GABI-KAT system

1
GABI-KAT system Plasmid map of pAC161 used for
tagging. Map of the binary transformation vector
pAC161 that contains the SULr ORF (for resistance
against the herbicide sulfadiazine (sul
4-amino-N-2-pyrimidinylbenzene sulfonamide)
driven by the 1 2 promoter. The 35S CaMV
promoter located at the right border can act as
an activation tagging element after T-DNA
integration. Structural parts and relevant
restriction sites of the vector are marked.
2
Site Preferences of Insertional Mutagenesis
Agents in Arabidopsis (Ref Plant Physiol.
137168, 2005)
Transposon-Based Agents Have Marked Preference
for High GC Content, whereas T-DNA-Based Agents
Show Preference for Low GC Content
Insertion frequency by GC content. Insertion
events have been normalized to 1,000 insertions
of each insertional mutagenesis agent to allow
for comparison between the agents.
3
Distribution of 1,000 insertions of each
insertional mutagenesis agent within genome
structure. Insertion events have been normalized
to 1,000 insertions of each agent to allow for
comparison between the agents.
4
Distribution of different insertions relating to
translation initiation codon.
5
T-DNA vectors for the PEF (plant exon finder)
system. Schematic diagrams of the pFJ8 T-DNA
construct with the 850-bp Arabidopsis HSP18.2
heat-shock promoter and the pFJ18 T-DNA construct
with the 800-bp CaMV 35S promoter. Both promoters
are fused to the HSP81-1 first exon and intron
partial sequences followed by the T-DNA left
border (LB). ATG indicates the start codon of
HSP81-1 in each construct.
6
T-DNA versus Transposon as insertion mutation
agent
The transposon-based agents show marked
preference for high GC content, whereas the
T-DNA-based agents show preference for low GC
content regions. The transposon-based agents show
a bias toward insertions near the translation
start codons of genes, while the T-DNAs show a
preference for the putative transcriptional
regulatory regions of genes. The transposon-based
agents also have higher insertion site densities
in exons than do the T-DNA insertions.
Each have unique features and therefore both
should be used for saturation insertion
mutagenesis. But transposon based strategy
requires a few starter lines, which will be a
great advantage for those plant systems, which
are difficult to transform.
7
A schematic representation of the T-DNA vector
harboring the plasmid pYS11 used for transforming
wild-type Arabidopsis Ws-0. LB, Left border
sequence of T-DNA hsp, heat shock promoter from
Glycine max Ac, activator element Ds,
dissociation element GT, gene trap StrpR,
streptomycin resistance gene A, triple-splice
acceptor I, Arabidopsis intron GUS,
 -glucuronidase gene KanR, kanamycin resistance
gene 35S, 35S cauliflower mosaic virus promoter
and RB, right border sequence of T-DNA.
8
(No Transcript)
9
(No Transcript)
10
(No Transcript)