It is shown here that the phosphate groups at the cos ends of phage λ DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native λ DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector λpGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine resstriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by λ terminase just before in vitro packaging.
- Medicago sativa (alfalfa) gene bank
- Recombinant DNA
- bacteriophage λ
- nodule specific genes)
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