Isolation of the kanamycin resistance region (Tn2350) of plasmid R1drd-19 as an autonomous replicon
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Abstract:
We have isolated a circular form of Tn2350, an IS1-flanked kanamycin resistance transposon forming part of the plasmid R1drd-19. This circle (pTn2350::9.6 kilobases) contains a single IS1 element and probably arises by recombination between the two directly repeated Is1 sequences of Tn2350. It can be used to transform Escherichia coli to kanamycin resistance. It is capable of autonomous replication but is not maintained stably in dividing cells and segregates under nonselective conditions. Cloning of a segment of pTn2350 on a conditional plasmid vector allowed us to assign the replication functions of this plasmid to a 1.6-kilobase restriction fragment. The plasmid R1drd-19 can thus be considered as a cointegrate between two replicons separated by IS1 sequences.Keywords:
Replicon
Kanamycin
Autonomously replicating sequence
T-DNA Binary system
Plasmid preparation
Cloning vector
Restriction map
Cloning vector
Plasmid preparation
In vitro recombination
Bacillus licheniformis
Multiple cloning site
T-DNA Binary system
BglII
Cloning (programming)
Shuttle vector
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We have constructed several cloning vectors which can be used in in vitro packaging and yeast transformation. These plasmids have been designed for the convenient cloning of large segments of DNA and their transfer to yeast. They contain bacterial plasmid DNA sequences for replication and selection in Escherichia coli, yeast 2-μm plasmid DNA sequences or chromosomal replicators and yeast markers necessary for replication and selection in yeast, and the cohesive ends of bacteriophage λ which allow packaging of recombinant molecules into λ phage heads. Large fragments (22-38 kb) of Klebsiella pneumoniae and Zea mays DNA were ligated into plasmid vector pBTI-1 to make complete genome libraries. One clone from the K. pneumoniae library was amplified in E. coli and the purified DNA used to transform yeast cells. Transformation of yeast by large DNA fragments occurred at high frequencies. The recombinant plasmid was stably maintained in yeast, provided selective pressure for Leu+ transformants was maintained. The structurally complete recombinant plasmid can be recovered from yeast by transforming E. coli to ampicillin resistance. Fewer than 5% of the recovered plasmids had undergone recombination with endogenous yeast 2-μm plasmid.
In vitro recombination
T-DNA Binary system
Plasmid preparation
Cloning vector
Library
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Derepression
Cloning vector
T-DNA Binary system
Plasmid preparation
Low copy number
In vitro recombination
Cloning (programming)
Multiple cloning site
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Cosmid
Multiple cloning site
Cloning vector
Subcloning
Cloning (programming)
Plasmid preparation
T-DNA Binary system
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Autonomously replicating sequence
T-DNA Binary system
Plasmid preparation
Pre-replication complex
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The use of recombinant DNA techniques in the analysis of the structure and replication of bacterial plasmids has provided much information on the properties of these genetic elements and has led to the construction of plasmid elements that are potentially very useful as gene cloning vehicles in Escherichia coli and other gram-negative bacteria. The genetic and molecular properties of plasmids mini-F, ColE1, and RK2 are described with particular emphasis on the origin and direction of replication and the identification of genetic regions essential for maintenance of these elements in the extra-chromosomal state. Low molecular weight derivatives of each of these plasmids have been obtained and a restriction enzyme map determined for these various derivatives. A hybrid DNA molecule consisting of a low molecular weight derivative of ColE1 joined to a segment of bacteriophage DNA has been constructed and shown to be capable of existing either as a plasmid element or packaged as an infectious viral particle. Finally, several of the low molecular weight derivatives of these plasmids described have certain advantages as vehicles for the cloning of DNA including derivatives of he broad host range plasmid RK2 that may be useful for gene cloning in gram-negative bacteria distantly related to E. coli.
ColE1
T-DNA Binary system
Plasmid preparation
Cloning vector
Rolling circle replication
Multiple cloning site
Cloning (programming)
Autonomously replicating sequence
In vitro recombination
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Plasmid preparation
T-DNA Binary system
Cloning vector
Cloning (programming)
Multiple cloning site
In vitro recombination
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In vitro recombination
Cloning vector
Plasmid preparation
T-DNA Binary system
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In order to develop practical recombinant DNA techniques in the industrially important yeast Candida utilis, at least six plasmids harboring autonomously replicating sequences (ARSs) were isolated from a C. utilis genomic library. Two ARSs were subjected to detailed analysis. Sequences of 1.9 and 1.8 kb were found to be necessary to exert ARS activity in a plasmid as assessed by transformation efficiency and mitotic stability. Both fragments were found to be rich in AT content (69.5% and 70.8% respectively), and to contain an 11-bp ARS consensus sequences (10 and 13 motifs with one base difference respectively). Using the ARS-containing plasmid as a promoter-cloning vector, several DNA fragments having promoter activities were cloned and characterized. Co-transformation of C. utilis with an integrating DNA fragment and a replicating plasmid yielded plasmid-free transformants harboring the fragment integrated into the C. utilis genome.
Autonomously replicating sequence
Cloning (programming)
Cloning vector
genomic DNA
T-DNA Binary system
Plasmid preparation
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Plasmid preparation
T-DNA Binary system
Selectable marker
In vitro recombination
Shuttle vector
Autonomously replicating sequence
Auxotrophy
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