Jan Leemans

Restoration of virulence of Vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains

SummaryThree avirulent Tn7 insertion mutants mapping in the vir E region of the Agrobacterium tumefaciens plasmid pTiB6S3 regain virulence by co-infection with several wildtype strains and with a number of strains carrying mutations in other regions of the Ti plasmid. This finding indicates that during tumour induction normal Agrobacterium strains produce a diffusable factor required for transformation and might allow the isolation of such a factor.

A general method for the transfer of cloned genes to plant cells

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells.

Broad-host-range cloning vectors derived from the W-plasmid Sa.

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (Mr 5.6−7.2 × 106), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,

Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements

SummaryDNA homology of thirteen R-plasmids of group P was examined by heteroduplex analysis and Southern blotting. Ten of these plasmids showed homology for extensive regions including all genes reported as necessary for replication and conjugational transfer. The differences between these plasmids could be explained by gain or loss of DNA sequences, many of which have been shown to be transposons.Of the other three plasmids, two showed unambiguous homology with the typical P-plasmids but this homology was imperfect, implying that these plasmids are products of lines which have evolved separately for long periods. One plasmid failed to produce heteroduplexes with the reference P plasmid.

Direct repetition of a 1.2 Md DNA sequence is involved in site-specific recombination by the P1 plasmid R68

R68.45, a mutant R68 plasmid, carries a 1.5 Md DNA insertion near its kanamycin-resistance region. This DNA consists of a 1.2 Md DNA repetition of neighbouring R68-DNA and a 0.3 Md foreign DNA fragment that is flanked by this direct DNA repeat. This fragment seems to be involved in the formation of R68.45 plasmids. Duplication of the 1.2 Md DNA sequence is also involved in site-specific recombination events of RP4. This 1.2 Md DNA fragment has the properties of an IS sequence and is denoted IS8.

Plasmid Mobilization as a Tool for in Vivo Genetic Engineering

Mutagenesis through the insertion of transposons has proved to be an invaluable technique for mapping the genes of complex plasmids1. No selection for a mutant phenotype has to be devised, but a straightforward selection for the antibiotic resistance markers, encoded by the transposon, is sufficient to identify the presence of a mutant plasmid.

The development of host vectors for directed gene transfers in plants

For the study of plant developmental biology nature has provided us with an unexpected system with the double advantage of being an efficient gene-vector that already contains a set of purified genes directly involved in the control of developmental processes in plants. This system is the crown gall tumor inducing Ti plasmid of Agrobacterium tumefaciens.

Transfer, Maintenance and Expression of Genes Introduced into Plant Cells via the Ti Plasmid

The capacity of a microorganism to establish itself successfully in a particular ecological niche often seems to depend upon the activities of a very small number of genes that are absent in competiting species. This additional DNA is frequently part of a plasmid that allows its host to metabolize rarely exploited carbon or nitrogen sources. Because of the presence of such genes, these plasmids have been called degradative or catabolic plasmids1. We believe that the Ti plasmids of Agrobacterium tumefaciens form a special class of catabolic plasmids2,3. In addition to encoding for proteins’ that catabolize several common amino acids4,5 and some polyphenols5, these plasmids also carry genes whose products catabolize compounds calles opines. Opines are unusual amino acids, such as nopaline6, octopine6 or agropine7 and phosphorylated sugars, such as the agrocinopines8. These opines have only been found in plant cells transformed by Ti plasmids into crown gall tumor cells. by inducing crown gall tumors, Agrobacterium tumefaciens forces a plant to synthesize compounds which only the same virulent strains can use.