Plant Molecular Biology WS 2017-2018
Lecture II
Chapter III Model Organisms (slides included in lecture I)
Chapter IV Mutant Analysis
Chapter V Genome Analysis Part 1.
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Chapter Topic Classification
I Food Security Introduction
II Plants as factories for the synthesis of biomaterials Technology
III Model Organisms Method
IV Mutant Analysis and Forward Genetics Method
V Genome Analysis Method
VI Gene Identification in the Post-genomic Era Method
VII Membrane Traffic Process
VIII Transcription Process
IX Proteolysis Process
X Cytoskeletal dynamics Process
XI Functional Genomics and Reverse Genetics Method
XII The Interactome Method
XIII Systems biology Method
XIV Take home lessons for green biotechnology Technology Conclusion
Course contents
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Arabidopsis research
1980 on
Identification and Development as a model organism
Forward genetic screens
Mutant analysis
Genetic maps
1990 -2000
Physical maps
Genome sequence and annotation
Gene Identification
2000-2010
Functional genomics
Reverse genetics
Transcriptomics
2010-2050 Systems biology 3
CHAPTER IV
MUTANT ANALYSIS AND FORWARD GENETICS
A. Forward Genetics: Definition
B. Important questions in plant biology
C. Example I: Light Response
D. Example II: Development and pattern formation
E. Mutant analysis
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Arabidopsis research
1980 on
Identification and Development as a model organism
Forward genetic screens
Mutant analysis
Genetic maps
199 -2000
Physical maps
Genome sequence and annotation
Gene Identification
2000-2010
Functional genomics
Reverse genetics
Transcriptomics
2010-2050 Systems biology
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Chapter IV – Mutant Analysis Definition
Forward genetics
phenotype genotype
Phenotype: the appearance of the plant. Genotype: what happens at the level of the DNA, mutant (knock out) versus wild-type.
Chapter IV – Mutant Analysis Definition
A. Definition
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Mutagenesis
• Chemical mutagenesis
– EMS (ethylmethane sulfonate, causes G/C-A/T transitions)
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Chapter IV – Mutant Analysis Definition
B. Some important questions in plant biology
• Light responses
• Water status
• Development
• Biotic and abiotic stress responses
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Chapter IV – Mutant Analysis Important questions in plant biology
a) Response to Light
• Intensity
• Duration
• Quality
• The time of day
• The season
• Shade
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Chapter IV – Mutant Analysis Important questions in plant biology
b) Development
• Pattern formation
The Arabidopsis seedling
cotyledons
hypocotyl
root
Shoot apical meristem
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Chapter IV – Mutant Analysis Important questions in plant biology
C. Light responses
Light dark
etiolation
response
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Chapter IV – Mutant Analysis Example: Light Response
Light dark
Design mutant screen
wt mut wt mut 15
Chapter IV – Mutant Analysis Example: Light Response
TASK 2: Design a mutant screen
What would a mutant look like that
A) does not understand that there is light?
B) does not understand that it is dark?
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Chapter IV – Mutant Analysis Example: Light Response
TASK 3: cop and hy mutants
• When is HY required (light or dark)?
• What is the role of HY in the wild-type?
• When is COP required?
• What is the role of COP in the wild-type?
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Chapter IV – Mutant Analysis Example: Light Response
Examples of forward genetic screens
Light responses
Development
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Chapter IV – Mutant Analysis Example: Development and pattern formation
D. Example: Development and Pattern
formation in the Arabidopsis seedling
cotyledons
hypocotyl
root
Shoot apical meristem
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Chapter IV – Mutant Analysis Example: Development and Pattern formation
wt
monopteros bodenlos
A basal deletion: no root
Berleth and Jürgens, 1993
Hamann et al., Genes Dev. 2002 22
Chapter IV – Mutant Analysis Example: Development and Pattern formation
Defective pattern formation
Additional phenotypes found in pattern formation screen
wt gnom
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Chapter IV – Mutant Analysis Example: Development and Pattern formation
fass
What is the primary defect?
E. Mutant analysis
1. First level of analysis
2. Summary of first level of analysis
3. Second level of analysis
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First division of the zygote
Apical cell
Basal cell
Embryo
Suspensor
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Chapter IV – Mutant Analysis Analysing Mutants – First step of analysis
N
Position of interphase nucleus determines division site
Marked by ring of microtubules at preprophase
Nascent cross wall guided to this site during cytokinesis
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Chapter IV – Mutant Analysis Analysing Mutants – First step of analysis
Establishment of polarity in the zygote
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Chapter IV – Mutant Analysis Analysing Mutants – First step of analysis
Wild-type
mutant
gnom fass
E
S
E
S
E
S
Mutant phenotypes: gnom and fass
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Chapter IV – Mutant Analysis Analysing Mutants – First step of analysis
Wild-type
mutant
gnom fass
E
S
E
S
E
S
Mutant phenotypes: gnom and fass
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Chapter IV – Mutant Analysis Analysing Mutants – First step of analysis
Establishment of cell polarity
Determination of division site
Summary: Identifying the major players
Mutant Identification Phenotypic analysis
Gene Biological Role Molecular function
COP1
HY5
MONOPTEROS
FASS/TONNEAU
GNOM
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Chapter IV – Mutant Analysis Analysing Mutants – Summary
Next level of analysis
• Antibody stains
• Confocal microscopy
• Electron microscopy
•Dissecting scope •Light microscopy
Phenotypic analysis
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Chapter IV – Mutant Analysis Analysing Mutants – Next level of analysis
First division of the zygote
Apical cell
Basal cell
Embryo
Suspensor
ring of microtubules at preprophase marks division plane, Can be seen with an anti-tubulin antibody stain.
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Chapter IV – Mutant Analysis Analysing Mutants – Next level of analysis
fass
Apical cell
Basal cell
Embryo
Suspensor
ring of microtubules at preprophase is missing Random division planes
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Chapter IV – Mutant Analysis Analysing Mutants – Next level of analysis
Identifying the major players
Mutant Identification Phenotypic analysis
Gene Biological Role Molecular function
COP1
HY5
MONOPTEROS
FASS/TONNEAU Cytoskeleton
GNOM
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Chapter IV – Mutant Analysis Analysing Mutants – Summary
Chapter IV: Mutant analysis take home lesson
An in depth cellular or physiological (for example wave lengths for light) analysis of
a mutant phenotype can point to the molecular function of the mutated gene
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Chapter IV – Mutant Analysis Take home lesson
Arabidopsis nomenclature
please observe that the Arabidopsis literature uses font “Schriftart” (regular versus italic) and case (lower versus upper) to distinguishes between wild-type versus mutant genes versus proteins (example, COP1) as follows:
•WILD-TYPE GENE or LOCUS (COP1)
•mutant gene (cop1)
•WILD-TYPE PROTEIN (COP1)
•mutant protein (cop1)
Extra information Arabidopsis nomenclature
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Chapter IV: Mutant analysis „Lernziele“
• Forward genetics: definition
• Mutant analysis: examples in light signaling and development
COP
HY
MONOPTEROS & BODENLOS
GNOM
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Chapter IV – Mutant Analysis Lernziele
examples of Arabidopsis mutants
What is your next level of analysis? How will you identify the gene if a mutant phenotype is
caused by a point mutation? Gene identification is greatly facilitated by genome
sequence information
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Chapter IV – V transition
Chapter V: The First Plant Genome Sequence
A. Events and advances that made it possible
B. Genome Sequencing
C. Genome Annotation
D. Lessons learnt
E. 2012 and beyond
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Arabidopsis research
1980 on
Identification and Development as a model organism
Forward genetic screens
Mutant analysis
Genetic maps
1990 -2000
Physical maps
Genome sequence and annotation
Gene Identification
2000-2010
Functional genomics
Reverse genetics
Transcriptomics
2010-2050 Systems biology 42
Chapter V: „Lernziele“ • Expressed sequence tags
• Genetic versus physical maps, molecular markers, SSLPs
• Linkage (Kopplung) and genetic distances
• Bacterial artificial chromosomes (Bauer)
• Shotgun versus ordered clone sequencing (Bauer)
• Structural versus functional annotation – Gene structure
– Protein domains and families
• Gene ontologies for classification of annotated genes
• Genome complexity
• Phylogenetic profiles and conserved neighbourhood methods
• Natural variation
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The First Plant Genome Sequence
A. Events and advances that made it possible
Arabidopsis Genome Project initiated in 1990.
Genome sequencing completed in 2000.
Cost: c. 80 million $
Continents/countries involved: Europe, US, Japan
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Arabidopsis has the smallest plant genome
species Genome size 1Mb =106bp
Repetitve
sequences
Arabidopsis 110 Mb 5%
Rice 420 Mb 30%a
Tomato 950 Mb
Maize 2,500 – 3,000 Mb 80%
Barley 5,000 Mb
Wheat 16,000 Mb c. 95%
a: in rice, 30% of all gene models encode transposable elements
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TASK 5
Get the backing of the community
What molecules can be sequenced
– rapidly and
– cheaply
– yet convey a lot of information?
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ESTs, expressed sequence tags
Gene A Gene B Gene C
intergenic DNA
transcription
Reverse transcription
mRNA
cDNA
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ESTs, expressed sequence tags
reverse transcription
mRNA
cDNA
EST contig
sequence
full length cDNA
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How is knowledge transmitted?
Database
EST sequences
How are resources shared?
Stock center (1991)
cDNA clones
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Advances that made the first plant genome sequence possible
• Genetic and molecular markers
• High resolution genetic map
• BAC (bacterial artificial chromosome)
• Physical Map (1997)
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Genetic versus physical maps
• The unit of a genetic map is in cM
cM: centi-Morgan, a function of recombination frequencies (RF), which assess linkage (Kopplung) between two markers
• The unit of a physical map is a bp
bp: base pair
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Genetic Maps
• Genetic maps were initially based on visible markers
• First detailed genetic map of Arabidopsis published in 1983
• Most mutations are “silent”. More abundant molecular markers were used for high resolution genetic maps.
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Molecular Markers
• A direct visualisation of DNA polymorphisms (difference in DNA sequence), via hybridisation or PCR.
• Different ecotypes of Arabidopsis are used in crosses for mapping.
• (Arabidopsis ecotypes Landsberg erecta Ler and Columbia Col typically used as both sequenced and have a high density of polymorphisms.)
• RFLP, CAP, SNP, SSLP
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SSLP Simple Sequence Length Polymorphism
CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA
slip
unequal crossing over
Simple sequence (also microsatellite sequence): repeats of a simple sequence
element
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SSLP CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA
CA CA CA CA CA CA
PCR using flanking sequence primers
Run agarose gel
Col Ler
Columbia, Col
Landsberg, Ler
20bp difference
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A. Advances that made genome sequencing possible
• Genetic and molecular markers
• High resolution Genetic map
• BAC (bacterial artificial chromosome)
• Physical Map (1997)
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Genomic Libraries using BAC vectors
• Long chromosomal fragments (~ up to 300 kb, usually c. 100kb) can be cloned
• Stable
• Easy to prepare large amounts of BAC DNA
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Genomic Libraries using BAC vectors
1. oriS and repE mediate the
unidirectional replication of the
F factor
2. Par A and B are partitioning
Genes that regulate copy number
3. CMR confers
chloramphenicol resitance,
positive selectable marker
4. The cloning strip is for
the insertion of foreign DNA
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Text Book Readings
• Modern Genetic Analysis Integrating genes and
genomes 2nd edition. Giffiths and Lewontin
Chapter 4 Mitosis vs Meiosis
Chapter 6 Recombination, Linkage
Chapter 9 Genomics
For next lecture see pages 303-305.
• Genes VIII, Benjamin Lewin paperback ISBN 013144946X
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