Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed phenotypes in T1 generation.

Plant genomic resources harbouring gain-of-function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation-tagging T-DNA was introduced into the genome of Arabidopsis creating 55,431 independent transformed lines. Of these T1 lines, 1,262 showed phenotypes different from those of wild-type plants. We called these lines 'AT1Ps' (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour, flowering time and fertility. Similar phenotypes re-appeared either in dominant or semi-dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor-PCR method was used to identify 1172 independent genomic loci of T-DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T-DNA knock-out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock-out populations (30-35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T-DNA integration sites by RT-PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left-border sequence of the T-DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the first ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/index.html).

[1]  M. Matsui,et al.  Selection of hygromycin-resistant Arabidopsis seedlings. , 2003, BioTechniques.

[2]  E. Ábrahám,et al.  Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. , 2002, The Plant journal : for cell and molecular biology.

[3]  Michael R Sussman,et al.  Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis. , 2002, Omics : a journal of integrative biology.

[4]  T. Ichikawa,et al.  Horizontal gene transfer: regulated expression of a tobacco homologue of the Agrobacterium rhizogenes rolC gene , 1995, Molecular and General Genetics MGG.

[5]  J. Levin,et al.  Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning. , 2001, Genetics.

[6]  Minami Matsui,et al.  Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. , 2003, The Plant journal : for cell and molecular biology.

[7]  C. Dean,et al.  Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis. , 1997, The Plant journal : for cell and molecular biology.

[8]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[9]  S. Goff,et al.  A High-Throughput Arabidopsis Reverse Genetics System Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004630. , 2002, The Plant Cell Online.

[10]  D. Bouchez,et al.  Arabidopsis gene knockout: phenotypes wanted. , 2001, Current opinion in plant biology.

[11]  D. G. Brown,et al.  The origins of genomic duplications in Arabidopsis. , 2000, Science.

[12]  C Camilleri,et al.  Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. , 1998, Genetics.

[13]  M. Matsui,et al.  Activation tagging, a novel tool to dissect the functions of a gene family. , 2003, The Plant journal : for cell and molecular biology.

[14]  Chris Somerville,et al.  Plant Biology in 2010 , 2000, Science.

[15]  T. Gojobori,et al.  The genome sequence and structure of rice chromosome 1 , 2002, Nature.

[16]  H. Morikawa,et al.  Nitrite reductase gene enrichment improves assimilation of NO(2) in Arabidopsis. , 2001, Plant physiology.

[17]  G. Coupland,et al.  A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. , 1996, The Plant cell.

[18]  Yujun Zhang,et al.  Sequence and analysis of rice chromosome 4 , 2002, Nature.

[19]  R. Dixon,et al.  Activation tagging in Arabidopsis. , 2000, Plant physiology.

[20]  L. Herrera-Estrella,et al.  Activation Tagging Using the En-I Maize Transposon System in Arabidopsis , 2002, Plant Physiology.

[21]  The Arabidopsis Genome Initiative.  Analysis of the genome sequence of the flowering plant Arabidopsis thaliana , 2000, Nature.

[22]  G. An,et al.  T-DNA Insertional Mutagenesis for Activation Tagging in Rice1 , 2002, Plant Physiology.

[23]  H. Hayashi,et al.  Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development , 1994, Plant Molecular Biology.

[24]  Huanming Yang,et al.  A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica) , 2002, Science.