Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis.
A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.
Krysan
Patrick J
PJ
Horticulture Department, University of Wisconsin-Madison 53706, USA. fpat@biotech.wisc.edu
Young
Jeffery C
JC
Jester
Peter J
PJ
Monson
Sean
S
Copenhaver
Greg
G
Preuss
Daphne
D
Sussman
Michael R
MR
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D032461:Chromosomes, Plant / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D017343:Genes, Plant; D018745:Genome, Plant; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA