Murals of Cacaxtla

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Murals of Cacaxtla 900 - 1200 A.C
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Maize Transposable Elements as a Flexible
Means to Disrupt Plant Genes in Heterologous
Systems
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Assessing Gene Function in Heterologous Systems

• Two general strategies for using transposons
• Large population of plants carrying each a single
  transposon insertion
• are generated each insertion site can be
  systematically sequenced
• transposons can be engineered to monitored
  the expression of target genes.
• - Ac/Ds transposon system
• - Enhancer Detection and Gene trapping
• - Forward and Reverse Genetics
• 2. Heavily mutagenized populations are used to
  identify by PCR
• plants that carry insertions into
  specific genes. - Mu and Spm/dSpm transposon
  systems
• - Forward and Reverse Genetics gt
  site-selected mutagenesis

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Cut and Paste Transposition Mechanisms Ac/Ds and
Smp/dSpm Copy and Paste Transposition
Mechanisms Mu/MuDr gt replicative transposons
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Determination of the embryonic origin of the
Arabidopsis root by clonal analysis
Single T-DNA insertions selection of F2 lines
homozygous for the T-DNA.
B. Scheres et al. Development 120, 2475-2487
(1994)
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Enhancer Detection and Gene Trapping in
Arabidopsis
-Transposon-based insertional mutagenesis to
identify genes based on their expression
patterns. -Depends on a genetic selection
scheme that allows for linked/unlinked
transposition recovery.
GUS
P min-GUS
Genomic regulatory element
Gene trap
Enhancer detector
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Gene Trap Ds Element within a T-DNA construct
A
uidA
1
IAAH
I
2
nptII
LB
RB
Promoterless reporter gene (GUS) within a Ds
element. Indole acetic acid hydrolase gene in
the launching pad (the T-DNA) confers
sensitivity to NAM (auxin precursor)
Selectable marker confers resistance to
kanamycin
uidA

I
A
Intron and triple splice acceptor to create
transcriptional fusions with endogenous genes
even if they land in introns.
IAAH
2
1
nptII
GUS
Ds
Transcriptional fusions
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Enhancer Detector Ds Element
uidA
-46 35S
1
IAAH
2
nptII
RB
LB
Minimal promoter does not drive transcription
without the influence of of endogenous regulatory
sequences
-46 35S
uidA
Promoterless reporter gene within a Ds
non-autonomous element. IAAH gene in the
launching pad (the T-DNA) confers sensitivity
to NAM (auxin precursor) Selectable marker
confers resistance to kanamycin
IAAH
2
1
nptII
Possibility of activation at a distance
P min-GUS
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Stable Activator Element
This is not a transposon (Rene Magritte)
35S
Ac transposase
nptII
IAAH
2
35S
RB
LB
It is a T-DNA construction
35S
Transposase gene under a constitutive promoter.
Ac transposase
Same as before.
nptII
IAAH
2
35S
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Ds KanR NamS

Ac KanR NamS
female
X

male
Ds KanR NamS
Ac KanR NamS
nptII
uidA
35S
Ac transposase
35S
IAAH
2
IAAH
nptII
Ac NamS Ds KanR NamS
Frequent linked transpositions. Unfrequent
non-linked transpositions.
F1

Selfed-pollination Medium with Kan and Nam
Ds KanR


F2



Maintain each line by self-pollination gt Screen
s
WTgt KanS Ac gt Stunted KanR Ac Ds gt
Stunted KanR Ds-linked gt Stunted
KanR Ds-unlinked gt NORMAL
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Examples of Genetic Screens with Gene Traps
GENE TRAPS / ENHANCER DETECTORS (Using
Sundaresan et al., 1995 CSHL) 3,000 MGTs and
METs
Intergenic insertions
Collaborative screens Osmotic
stress (Covarrubias lab) viral
infection (Riveras lab) root development (Herrer
as lab) secondary metabolism (Leons lab)
microRNA precursors
Sterility male/female
GUS expression in ovules
Segregation Distortion KanRKanS
Gametophytic lethals
RNA interference (RNAi)
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Genetics screen distorted segregation following
KanRKanS
1 000 Lines screened (MGT or MET) Segregation
Ratios (KanRKanS) in F3 31 46.7 21
6.1 11 1.1 lt11 0.8 31 ltxlt
151 15.34 151 2.8 100 KanR 21.5
KanR seedlings
Kan S Kan R
Putative gametophytic lethals
?2lt3.841(1) 0.05
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Degenerated primer
Ds
Liu et al 1995
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TLAZOLTEOTL encodes a GATA-like transcription
factor essential for ovule growth
tlz/
tlz/tlz

• Morphological defect 60embryo sacs extruded
• Expression pattern associated
• Single Ds insertion within first exon

Tlazolteotl codex Borgia
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Reversion analysis facilitates phenotype
confirmation
Crossing back to transposase source allows new Ds
transposition often causing revertant sectors in
homozygous mutant plants.
Sequencing the original insertion site allows
confirmation of the molecular nature of
the reversion
mutant sil.
tlz/tlz
revertant sector.
tlz-1 CGTTTACTCTAGT Ds
ACTCTAGTCCAAG WT
CGTTTACTCTAGT . . . . . . . . . . . . . . . . .
.CCAAG revertant CGTTTACTCTAGT
. . . . . . . . . . . . . . . . . .CCAAG
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Simultaneous analysis of genetic
interactions Morphology Gene expression
tlz y ino act synergistically to ensure inner
integument development. TLZ and INO activity is
necessary to restrict TLZ expression basally.
.
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coatlicue , a gene trap (GT) line defective in
male and female gametophyte development
Inverted orientation of reporter gene
trasnscription with respect to COA transcription
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Identification of enhancer detector lines acting
during female gametophyte and ovule development
Close to 2 of our MET lines show early GUS
expression
9 of the MET lines show GUS expression in
mature ovules gt 5 female gametophyte
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Many genes identified, but only a few show a
corresponding mutant phenotype

LRR-protein kinase
myc-like p.
sugar isomerase
ß 1-3 glucanase
chromatin remodelling factors AGPs 14-3-3
proteins cell cycle regulators RNA binding
proteins unknown proteins

• Insertions within non-coding regions (intergenic
  vicinity, introns, 3end)
• Subtle mutant phenotypes difficult to discover
• Detection of enhancers acting in trans
• - Other (mysterious) reasons

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2 Ds insertions within the putative regulatory
region of a gene encoding an arabinogalactoprotei
n (AGP)
3
3
5
5
CODING REGION
INTERGENIC REGION
1
-139
-880
1685 pb

• -MET333 encodes a classical AGP
• 2 Ds insertions within the putative regulatory
  region.
• No mutant phenotype identified in
• homozygous individuals

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Reverse Genetics
dsRNA-Mediated Postranscriptional Silencing of
Selected Genes
intron splicing
dsRNA
Postranscriptional gene silencing. (mRNA
degradation/traductional repression)
pFGC5941
Rich Jorgensen, University Arizona http//ag.arizo
na.edu/chromatin/fgc5841
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Some lines show an extremely specific pattern
Ex specification of the functional megaspore
(single cell).
Insertion 853 bp upstream of START codon gt no
mutant phenotype
Valuable tools to attempt the manipulation of
sexual developmental pathways
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Spm/dSpm Systems for Dicots
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Multiple Independent Defective Suppressor-mutator
Transposon Insertions in Arabidopsis A Tool for
Functional Genomics Alain F. Tissiera,
Sylvestre Marillonneta, Victor Klimyuka, Kanu
Patela, Miguel Angel Torresa, George Murphyb,
and Jonathan D. G. Jones a Sainsbury Laboratory,
John Innes Centre, Colney Lane, Norwich NR4 7UH,
United Kingdom b John Innes Centre, Colney Lane,
Norwich NR4 7UH, United Kingdom
LB and RB, left and right borders, respectively
P, promoter driving the expression of the
transposase (Spm, 35S, or AtDMC1 promoters)
Spec, spectinomycin resistance gene for selection
in bacteria SU1, counterselectable marker. It
carries a phosphi-nothricin (PPr) resistance gene
(BAR)
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Different possibilities in the segregation of
the T-DNA and dSpm red box, counterselectable
marker long white box, transposase
cassette short white box, GUS gene triangle,
dSpm
Represented for a hypothetical 2-chromosome
individual. 1, loss of the complete T-DNA. 2, no
dSpm excision 3, transposition of the dSpm to a
linked site 4, transposition of the dSpm inside
the T-DNA 5, transposition of the dSpm inside
the counterselectable marker 6, transposition of
the dSpm to an unlinked site and segregation from
the T-DNA
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Table 1. Frequency of Excision Events in the
Progeny of Primary Transformants
-------------------------------------------------
----------------------- Transformant Total
Seed GUSa GEb ----------------------------------
-------------------------------------- 8337 No.
1 1160 55 4.7 8337 No. 3 1120 0 lt0.0009 8337 No.
4 1200 18 1.5 8337 No. 5 1160 1 0.1 8337 No.
6 1080 16 1.5 8337 No. 7 1200 3 0.3 8337 No.
8 1080 0 lt0.0006 8337 No. 9 1080 7 0.6 8313 No.
1 1320 24 1.8 8313 No. 2 1240 2 0.2 8313 No.
4 1240 10 0.8 8353 No. 2 1280 0 lt0.0008 8353 No.
4 1440 5 0.3 8353 No. 5 1560 0 0 8353 No.
7 1200 31 2.6 a Number of completely blue
seedlings after GUS staining. b Percentage of
germinal excision events as estimated from the
GUS st
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Table 2. Frequency of DR Plants and Independent
Transposition Events in the Progeny of Primary
Transformants -----------------------------------
--------------------------------------------------
--------------------------------------------------
------------ Transf Total Seed DR Frequency
GUSa DNA Independent
Independent gel blot-b /Total ------------
--------------------------------------------------
--------------------------------------------------
-------------------------------------- No.
1 16,000 28 1.75 x 10-3 2 4 14.3 0.025 No.
2 4,000 1 2.5 x 10-4 0 1 100 0.025 No.
4 16,000 30 1.88 x 10-3 28 0 0 0 No.
6 17,600 6 3.4 x 10-4 4 2 33.3 0.011 No.
9 17,600 1 6 x 10-5 0 1 100 0.006 . a
Number of GUS-staining plants. b Number of
independent insertions as estimated by DNA gel
blot analysis.
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Strategy for Large-Scale Production of Double
Resistant (DR) Individuals. The numbers at right
represent the number of plants at the
corresponding stage of the selection procedure.
From the F2 generation, the numbers of
heterozygous and homozygous plants are given
separately, although the plants were actually
grown together.
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Pooling Strategy and Organization of a
Transposant Library Seeds and DNA from plants
that survived the double selection are harvested
in pools of 50 individuals. These pools of 50
were then assigned to a superpool of 48 pools.
Each pool can thus be defined according to a
two-coordinate address (x.y e.g, 01.01), where
x is the superpool number and y the pool
number. Superpools are collected, which
represent a total number of 48,000 DR
individuals in 960 pools.
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An Example of Adapter PCR Performed with Various
DNA Pools.
Each band represents a different insertion and
allows for a minimal estimate of the number of
independent insertions in the pool. The
intensity of the bands may reflect the relative
abundance of the insertion within the pool
(i.e., some events are clonal) and/or the
relative efficiency of the amplification. These
bands can then be excised and the DNA reamplified
for direct sequencing. Each lane is the result
of adapter PCR with a different pool. The pools
used are (03.01) to (03.28). Molecular length
markers are given at right in base pairs.
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Distribution of Sequenced Insertion Sites and
T-DNAs on the Arabidopsis Chromosomes.
Black and white triangles represent insertions
from line 8313 plant 1 and mixed lines,
respectively. The two mapped T-DNA inserts (8313
plant 1 and 8337 plant 6) appear as hatched
triangles. cM, centimorgans.
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Activation Tagging with Spm/dSpm
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Activation Tagging Using the En-I Maize
Transposon System in Arabidopsis Marsch-Martínez
et al. Plant Physiol, August 2002, Vol. 129,
pp. 1544-1556
En-TPase
4X 35S Enh.
BAR
nptII
SSU3
SU1
Pnos
Psu
Pnos
Tnos
P35S
T35S
Tnos
LB
RB
dSpm
The construct used for plant transformation.
Relevant EcoRI sites used for the molecular
analysis are indicated. LB, Left border RB,
right border 35SP, 35ST, CaMV 35S promoter and
terminator, respectively EnTPase, En immobile
transposase source ILtir, IRtir, I-element left
and right terminal-inverted repeat, respectively
4 Enh., tetramer of the CaMV 35S enhancer Pnos,
Tnos, promoter and terminator sequences from the
nopaline synthase gene, respectively SSU5',
SSU3', promoter and transit signal peptide to the
chloroplast and terminator of the small subunit
of Rubisco gene, respectively. The gene specific
probes (BAR and SSU3') used for blot
hybridization are indicated as bars above or
below the figure.
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Table I.   Stable and independent transposition
frequencies of sprayed progeny -----------------
--------------------------------------------------
----- Line Plants Plants Analyzed Different
Insertions STFc ITFd -----------------------------
------------------------------------------- Single
plant analysisa   WAT 1 23 23
2 13.33 8.9   WAT 2
18 18 9 0.23 50   WAT 3 11 11 2 3.8 18.2   W
AT 3 18 18 4 4.82 25   WAT 5
18 18 6 2.83 31.5 Pooled plantsb   WAT 3
19 6 1 4.52 16.67    WAT 21
9 9 6 1.18 66.6   WAT 23 10 10 3 0.82 30.0   
WAT 24 32 7 2 1.31 28   WAT 29
3 3 3 0.92 100.0   WAT 30 3 3 2 0.68 66.7  
a  Data obtained with single plant Southern
analysis.     b  Data obtained using pooled
samples for the Southern analysis.     c  The STF
was calculated as the ratio between the number of
double resistant plants and the number of
double-sprayed seeds.     d  The ITF was
estimated as the ratio of the number of different
fragments observed to the total number of
plants analyzed from a family.    
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Table II.   Number of inserts in each chromosome
(Chr.) -----------------------------------------
--------------------------------------------------
---- Line Chr. I Chr. II Chr. III Chr. IV Chr.
V Total Insertionsa ------------------------------
--------------------------------------------------
-------------------------------- WAT2 1 2 7 6 2 18
WAT3 1 1 2 WAT8 2 5 4 1 12 WAT10 2 2 3 7 WAT13 1
2 1 2 6 WAT14 1 4 1 6 WAT17 2 1 1 1 5 WAT19 2 1 2
1 6 WAT21 2 1 3 WAT24 1 1 2 WAT30 1 1 WAT32 1 1 To
tal 11 14 20 17 7 69
Marsch-Martinez et al. 2002
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Marsch-Martinez et al. 2002
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New Perspectives?
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Implementing Enhancer Detection and Gene Trapping
in Maize
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Laboratory of Reproductive Development and
Apomixis CINVESTAV - Unidad Irapuato