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. 1991 Jan 2;112(2):203–213. doi: 10.1083/jcb.112.2.203

Developmental regulation of Drosophila DNA topoisomerase II

PMCID: PMC2288815  PMID: 1846370

Abstract

Affinity-purified polyclonal antibodies were used to quantitate steady- state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi Y., Käs E., Laemmli U. K. Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions. EMBO J. 1989 Dec 20;8(13):3997–4006. doi: 10.1002/j.1460-2075.1989.tb08582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aebi U., Cohn J., Buhle L., Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986 Oct 9;323(6088):560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
  3. Allis C. D., Waring G. L., Mahowald A. P. Mass isolation of pole cells from Drosophila melanogaster. Dev Biol. 1977 Apr;56(2):372–381. doi: 10.1016/0012-1606(77)90277-9. [DOI] [PubMed] [Google Scholar]
  4. Avrameas S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry. 1969 Jan;6(1):43–52. doi: 10.1016/0019-2791(69)90177-3. [DOI] [PubMed] [Google Scholar]
  5. Berrios M., Fisher P. A. A myosin heavy-chain-like polypeptide is associated with the nuclear envelope in higher eukaryotic cells. J Cell Biol. 1986 Sep;103(3):711–724. doi: 10.1083/jcb.103.3.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berrios M., Osheroff N., Fisher P. A. In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4142–4146. doi: 10.1073/pnas.82.12.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berrios S., Fisher P. A. Thermal stabilization of putative karyoskeletal protein-enriched fractions from Saccharomyces cerevisiae. Mol Cell Biol. 1988 Oct;8(10):4573–4575. doi: 10.1128/mcb.8.10.4573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  9. Bravo R., Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol. 1987 Oct;105(4):1549–1554. doi: 10.1083/jcb.105.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  11. Brill S. J., Sternglanz R. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell. 1988 Jul 29;54(3):403–411. doi: 10.1016/0092-8674(88)90203-6. [DOI] [PubMed] [Google Scholar]
  12. Burgoyne L. A., Wagar M. A., Atkinson M. R. Calcium-dependent priming of DNA synthesis in isolated rat liver nuclei. Biochem Biophys Res Commun. 1970 Apr 24;39(2):254–259. doi: 10.1016/0006-291x(70)90786-2. [DOI] [PubMed] [Google Scholar]
  13. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  14. Chung T. D., Drake F. H., Tan K. B., Per S. R., Crooke S. T., Mirabelli C. K. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9431–9435. doi: 10.1073/pnas.86.23.9431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. DiNardo S., Voelkel K., Sternglanz R. DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci U S A. 1984 May;81(9):2616–2620. doi: 10.1073/pnas.81.9.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Drake F. H., Hofmann G. A., Bartus H. F., Mattern M. R., Crooke S. T., Mirabelli C. K. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry. 1989 Oct 3;28(20):8154–8160. doi: 10.1021/bi00446a029. [DOI] [PubMed] [Google Scholar]
  17. Drake F. H., Zimmerman J. P., McCabe F. L., Bartus H. F., Per S. R., Sullivan D. M., Ross W. E., Mattern M. R., Johnson R. K., Crooke S. T. Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. J Biol Chem. 1987 Dec 5;262(34):16739–16747. [PubMed] [Google Scholar]
  18. Fairman R., Brutlag D. L. Expression of the Drosophila type II topoisomerase is developmentally regulated. Biochemistry. 1988 Jan 26;27(2):560–565. doi: 10.1021/bi00402a009. [DOI] [PubMed] [Google Scholar]
  19. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  20. Fisher P. A., Berrios M., Blobel G. Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster. J Cell Biol. 1982 Mar;92(3):674–686. doi: 10.1083/jcb.92.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fisher P. A. Chromosomes and chromatin structure: the extrachromosomal karyoskeleton. Curr Opin Cell Biol. 1989 Jun;1(3):447–453. doi: 10.1016/0955-0674(89)90004-5. [DOI] [PubMed] [Google Scholar]
  22. Fisher P. A., Smith D. E. Affinity purification of antibodies using antigens immobilized on solid supports. Biochem Soc Trans. 1988 Apr;16(2):134–138. doi: 10.1042/bst0160134. [DOI] [PubMed] [Google Scholar]
  23. Gasser S. M., Laroche T., Falquet J., Boy de la Tour E., Laemmli U. K. Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol. 1986 Apr 20;188(4):613–629. doi: 10.1016/s0022-2836(86)80010-9. [DOI] [PubMed] [Google Scholar]
  24. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  25. Gruenbaum Y., Landesman Y., Drees B., Bare J. W., Saumweber H., Paddy M. R., Sedat J. W., Smith D. E., Benton B. M., Fisher P. A. Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene. J Cell Biol. 1988 Mar;106(3):585–596. doi: 10.1083/jcb.106.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hadlaczky G., Praznovszky T., Sófi J., Udvardy A. Intracellular forms of Drosophila topoisomerase II detected with monoclonal antibodies. Nucleic Acids Res. 1988 Nov 11;16(21):10013–10023. doi: 10.1093/nar/16.21.10013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Heck M. M., Earnshaw W. C. Topoisomerase II: A specific marker for cell proliferation. J Cell Biol. 1986 Dec;103(6 Pt 2):2569–2581. doi: 10.1083/jcb.103.6.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heck M. M., Hittelman W. N., Earnshaw W. C. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1086–1090. doi: 10.1073/pnas.85.4.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heck M. M., Hittelman W. N., Earnshaw W. C. In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis. J Biol Chem. 1989 Sep 15;264(26):15161–15164. [PubMed] [Google Scholar]
  30. Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
  31. Hsiang Y. H., Wu H. Y., Liu L. F. Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells. Cancer Res. 1988 Jun 1;48(11):3230–3235. [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lin L., Fisher P. A. Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms. J Biol Chem. 1990 Jul 25;265(21):12596–12601. [PubMed] [Google Scholar]
  34. Luke M., Bogenhagen D. F. Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis. Dev Biol. 1989 Dec;136(2):459–468. doi: 10.1016/0012-1606(89)90271-6. [DOI] [PubMed] [Google Scholar]
  35. McConnell M., Whalen A. M., Smith D. E., Fisher P. A. Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ. J Cell Biol. 1987 Sep;105(3):1087–1098. doi: 10.1083/jcb.105.3.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McGadey J. A tetrazolium method for non-specific alkaline phosphatase. Histochemie. 1970;23(2):180–184. doi: 10.1007/BF00305851. [DOI] [PubMed] [Google Scholar]
  37. Mirkovitch J., Gasser S. M., Laemmli U. K. Relation of chromosome structure and gene expression. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):563–574. doi: 10.1098/rstb.1987.0081. [DOI] [PubMed] [Google Scholar]
  38. Ng L., Prelich G., Anderson C. W., Stillman B., Fisher P. A. Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart. J Biol Chem. 1990 Jul 15;265(20):11948–11954. [PubMed] [Google Scholar]
  39. Nolan J. M., Lee M. P., Wyckoff E., Hsieh T. S. Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3664–3668. doi: 10.1073/pnas.83.11.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. O'Connell M. L., Canipari R., Strickland S. Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells. J Biol Chem. 1987 Feb 15;262(5):2339–2344. [PubMed] [Google Scholar]
  41. Paddy M. R., Belmont A. S., Saumweber H., Agard D. A., Sedat J. W. Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell. 1990 Jul 13;62(1):89–106. doi: 10.1016/0092-8674(90)90243-8. [DOI] [PubMed] [Google Scholar]
  42. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  43. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  44. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Smith D. E., Fisher P. A. Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis. J Cell Biol. 1989 Feb;108(2):255–265. doi: 10.1083/jcb.108.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sternglanz R. DNA topoisomerases. Curr Opin Cell Biol. 1989 Jun;1(3):533–535. doi: 10.1016/0955-0674(89)90016-1. [DOI] [PubMed] [Google Scholar]
  47. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  48. Sundin O., Varshavsky A. Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell. 1981 Sep;25(3):659–669. doi: 10.1016/0092-8674(81)90173-2. [DOI] [PubMed] [Google Scholar]
  49. Sundin O., Varshavsky A. Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell. 1980 Aug;21(1):103–114. doi: 10.1016/0092-8674(80)90118-x. [DOI] [PubMed] [Google Scholar]
  50. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  53. Wyckoff E., Natalie D., Nolan J. M., Lee M., Hsieh T. Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. J Mol Biol. 1989 Jan 5;205(1):1–13. doi: 10.1016/0022-2836(89)90361-6. [DOI] [PubMed] [Google Scholar]

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