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Mutants: Their Uses

Pick Kuen Chan

What is a Mutation?
permanent change in the DNA sequence Range from single DNA base to a large segment of a chromosome

What Causes DNA Mutations?


DNA damage from environmental agents - ultraviolet light (sunshine) - nuclear radiation - certain chemicals Mistakes that occur when a cell copies its DNA in preparation for cell division

Types of Mutations
Point mutations
single nucleotide base changes in a gene's DNA sequence
- Missense mutations - Nonsense mutations - Silent mutations

Insertion and deletion mutations


add or remove one or more DNA bases. Insertion and deletion mutations cause frameshift mutations

The Central Dogma of Biology


DNA
Transcription

RNA
Translation

Protein

Genome to Gene Function


Genome sequence Gene discovery Gene functional annotation
In silico prediction Gene expression Protein-protein interaction Mutant analysis

Mutant Analysis
Known as forward genetics Phenotype-centric process Reliable means to assign gene function Enormous effort to identify each gene responsible for a particular phenotype

Mutant Analysis
Reverse genetics Prediction of function based on DNA sequence Required large collections gene-indexed mutation

Mutant Analysis
Forward Genetics
Select a biological process Generate highly redundant mutant populations Screen for mutants with a desired phenotype Map and clone the gene responsible for the phenotype

Reverse Genetics
Select a gene or genes of interest Generate highly redundant mutant populations Develop and conduct sequence-based mutant screens Analyse the phenotype of the mutants

Alfonso and Ecker, 2006

Generation of Mutant Population


Chemical agents Ethyl methanesulphonate (EMS) and nitrosomethylurea (NMU) Physical agents Fast neutrons, X-ray and carbon ions Biological agents Transposons and transferred-DNA (T-DNA)

EMS Mutagenesis
Efficient mutagens in plant - Soybean, pea, Lotus japonicus, Medicago truncatula Generate a broad range of DNA alteration - Point mutation Unbiased distribution of mutation

Supernodulation mutant

EMS

Wild type

nts1007

Ethylene insensitive mutants

Wild type

Skl Penmetsa et al., 2003

Fast Neutron Mutagenesis


Intermediate efficiency Produce double stranded DNA breaks that are then repaired DNA alteration - Big chromosomal insertions, deletions and rearrangement

Examples

ga1-3 transgenic mutant ga1-3

wild type

FN37

wild type Men et al., 2002

Sun et al., 1992

Biological agents
DNA or RNA molecules as mutagen Introduction of engineered DNA/RNA molecule into living cell. - Agrobacterium tumefaciens as shuttle - Plant-associated bacteria - DNA/RNA viruses - Particle bombardment

Application in Plant Transformatiom


Insertional mutagenesis Ectopic expression Gene silencing Homologous recombination

Criteria for T-DNA


Selectable marker - Herbicide resistance gene, antibiotic resistance gene Reporter genes - GFP, -glucuronidase (GUS), luciferase Gene/s of interest

Schematic Diagram on Transformation

Examples

Ottenschlger et al., 2003

Workshop on Mycorhizal