Charon Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning

Abstract

The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

“…1 and 2, the sequence of the soya bean 4.5-5S rRNA gene region was determined primarily from restriction endonuclease digestion fragments of a larger 3.1 Kb EcoRI fragment which was initially isolated from whole cell DNA using a X Charon 3A vector (23) and subcloned in pBR322 (24) or M13mp9 (26). Hybridization analysis on isolated chloroplast DNA confirmed the presence of this 3.1 Kb EcoRI fragment (results not shown).…”

“…The DNA was then purified by hydroxylapatite column chromatography (22), which resulted in DNA that was significantly free from contaminating RNA and polysaccharides. A genomic library of EcoRI fragments was prepared by using the X Charon 3A vector (23) Fragments that were complementary to the chloroplastid 5S rRNA were characterized and identified for DNA sequence analysis by electroblothybridization techniques (24,25). The complementary DNA was prepared by digesting the hybrid plasmid with EcoRI restriction endonuclease and purifying the inserted DNA on a 0.8% agarose slab gel (24).…”

Detection of non-homology-containing heteroduplex molecules

“…A physical map of X, showing the positions of the relevant deletions and restriction sites, is shown in Figure 1. Preparation of bacteriophage DNA Phage stocks were grown in MJL266, a pro::Tn5 lacAU169 derivative of BNN45 (12) using the NZC broth method (13). Phage lysates were concentrated 50-fold by polyethylene glycol (PEG) precipitation (14) followed by resuspension of the PEG precipitate in SM buffer (15).…”

Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors

“…The most widely used method relies on bacteriophage lambda as a vector. It can accommodate at most 18-21 kb of foreign DNA (1). More recently, J. Collins and B. Hohn developed a new type of vector, termed cosmid (2), which combines some of the features of lambda cloning (efficiency of transfection with packaged "phage" particles)with some of the advantages of using a plasmid replicon (acceptance of a larger segment of foreign DNA).…”

“…More recently, J. Collins and B. Hohn developed a new type of vector, termed cosmid (2), which combines some of the features of lambda cloning (efficiency of transfection with packaged "phage" particles)with some of the advantages of using a plasmid replicon (acceptance of a larger segment of foreign DNA). This procedure, although well suited for the cloning of large DNA ©) IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K.…”