We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. former include an ability to clone DNA fragments that are up to 95 kb in size in an Escherichia coli host, an ability easily to use those clones to isolate and manipulate microgram quantities of recombinant DNA, an ability to produce libraries with a minimal effort (compared to YACs), and an ability to produce libraries that faithfully represent the genomic source DNA (9-12). In addition, the P1 cloning vector, pADl0sacBII, contains a positive selection for cloning and the vector is organized such that cloned inserts are flanked by rare-cutting restriction enzymes as well as by T7 and Sp6 promoters (4). To determine how well the P1 system is suited for genome mapping and sequencing it is important to produce and evaluate multicoverage P1 libraries. Toward this end a 3-fold coverage mouse library was constructed from cell line C127
The packaging of bacteriophage P1 DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full. We have previously shown that pac is-a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of pac is methylation sensitive. Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not. Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase. Comparison of that uncutpac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated. In contrast, virion DNA containing a cut pac site was not undermethylated. We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle.Bacteriophage P1 packages its DNA by a processive headful mechanism that uses a concatemeric DNA substrate consisting of repeating units of viral DNA (1-3). Packaging is initiated when a specific 162-base-pair (bp) pac sequence on P1 DNA (Fig. 1) is recognized and cleaved (1, 3,4). The DNA is then packaged unidirectionally from the cleaved pac end into an empty phage prohead until that head is full (3, 5). When packaging has been completed, the DNA inside the head is separated from that outside of the head by a cutting process that appears independent of DNA sequence. The DNA end that remains outside ofthe head after the cut is then used to initiate the next P1 sequential packaging event (1,3,6). In this way packaging proceeds down the concatemer in a processive series with pac being recognized and cleaved only once, to initiate the series.For P1, a headful contains '110-115 kilobase pairs (kbp) of DNA (7, 8) or -10-15% more DNA than is present in the viral genome. Because the virus packages this headful from a concatemer, the DNA present in each virus particle contains the same DNA sequences at both ends: it is terminally redundant (6, 9). That redundancy is critical for the vegetative growth of the virus because it permits the viral DNA to cyclize after its injection into a host cell. Cyclization is mediated by the homologous recombination system of the host (10, 11) or in a recombination-deficient host by the P1-lox-Cre site-specific recombination system, when loxP sites are present in the terminally redundant regions (12). For P1 to generate terminally redundant DNA it is important to prevent the cleavage of concatemeric P1 DNA at each pac site. We show here that pac cleavage depends on adenine methylation and suggest that this dependency allows the cleavage process to be regulated in the cell, so as to permit the production of terminally redundant viral molecules.
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