Plant Molecular Biology 25: 339-342, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 339 News and views Identification of unexplained DNA fragments within the T-DNA borders of the Bin 19 plant transformation vector Rupert G. Fray, Andrew D. Wallace and Donald Grierson* Department of Physiology and Environmental Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Loughborough, LE12 5RD, UK (*author for correspondence) Received 21 March 1994; accepted 31 March 1994 Bin 19 is a popular and widely used plant binary transformation vector, that uses T-DNA borders from pTiT37 and pTiT7 [1 ]. Unexplained DNA fragments originating from Bin 19 have been in- vestigated. This has shown that there are previ- ously unsuspected M13 sequences present in the vector T-DNA which would be transferred to plants during transformation (Fig. 1). Within the T-DNA of Bin 19 there is a single Sac II restriction site; it lies close to the right border in the nopaline synthase (nos) promoter that drives the expression of the plant selectable marker gene. There is also a second Sac II site located at an unspecified position in the non- transferred portion of the plasmid [ 1]. From the description of the construction of Bin 19 [1], a double digest using Sac II and a second restric- tion enzyme cutting only in the multiple cloning site of the T-DNA should release a fragment of 1750 bp plus two fragments of unknown size. When Bin 19 was digested with Sac II alone, two fragments were produced, one of 2650 bp and one of ca. 8000 bp. When a double digest using Sac II and Hind III was performed, bands of 2300 bp, 2650 bp and 6000 bp resulted (Fig. 2). Hind III cuts Bin 19 only once (within the multiple clon- ing site of the T-DNA) so the band of 2300 bp must correspond to the fragment internal to the T-DNA from the multiple cloning site to the nos promoter. However, it is ca. 550 bp larger than predicted from the description of its construction (Fig. 1). This extra 550 bp was also found to be present in a different stock of Bin 19 plasmid and in three Bin 19-based constructs that had been successfully used for plant transformation and transgene expression. During the original construction of Bin 19, a 440 bp Hae II fragment from M13mp19 was in- serted into the T-DNA, replacing a 1.6 kb Agro- bacterium sequence [1]. This 440 bp sequence contains the e-complementing region of fl-galactosidase and the pUC19 multiple cloning site within it. In order to resolve uncertainties in the structure of the vector the entire Bin 19 T-DNA was sequenced. Two additional DNA fragments were indeed found between the right border and the multiple cloning site. The first of these, a 440 bp fragment, is located between the nos polyadenylation signal and the fl-galactosidase gene. This extra DNA (fragment A in Fig. 1) was found to correspond entirely to a 440 bp fragment of Ml3mpl9, extending from positions 2269 to 2709 of the phage (the 440 bp fl-galactosidase fragment lies between coordi- nates 6005 and 6282). It seems probable that this 440bp M13mpl9 fragment was inadvertently cloned into Bin 19 at the same time as the 440 bp fl-galactosidase M13mp19 fragment, and was overlooked in subsequent analysis (though how such a fragment could be generated from a simple Hae II digest is unclear). The second fragment (fragment B in Fig. 1), of 200 bp, is located be- tween the 3' end of the neomycin phosphotrans- ferase II (NPTII) transcribed sequence and the nos polyadenylation signal. The sequence matches a portion of the coding sequence of the ornithine