Further characterization of En/Spm transposition in tobacco
--Guillermo H. Cardon, Monika Frey, Heinz Saedler and Alfons Gierl

We have determined the frequency of germinal excision and reintegration, and the frequency of linked transposition of the maize transposable element En/Spm in an artificial transgenic tobacco system (Frey et al., EMBO J. 9:4037-4044). In this transposition system, TnpA and TnpD, the two En-encoded trans-acting factors necessary to promote excision of an I/dSpm receptor element, are expressed from their cDNAs fused to the CaMV 35S promoter. Two different receptor elements were tested: 1 2.2kb native dSpm (dSpm standard or dSpm-S) and a 4.2kb artificial receptor which carries a DHFR marker gene conferring resistance to methotrexate (dSpm-DHFR). Excision and reinsertion of these receptors take place with similar characteristics as in the homologous host maize. Excision reporter constructs were made in which the dSpm is inserted in the 5' untranslated leader of the bar gene, blocking its expression. Germinal revertants could therefore be isolated by seed germination on L-PPT containing medium. dSpm excision leading to the generation of bar expressing gametes (germinal excision) was measured in three independent single copy transformants of each bar-based excision reporter construct. The average frequency of germinal excision (female germline) was 10.1% for dSpm-S and 1.3% for dSpm-DHFR. The frequency of germinal excision transmitted by pollen seems to be similar. Southern analysis of dSpm-S germinal revertants showed that dSpm-S reintegrates with high frequency (up to 90%). Generally, several independent transposition events were detected within a single seed capsule. The bar excision reporter construct carrying the marked receptor dSpm-DHFR allows detection of reinsertion after germinal excision by plating seed on culture medium containing L-PPT and methotrexate. Even though dSpm-DHFR seems to undergo loss more frequently than dSpm-S, it proved to be a valuable tool for the selection of revertants carrying a transposed element. dSpm-DHFR was also instrumental for the estimation of the fraction of transposed elements which reinserted in the proximity of the donor site. The study of the segregation of the L-PPTTR and MtxR markers (empty donor site and transposed dSpm-DHFR, respectively) in outcross progeny of 18 independent germinal revertants showed that in tobacco about 44% of the reinsertions take place into linked chromosomal locations. In maize, up to 60% of the transpositions are into linked sites on the same chromosome (P. A. Peterson, PA, pers. comm.). Therefore, similarly to maize, the probability of tagging a gene will be increased if the receptor is inserted at a linked location. All parameters analyzed indicate that this En/Spm three component tagging system has the potential of generating insertion mutants. If the components of the system are kept at low copy, the molecular characterization of the mutants and later isolation of the tagged genes should be straightforward. 

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