Title: 2002 Plant BIOLOGY SUMMER COURSE August 13, 2002 Xing Wang Deng (xingwang.deng@yale.edu) Professor of MCDB Dept. Yale University http:/www.yale.edu/Denglab or http:/plantgenomics.yale.biology.edu/ Phone: 203-432-8908
12002 Plant BIOLOGY SUMMER COURSE August 13,
2002Xing Wang Deng (xingwang.deng_at_yale.edu)Pro
fessor of MCDB Dept. Yale Universityhttp/www.yal
e.edu/Denglab orhttp/plantgenomics.yale.bi
ology.edu/Phone 203-432-8908
(direct)Fax 203-432-5726 (direct)Director of
Peking-Yale Joint Center for Plant Molecular
Genetics AgrobiotechnologyAt Beida Phone
010-6275-3018 Fax 010-6275-3339At
Yale Phone 203-432-3117 Fax 203-432-3204
2Lecture 1
- Genetic vs. reverse genetic approaches
- Genetic transformation
- Dominant affecting mutant
- Protein Tagging systems
- Insertional mutagenesis
3Understanding Development
- 1. What Genes involved in a given development
process - 2. How those genes (and proteins) work at
cellular/molecular level - 3. How those genes (and proteins) activities
are regulated
4How to identify Genes critical in a given
development process
- 1. Genetics What wrong with a mutant lacking a
functional gene tells the normal role of the
gene. Start with mutants, then the gene how it
works - 2. Reverse genetics Starting with a gene of
known identity, modifying its expression or
activity to reveal its development function
5Genetics generic steps in identifying
mutant and then the gene.
- 1. Choose your favorite development process
model organism - 2. Mutagenizing a population create mutations
in all genes with all possible phenotypes - 3. Select individuals with disirable phenotypes
in the chosen development process - 4. Identify the gene that mutated
- 5. Figure out how the gene and its encoded
protein work and is regulated
6Requirements for a genetic model organism
- 1. Easy to handle large number of individuals
- 2. Short generation time
- 3. Sexual life cycle and genetic cross possible
- 4. Small genome and easy in cloning gene with
known mutations - 5. Stable genetic transformation available
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8 Drosophila mutant affects WING development
Normal (wild type, WT)
Mutant
9 Wild type
Mutant
10A common route of identifying gene based on
mutation
- 1. Precisely map the mutation to a small genomic
region (may still contain several genes) - 2a. Directly sequence the genes in mutants to
reveal the one with mutation - OR (AND)
- 2b. Take each corresponding wild type genes in
the region and transform it into mutant to rescue
the phenotype defect
11Transformation
- Put a gene, e.g., DNA, back into the genome of an
organism, enable it become an inheritable part of
the genetic information
12Transformation in higher plants
copied
Agrobacterium Ti-plasmid T-DNA transfer DNA
Ti Transfer integration
13Transformation in higher plants
14A Common Transformation protocol
15An Easy Transformation protocol for Arabidopsis
Arabidopsis
Arabidopsis
The easiest and most efficient transformation
protocol for any multicellular organism
16Genetics generic steps in identifying
mutant and then the gene.
1. Choose your favorite development process
model organism
2. Mutagenizing a population create
mutations in all genes with all possible
phenotypes
3. Select individuals with desirable phenotypes
in the chosen development process
4. Identify the gene that mutated
5. Figure out how the gene and its encoded
protein work and is regulated.
17A few points of genetics or Mutant-to-gene
approach
- 1. No need of knowledge of the genes involved
generic applicable - 2. Its the most reliable powerful approach to
investigate any new development system - 3. Its hard to control of the precise nature of
the mutation in the genes (select whats
available) - 4. Its time consuming even in model organisms
and unpractical to do in many organisms - 5. Some genes, such as redundant ones, can not be
identified by phenotype screen
18How to identify Genes critical in a given
development process
- 1. Genetics What wrong with a mutant lacking a
functional gene tells the normal role of the
gene. Start with mutants, then the gene how it
works - 2. Reverse genetics Starting with a gene of
known identity, specifically modifying its
expression or activity to reveal its development
function (modifying in test tube and transform
back to organism)
19Why Reverse genetics (From gene to
phenotype/function)
- 1. Many undefined genes are sequenced due to the
advancement of sequence technology - 2. Can control where, when, and how much an
active gene product to be made - 3. Easy to design specific mutations into the
gene product - 4. Add TAGs to a gene product to simplify its
study within the cell - 5. Create mutation on gene that has no available
mutation or not feasible to have
20Why Reverse genetics (From gene to
phenotype/function)
1. Many unknown genes are sequenced due to the
advancement of sequence technology
2. Can control where, when, and how much an
active gene product to be made
3. Easy to design specific mutations into the
gene product
4. Add TAGs to a gene product to simplify its
study within the cell
5. Create mutation on gene that has no
available mutation
21Design specific mutations into the gene product
- 1. Make mutant protein with desirable change in
amino acid sequence - 2. Make gross domain deletion, addition, or
substitution
There are unlimited options and can be precisely
designed. That is not possible in mutagenesis
and mutant screen.
22Why Reverse genetics (From gene to
phenotype/function)
1. Many unknown genes are sequenced due to the
advancement of sequence technology
2. Can control where, when, and how much an
active gene product to be made
3. Easy to design specific mutations into the
gene product
4. Add TAGs to a gene product to simplify its
study within the cell
5. Create mutation on gene that has no
available mutation
233 common ways to control where, when, andhow
much an active gene product in a cell
1. Utilization of desirable regulatory sequence
to drive the expression of your gene product
- 2. Gene Silencing or Antisense approach to
control the level of mRNA of a given gene - 3. Expression of desirable Dominant Affecting
Mutant form
24How to make Antisense RNA
mRNA and asRNA can form RNA/RNA duplex, which
actually results in formation of 20-22 nts small
RNA that trigger the degradation of mRNA
25How to Silencing a Gene
mRNA
Form double stranded hairpin RNA (dsRNA)
The dsRNA results in formation of random 20-22
nts small RNA, which then triggers the
degradation of the mRNA where the homologous
derived from.
26An antisense approach to control mRNA level
- Other ways
- Co-suppression
- RNAi
27How Dominant Affecting Mutant works
Introduction of a mutated version renders the
active one also inactive
28Dominant Affecting Mutant an example
29Injection of the Dominant negative FGFR mutant
mRNA into two-cell stage frog embryo resulted in
tadpole without tail
30Why Reverse genetics (From gene to
phenotype/function)
1. Many unknown genes are sequenced due to the
advancement of sequence technology
2. Can control where, when, and how much an
active gene product to be made
3. Easy to design specific mutations into the
gene product
4. Add TAGs to a gene product to simplify its
study within the cell
5. Create mutation on gene that has no
available mutation
31TAGs are useful tools for monitoring a protein
within the cell
- By monitoring the TAG, you achieve the job of
observing the protein (X) of interest.
32TAGs 3 common types
- 1. Proteins with easily assayable biochemical
activity - LacZ (?-galactosidase) for all animal and
microbes - GUS (?-glucuronidase) for all plants
- Both convert a small colorless substrate into a
colored and insoluble product - 2. Proteins which have its own color (emitting
light under specific conditions) - green fluorescence protein (GFP) from Jellyfish,
no substrate is needed and can observe in living
cells - 3. Small peptides tags
- epitope tages monoclonal antibody recognition
sites - His tag six HIS residues in a row to add
purification
33TAGs are useful tools for monitoring a protein
within the cell
- Ideally, the tagged protein should be introduced
into a mutant background where there is no
protein X, thus ensure the tagged protein X is
fully functional - Large tag or reporter protein has greater
tendency to affect the function of the protein X,
while smaller ones have better chance of keeping
full functionality of the protein X
34TAGs are useful tools for determining the
functional region of a protein
loss-of-function
gain-of-function
Both loss-of-function and gain-of-function tests
are important to substantiate the conclusion
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36Why Reverse genetics (From gene to
phenotype/function)
1. Many unknown genes are sequenced due to the
advancement of sequence technology
2. Can control where, when, and how much an
active gene product to be made
3. Easy to design specific mutations into the
gene product
4. Add TAGs to a gene product to simplify its
study within the cell
5. Create mutation on gene that has no
available mutation
37Insertional mutagenesis
- Generating large collection of lines with T-DNA
or transposon insertion - Identification of a line with an insertion (gene
disruption) in your favorable gene.