduce a three-nucleotide deletion that will result 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 central mismatch. Larger mutations can be introduced by using cassette mutagenesis in which case a specific region of the original sequence of the original gene is deleted and replaced by oligonucleotide cassettes.

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.

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.

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.

Mismatched primer mutagenesis

The primer is designed to be only partially complementary to the target site but in such a way that it will still bind specifically to the target.
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 chosen sequence 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.