Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

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Studies on transformation of Escherichia coli with plasmids

We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

Use of T7 RNA polymerase to direct expression of cloned genes.

The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Transformation of E. coli cells treated with CaCl2 to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance, fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R-factor DNA in this system.

https://www.pnas.org/content/pnas/69/8/2110.full.pdf

Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.

Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor.

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.

https://www.pnas.org/content/pnas/70/11/3240.full.pdf

Construction of Biologically Functional Bacterial Plasmids In Vitro

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the anti-metabolic drug trimethoprim.

Annie C. Y. Chang

Papers

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA

Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor.

Construction of Biologically Functional Bacterial Plasmids In Vitro

Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase