Ds insertion lines with sequenced positions of the Ds elements

These Arabidopsis thaliana (ecotype Landsberg) lines contain mostly single copy insertions of modified maize transposable Dissociation (Ds) elements inserted into different positions within the genome.

Each Ds element carries a NPTII gene, and therefore plants with Ds insertions are resistant to kanamycin. The Ds elements in these lines are stable, but capable of remobilization if the Ac transposase is provided, e.g., by crossing with one of the Ac starter lines (CS/N8043 – 8049). Ds elements also carry the GUS reporter gene designed to act as gene trap (SGT or GT) or enhancer trap (SET). For gene trap lines GUS expression requires that the orientation of the GUS reporter gene lie in the same direction as the disrupted gene. For details of the Ds elements and the transposon mutagenesis system please see Sundaresan et al. (1995).

Sequences flanking the Ds insertions in the following transposant lines were amplified using TAIL-PCR (Liu et al., 1995). Sequences were analyzed by BLAST searches in two public databases: NCBI GenBank and Arabidopsis GenBank in Stanford.   For further details please see Parinov et al. (1999).

How to find your gene in the database:

• Identify the gid numbers of sequences in GenBank identical to your sequence:
  Run a BLASTN search with the nucleotide sequence of your gene at http://www.arabidopsis.org/blast/, and record the names or gi / gid numbers of the GenBank entries that are identical to your gene
• Search the ima page for these names / gid numbers:
  Use the "Find" (Ctrl-F) option on your Internet browser to search for matches within the table on the ima lines web page. Try all names and/or numbers (BAC and P1 clones, BAC Ends, EST and other nucleotide entries of GenBank) that are identical to your gene of interest since only one of them may be represented in this database (preference is given to BAC and P1 clones).
• This will reveal seed lines with insertions in your gene.
• Order the line by following the link in the order column.

IMPORTANT: We detected up to 7% cross-contamination in the sequencing data in our lines, that result mainly from PCR-contamination, but also occasionally from seed contamination. Since the flanking sequence data was obtained from single runs, each line has to be independently confirmed. This confirmation is accomplished most simply by performing PCR amplification of DNA from the line, using gene specific primers and Ds-specific primers. The presence of PCR products of the correct size on a gel is usually sufficient confirmation, although sequencing of the products can be performed if necessary.

Primers

The following primers complimentary to the ends of Ds element can be used for PCR amplification.
Distance from the 5'-end of each primer to the corresponding end of transposon is indicated.

For 3'-end of Ds:
Ds3'-1	ggTTCCCgTCCgATTTCgACT	179 bp
Ds3'-2	CgATTACCgTATTTATCCCgTTC	131 bp
Ds3'-3	TCgTTTCCgTCCCgCAAgT	82 bp
For 5'-end of Ds:
Ds5'-1	ACggTCgggAAACTAgCTCTAC	173 bp
Ds5'-2	TCCgTTCCgTTTTCgTTTTTTAC	114 bp
Ds5'-3	CggTCggTACgggATTTTCC	39 bp

References

Sundaresan, V., Springer, P., Volpe, T., Haward, S., Jones, J.D., Dean, C., Ma, H., and Martienssen, R. (1995). Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev. 9, 1797-1810.

Liu, Y.G., and Whittier, R.F. (1995). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674-681.

Parinov, S., Sevugan, M., Ye, D., Yang, W.-C., Kumaran, M., and Sundaresan, V. (1999) Analysis of Flanking Sequences from Ds Insertion Lines: A Database for Reverse Genetics in Arabidopsis. Plant Cell In Press