Mutants and Their Uses - Pick Kuen

7/29/2019 Mutants and Their Uses - Pick Kuen

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Mutants: Their Uses

Pick Kuen Chan


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What is a Mutation? permanent change in the DNA sequence

Range from single DNA base to a largesegment of a chromosome


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What Causes DNA Mutations? DNA damage from environmental agents

- ultraviolet light (sunshine)

- nuclear radiation

- certain chemicals

Mistakes that occur when a cell copies itsDNA in preparation for cell division


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Types of Mutations Point mutations

single nucleotide base changes in a gene's DNA

sequence- Missense mutations

- Nonsense mutations

- Silent mutations

Insertion and deletion mutationsadd or remove one or more DNA bases. Insertion and

deletion mutations cause frameshift mutations


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The Central Dogma of Biology

Transcription

Translation

DNA

RNA

Protein


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Genome to Gene Function

Genome sequence

Gene discovery

Gene functional annotation In silico prediction

Gene expression Protein-protein interaction

Mutant analysis


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Mutant Analysis Known as forward genetics

Phenotype-centric process

Reliable means to assign gene function

Enormous effort to identify each generesponsible for a particular phenotype


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Mutant Analysis

Reverse genetics

Prediction of function based on DNA

sequence

Required large collections gene-indexed

mutation


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Mutant Analysis

Forward Genetics

Select a biological process

Generate highly redundant

mutant populations

Screen for mutants with adesired phenotype

Map and clone the generesponsible for the phenotype

Reverse Genetics

Select a gene or genes ofinterest

Generate highly redundantmutant populations

Develop and conductsequence-based mutantscreens

Analyse the phenotype ofthe mutants

Alfonso and Ecker, 2006


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Generation of Mutant Population Chemical agents

Ethyl methanesulphonate (EMS) andnitrosomethylurea (NMU)

Physical agents Fast neutrons, X-ray and carbon ions

Biological agents Transposons and transferred-DNA (T-DNA)


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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


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Wild type nts1007

EMS

Supernodulation mutant


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Ethylene insensitive mutants

Wild type

Skl

Penmetsa et al., 2003


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Fast Neutron Mutagenesis Intermediate efficiency

Produce double stranded DNA breaks thatare then repaired

DNA alteration

- Big chromosomal insertions, deletionsand rearrangement


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Examples

wildtype

ga1-3mutant

transgenicga1-3

Sun et al., 1992

FN37 wild type

Men et al., 2002


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Biological agents DNA or RNA molecules as mutagen

Introduction of engineered DNA/RNAmolecule into living cell.

- Agrobacterium tumefaciens as shuttle

- Plant-associated bacteria

- DNA/RNA viruses

- Particle bombardment


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Application in Plant Transformatiom

Insertional mutagenesis

Ectopic expression

Gene silencing

Homologous recombination


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Criteria for T-DNA Selectable marker

- Herbicide resistance gene, antibioticresistance gene

Reporter genes

- GFP, -glucuronidase (GUS), luciferase

Gene/s of interest


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Schematic Diagram on

Transformation


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Examples

Ottenschlger et al., 2003


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Workshop on Mycorhizal

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Mutants and Their Uses - Pick Kuen