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The construction of new plasmid DNA species by in vitro joining of restriction endonucleasegenerated 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.Controlled shearing of antibiotic resistance (R) factor DNA leads to formation of plasmid DNA segments that can be taken up by appropriately treated Escherichia coli cells and that recircularize to form new, autonomously replicating plasmids (1). One such plasmid that is formed after transformation of E. coli by a fragment of sheared R6-5 DNA, pSC101 (previously referred to as Tc6-5), has a molecular weight of 5.8 X 106, which represents about 10% of the genome of the parent R factor. This plasmid carries genetic information necessary for its own replication and for expression of resistance to tetracycline, but lacks the other drug resistance determinants and the fertility functions carried by R6-5 (1).Two recently described restriction endonucleases, EcoRI and EcoRII, cleave double-stranded DNA so as to produce short overlapping single-stranded ends. The nucleotide sequences cleaved are unique and self-complementary (2-6) so that DNA fragments produced by one of these enzymes can associate by hydrogen-bonding with other fragments produced by the same enzyme. After hydrogen-bonding, the 3'-hydroxyl and 5'-phosphate ends can be joined by DNA ligase (6). Thus, these restriction endonucleases appeared to have great potential value for the construction of new plasmid species by joining DNA molecules from different sources. The EcoRI endonuclease seemed especially useful for this purpose, because on a random basis the sequence cleaved is expected to occur only about once for every 4,000 to 16,000 nucleotide pairs (2); thus, most EcoRI-generated DNA fragments should contain one or more intact genes. We describe here the construction of new plasmid DNA species by in vitro association of the EcoRI-derived DNA fragments from separate plasmids. In one instance a new plasmid has been constructed from two DNA species of entirely different origin, while in another, a plasmid which has itself been derived from EcoRI-generated DNA fragments of a larger parent plasmid genome has been joined to another replicon derived independently from the same parent plasmid. Plasmids that have been constructed by the in vitro joining of 3240EcoRI-generated fragments have been inserted into appropriately-treated E. coli by transformation (7) and have been shown to form biologically functional replicons that possess genetic properties and nucleotide base sequences of both parent DNA species. MATERIALS AND METHODSE. coli strain W1485 containing the RSF1010 plasmid, which carrie...

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.