DNA polymerase ␣-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim 2 ) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim 2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2⅐10 ؊8 M and 1.3⅐10 ؊8 M, respectively.In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process (reviewed in Ref. 1). For each reaction a specific set of enzymes and accessory proteins is required to replicate chromosomal DNA (1-4). The first step in leading strand DNA replication is accomplished by the synthesis of oligoribonucleotides, called RNA primers, at the origin of DNA replication. This process is carried out by a special enzyme, DNA primase (5-7). The eukaryotic enzyme consists of two subunits, the catalytic subunit p48 and p58, which serves to stabilize the primase activity (8 -13). In eukaryotic cells these two subunits assemble together with the catalytic DNA polymerase subunit, p180, and the p68 polypeptide into a heterotetrameric DNA polymerase ␣-primase complex (pol-prim) 1 (1-7, 14, 15).The initiation of DNA replication requires the interaction of several proteins in vivo and in vitro (1). The start of DNA synthesis de novo occurs by two independent processes: the singular priming event on the leading strand at the origin and the multiple priming steps for Okazaki fragment synthesis on the lagging strand. Both tasks are carried out by the primase activity (1-3, 14 -17). Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded (ss) DNA templates bound by replication protein A (RPA) (17, 18). Using these cell-free systems, it was shown that the initiation of SV40 DNA replication at the origin is species-specific and requires the activity of the p180 subunit of primate pol-prim (19 -21). In contrast to these results, the initiation of Okazaki fragments during lagging strand DNA synthesis is not species-specific and the human as well as the bovine pol-prim can perform lagging strand initiat...

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Jasmonic acid (JA) and its derivatives are well-characterized signaling molecules in plant defense and development, but the site of their localization within plant tissue is entirely unknown. To address the question whether applied JA accumulates extracellularly or intracellularly, leaves of tomato and barley were fed with 14 C-labeled JA and the label was localized in cryo¢xed and lyophilized leaf tissues by microautoradiography. In tomato the radioactivity was detectable within the apoplast, but no label was found within the mesophyll cells. By contrast, in barley leaf tissues, radioactivity was detected within the mesophyll cells suggesting a cellular uptake of exogenously applied JA. JA, applied to leaves of both plants as in the labeling experiments, led in all leaf cells to the expression of JAinducible genes indicating that the perception is completed by JA signal transduction.