No abstract

Similar 23-base-pair (bp) direct repeats occur at the ends of two adjacent but noncontiguous T-DNAs, TL and TR (left and right T-DNA), in the tumor-inducing plasmid pTiA6NC. Thus, three border repeats lie right and one lies left of TL, which carries the genes needed for tumor maintenance. To determine whether T-DNA transfer and integration (subsequently called T-DNA transmission) require sequences in addition to the 23-bp border repeat, we constructed a deletion removing the three potential TL right borders (the TL right border and both TR borders (kb)] tumor-inducing (Ti) plasmid that carries genes essential for tumorigenesis (2). During tumorigenesis a specific segment of the Ti plasmid, the T-DNA, integrates into plant nuclear DNA (1). Plant tumor cells express T-DNA genes responsible for tumorous growth, but T-DNA transmission into plant cells does not require T-DNA-encoded proteins and it can occur in the absence of tumor growth (3, 4). Mutations in virulence (vir) genes, located outside the T region, block T-DNA transmission into plant nuclear DNA (5). Presumably, some vir proteins play a direct role in T-DNA integration, but the mechanism of T-DNA integration remains unknown.Specific sequences apparently signal T-DNA borders, because T-DNA ends usually occur at specific regions of the Ti plasmid (6-8). Similar 23-base-pair (bp) direct repeats lie at both ends of three different T regions, and T-DNA ends occur in or near these repeats in several different tumors (9-13). Although deletions that remove the left end of the T region do not affect virulence (14-16), deletions that remove the right end severely attenuate virulence (14)(15)(16)(17)(18). This implies that T-DNA transmission requires the right border repeat. Alternatively4 these deletions might abolish virulence by removing other necessary sequences.We conducted experiments to verify that T-DNA transmission requires the right border repeat and to determine whether this process requires additional sequences. We also tested the influence of orientation, location, and flanking sequences on border repeat function. To identify sequences that form a functional right T-DNA border, we introduced different restriction fragments, each containing a border repeat, into a Ti plasmid with the right borders deleted and tested their ability to restore virulence. These experiments defined an 80-bp region that contains a functional right T-DNA border and identified nonessential sequences outside the border repeat that stimulate T-DNA transmission. The right border repeat functioned normally when moved to a new location in the Ti plasmid only when it remained in its wild-type orientation. MM294, pUC8 (26) into JM103 or JM105, and pRK290 (27) into SF800. MATERIALS AND METHODSMedia. We cultured A. tumefaciens on AB minimal agar or YEP broth (28) and E. coli on L agar or L broth (29). To select drug-resistant E. coli, we used tetracycline (10 ,ug/ml), kanamycin (25 ,ug/mnl), or ampicillin (50 pug/ml). To select drug-resistant A. tumefaciens, we used gentamici...

Three genetic loci affecting tumor morphology lie within pTiA6NC T-DNA: tins, tmr, and tin. Using deletions and multiple transposon insertions, we constructed tumor-inducing (Ti) plasmids representing every possible double and triple mutant combination. tins tmr and tms tmr tml mutants did not incite tumors on most plants and produced a very weak response on a few other hosts but tins tin and tmr tin mutants were virulent.Thus, either tms' or tmr' alone can promote significant tumor growth but tml+ by itself is not sufficient. On hosts where tis mutants induce tumors accompanied by shoot proliferation, addition of a tmn mutation reduces or eliminates shoot proliferation, suggesting that tnl+ promotes shoot development. The small calli incited by tins tmr and tns tmr tin mutants contain agropine, an indication that these plant cells incorporate T-DNA in the absence of substantial tumor growth.During crown gall tumor induction by Agrobacterium tumefaciens, a specific segment of the tumor-inducing (Ti) plasmid called the T-DNA integrates into plant nuclear DNA (1-3).

We analyzed the nucleotide sequence of a 1.325-kilobase region of wild-type Escherichia coli containing a functional recF gene and six Tn3 mutations that inactivate recF. The analysis shows a potentially translatable reading frame of 1071 nucleotides, which is interrupted by all six insertions. A protein of 40.5 kilodaltons would result from translation of the open reading frame, and a radioactive band of protein of an apparent molecular weight of %40 kilodaltons was seen by the maxicell method using a recF+ plasmid. Putative truncated peptides were seen when two recF::Tn3 mutant plasmids were used. Differential expression of dnaN and recF from a common promoter was noted. recF332::Tn3 was transferred to the chromosome where, in hemizygous condition, it produced UV sensitivity indistinguishable from that produced by two presumed recF point mutations.The recF gene of Escherichia coli lies within a cluster of genes involved in DNA metabolism, the gene order being gyrB recF dnaN dnaA (1). gyrB has been shown to code for one of the subunits of DNA gyrase (2), dnaN determines the 13 subunit of DNA polymerase III holoenzyme (3), and dnaA encodes a 52-kilodalton (kDa) protein (4,5) involved with the initiation of replication of the Escherichia coli chromosome (6).The phenotype of a recF mutant indicates that recF also is involved in DNA metabolism and possibly with replication.

Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of lambda tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.

No abstract

Agrobacterium tumefaciens incites crown gall tumors when bacterial DNA integrates into plant nuclear DNA. Plant cells can express these integrated bacterial genes. Following insertion of desired genes into bacterial DNA using recombinant DNA techniques, this system permits introduction of these new genes into plant DNA. We discuss the potential for genetic manipulation of plants using Agrobacterium tumefaciens and the related organism Agrobacterium rhizogenes.

No abstract