plant molecular biology ws 2017-2018 lecture ii

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.

1

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

2

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

TASK I. Criteria for assessing gene function in the absence of sequence information?

4

CHAPTER IV

MUTANT ANALYSIS AND FORWARD GENETICS

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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

6


1980 on



Mutant analysis

Genetic maps

199 -2000

Physical maps


Gene Identification

2000-2010

Functional genomics

Reverse genetics

Transcriptomics

2010-2050 Systems biology

7

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.


A. Definition

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Mutagenesis

• Chemical mutagenesis

– EMS (ethylmethane sulfonate, causes G/C-A/T transitions)

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Forward genetics starts with

a biological question

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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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b) Development

• Pattern formation

The Arabidopsis seedling

cotyledons

hypocotyl

root

Shoot apical meristem

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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


HY

WT hy

Germinated in the light

16


COP

wt cop

Germinated in the dark

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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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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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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


Defective pattern formation

Additional phenotypes found in pattern formation screen

wt gnom

23


fass

What is the primary defect?

Are these patterning mutants?

What is your next level of analysis?

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E. Mutant analysis

1. First level of analysis

2. Summary of first level of analysis

3. Second level of analysis

25

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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Establishment of polarity in the zygote

28


Wild-type

mutant

gnom fass

E

S

E

S

E

S

Mutant phenotypes: gnom and fass

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TASK 4

What are the primary defects in gnom and fass mutants?

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Wild-type

mutant

gnom fass

E

S

E

S

E

S

Mutant phenotypes: gnom and fass

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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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fass

Apical cell

Basal cell

Embryo

Suspensor

ring of microtubules at preprophase is missing Random division planes

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Identifying the major players

Mutant Identification Phenotypic analysis

Gene Biological Role Molecular function

COP1

HY5

MONOPTEROS

FASS/TONNEAU Cytoskeleton

GNOM

36

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

37

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

41


1980 on



Mutant analysis

Genetic maps

1990 -2000

Physical maps


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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1996

50

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 linkage analysis

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Distances in a genetic map are measured in centimorgans

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The mapping function

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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

slip

unequal crossing over

Simple sequence (also microsatellite sequence): repeats of a simple sequence

element

58

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

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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References

• Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. THE

ARABIDOPSIS GENOME INITIATIVE Nature 408: 796-815. 2000.

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plant molecular biology ws 2017-2018 lecture ii

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