Abstract
Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as β-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.
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References
Casadaban, M. J. and Cohen, S. N. (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA 76, 4530–4533.
Bellen, H. J. (1999) Ten years of enhancer detection: lessons from the fly. Plant Cell 11, 2271–2281.
Springer, P. S. (2000) Gene traps: tools for plant development and genomics. Plant Cell 12, 1007–1020.
Stanford, W. L., Cohn, J. B., and Cordes, S. P. (2001) Gene trap mutagenesis: past, present and beyond. Nat. Rev. Genet. 2, 756–768.
Chin, H. G., Choe, M. S., Lee, S. H., et al. (1999) Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. Plant J. 19, 615–623.
Martirani, L., Stiller, J., Mirabella, R., et al. (1999) T-DNA tagging of nodulation-and root-related genes in Lotus japonicus: expression patterns and potential for promoter trapping and insertional mutagenesis. Mol. Plant Microbe Interact. 12, 275–284.
Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M., and Hasebe, M. (2000) Tagged mutagenesis and gene trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res. 7, 9–17.
Jeon, J. S., Lee, S., Jung, K. H., et al. (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22, 561–570.
Martienssen, R. A. and Springer, P. S. (2000) Enhancer and gene trap transposon mutagenesis in Arabidopsis, in (http://www.arabidopsis.org/info/springer.html).
Weigel, D. and Glazebrook, J. (2002) Arabidopsis: A Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor, NY.
Tsugeki, R., Kochieva, E. Z., and Fedoroff, N. V. (1996) A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation. Plant J. 10, 479–489.
Liu, Y.-G., Mitsukawa, N., Oosumi, T., and Whittier, R. F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463.
Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995.
Shure, M., Wessler, S., and Fedoroff, N. (1983) Molecular identification and isolation of the Waxy locus in maize. Cell 35, 225–233.
Martienssen, R. A. (1998) Functional genomics: probing plant gene function and expression with transposons. Proc. Natl. Acad. Sci. USA 95, 2021–2026.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor, NY.
Ride, J. P., Davies, E. M., Franklin, F. C., and Marshall, D. F. (1999) Analysis of Arabidopsis genome sequence reveals a large new gene family in plants. Plant Mol. Biol. 39, 927–932.
Cock, J. M. and McCormick, S. (2001) A large family of genes that share homology with CLAVATA3. Plant Physiol. 126, 939–942.
MacIntosh, G. C., Wilkerson, C., and Green, P. J. (2001) Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs. Plant Physiol. 127, 765–776.
Llave, C., Kasschau, K. D., Rector, M. A., and Carrington, J. C. (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14, 1605–1619.
Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002) MicroRNAs in plants. Genes Dev. 16, 1616–1626.
Seki, M., Narusaka, M., Kamiya, A., et al. (2002) Functional annotation of a full-length Arabidopsis cDNA collection. Science 296, 141–145.
Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907.
Sieburth, L. E. and Meyerowitz, E. M. (1997) Molecular dissection of the AGA-MOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9, 355–365.
Brand, U., Grünewald, M., Hobe, M. and Simon, R. (2002) Regulation of CLV3 expression by two homeobox genes in Arabidopsis. Plant Physiol. 129, 565–575.
Lohmann, J. U., Hong, R. L., Hobe, M., et al. (2001) A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell 105, 793–803.
Xiang, C, Han, P., Lutziger, I., Wang, K., and Oliver, D. J. (1999) A mini binary vector series for plant transformation. Plant Mol. Biol. 40, 711–717.
Nagel, R., Elliott, A., Masel, A., Birch, R. G., and Manners, J. M. (1990) Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol. Lett. 67, 325–328.
Clough, S. J. and Bent, A. J. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Baxter-Burrell, A., Yang, Z., Springer, P. S., and Bailey-Serres, J. (2002) RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296, 2026–2028.
Feldmann, K. A. (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J. 1, 71–82.
Bancroft, I., Jones, J. D. G., and Dean, C. (1993) Heterologous transposon tagging of the DRL1 locus in Arabidopsis. Plant Cell 5, 631–638.
Sundaresan, V., Springer, P., Volpe, T., et al. (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev. 9, 1797–1810.
Benfey, P. N., Ren, L., and Chua, N. H. (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J. 8, 2195–2202.
Cocherel, S., Perez, P., Degroote, F., Genestier, S., and Picard, G (1996) A promoter identified in the 3′ end of the Ac transposon can be activated by cis-acting elements in transgenic Arabidopsis lines. Plant Mol. Biol. 30, 539–551.
Fobert, P. R., Labbé, H., Cosmopoulos, J., et al. (1994) T-DNA tagging of a seed coat-specific cryptic promoter in tobacco. Plant J. 6, 567–577.
Foster, E., Hattori, J., Labbé, H., et al. (1999) A tobacco cryptic constitutive promoter, tCUP, revealed by T-DNA tagging. Plant Mol. Biol. 41, 45–55.
Plesch, G, Kamann, E., and Mueller-Roeber, B. (2000) Cloning of regulatory sequences mediating guard-cell-specific gene expression. Gene 249, 83–89.
Mollier, P., Hoffmann, B., Orsel, M., and Pelletier, G (2000) Tagging of a cryptic promoter that confers root-specific gus expression in Arabidopsis thaliana. Plant Cell Reports 19, 1076–1083.
The Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.
Ruiz-Medrano, R., Xoconostle-Cazares, B., and Lucas, W. J. (1999) Phloem longdistance transport of CmNACP mRNA: implications for supracellular regulation in plants. Development 126, 4405–4419.
Koltai, H. and Bird, D. M. (2000) High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections. Plant Physiol. 123, 1203–1212.
Hellens, R., Mullineaux, P., and Klee, H. (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.
Coupland, G., Plum, C., Chatterjee, S., Post, A., and Starlinger, P. (1989) Sequences near the termini are required for transposition of the maize transposon Ac in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 86, 9385–9388.
Baker, B., Schell, J., Lörz, H., and Fedoroff, N. (1986) Transposition of the maize controlling element “Activator” in tobacco. Proc. Natl. Acad. Sci. USA 83, 4844–4848.
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© 2003 Humana Press Inc., Totowa, NJ
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Rojas-Pierce, M., Springer, P.S. (2003). Gene and Enhancer Traps for Gene Discovery. In: Grotewold, E. (eds) Plant Functional Genomics. Methods in Molecular Biology™, vol 236. Humana Press. https://doi.org/10.1385/1-59259-413-1:221
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DOI: https://doi.org/10.1385/1-59259-413-1:221
Publisher Name: Humana Press
Print ISBN: 978-1-58829-145-5
Online ISBN: 978-1-59259-413-9
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