Skip to main content
SpringerLink
Log in

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

Characteristics of incorporation and inhibition by α-amanitin

  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and α-amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs.-Autoradiographic studies carried out in the presence or absence of the toxin α-amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in α-amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Armelin, H.A., Marques, N.: Transcription and processing of Ribonucleic acid in Rhynchosciara salivary glands. I. Rapidly labelled Ribonucleic acid. Biochemistry 11, 3663–3672 (1972)

    Google Scholar 

  • Armelin, H.A., Meneghini, R., Lara, F.J.S.: Patterns of Ribonucleic acid synthesis in salivary gland of Rhynchosciara angelae during development. Genetics Suppl. 61, 351–360 (1969)

    Google Scholar 

  • Beermann, W.: Effect of α-amanitin on puffing and intranuclear RNA synthesis in Chironomus salivary glands. Chromosoma (Berl.) 34, 152–167 (1971)

    Google Scholar 

  • Berendes, H.D., Boyd, J.B.: Structural and functional properties of polytene nuclei isolated from salivary glands of Drosophilia hydei. J. Cell Biol. 41, 591–599 (1969)

    Google Scholar 

  • Bray, G.A.: A simple efficient liquid scintillation for counting aqueous solution in a liquid scintillation counter. Ann. Biochem. 1, 279–285 (1960)

    Google Scholar 

  • Breuer, M.E.: Revision of the genus Rhynchosciara Rübsaamen (Diptera, Sciaridae) in the neotropical region. Archivo Zool.) (S. Paulo) 17, 167–198 (1969)

    Google Scholar 

  • Breuer, M.E., Pavan, C.: Behavior of polytene chromosomes of Rhynchosciara angelae at different stages of larval development. Chromosoma (Berl.) 7, 317–386 (1955)

    Google Scholar 

  • Brown, D.O., David, J.: Specific gene simplification in oocytes. Science 160, 272–280 (1968)

    Google Scholar 

  • Cohen, L.H., Gotchel, B.V.: Histones of polytene and non polytene nuclei of Drosophila melanogaster. J. biol. Chem. 246, 1811–1848 (1971)

    Google Scholar 

  • Daneholt, B., Edström, J.E., Egyhazi, E., Lambert, B., Ringborg, U.: Chromosomes RNA synthesis in polytene chromosomes of Chironomus tentans. Chromosoma (Berl.) 28, 399–417 (1969)

    Google Scholar 

  • Edstrom, J.E., Daneholt, B.: Sedimentation properties of newly synthesized RNA from isolated nuclear components of Chironomus tentans salivary gland cells. J. molec. Biol. 28, 331–343 (1967)

    Google Scholar 

  • Egyhazi, E.: Inhibition of Balbiani ring RNA synthesis at the initiation level. Proc. nat. Acad. Sci. (Wash.) 72, 947–950 (1975)

    Google Scholar 

  • Egyhazi, E., Daneholt, B., Edström, J.E., Lambert, B., Ringborg, U.: Low molecular weight RNA in cell components of Chironomus tentans salivary glands. J. molec. Biol. 44, 517–532 (1969)

    Google Scholar 

  • Egyhazi, E., D'Monte, B., Edström, J.E.: Effects of α-amanitin on in vitro labelling of RNA from defined nuclear components in salivary gland cells from Chironomus tentans. J. Cell Biol. 53, 523–531 (1972)

    Google Scholar 

  • Evans, D., Birnstiel, M.L.: Localization of amplified ribosomal DNA in the oocyte of Xenopus laevis. Biochim. biophys. Acta (Amst.) 166, 274–276 (1968)

    Google Scholar 

  • Ficq, A., Pavan, C.: Autoradiography of polytene chromosomes of Rhynchosciara angelae at different stages of larval development. Nature (Lond.) 180, 983–984 (1957)

    Google Scholar 

  • Gabrusewycz-Garcia, N.: Further studies of the nucleolar material in salivary gland nuclei of Sciara coprophila. Chromosoma (Berl.) 38, 237–254 (1972)

    Google Scholar 

  • Gall, J.G.: Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. nat. Acad. Sci. (Wash.) 60, 553–560 (1968)

    Google Scholar 

  • Grummt, J., Lindigkeit, R.: Pre-ribosomal RNA synthesis in isolated rat liver nucleoli. Europ. J. Biochem. 36, 244–249 (1973)

    Google Scholar 

  • Gude, V.D.: Autoradiographic Techniques. Localization of radioisotopes in biological material. Englewood Cliffs, N.J.: Prentice-Hall 1968

    Google Scholar 

  • Harris, S.E., Forrest, H.S.: RNA polymerase activity in isolated nuclei from eggs of Oncopeltus fasciatus. J. Insect Physiol. 17, 303–312 (1971)

    Google Scholar 

  • Hunjo, S., Reeder, R.H.: Transcription of Xenopus chromatin by homologous ribonucleic acid polymerase: Aberrant synthesis of Ribosomal and 5S ribonucleic acid. Biochemistry 13, 1896–1899 (1974)

    Google Scholar 

  • Jacob, S.T., Sadjel, E.M., Munro, H.N.: Specific action of α-amanitin on mammalian RNA polymerase protein. Nature (Lond.) 225, 60–62 (1970)

    Google Scholar 

  • Johnson, J.D., Jant, B.A., Kaufman, S., Sokoloff, L.: Effects of ionic strength on the RNA-polymerase activities of isolated nuclei and nucleoli of rat liver. Arch. Biochem. Biophys. 142, 489–500 (1971)

    Google Scholar 

  • Kedinger, C., Gniazdowski, M., Mandel, Jr., J.L., Gissinger, F., Chambon, P.: α-Amanitin: a specific inhibitor of one of two DNA dependent RNA polymerases activities from calf thymus. Biochem. biophys. Res. Commun 38, 165–171 (1970)

    Google Scholar 

  • Lara, F.J.S., Hollander, F.M.: Changes in RNA metabolism during development of Rhynchosciara angelae. Nat. Cancer Inst. Monogr. 127, 235–242 (1967)

    Google Scholar 

  • Lara, F.J.S., Tamaki, H., Pavan, C.: Laboratory culture of Rhynchosciara angelae. Amer. Naturalist 99, 189–191 (1965)

    Google Scholar 

  • Lee, M.J., Penman, M., Malt, R.A.: The use of deoxyribonuclease in the isolation of nucleoli and purification of nuclear RNA caution. Biochim biophys. Acta (Amst.) 294, 461 (1973)

    Google Scholar 

  • Lindell, T.J., Weinberg, F., Morris, P.W., Roeder, R.G., Rutter, W.J.: Specific inhibition of nuclear RNA polymerase II by α-amanitin. Science 170, 447–445 (1970)

    Google Scholar 

  • Marzluff, W.F., Murphy, E.C., Jr., Huang, R.C.: Transcription of the genes for 5S ribosomal RNA and transfer RNA in isolated mouse myeloma cell nuclei. Biochemistry 13, 3689–3696 (1974)

    Google Scholar 

  • Maul, G.G., Hamilton, T.H.: The intranuclear localization of two DNA dependent RNA polymerase activities. Proc. nat. Acad. Sci. (Wash.) 57, 1371–1378 (1967)

    Google Scholar 

  • Meneghini, R., Armelin, H.A., Balsamo, J., Lara, F.J.S.: Indication of gene amplification in Rhynchosciara by RNA-DNA hybridization. J. Cell Biol. 49, 913–916 (1971)

    Google Scholar 

  • McReynolds, L., Penman, S.: A polymerase activity forming 5S and pre-4S RNA in isolated HeLa cell nuclei. Cell 1, 139–145 (1974)

    Google Scholar 

  • Nonato, E., Pavan, C.: A new species of Rhynchosciara Rübsaamen 1894 (Diptera, Mycetophilidae). Rev. bras. Biol. 11, 435–437 (1951)

    Google Scholar 

  • O'Brien, B.R.A.: The partial cytolysis of the amphibian erythrocyte and liver parenchyma cell by a non-ionogenic surface active agent. J. Cell Biol. 20, 521–524 (1964a)

    Google Scholar 

  • O'Brien, B.R.A.: A rapid method for the isolation and collection of nuclei from whole cells suspensions. J. Cell Biol. 20, 525–528 (1964b)

    Google Scholar 

  • Pardue, M.L., Gall, J.G.: Molecular cytogenetics. In: Molecular genetics and developmental biology (M. Sussman, ed.), p. 66–99. Englewood Cliffs, N.J.: Prentice-Hall Inc. 1972

    Google Scholar 

  • Pardue, M.L., Gerbi, S.A., Eckardt, R.A., Gall, J.G.: Cytological localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera. Chromosoma (Berl.) 29, 268–290 (1970)

    Google Scholar 

  • Pavan, C.: Nucleic acid metabolism in polytene chromosomes and the problem of differentiation. Brookhaven Symp. Biol. 18, 224–241 (1965)

    Google Scholar 

  • Pogo, A.O., Littau, V.C., Allfrey, U.G., Mirsky, A.E.: Modification of ribonucleic acid synthesis in nuclei isolated from normal and regenerating liver. Some effects of salt and specific divalent cations. Proc. nat. Acad. Sci. (Wash.) 57, 743–750 (1967)

    Google Scholar 

  • Price, R., Penman, S.: A distinct RNA polymerase activity, synthesizing 5.5S, 5S and 4S RNA in nuclei from Adenovirus 2-infected HeLa cells. J. molec. Biol. 70, 435–450 (1972)

    Google Scholar 

  • Reeder, R.H., Roeder, R.G.: Ribosomal RNA synthesis in isolated nuclei. J. molec. Biol. 67, 433–441 (1972)

    Google Scholar 

  • Roeder, R.G., Rutter, W.J.: Multiple forms of DNA-dependent RNA polymerase in eukariotic organisms. Nature (Lond.) 224, 234–237 (1969)

    Google Scholar 

  • Roeder, R.G., Rutter, W.J.: Multiple Ribonucleic acid polymerases and ribonucleic acid synthesis during sea urchin development. Biochemistry 12, 2543–2553 (1970)

    Google Scholar 

  • Rudkin, G.T., Corlette, S.L.: Disproportionate synthesis of DNA in a polytene chromosome region. Proc. nat. Acad. Sci. (Wash.) 43, 964–968 (1957)

    Google Scholar 

  • Santelli, R.V., Machado-Santelli, G.M., Lara, F.J.S.: Transcrição em glândulas salivares de Rhynchosciara: efeito inibidor de α-amanitina. Anais Acad. Bras. Ciênc. 43, 833 (1971)

    Google Scholar 

  • Serfling, E., Wobus, U., Panitz, R.: Effect of α-amanitin on chromosomal and nuclear RNA synthesis in Chironomus thummi polytene chromosomes. FEBS Letters 20, 148–152 (1972)

    Google Scholar 

  • Shaaya, E., Sekeris, C.E.: Inhibitory effects of α-amanitin on RNA synthesis and induction of DOPA decarboxilase by B-ecdysone FEBS Letters 16, 333–336 (1971)

    Google Scholar 

  • Stirpe, F., Fiume, L.: Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanition on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei. Biochem. J. 105, 779–782 (1967)

    Google Scholar 

  • Tata, J.R., Hamilton, M.J., Shields, D.: Effects of α-amanitin in vivo on RNA polymerase and nuclear RNA synthesis. Nature (Lond.) New Biol. 238, 161–164 (1972)

    Google Scholar 

  • Terra, E.R., Bianchi, A.G., Gambarini, A.G., Lara, F.J.S.: Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly Rhynchosciara americana. J. Insect Physiol. 19, 2097–2106 (1973)

    Google Scholar 

  • Terra, W.R., Bianchi, A.G., Gambarini, A.G., Pueyo, M.T., Lara, F.J.S.: A medium for short-term incubation of salivary glands based on the haemolymph composition of the Rhynchosciara americana, Ciência e Cultura (in Press, 1976)

  • Widnell, C.C., Tata, J.R.: Evidence for two DNA-dependent RNA polymerase activities in isolated rat liver nuclei. Biochim. biophys. Acta (Amst.) 87, 531–533 (1964)

    Google Scholar 

  • Wieland, T.: Poisonous principles of mushrooms of the genus Amanita. Science 159, 946–952 (1968)

    Google Scholar 

  • Zylber, E.A., Penman, S.: Products of RNA polymerases in HeLa cell nuclei. Proc. nat. Acad. Sci. (Wash.) 68, 2861–2865 (1971)

    Google Scholar 

Download references

Author information

Author notes

  1. Roberto V. Santelli, Gláucia Maria Machado-Santelli & Francisco J. S. Lara

    Present address: Laboratório de Oncologia Experimental, Faculdade de Medicina, Universidade de São Paulo, Caixa Postal 2921, São Paulo, Brasil

Authors and Affiliations

  1. Institute de Quimica, Universidade de São Paulo, Caixa Postal 20780, São Paulo, Brasil

    Roberto V. Santelli, Gláucia Maria Machado-Santelli & Francisco J. S. Lara

Authors
  1. Roberto V. Santelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gláucia Maria Machado-Santelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francisco J. S. Lara
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Santelli, R.V., Machado-Santelli, G.M. & Lara, F.J.S. In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands. Chromosoma 56, 69–84 (1976). https://doi.org/10.1007/BF00293731

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00293731

Keywords

Search

Navigation