A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.
Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase alpha and DNA polymerase delta, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase alpha was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA.
A comparison of the 35S-methionine metabolically labelled immunoreactive glycoproteins of immature and mature F. hepatica was carried out by one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of rabbits infected for 3 weeks reacted much more strongly with glycoproteins of immature flukes than with glycoproteins of mature flukes as compared to sera of rabbits infected for 9 weeks. Several of the immunoreactive glycoproteins were also released by immature F. hepatica into the culture medium. At least one was a component of the T1 type granules. Analysis of the in vitro translation products of mature F. hepatica indicated that the initial humoral immune response of rabbit hosts may be directed against carbohydrate moieties.
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