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Mutants: Their Uses

Pick Kuen Chan

Mutants: Their Uses Pick Kuen Chan
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

Mutant Analysis Forward Genetics Select a biological process Generate highly redundant mutant populations Screen

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

Supernodulation mutant Wild type EMS nts1007

Wild type

Supernodulation mutant Wild type EMS nts1007

EMS

Supernodulation mutant Wild type EMS nts1007

nts1007

Ethylene insensitive mutants

Wild type Skl Penmetsa et al., 2003
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 wild FN37 wild type mutant ga1-3 type Sun et al., 1992 Men et
ga1-3
transgenic
wild
FN37
wild type
mutant
ga1-3
type
Sun et al., 1992
Men et al., 2002

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, β-glucuronidase (GUS), luciferase

• Gene/s of interest

Schematic Diagram on Transformation

Schematic Diagram on Transformation

Examples

Ottenschläger et al., 2003
Ottenschläger et al., 2003
Ottenschläger et al., 2003
Ottenschläger et al., 2003
Ottenschläger et al., 2003
Ottenschläger et al., 2003

Ottenschläger et al., 2003

Workshop on Mycorhizal