Skip to main content
The Plant Cell logoLink to The Plant Cell
. 1999 Dec;11(12):2263–2270. doi: 10.1105/tpc.11.12.2263

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis.

S Parinov 1, M Sevugan 1, D Ye 1, W C Yang 1, M Kumaran 1, V Sundaresan 1
PMCID: PMC144131  PMID: 10590156

Abstract

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

Full Text

The Full Text of this article is available as a PDF (121.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benson D. A., Boguski M. S., Lipman D. J., Ostell J., Ouellette B. F. GenBank. Nucleic Acids Res. 1998 Jan 1;26(1):1–7. doi: 10.1093/nar/26.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bevan M., Bancroft I., Bent E., Love K., Goodman H., Dean C., Bergkamp R., Dirkse W., Van Staveren M., Stiekema W. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature. 1998 Jan 29;391(6666):485–488. doi: 10.1038/35140. [DOI] [PubMed] [Google Scholar]
  5. Bevan M., Bancroft I., Mewes H. W., Martienssen R., McCombie R. Clearing a path through the jungle: progress in Arabidopsis genomics. Bioessays. 1999 Feb;21(2):110–120. doi: 10.1002/(SICI)1521-1878(199902)21:2<110::AID-BIES5>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
  6. Copenhaver G. P., Pikaard C. S. RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4. Plant J. 1996 Feb;9(2):259–272. doi: 10.1046/j.1365-313x.1996.09020259.x. [DOI] [PubMed] [Google Scholar]
  7. Copenhaver G. P., Pikaard C. S. Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution. Plant J. 1996 Feb;9(2):273–282. doi: 10.1046/j.1365-313x.1996.09020273.x. [DOI] [PubMed] [Google Scholar]
  8. Das L., Martienssen R. Site-selected transposon mutagenesis at the hcf106 locus in maize. Plant Cell. 1995 Mar;7(3):287–294. doi: 10.1105/tpc.7.3.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dooner H. K., Belachew A., Burgess D., Harding S., Ralston M., Ralston E. Distribution of unlinked receptor sites for transposed Ac elements from the bz-m2(Ac) allele in maize. Genetics. 1994 Jan;136(1):261–279. doi: 10.1093/genetics/136.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flanders D. J., Weng S., Petel F. X., Cherry J. M. AtDB, the Arabidopsis thaliana database, and graphical-web-display of progress by the Arabidopsis Genome Initiative. Nucleic Acids Res. 1998 Jan 1;26(1):80–84. doi: 10.1093/nar/26.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grossniklaus U., Vielle-Calzada J. P., Hoeppner M. A., Gagliano W. B. Maternal control of embryogenesis by MEDEA, a polycomb group gene in Arabidopsis. Science. 1998 Apr 17;280(5362):446–450. doi: 10.1126/science.280.5362.446. [DOI] [PubMed] [Google Scholar]
  12. Hui E. K., Wang P. C., Lo S. J. Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants. Cell Mol Life Sci. 1998 Dec;54(12):1403–1411. doi: 10.1007/s000180050262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iba K., Gibson S., Nishiuchi T., Fuse T., Nishimura M., Arondel V., Hugly S., Somerville C. A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem. 1993 Nov 15;268(32):24099–24105. [PubMed] [Google Scholar]
  14. Jacobs D. M., Sano M., Albert S., Schofield P., Dooneief G., Stern Y. Cross-cultural neuropsychological assessment: a comparison of randomly selected, demographically matched cohorts of English- and Spanish-speaking older adults. J Clin Exp Neuropsychol. 1997 Jun;19(3):331–339. doi: 10.1080/01688639708403862. [DOI] [PubMed] [Google Scholar]
  15. Kotani H., Sato S., Fukami M., Hosouchi T., Nakazaki N., Okumura S., Wada T., Liu Y. G., Shibata D., Tabata S. A fine physical map of Arabidopsis thaliana chromosome 5: construction of a sequence-ready contig map. DNA Res. 1997 Dec 31;4(6):371–378. doi: 10.1093/dnares/4.6.371. [DOI] [PubMed] [Google Scholar]
  16. Liebman S. W., Newnam G. A ubiquitin-conjugating enzyme, RAD6, affects the distribution of Ty1 retrotransposon integration positions. Genetics. 1993 Mar;133(3):499–508. doi: 10.1093/genetics/133.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu Y. G., Mitsukawa N., Oosumi T., Whittier R. F. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 1995 Sep;8(3):457–463. doi: 10.1046/j.1365-313x.1995.08030457.x. [DOI] [PubMed] [Google Scholar]
  18. Liu Y. G., Whittier R. F. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics. 1995 Feb 10;25(3):674–681. doi: 10.1016/0888-7543(95)80010-j. [DOI] [PubMed] [Google Scholar]
  19. Machida C., Onouchi H., Koizumi J., Hamada S., Semiarti E., Torikai S., Machida Y. Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8675–8680. doi: 10.1073/pnas.94.16.8675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sato S., Kaneko T., Kotani H., Nakamura Y., Asamizu E., Miyajima N., Tabata S. Structural analysis of Arabidopsis thaliana chromosome 5. IV. Sequence features of the regions of 1,456,315 bp covered by nineteen physically assigned P1 and TAC clones. DNA Res. 1998 Feb 28;5(1):41–54. doi: 10.1093/dnares/5.1.41. [DOI] [PubMed] [Google Scholar]
  21. Smith D., Yanai Y., Liu Y. G., Ishiguro S., Okada K., Shibata D., Whittier R. F., Fedoroff N. V. Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis. Plant J. 1996 Oct;10(4):721–732. doi: 10.1046/j.1365-313x.1996.10040721.x. [DOI] [PubMed] [Google Scholar]
  22. Spradling A. C., Stern D. M., Kiss I., Roote J., Laverty T., Rubin G. M. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10824–10830. doi: 10.1073/pnas.92.24.10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sundaresan V., Springer P., Volpe T., Haward S., Jones J. D., Dean C., Ma H., Martienssen R. Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev. 1995 Jul 15;9(14):1797–1810. doi: 10.1101/gad.9.14.1797. [DOI] [PubMed] [Google Scholar]
  24. Tissier A. F., Marillonnet S., Klimyuk V., Patel K., Torres M. A., Murphy G., Jones J. D. Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell. 1999 Oct;11(10):1841–1852. doi: 10.1105/tpc.11.10.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Townley D. J., Avery B. J., Rosen B., Skarnes W. C. Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice. Genome Res. 1997 Mar;7(3):293–298. doi: 10.1101/gr.7.3.293. [DOI] [PubMed] [Google Scholar]
  26. Tsugeki R., Kochieva E. Z., Fedoroff N. V. A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation. Plant J. 1996 Sep;10(3):479–489. doi: 10.1046/j.1365-313x.1996.10030479.x. [DOI] [PubMed] [Google Scholar]
  27. Zambrowicz B. P., Friedrich G. A., Buxton E. C., Lilleberg S. L., Person C., Sands A. T. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature. 1998 Apr 9;392(6676):608–611. doi: 10.1038/33423. [DOI] [PubMed] [Google Scholar]
  28. Zwaal R. R., Broeks A., van Meurs J., Groenen J. T., Plasterk R. H. Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7431–7435. doi: 10.1073/pnas.90.16.7431. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Data Supplement]
plntcell_11_12_2263__1.html (249.2KB, html)

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES