Cointegrate

Abstract: The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

Abstract: Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutnat phenotypes. Whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicells by ColE1 and ColE1 :: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be beta-lactamase. ColE1 :: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.

Abstract: A derivative of the octopine-type Ti plasmid, pTiB6S3, was constructed in which the TL-DNA oncogenic functions, the TL-DNA right border sequence and all of TR-DNA were deleted and replaced with the kanamycin antibiotic resistance marker from Tn903 (601). The resulting avirulent plasmid, pTiB6S3–SE contains only the TL-DNA left border sequence and a region (1.6 kb) of homologous DNA (LIH) to allow recombination with intermediate vectors such as pMON120 or pMON200. Cointegrate formation between pTiB6S3–SE and pMON200, which contains a chimeric plant kanamycin resistance gene (Kmr), an intact nopaline synthase gene (NOS), a poly linker region to facilitate the insertion of foreign genes into the vector and a functional T-DNA right border sequence, results in the formation of a selectable, avirulent T-DNA. This vector system is referred to as the SEV system (Split End Vector) since the T-DNA border sequences are present on separate plasmids prior to recombination. Plant cells (tobacco, petunia and tomato) transformed with SEV T-DNA are easily identified by their resistance to kanamycin and their ability to synthesize nopaline. A major advantage of the new disarmed system is that all transformed colonies can potentially be regenerated into intact plants that normally express and segregate the inserted foreign DNA sequences.

Abstract: Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.