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  • Title: Origin and initiation sites of lambda dv DNA replication in vitro.
    Author: Tsurimoto T, Kouhara H, Matsubara K.
    Journal: Basic Life Sci; 1985; 30():151-72. PubMed ID: 2990406.
    Abstract:
    The sequence of lambda DNA essential for the unique initiation of replication was analyzed in an in vitro replication system. Fragments of lambda DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests. A 165-bp region left of the EcoRI site in the O gene of the lambda genome was defined as the functional origin. This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the O protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure. Regions that have long been suspected to participate in lambda DNA replication initiation, ice and oop were not required for the O, P-dependent lambda-specific replication initiation. The lambda dv and the "ori region plus" recombinant plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of lambda-coded initiators, O and P proteins. Early replicative intermediates of lambda dv were prepared in an in vitro replication system in the presence of ddCTP, an inhibitor of DNA chain elongation. This system allows only that DNA synthesis that is a result of replication initiation. Using this system, the initiation site(s) of the DNA synthesis was finely analyzed by mapping the transition sites from primer RNA to DNA synthesis. Short-chain DNAs produced from regions near the ori region were purified from the intermediates. A fraction of the short-chain DNAs was covalently linked to primer RNA. The 5'-ends that had been linked to RNA (transition sites) were exposed by alkaline hydrolysis, labeled with 32P, and the transition sites were mapped along the nucleotide sequence of the genome. Two striking features emerged from this analysis: (i) The transition sites are located on both sides of the ori region, and no transition arose within the 165-bp ori region; (ii) The transition sites on both sides are not unique, but multiple, and are clustered in one of the 2 strands. Furthermore, their orientation demonstrates that the DNA synthesis in initiation of replication from the 2 sides of the ori region converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis. These results reflect asymmetric bidirectional replication of lambda dv DNA, and may also reflect replication of lambda phage DNA.
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