A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.

We have determined the complete primary structure (13 637 bp) of the TL‐region of Agrobacterium tumefaciens octopine plasmid pTiAch5 . This sequence comprises two small direct repeats which flank the TL‐region at each extremity and are involved in the transfer and/or integration of this DNA segment in plants. TL‐DNA specifies eight open‐reading frames corresponding to experimentally identified transcripts in crown gall tumor tissue. The eight coding regions are not interrupted by intervening sequences and are separated from each other by AT‐rich regions. Potential transcriptional control signals upstream of the 5′ and 3′ ends of all the transcribed regions resemble typical eukaryotic signals: (i) transcriptional initiation signals (‘TATA’ or Goldberg‐ Hogness box) are present upstream to the presumed translational start codons; (ii) ‘ CCAAT ‘ sequences are present upstream of the proposed ‘TATA’ box; (iii) polyadenylation signals are present in the 3′‐untranslated regions. Furthermore, no Shine‐Dalgarno sequences are present upstream of the presumed translational start codons.

Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.

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Vectors were constructed for the isolation of random transcriptional and translational fi-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli .3-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P355-nptlI-3' ocs) kanamycinresistance gene present in the same T-DNA. Subsequent screening of these for 13-glucuronidase expression allows the identification of clones harboring a fusion of the fi-glucuronidase coding sequence with plant 5' regulatory sequences. In particular, the histochemical staining using 5-bromo-4-chloro-3-indolyl 3-Dglucuronic acid (X-GlcA) facilitates the detailed analysis of tissue or cell-type specificity of the gene fusions. T-DNA-mediated gene fusions were reported to occur at high frequency, independently of genome size (3). Therefore, this method can in principle be applied to all plant species that can be transformed efficiently by Agrobacterium. The small cruciferous plant Arabidopsis thaliana is an ideal model system for molecular genetics because of its small genome size, low repetitive DNA content, short generation time, and well-known genetics (9). Efficient Agrobacterium-mediated transformation of Arabidopsis has been described (10-13).The aim of the present work was to combine the advantages of T-DNA insertion mutagenesis and gus gene fusion technology. By transformation of Arabidopsis with the appropriate T-DNA vectors, gus fusions were obtained at a high frequency. Different types of organ-and tissue-specific GUS expression patterns were found. The usefulness ofthe system was further proved by the cloning of a DNA fragment mediating phloem-specific gene expression in homologous and heterologous systems. § MATERIALS AND METHODSBacterial Strains and Vector Constructions. DNA manipulations were performed as described (14). E. coli JM101 (15) and K514 (16) were used for plasmid transformation. E. coli NM430 (17) and MC1061 (18) were used for growing bacteriophage EMBL4. Plasmid pUC8 (19) was used for subcloning.The pGV1030 was derived from pGV943 (20) by deleting the unique Pst I/Sca I fragment of pGV943, thereby inactivating the 8-lactamase gene.The binary vectors were mobilized (21) Abbreviations: T-DNA, transferred DNA; X-GlcA, 5-bromo-4-chloro-3-indolyl 1-D-glucuronic acid.