Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue

Abstract

We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the gehome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the D N A structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic D N A sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

“…However, up to the present, comprehensive information about the status of the transgene copies in F4 transgenic fish has been lacking. Since the technique of plasmid rescue was firstly described by Perucho et al [13]), it has been utilized in the study of transgenic mice [14][15][16][17], transgenic tomato [18] and transgenic Drosophila [19], but it has rarely, to our knowledge, been employed previously in the study of transgenic fish. Recently, we briefly reported three integration-site sequences in F4 transgenic fish [20]; however, the detailed integration pattern of transgene in F4 transgenic fish needs to be clarified.…”

Crop Improvement through Modification of the Plant's Own Genome

“…Blotting, hybridization, and autoradiography were performed as described previously (Rommens et al, 1992). Primers used to amplify 0.5-kb DNA fragments that were used to generate probes to visualize the presence of backbone DNA flanking the P-DNA were 5#-CAC AGG AAA GAC GAC GAC CG-3# and 5#-CCT CAG CCG AGG TTC ATT CC-3# (for BBL), and 5#-CCG TTC GTC CAT TTG TAT GTG GTC-3# and 5#-CGT AGG TGG TCA AGC ATC CTG-3# (for BBR).…”

Transposition pattern of a modified Ds element in tomato

“…The four F1 tomato plants used for our studies (designated AAT6515-02, -30, -33 and -64) were derived from a cross between primary transformants, harbouring a single and mapped Ds-rcontaining T-DNA (AAT6514-02, -30, -33 and -64; [47] and B. Overduin, unpublished results), and a transgenic plant homozygous for two SLJ1515 T-DNAs, containing a modified Ac element [34]. The Ds-r element contains both the GUS gene driven by a 0.8 kb fragment of the CaMV 35S promoter and parts of plasmid pA-CYC 184 (origin of replication and chloramphenicol resistance gene) between the 586 bp 5' Ac terminal sequence and the 448 bp 3' Ac terminal sequence [46].…”

“…This crop species contains many agronomically important genes that could be targets for transposon tagging experiments. Initial experiments have shown that Ac maintains its capacity to transpose and transactivate Ds in this host in a similar way as described for maize [37,42,47,48]. The availability of a tomato RFLP map [7] made it possible to map transposed Ac's at any location in the tomato genome.…”

“…This element has the origin of replication of plasmid pACYC184 and the chloramphenicol resistance gene (functional in Escherichia coli) inserted between 0.5 kb Ac terminal sequences. The presence of these bacterial plasmid sequences allow the Ds element, designated Ds-r, to be rescued from the plant genome [47]. Since the size of Ds-r is 7.8 kb, it is larger than Ac itself (4.6 kb) or any well characterized Ds element (0.4-4.4 kb) [21].…”