Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.

https://www.pnas.org/content/pnas/76/1/106.full.pdf

Expression in Escherichia coli of chemically synthesized genes for human insulin

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

Methotrexate, a folate antagonist, blocks import into mitochondria of mouse dihydrofolate reductase fused to a mitochondrial presequence. Methotrexate does not mask the presequence, but stabilizes the dihydrofolate reductase moiety. It does not inhibit import of the authentic precursor from which the presequence is derived. This suggests that dihydrofolate reductase must at least partly unfold in order to be transported across mitochondrial membranes.

Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria

Publisher Summary This chapter discusses the plasmids of Escherichia coli ( E. coli ) as cloning vectors. The essence of molecular cloning or recombination in vitro is the joining together in vitro of two or more deoxyribonucleic acid (DNA) fragments. One fragment, called the “vector” or “vehicle,” is capable of replication in some host organism and the other(s), referred to as the “cloned or passenger fragment(s),” can be passively replicated when joined to the vector. Hybrid molecules are then put into a host organism by a process called “transformation,” and the clones that contain various arrangements of the joined fragments are isolated. The chapter discusses the procedures that can be used for the generation of DNA fragments and presents the methods for joining the DNA fragments. The chapter discusses the properties of plasmid cloning vectors of E. coli as well as the techniques that can be used to select from a population of bacterial cells the ones carrying recombinant DNA molecules of interest. The elution of DNA fragments from polyacrylamide gels is also discussed in the chapter.

Plasmids of Escherichia coli as cloning vectors.

Construction and characterization of new cloning vehicles IV. Deletion derivatives of pBR322 and pBR325

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.

A general method for the purification of restriction enzymes.

In vivo events in the generation of pBR322.

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Plasmids of Escherichia coli as cloning vectors.

A general method for the purification of restriction enzymes.

In vivo events in the generation of pBR322.