THE SITE-SPECIFIC RECOMBINATION OF ISXC5 RESOLVASE
ISXc5 is a transposable elements isolated from gram-negative phytopathogen Xanthomonas campestris pv. citri. It shares a common feature of two-step transposition with other class II transposable elements. The gene structure and gene products of the elements have been analyzed. The element is composed of 6,938 bp, and encodes a 48-kD transposase for transposition and a 40-kD resolvase for resolution. Since 1997 we have focused our work of ISXc5 on its site-specific recombination system. Conservative sitespecific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalyzed by transposon-encoded resolvase that belongs to resolvase/DNA invertase family of recombinases. Resolvases and DNA invertases are likely to employ similar reaction mechanisms during recombination. There are still important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated sites. DNA invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated sites. The ISXc5 resolvase shows two unique features amongst the known resolvases. First, it is phylogenetically more close to DNA invertases than to other resolvases in amino acid sequence analysis. Second, the enzyme exhibits a large extension of its C-terminal domain with unknown function. However, biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by gamadelta resolvase. Despite its apparent similarity to invertases, recombination on standard substrates for DNA inversion was not found. Recently our results showed that a deletion of 99 amino acids (about 1/3 of the polypeptide) in the C-terminal of this enzyme did not influence its resolvase activity. More analyses of the enzyme are carried out in our laboratory.
CONSTRUCTION OF INDUCIBLE TRANSPOSONS FOR GENE TAGGING SYSTEM IN HIGHER PLANTS
We have constructed an inducible transposable element named INAc (inducible Activator). Compared with the native Ac element, an inducible transposable element is expected to be induced any time during the development of the plants and the resulting mutants are stable after removal of the inducer. The Ac element was deleted to retain approximately 400-bp of each end and was inserted with a PR-1a::TPase fusion and a hygromycin resistance gene, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high spontaneous transposition efficiency was observed in primary transformed tomato calli but not in tobacco calli. After treatment of tobacco plants with salicylic acid (SA), the induced transposition events were detected in somatic tissues and germinal tissues. Germinal transposition events were observed in the progeny of transformed tobacco containing INAc. According to the behavior of Ac/Ds elements in various plants, the development of an inducible transposon system based on INAc may have important implication for gene isolation in heterologous plant species.
Cheng, C. M., Tu, J., Yang, P. C., and Kuo, T. T. 1996. Rifampicin: an inhibitor of Xp12-specific protein phosphorylation in Xanthomonas campestris pv. oryzae. FEMS Microbiol. Lett. 143: 141-149.
Charng, Y. C., Ma, C., Tu, J., and Kuo, T. T. 1997. A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives. Plant Sci. 130: 73-86.
Liu, C. C., Huhne, R., Tu, J., Lorbach, E., and Droge, P. 1998. The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases. Genes Cells 3: 221-233.
Charng, Y. C., Pfitzner, A. J. P., Pfitzner, U. M., Charng-Chang, K. F., Chen, C. M., Tu, J., and Kuo, T. T. 2000. Construction of an inducible transposon, INAc, to develop a gene tagging system in higher plants. Mol. Breed. 6: 353-367.
Yang, Y. C., Yang, M. K., Kuo, T. T., and Tu, J. 2001. Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri. Mol. Gen. Genet. 265: 316-326.
Tsai, P.-J., Tu, J., and Chen, T.-H. 2002. Cloning of a Ca2+/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides. FEMS Microbiol. Lett. 212: 7-13.
Charng, Y. C., Li, H., Ping., Li, K. T., Hseu, T. H., and Tu, J. 2004. Fusion of the transposase with a classical nuclear localization signal to increase the transposition efficiency of Ac transposon. Bot. Bull. Academia Sinica 45:267-274.
