The mγδ-1 element, a small γδ (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing

Berg, Claire M.; Vartak, Narendra B.; Gan, Wang; Xu, Xiaoxin; Liu, Lin; MacNeil, Douglas J.; Gewain, Keith M.; Wiater, Lawrence A.; Berg, Douglas E.

doi:10.1016/0378-1119(92)90664-b

Cited by 36 publications

(27 citation statements)

References 28 publications

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“…A set of mini-␥␦ transposon insertions which carry kan (m␥␦-1) was generated in pBS2.7, using the strains and method developed by Berg et al (6). To enhance the number of independent insertions, the procedure was repeated five times.…”

Section: Methodsmentioning

confidence: 99%

AinS and a new family of autoinducer synthesis proteins

1995

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In Vibrio fischeri, the autoinducer N-3-oxohexanoyl-L-homoserine lactone (AI-1) governs the cell densitydependent induction of the luminescence operon via the LuxR transcriptional activator. The synthesis of AI-1 from bacterial metabolic intermediates is dependent on luxI. Recently, we found a second V. fischeri autoinducer molecule, N-octanoyl-L-homoserine lactone (AI-2), that in E. coli also activates the luminescence operon via LuxR. A locus independent of luxI was identified as being required for AI-2 synthesis. This 2.7-kb ain (autoinducer) locus was characterized by transposon insertion mutagenesis, deletion and complementation analysis, and DNA sequencing. A single 1,185-bp gene, ainS, was found to be the sole exogenous gene necessary for the synthesis of AI-2 in Escherichia coli. In addition, a V. fischeri ainS mutant produced AI-1 but not AI-2, confirming that in its native species ainS is specific for the synthesis of AI-2. ainS is predicted to encode a 45,580-Da protein which exhibits no similarity to LuxI or to any of the LuxI homologs responsible for the synthesis of N-acyl-L-homoserine lactones in a variety of other bacteria. The existence of two different and unrelated autoinducer synthesis genes suggests the occurrence of convergent evolution in the synthesis of homoserine lactone signaling molecules. The C-terminal half of AinS shows homology to a putative protein in Vibrio harveyi, LuxM, which is required for the synthesis of a V. harveyi bioluminescence autoinducer. Together, AinS and LuxM define a new family of autoinducer synthesis proteins. Furthermore, the predicted product of another gene, ainR, encoded immediately downstream of ainS, shows homology to LuxN, which is similarly encoded downstream of luxM in V. harveyi and proposed to have sensor/regulator functions in the bioluminescence response to the V. harveyi autoinducer. This similarity presents the possibility that AI-2, besides interacting with LuxR, also interacts with AinR under presently unknown conditions.

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Section: Methodsmentioning

confidence: 99%

AinS and a new family of autoinducer synthesis proteins

1995

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“…L agar (LA) and L broth (LB) plus thymine (10 ,ug/ml) and Vogel and Bonner medium E plus 0.5% Casamino Acids with the indicated additives were used for growth and selection (4,6). Ampicillin (100 ,ug/ml), chloramphenicol (30 j±g/ml), kana- DNA manipulations and sequencing.…”

Section: Materuils and Methodsmentioning

confidence: 99%

“…The recA rpsL Escherichia coli K-12 strains used in this study were CBK884 (4); CBK929, a AthyA derivative of CBK884 (this work); and DH1OB (12). The conditions for bacterial growth and transposition were as described previously (4,6). All incubations were at 37°C.…”

Section: Materuils and Methodsmentioning

confidence: 99%

“…Specialized chemicals were from Sigma Chemical Co., St. Louis, Mo. Agar and components of Lennox (L) medium were from Difco Laboratories, Detroit, Mich. Agarose, restriction enzymes, T4 polymerase, DNA size standards, and the double-stranded DNA cycle sequencing kit were from Bethesda Research Laboratories (BRL), Gaithersburg, Md.L agar (LA) and L broth (LB) plus thymine (10 ,ug/ml) and Vogel and Bonner medium E plus 0.5% Casamino Acids with the indicated additives were used for growth and selection (4,6). Ampicillin (100 ,ug/ml), chloramphenicol (30 j±g/ml), kana-1332 …”

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confidence: 99%

“…Recently, small, engineered derivatives of y8 have been constructed to facilitate molecular analysis of cloned fragments (4,6,28) and marker exchange (4,6). -y generally moves by a replicative mechanism that is believed to involve transposasecatalyzed single-stranded breaks at each transposon end, a five-base staggered cut in the target DNA, ligation of the free transposon ends to target DNA, repair of the five-base gaps, and replication of the element from these newly generated forks.…”

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confidence: 99%

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Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon

Gan

Chen

et al. 1994

J Bacteriol

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Intramolecular transposition by an engineered derivative of the transposon yB (TnlOOO) is described. This 1-kb element contains inverted repeats of the 40 bp of the { end of y8, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-y8 element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [qpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-y8 inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.Transposon y5, a member of the Tn3 family (21), has been used extensively for molecular genetic analysis of cloned DNAs (2, 13). Recently, small, engineered derivatives of y8 have been constructed to facilitate molecular analysis of cloned fragments (4, 6, 28) and marker exchange (4, 6). -y generally moves by a replicative mechanism that is believed to involve transposasecatalyzed single-stranded breaks at each transposon end, a five-base staggered cut in the target DNA, ligation of the free transposon ends to target DNA, repair of the five-base gaps, and replication of the element from these newly generated forks. The transposon is duplicated, and no DNA is lost in the transposition process.When -y8 moves from one molecule to another (intermolecular transposition), the product is a cointegrate in which donor and target molecules are joined by direct repeats of the transposon, with the five-base target site duplicated at the new transposon-target junctures. If the resolvase gene is functional and the resolution site is present in the transposon, these cointegrates are broken down into a target molecule containing a copy of the element (bracketed by the 5-bp target site duplication) plus an unchanged donor molecule, even in recA cells. However, when yi moves to a site in the same molecule (intramolecular transposition), the product is either a deletion or an inversion, depending on how DNA between the donor and target sites was twisted (21) (Fig. 1). As described below, an inversion plasmid has the predicted duplication of y8 and of the 5-bp target sequence. On the other hand, only one of the ...

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