Viable Molecular Hybrids of Bacteriophage Lambda and Eukaryotic DNA

Abstract

A bacteriophage X strain has been constructed and a method developed by which DNA from potentially any source can be covalently inserted through EcoRI cohesive ends into the middle of the X DNA. These hybrid DNAs can infect nonrestricting Escherichia coli cells and can then propagate as plaque-forming phage. A unique feature of this X strain is that extra DNA in the middle of its genome is required for plaque formation. A large number of such phages have been produced with E. coli DNA and Drosophila melanogaster DNA.Hybrid DNA molecules can be constructed in vitro by joining any two or more DNA molecules through the cohesive ends that are generated by cleavage with EcoRI endonuclease (1). This discovery has made possible the formation of hybrid DNA molecules that can be propagated in Escherichia coli. Such hybrids may be formed between a vector molecule, which can be any molecule cleaved by EcoRI endonuclease capable of self propagation in E. coli, and potentially any other DNA molecule with cohesive ends generated by EcoRI endonuclease.There already exist in bacteriophage X (Fig. la) viable deletion mutants that lack most of the center two EcoRI-endonuclease-generated B and C fragments (2, 3). It was established that fragments B and C are in fact not required for lytic growth by cleaving X DNA with EcoRI endonuclease, randomly rejoining the fragments with DNA ligase, and, after DNA infection, selecting viable phage that lack the B and C fragments (Abraham, Thomas, and Davis, in preparation). Therefore, it should be possible to construct viable hybrid DNA molecules by inserting foreign DNA into X DNA in place of the B and C fragments. To facilitate such an insertion, the two rightmost EcoRI restriction sites were eliminated by mutation.An advantage of X DNA as a vector molecule is that an insertion of foreign DNA can be made to be essential for plaque formation. The shortest X DNA molecules that produce plaques of nearly normal size are 25% deleted (J. S. Parkinson, unpublished observation). Apparently, if too much of even nonessential DNA is deleted from the X genome, it cannot be packaged into virus particles. The EcoRI-B and -C fragments represent 21.1% of the X DNA (Thomas and Davis, submitted to J. Mol. Biol.). Deletion of the nonessential nin5 region (6.1%o of ? DNA) (2) as well as the EcoRI-B and -C fragments yields a DNA that would not be expected to produce plaque-forming phage even though no essential genes have been deleted. However, this deleted X DNA can be made to produce plaques if a new DNA segment is inserted into the remaining central EcoRI restriction site. This constitutes a positive selection for X phage carrying a DNA insertion. The X strain used for the construction of these hybrid phages, hereafter referred to as Xgt-XC, contains the X EcoRI-C fragment in this central region in order to retain sufficient DNA that it may be propagated. This segment is removed after EcoRI endonuclease cleavage, and EcoRI-endonuclease-cleaved foreign DNA is inserted. MATERIALS AND METHODSTerminology....

Determination of genes, restriction sites, and DNA sequences surrounding the 6S RNA template of bacteriophage lambda.

“…By hybridization, radioactive 6S RNA was found to be complementary to EcoRI fragment no. 3, the subterminal fragment from the right-hand half of the lambda chromosome (17,18), and to fragments Hind-2 and Hae-10 ( Fig. 1A and B).…”

DNA cloning: A personal view after 40 years

“…Attaching the Drosophila DNA to the pSC101 plasmid enabled Hogness and his coworkers to confirm in late 1974 the cloning of eukaryotic cell genes in bacteria and to use DNA cloning to map sequences in Drosophila chromosomes (63). Concurrently, other laboratories focused on constructing new bacteriophage λ variants able to produce viable molecular hybrids (64,65), and, in November 1974, Ron Davis and his coworkers at Stanford reported that such hybrids can be propagated as plaque-forming phage (66).…”

“…Ironically with regard to Pollack's scenario, Mertz's 1975 PhD dissertation (67) stated that "scientific problems have been encountered during attempts to use λdvgal as a vector for replicating other DNA molecules" and that "the plasmid is unstable and readily lost from its E. coli host" (67). It was later learned that insertion of foreign DNA into the λdvgal site that the Berg team had used for construction of hybrid DNA molecules (52, 54) disrupts a gene essential for λdvgal replication in bacteria (54,66,69), possibly explaining the lack of success of the DNA cloning attempts reported in Mertz's dissertation (67). But Pollack's concerns and Berg's decision had importantly raised awareness about possible biohazardous consequences of creating novel DNA combinations (54).…”

Molecular Cloning of a Cellulase Gene fromBacillus subtilisand Its Expression inEscherichia coli

“…E. coli JMI05 f1lacpro, thi, strA, endA, sbcB15, hsdR4 (F', traD36, proAB, lacIQ, lacZf1M15) was purchased from Amersham Co. E. coli C600 r k -,m k -, F-, thi1, thr1, leuB6, lacY1, tonA21, supE44, A-was obtained from R. Davis. 9 ) Plasmids pBD64,t°) pUBl10 ll ) and pBR322 12 ) were used as vectors.…”