Transcript Lecture 13

Gene identification and functional analysis by transgenic
approach
T-DNA mediated insertional mutagenesis
LB
T-DNA
RB
Wild type gene
Why T-DNA?
1. Homologous Recombination in plants is inefficient.
2. Active transposons do not exist in every species including Arabidopsis.
3. Agrobacterium-mediated transformation is simpler: floral dip, leaf disk transformation.
4. More likely to generate simpler full-length integration as compared to other methods.
5. T-DNA insertion mainly occurs in transcriptionally active area of the genome.
6. The mutated locus gets tagged.
Effect of tissue culture process on T-DNA tagging
Root explant
Somaclonal
variation
callus
plant
Somaclonal variation
Root explant
cocultivation
callus
plant
T-DNA
insertion
Preliminary data
60 transformants: none of the visible mutation were linked to T-DNA.
101 regenerants (non-transformed) had 7 visible recessive mutants (identified by
progeny analysis).
Suggestion by the authors: high number of mutations due to somaclonal variation
rather than T-DNA insertion.
Isolation of a plant gene by T-DNA tagging:
The Plant Cell, Vol. 6, 1211-1225
Control of Arabidopsis Flower and Seed Development by the Homeotic
Gene APETALA2
K. Diane Jofuku, Bart G. W. den Boer, Marc Van Montagu and Jack K. Okamuroa
Tissue
Leaf
Stems
Seeds
Callus
Roots
Cotyledons
Inflorescence
Inflorescence
ref.
1986
1986
1987
1987
1988
1988
1993
1994
problem
somaclonal variation
ditto
real mutants!!! Irreproducible.
somaclonal variation
ditto
ditto
Best procedure
Labor intensive
Seed Infection
1.
2.
3.
4.
5.
6.
Germinating seeds were soaked in agro suspension.
In 1985, a group showed that exogenous DNA is able to get into the cells of
imbibing seeds of Arabidopsis and integrate into its genome.
In 1987, Feldman and Marks followed it up by soaking arabidopsis seeds in
Agro suspension. They recovered several transformed seedlings.
More than 14000 transformants have been generated by this method.
T-DNA contained kanamycin resistant gene.
57% contain single locus, 25% two linked or unlinked locus, 5% 3 locus,
5% 4 or more, 9% becoming KanS. Average number of insert/ locus= 1.5.
LB
pBR322
neo
pBR322
RB
Structure of inserts
10 lines were characterized: 7 were concatameric, each containing 2-5 T-DNA or parts thereof.
6/7 contained rearranged T-DNA. 6/10 were inverted repeats of RB region and 3/10 were
inverted repeats of LB region. 70% contain LB-plant DNA junction or RB-plant DNA
junction or both. 30% contain internal T-DNA/ plant DNA junction.
Mutants
Progeny analysis
Dominant phenotype will segregate as 3 mutant:1wild-type
Recessive phenotype will segregate as 1 mutant: 3 wild-type.
15-26% lines show visible phenotype (under normal growth condition)
Genetic redundancy in higher organisms
Majority of the mutants were recessive
Are mutants tagged?
Co-segregation analysis indicates that only 35-40% mutants are due to T-DNA
insertion i.e. they are tagged.
Untagged lines
Untagged lines were used for cloning of the mutant gene by other techniques such
as map based cloning technique. It has been found that all untagged lines contain
short deletion in the mutant gene. This indicates abortive T-DNA integration
resulting in deletion and phenotype.
Utility of T-DNA tagged population in Reverse Genetics
Have the gene but don’t know the phenotype? So design a
PCR primer in T-DNA border and another in the gene-ofinterest and identify your mutant by PCR screening. Need
following:
1. Large number of T-DNA tagged mutants.
2. Random insertions
3. Pooled DNA.
Transposon tagging
Ac-Ds system: contain 11 bp terminal inverted repeat (TIR), create 8 bp target site
duplication. The 4.5 kb Ac element codes for 3.5 kb mRNA for transposase.
En-Spm system: contain a 13 bp TIR and create a 3 bp target site duplication. En
element is 8.3 kb long and contains 2 alternately spliced gene products, TnpA and
TnpD. Both are required for transposition.
Robertson’s mutator (Mu)/ MuDR: Very mobile, short (1.5 or 1.7 kb) in maize.
Cause high rate of mutation, somatic instability. MuDR encodes the MURA
transposase required for Mu transposition and MURB, a helper protein implicated
in insertion. All Mu elements share 215-bp terminal inverted repeat (TIR)
sequences and the mobile Mu elements contain a highly conserved 32-bp MURA
transposase binding site. Characteristic 9-bp host sequence duplications are
generated during MuDR/Mu germinal insertion.
Maize Gene Discovery Project: RescueMu
http://www.maizegdb.org/documentation/mgdp/education/
Tagging strategies
Random vs Targeted tagging.
Dilute somaclonal variation effect.
Use gametophytic promoters to drive transposase expression.
Proof of tagging
1. In case of excision system: phenotypic reversion.
2. Variegation in the presence of transposase
3. In case of non-excision system: co-segregation of mutant
phenotype and specific element.
Classical examples:
Bz was isolated in 1984 by Ac tagging. Ac was present in very few copies.
A1 was isolated in 1985 by En and Mu tagging. Each of which were present in
high copies, so a cross hybridization strategy was used to identify which En-tag or
Mu-tag contained A1 gene.
Once the tag is identified, it can be used to probe genomic library to isolate the
wild-type gene.
Heterologous transposition
Ac-Ds in tobacco, rice, potato, tomato etc.
Ds
or
Spm
promoter
Excision marker
X
Pro-transposase
Pro-codA
[Pro: promoter; codA: negative selection marker]
Entrapment strategies: for the gene of unknown function
Genetrap.cshl.org/traps.htm
Enhancer Trap
P gene
TIR MP
marker
TIR
TIR: terminal inverted repeat
MP: minimal promoter
enhancer
Promoter Trap
Native
promoter
marker
ATG
Native ORF
Trn stop
Gene Trap
P
ex1
ex2
ex3
Fusion protein
TIR
marker
Splice acceptor
site
TIR
Transcription
stop
ex4
Activation tagging
1.
2.
Based on over-expression of native genes: gain-of-function.
Addresses the problem of insertional mutagenesis:
i) genetic redundancy
ii) multiple inserts make gene isolation difficult.
iii) large number of T-DNA insertions are not linked with the mutant
phenotype.
Bar selection
gene
4X 35S
RB
Native ORF
Advantages of entrapment
1.
2.
3.
4.
Can identify very low abundance transcripts. cDNA libraries do not
well represent low abundance and very low abundance mRNA.
Tissue specific transcripts can be identified: seedling cDNA libraries
do have good representation of tissue specific transcripts.
Temporally expressed transcripts can be identified: ditto problem with
cDNA libraries.
High frequency of promoter traps (30% in arabidopsis and similar frequencies
in other species) suggests that T-DNA insertion is not random, that it is
in transcriptional regions of genome.
Isolation of the tagged- or trapped gene
1. Plasmid rescue
2. Inverse PCR
3. TAIL-PCR
Gene function study of isolated unknown gene
1. Over-expression
2. Suppression
Stable transgenics
i) Antisense
ii) RNAi
Transient transgenics: VIGS
Applications of Gene Silencing
Antisense RNA approach: Antisense approach has been successfully used to
down regulate or inhibit gene expression in E.coli, C. elegans, D. discoideum, plants
and vertebrates. Several mechanisms have been suggested based on studies:
1. In C. elegans lin4 antisense RNA inhibits translation of the lin4 mRNA.
2. Nuclear destabilization of the human eIF2a mRNA by an antisense transcript.
3. Cytoplasmic destabilization of D. discoideum PSV-A mRNA by the antisense RNA
4. Inhibition of the splicing of erbA gene transcript by antisense RNA in mammalian
cells.
5. In prokaryotes: inhibition of translational coupling by antisense RNA.
In principal, the loss of gene expression may or may not be accompanied by the loss of
the respective mRNA.
Additional mechanism: Methylation of the respective gene copy in response to
antisense expression.
It is unclear whether or not these mechanisms function simultaneously or function
in different cell types.
Differential regulation of antisense machinery
Some genes are regulated by endogenous antisense transcript such as
D. discoideum prespore gene.
1. With the induction of antisense transcription, the half life of the
mRNA is reduced from several hours to 15 min.
2. Inhibition of transcription (general) stabilizes the previously
accumulated mRNA.
2. Antisense transcripts are accumulated only after the majority of the
mRNA has been degraded.
These observations suggest: hybrid formation between sense and
antisense and rapid degradation. It implies that sense and antisense
transcripts can never accumulate simultaneously unless antisense
machinery of hybrid formation and degradation is under differential
control. Targets for this control would be the proteins that promote
hybrid formation, proteins which resolve duplexes (helicase) and
nucleases that degrade dsRNA.
Formation of dsRNA and degradation by dsRNases. In principle, an
RNA complementary to the target RNA should be able to form a
duplex and if this duplex sequesters translation initiation signal or
become substrate of dsRNases, inhibition of gene expression is
expected. However, ineffectiveness of antisense approach has
commonly been experienced. General factors that determine the
effectiveness may be: physico-chemical principles that determine
whether structural complementarity of folded RNAs is possible and
rate at which pairing occurs and the binding pathway specificity.
Further mechanism of degradation may be a key factor in the
success or failure of this technology.
RNAi
PTGS for functional genomics
Injection of dsRNA into nematodes can trigger specific (homologous) RNA
degradation. RNAi has been utilized to study 4000 genes in C. elegans. In plants,
dsRNA and sshairpin RNA have similar effect.
The predicted RNA structure and efficacy of gene-silencing constructs.
(Ref: Wesley et al., 2001, Plant J. 27: 581-590)
Constructs, controlled by Ubi1 promoter, silencing GUS in rice. Thick lines indicate a
560 nt GUS sequence; grey lines indicate non-GUS sequences; dashed grey lines
indicate intron-junction sequences left after splicing; and short lines within the stem
of hairpin structures indicate base pairing. Numbers in PTGS column indicate the
percentage of plants showing GUS silencing; n = number of plants in each treatment.
From Ref: Wesley et al.
(Ref: Wesley et al., 2001, Plant J. 27: 581-590)
Using hpRNA constructs, we have obtained silenced plants for every
gene that we targeted, irrespective of whether it was a viral gene,
transgene or endogenous gene, and the silencing appears to be uniform
within tissues in which the hpRNA is expressed. With ihpRNA
constructs the efficiency averaged about 90%, and arms of 400±800 nt
appear to be stable and effective. High levels of silencing were obtained
with constructs having unmatched arm lengths, with arms as long as 853
nt or as little as 98 nt, and with arm sequences derived from coding, 3’
or 5’ untranslated regions of the target gene. These results suggest that
ihpRNA constructs will be effective in a wide range of circumstances,
and augur well for the generic use of the technology. The silencing was
much more profound with ihpRNA constructs than either anti-sense or
co-suppression constructs; some ihpRNA transformants were close to
exhibiting a complete knockout of the target endogenous gene.
However, most of the ihpRNA plants showed dramatically reduced
but detectable levels of target gene activity.
Virus induced gene silencing
RNA mediated defense mechanism is related to PTGS in which RNA
are specifically degraded if they are similar in sequence to a transgene,
especially if it is designed to produce dsRNA. This process is
mechanistically similar between plants, animals and fungi. This approach
can be applied to carry out Gene silencing of the native gene and study
their function.
Procedure: Agro-infiltration
Vectors
based on
TMV, PVX,
TGMV and
TRV.
Problems:
1. VIGS phenotype is superimposed on, and complicated by, chlorosis, leaf
distortion, and necrosis.
2. Virus is not able to invade every cell, such that cells in which the target
gene is not silenced may obscure VIGS phenotype.
3. Viruses commonly used in VIGS such as TMV, PVX and TGMV don't
infect growing points of plant and therefore remain uninformative about
genes effecting identity and development of plant tissue.
4. Variation between experiments in proportion of plants showing VIGS
phenotype.
Better vector (Tobacco Rattle Virus): TRV is better because it induces mild
symptoms, infects large area of adjacent cells and infects growing points.
TRV persists in ‘recovered’ plants. TRV is shown to be successful in
Nicotiana and Tomato. Its potential lies in potato and oats as well.
Plasmid Rescue
E
H
P1
H
pUC19
P2
E
Restriction
H
H
P1
pUC19
E
pUC19
E
P2
Ligation
P1
P2
Flank
Flank
pUC19
Flanking genomic seq.
P1 and P2 = vector sequences
pUC19
Transform into E. coli
Inverse PCR
H
H
P1
H
P2
HindIII cut followed by ligation
P1
H
PCR primer
P2
H
Left flank
Right flank
Cloning and sequencing
TAIL-PCR (Thermal Asymmetric Interlaced PCR)
P1
RP3
RP2
RP1
LP1
LP2
LP3
AD
P2
LP1-AD or RP1-AD reaction
Target + non-target amplification
LP2-AD or RP2-AD reaction
LP3-AD or RP3-AD reaction
Target product
AD