Alterations in the genetic material

• Alternation in the genetic material can arise as a consequence of any one of several kinds of events, the most important of which are mutation and recombination

Mutation

• Any alteration in the nucleotide sequence of a DNA molecule

• Generally refer to a relatively small-scale change

Recombination

• The exchange of segments of polynucleotides between different DNA molecules and can give rise to quite substantial rearrangements

• Silence: mutation occurs in an intergenic region and have no discernible effect on the cell

• Phenotype: mutation occurs in a gene it may alter the gene product and generate an observable change Genotype: genetic constitution

• Wild type: An organism displaying the usual phenotype

• Mutant; phenotype has been altered by mutation

• Mutagen: chemical or physical agent that cause mutation

Different types of mutation

• Point mutation • Insertion or Deletion • Inversion

Point Mutation

• A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair.

Point Mutation

• Sickle-cell disease is caused by a single point mutation (a missense mutation) in the beta-hemoglobin gene that converts a GAG codon into GUG, which encodes the amino acid valine rather than glutamic acid. This is an example of a non-conservative (missense) mutation.

Point Mutation • nonsense mutations: code for a stop, which can truncate the protein

Point Mutation • silent mutations: code for the same amino acid

Point Mutation

• Conservative mutation: result in an amino acid change; however, the properties of the amino acid remain the same (e.g. hydrophobic, hydrophilic, etc).

• Non conservative mutation: result in an amino acid change that has different properties than the wild type.

Non conservative mutation

Insertion Mutation • an insertion (also called an

insertion mutation) is the addition of one or more nucleotide base pairs into a DNA sequence

Frameshift mutation

• A frameshift mutation (also called a framing error or a reading frame shift) is a genetic mutation caused by indels (insertions or deletions) of a number of nucleotides that is not evenly divisible by three from a DNA sequence. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame (the grouping of the codons), resulting in a completely different translation from the original

Mutangens • a mutagen (Latin, literally origin of change) is a

physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are typically also carcinogens. Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to errors in DNA replication, repair and recombination.

Mutagens • Ionizing radiation, for example X-rays, gamma rays and alpha particles • Ultraviolet, electromagnetic radiation with a wavelength shorter than that of

visible light but longer than x-rays • Base analog, which can substitute for DNA bases and cause copying errors • Deaminating agents such as nitrous acid • Intercalating agents such as ethidium bromide • Alkylating agents such as ethylnitrosourea • Transposon, a section of DNA that undergoes autonomous fragment

relocation/multiplication • Alkaloid plants, such as those from Vinca species • Bromine and some compounds that contain bromine in their chemical structure • Sodium azide, an azide salt that is a common reagent in organic synthesis and a

component in many car airbag systems • Psoralen combined with ultraviolet radiation causes DNA cross-linking and hence

chromosome breakage • Benzene, an industrial solvent and precursor in the production of drugs, plastics,

synthetic rubber and dyes

Mutagens:Ethidium bromide

• Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis.

Mutagens: EtBr Dimension of three-ringed molecules are similar to those of a purine-pyrimidine base pair so that the compounds can intercalate into a double helix Insertion of a single nucleotide is likely to occur at the intercalation position and cause a frameshift mutation if the position lies within a gene Because frameshifts generally give rise to phenotypic changes, intercalating agents are popular mutagens fro generating mutants to be used in research

3.4Å

Mutagens: EtBr

Ethidium bromide is a flat molecule that fits between adjacent base pairs (intercalates) in the DNA double helix. It has UV absorbance maxima at 300 and 360nm, and can also absorb energy from nucleotides excited at 260nm. The absorbed energy is emitted as orange/yellow light at 590nm. The fluoresence of EtBr is significantly higher when intercalated than it is in aqueous solution

Ultraviolet radiation is mutagenic

• Ultraviolet radiation of about 260 nm wavelength is absorbed by purine and pyrimidines and can cause chemical chnages in their structure, in particular by the formation of dimers between adjacent pyrimidine rings in a polynucleotides, most often between thymines.

Thymine and Cytosine dimer

Dimerization causes the bases to stack closer together and can give rise to deletion during DNA replication. An efficient mechanism exists, at least in bacteria, to repair thymine dimers, no doubt reflecting the high probability that UV will be encountered as a mutagen in the natural environment. A group of repair enzymes can recognize a thymine dimer, remove it by endonuclease action, and replace the missing nucleotides (DNA polymerase I).

Making Mutants

• Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

Overlap PCR tool for Point Mutation The Overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR which can a) insert mutations at specific points in a sequence, further than ~55 nucleotides from either end and/or b) produce polynucleotides from smaller fragments. To insert a mutation into a sequence, a primer is generated depending on requirements. A single substitution may be included or a totally new sequence added at the 5' end. If a deletion is required, then the continuation of the sequence 5' of the deletion effectively becomes an addition. The 3' end must be complementary to the template strand so that DNA polymerase can copy the template from the primer (DNA polymerase can only copy DNA in one direction; 5' to 3').

Recombination

Recombination • Recombination results in a

rearrangement in the genetic material of the cell

• Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining.

Homologous recombination The Holliday model of DNA crossover. (a) Two homologous DNA molecules line up (e.g., two nonsister chromatids line up during meiosis). (b) Cuts in one strand of both DNAs. (c) The cut strands cross and join homologous strands, forming the Holliday structure (or Holliday junction). (d) Heteroduplex region is formed by branch migration. (e) Resolution of the Holliday structure. Figure 8-D-2e is a different view of the Holliday junction than Figure 8-D-2d. DNA strands may be cut along either the vertical line or horizontal line. (f) The vertical cut will result in crossover between f-f' and F-F' regions. The heteroduplex region will eventually be corrected by mismatch repair. (g) The horizontal cut does not lead to crossover after mismatch repair. However, it could cause gene conversion.

Recombination between non-homologous molecules

• In some circumstance processes recombination can occur between non-homologous DNA molecules which have no apparent regions of nucleotide sequence similarity

• Has been observed between chromosomal DNA and small circular DNA in higher organism such as fungi and plant

• The mechanism by which heterologous recombination occurs is still a mystery

B cell Rearrangement (reconbination) ; Generate Antibody Diversity

• Susumu Tonegawa (利根川 進 Tonegawa Susumu, born September 6, 1939) is a Japanese scientist who won the Nobel Prize for Physiology or Medicine in 1987 for his discovery of the genetic mechanism that produces antibody diversity. Although he won the Nobel Prize for his work in immunology, Tonegawa is a molecular biologist . In his later years, he has turned his attention to the molecular and cellular basis of memory formation.