Transcript Lecture 15

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.
Site Preferences of Insertional Mutagenesis
Agents in Arabidopsis (Ref: Plant Physiol. 137:168, 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.
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.
Distribution of different insertions relating to translation
initiation codon.
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.
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.
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.