Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum.
Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions. However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends. During the study of this phenomenon, we obtained KmR Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA. Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region. The most conspicuous result from Southern analyses on four such KmR plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence. Four truncated T-DNA insertions have been cloned. Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA. Another is missing the left T-DNA part and the last T-DNA is lacking both ends. Sequence analysis of the T-DNA::plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence. S1-type mapping of the most strongly expressed plant genome::nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic Pnos-nptII plants.
Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium.
G
G
Gheysen
L
L
Herman
P
P
Breyne
J
J
Gielen
M
M
Van Montagu
A
A
Depicker
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C036483:T-DNA; D012313:RNA