A 130 kDa Polypeptide Immunologically Related to the 180 kDa Catalytic Subunit of DNA Polymerase α-Primase Complex Is Detected in Early Embryos of Drosophila

• Published in: Journal of biochemistry (Tokyo) 1995-04, Vol.117 (4), p.809-818
• Main Authors: Kuroda, Kazufumi, Ueda, Ryu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibody Specificity; Blotting, Western; Cell Division - physiology; Cell Nucleus - enzymology; Cell Nucleus - physiology; Chromatography; DNA polymerase α; DNA Primase; Drosophila embryo; Drosophila melanogaster - embryology; Drosophila melanogaster - enzymology; extract from single embryo; immunocytochemical method; Immunohistochemistry; Interphase; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Peptides - analysis; Peptides - immunology; RNA Nucleotidyltransferases - analysis; RNA Nucleotidyltransferases - immunology; Staining and Labeling - methods; Western blotting
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a124780

An immunocytochemical method using specific antibodies was employed to detect DNA polymerase α-primase complex in Drosophila melanogaster embryos during the first 13 nuclear division cycles. A monoclonal antibody specific to the 72 kDa polypeptide stained interphase nuclei, but not metaphase chromosome, while at late anaphase and thereafter staining of the chromosome was regained. On the other hand, a polyclonal antibody specific to the 180 kDa polypeptide stained not only the interphase nuclei but also the cytoplasmic regions surrounding interphase nuclei. These results suggest that the distributions of the 180 kDa and the 72 kDa polypeptides of DNA polymerase α-primase complex are different. We detected the 180 kDa and the 72 kDa polypeptides in the extract prepared from a single Drosophila embryo by Western blotting, and a 130 kDa polypeptide immunologically related to the 180 kDa polypeptide was also detected in the extract. These polypeptides (180, 130, and 72 kDa) in the embryos were detected at similar levels at interphase and at the mitotic phase. These three polypeptides were also detected in unfertilized eggs, showing that they were maternally stored. The 130 kDa polypeptide was detected till cycle 10, then began to decrease, and finally disappeared at cycle 14, whereas the 180 kDa and the 72 kDa polypeptides were present without marked fluctuation in quantity throughout the developmental stages. Even in unfertilized eggs, the level of the 130 kDa polypeptide decreased gradually with a similar time course to that in fertilized ones, but the levels of the 180 kDa and the 72 kDa polypeptides remained unchanged. This is the first report suggesting the existence of the 130 kDa polypeptide in vivo in the early embryos of Drosophila. The significance of the 130 kDa polypeptide is discussed