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To study the functions of the mini-Pl replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer deteetable.P1 prophage is a stringently controlled plasmid replicon that is maintained at a copy number approximately equal to the number of Escherichia coli chromosomes (16). The basic plasmid replicon, the minimal region required to maintain the plasmid at its normal copy number, consists of an origin, the gene for the initiator protein RepA, and a control locus. The salient feature of the basic replicon is the presence of two sets of repeated, nearly identical 19-base-pair sequences. A set of five repeats is an essential part of the origin. A separate set of nine repeats constitutes the control locus (3, 4). The purified RepA protein binds to both sets of repeats (1). Extra copies of the repeats decrease the replication frequency, whereas deletion of the control locus increases the frequency (4, 14). Similar results were obtained for mini-F plasmids, whose genetic organization is very similar to that of mini-Pl (25). It has been proposed (25) that the origin and the control repeats compete for RepA, which has been shown to be limiting for replication (13,18), implying that the physiological concentration of the protein is so low that sequestration of a few molecules would have a significant effect on the rate of replication. In other words, the number of repeats and the number of RepA molecules are expected to be comparable in the cell. In this paper we report the measurement of RepA concentration by two approaches. These measurements are consistent with the sequestration hypothesis.The protein is also known to have two other functions. It inhibits the synthesis of the repA transcript and, when overproduced, inhibits replication (6). Knowledge of the protein concentration will thus be helpful in understanding how RepA functions. A preliminary report of some of this work has been presented (5). Protein determination. The protein concentration was determinated by using amido black as described by Schaffner and Weissman (20). All samples were brought to 1% SDS, and BSA was-used as the protein standard. The protein concentration of purified RepA as determined by amino acid analysis was about 30% less than the concentration determined by the amido black method with BSA as standard. The discrepanc...

A monoclonal antibody against purified calf DNA polymerase a (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Most attempts to elucidate the subunit structure of DNA polymerase a (a-polymerase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) have relied upon NaDodSO4/polyacrylamide gel electrophoretic analysis of highly purified preparations. However, identification of enzyme subunits by this approach is complicated by the possibilities of both proteolytic degradation and elimination of important enzyme polypeptides during laborious isolation procedures. Clearly, additional methods, not involving laborious purification, are needed for the study of apolymerase polypeptides. Recently, "activity gel" analysis (1-3) of crude homogenates resulted in the identification of a Mr 110,000-120,000 a-polymerase catalytic polypeptide in a number of eukaryotic tissues; in addition, putative a-polymerase catalytic polypeptides also were detected at about Mr 70,000 (1-3). Nevertheless, use of the activity gel method is complicated by the fact that the detection efficiency of apolymerases is low, and some purified a-polymerases are completely unable to produce an activity signal (2). A promising immunological approach, based upon solid-phase immunobinding, was recently reported by Sauer and Lehman (4). Those workers observed a Mr 182,000 polypeptide in crude extracts of Drosophila embryo that cross-reacted with an antiserum raised against the Mr 148,000 polypeptide of purified Drosophila a-polymerase; generally, a-polymerase subunits of Mr > 160,000 had not been reported prior to this work. Lehman and co-workers (5, 6) subsequently showed that the Mr 182,000 polypeptide itself was capable of DNA polymerase catalytic activity.Studies of a-polymerases have been facilitated recently by the development of monoclonal antibodies to these enzymes. Kom and co-workers (7, 8) used a monoclonal antibody to KB cell a-polymerase to localize the enzyme by immunocytofluorescence, and this group and Wahl et al. used a monoclonal antibody to purify the enzyme by immunoaffinity chromatography (9, 10). Masaki et al. (11) found that a monoclonal antibody to calf a-polymerase could distinguish individual species of the enzyme in partially purified preparations from calf thymus, and Matsukage et al. (12) used a monoclonal antibody to chicken a-polymerase to study tissue-specific expression of the enzyme as a function of embryonic development. In the present study, we used a monoclonal antibody approach to elucidate components of mammalian cell lines that share immunological determinants with purified a-polymerase. Proteins in a crude soluble extract from growth-phase monkey cells were subjected to immunoprecipitation with one of our monoclonal antibodies to apolymerase. Immunoprecipitated polypeptides were electrophoresed in NaDodSO4/polyacrylamide gels and then examined for DNA polymerase activ...

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A 21-mer synthetic peptide (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1(58-78), was constructed from the primary structure of the toxic shock syndrome toxin 1 (TSST-1). The peptide reacted with a panel of neutralizing monoclonal antibodies (MAbs) to whole TSST-1 in solid-phase immunoassays. TSST-1(58-78) promoted the in vitro proliferation of human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Minimum dose required for stimulation (P less than or equal to .05 microM) was 0.75 microM peptide. This mitogenic effect was abrogated by incubation of the peptide with MAbs to whole TSST-1 before addition to PBMC. The ability of TSST-1(58-78) to stimulate the proliferation of highly purified resting human T cells was analyzed. Significant proliferation (P less than or equal to .01) was observed only in the presence of increasing populations of monocytes added to the cultures. Adherent human monocytes exposed to TSST-1(58-78) released tumor necrosis factor. Thus, some of the immunoregulatory properties attributed to TSST-1 are demonstrated by the region of the toxin represented by the peptide TSST-1(58-78).