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The Nucleus pp 29-54 | Cite as

Gene Expression in Polytene Nuclei

  • Petra Björk
  • Lars Wieslander
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

Gene expression in eukaryotic cells is a multi-step process. Many of the steps are both co-ordinated and quality controlled. For example, transcription is closely coupled to pre-messenger RNA (mRNA)–protein assembly, pre-mRNA processing, surveillance of the correct synthesis of messenger ribonucleoprotein (mRNP), and export. The coordination appears to be exerted through dynamic interactions between components of the transcription, processing, surveillance, and export machineries. Our knowledge is so far incomplete about these molecular interactions and where in the nucleus they take place. It is therefore essential to analyze the intranuclear steps of gene expression in vivo. Polytene nuclei are exceptionally large and contain chromosomes and individual genes that can be structurally analyzed in situ during ongoing transcription. Furthermore, they contain gene-specific pre-mRNPs/mRNPs that can be visualised and analyzed as they are synthesised on the gene and then followed on their path to the cytoplasm. We describe methods for investigating the structure and composition of active chromatin and gene-specific pre-mRNPs/mRNPs in the context of analyses of gene expression processes in the nuclei of polytene cells.

Keywords

Polytene chromosomes Balbiani ring genes Chromatin Gene expression transcription mRNA processing Nucleocytoplasmic export Immunolabelling Electron microscopy 

Notes

Acknowledgments

We are grateful for helpful comments from Prof. B. Daneholt and Prof. N. Visa. We also acknowledge the assistance of K. Bernholm.

References

  1. 1.
    Case, S.T. and Wieslander, L. (1992) Secretory proteins of Chironomus salivary glands: structural motifs and assembly characteristics of a novel biopolymer. Results Probl. Cell Differ. 19, 187-226PubMedGoogle Scholar
  2. 2.
    Wieslander, L. (1994) The Balbiani ring multigene family: coding repetitive sequences and evolution of a tissue-specific cell function. Progr. Nucleic Acid Res. Mol. Biol. 48, 275-313CrossRefGoogle Scholar
  3. 3.
    Cremer, T., Cremer, M., Dietzel, S., Müller, S., Solovei, I., and Fakan, S. (2006) Chromosome territories—a functional nuclear landscape. Curr. Opin. Cell Biol. 18, 307-316PubMedCrossRefGoogle Scholar
  4. 4.
    Carter, D., Chakalova, L., Osbourne, C.S., Dai, Y.-F., and Fraser, P. (2002) Longe-range chromatin regulatory interactions in vivo. Nature Genet. 32, 623-626PubMedCrossRefGoogle Scholar
  5. 5.
    Udvardy, A., Maine, E., and Schedl, P. (1985) The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J. Mol. Biol. 185, 341-358PubMedCrossRefGoogle Scholar
  6. 6.
    Blanton, J., Gaszner, M., and Schedl, P. (2003) Protein:protein interactions and the pairing of boundary elements in vivo. Genes Dev. 17, 664-675PubMedCrossRefGoogle Scholar
  7. 7.
    O’Sullivan, J.M., Tan-Wong, S.M., Morillon, A., Lee, B., Coles, J., Mellor, J., and Proudfoot, N.J. Nat. Genet. 36, 1014-1018Google Scholar
  8. 8.
    Saiz, L., and Vilar, J.M.G. (2006) DNA looping: the consequences and its control. Curr. Opin. Cell Biol. 16, 344-350CrossRefGoogle Scholar
  9. 9.
    Chambeyron, S. and Bickmore, W.A. (2004) Does looping and clustering in the nucleus regulate gene expression? Curr. Opin. Cell Biol. 16, 256-262PubMedCrossRefGoogle Scholar
  10. 10.
    Eissenberg, J.C. (2006) Functional genomics of histone modification and non-histone chromosomal proteins using the polytene chromosomes of Drosophila. Methods 40, 360-364PubMedCrossRefGoogle Scholar
  11. 11.
    Andersson, K., Björkroth, B., and Daneholt, B. (1980) The in situ structure of the active 75 S RNA genes in Balbiani rings of Chironomus tentans. Exp. Cell Res. 130, 313-326PubMedCrossRefGoogle Scholar
  12. 12.
    Beermann, W. (1973) Directed changes in the pattern of Balbiani ring puffing in Chironomus: effects of a sugar treatment. Chromosoma 41, 297-326PubMedCrossRefGoogle Scholar
  13. 13.
    Egyházi, E. (1975) Inhibition of Balbiani ring RNA synthesis at the initiation level. Proc. Natl. Acad. Sci. USA 72, 947-950PubMedCrossRefGoogle Scholar
  14. 14.
    Ericsson, C., Mehlin, H., Björkroth, B., Lamb, M.M., and Daneholt, B. (1989) The ultrastructure of upstream and downstream regions of an active Balbiani ring gene. Cell 56, 631-639PubMedCrossRefGoogle Scholar
  15. 15.
    Björkroth, B., Ericsson, C., Lamb, M.M., and Daneholt, B. (1988) Structure of the chromatin axis during transcription. Chromosoma 96, 333-340CrossRefGoogle Scholar
  16. 16.
    Belikov, S., Paulsson, G., and Wieslander, L. (1998) Promoter regions of four Balbiani ring genes in Chironomus tentans exhibit a common salivary gland-specific chromatin organisation, which is dependent of the rate of transcriptional initiation. Mol. Gen. Genet. 258, 420-426PubMedCrossRefGoogle Scholar
  17. 17.
    Andersson, K., Björkroth, B., and Daneholt, B. (1984) Packaging of a specific gene into higher order structures following repression of RNA synthesis. J. Cell Biol. 98, 1296-1303PubMedCrossRefGoogle Scholar
  18. 18.
    Ericsson, C., Grossbach, U., Björkroth, B., and Daneholt, B. (1990) Presence of histone H1 on an active Balbiani ring gene. Cell 60, 73-83PubMedCrossRefGoogle Scholar
  19. 19.
    Ringrose, L., Ehret, H., and Paro, R. (2004) Distinct contributions of histone H3 lysine 9 and 27 methylation to locus-specific stability of polycomb complexes. Mol. Cell 16, 641-653PubMedCrossRefGoogle Scholar
  20. 20.
    Westwood, J.T., Clos, J., and Wu, C. (1991) Stress-induced oligomerization and chromosomal relocalization of heat-shock factor. Nature 353, 822-827PubMedCrossRefGoogle Scholar
  21. 21.
    Sjölinder, M., Björk, P., Söderberg, E., Sabri, N., Farrants, A.K., and Visa, N. (2005) The growing pre-mRNA recruits actin and chromatin-modifying factors to transcriptionally active genes. Genes Dev. 19, 1871-1874PubMedCrossRefGoogle Scholar
  22. 22.
    Osborne, C.S., Chakalova, L., Brown, K.E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J.A., Lopes, S., Reik, W., and Fraser, P. (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 10, 1065-1071CrossRefGoogle Scholar
  23. 23.
    Daneholt, B. (1992) The transcribed template and the transcription loop in Balbiani rings. Cell Biol. Int. Rep. 16, 709-715PubMedCrossRefGoogle Scholar
  24. 24.
    Daneholt, B. (2001) Assembly and transport of a premessenger RNP particle. Proc. Natl. Acad. Sci. USA. 98, 7012-7017PubMedCrossRefGoogle Scholar
  25. 25.
    Skoglund, U., Andersson, K., Björkroth, B., Lamb, M.M., and Daneholt, B. (1983) Visualization of the formation and transport of a specific hnRNP particle. Cell, 34, 847-855PubMedCrossRefGoogle Scholar
  26. 26.
    Lönnroth, A., Alexciev, K., Mehlin, H., Wurtz, T., Skoglund, U., and Daneholt, B. (1992) Demonstration of a 7 nm RNP fiber as the basic structural element in a premessenger RNP particle. Exp. Cell Res. 199, 292-296PubMedCrossRefGoogle Scholar
  27. 27.
    Wetterberg, I., Zhao, J., Masich, S., Wieslander, L., and Skoglund, U. (2001) In situ transcription and splicing in the Balbiani ring 3 gene. EMBO J. 20, 2564-2574PubMedCrossRefGoogle Scholar
  28. 28.
    Björk, P., Wetterberg-Strandh, I., Baurén, G., and Wieslander, L. (2006) Chironomus tentans repressor splicing factor represses SR protein function locally on pre-mRNA exons and is displaced at correct splice sites. Mol. Biol. Cel.l 17, 32-42CrossRefGoogle Scholar
  29. 29.
    Wurtz, T., Kiseleva, E., Nacheva, G., Alzhanova-Ericsson, A.T., Rosen, A., and Daneholt, B. (1996) Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles. Mol. Cell. Biol. 16, 1425-1435Google Scholar
  30. 30.
    Sun, X., Zhao, J., Kylberg, K., Soop, T., Palka, K., Sonnhammer, E., Visa, N., AlzhanovaEricsson, A.T., and Daneholt, B. (2004) Conspicuous accumulation of transcription elongator repressor hrp130/CA150 on the intron-rich Balbiani ring 3 gene. Chromosoma 113, 244-257PubMedCrossRefGoogle Scholar
  31. 31.
    Singh, O.P., Visa, N., Wieslander, L., and Daneholt, B. (2006) A specific SR protein binds preferentially to the secretory protein gene transcripts in salivary glands of Chironomus tentans. Chromosoma 115, 449-458PubMedCrossRefGoogle Scholar
  32. 32.
    Visa, N., Izaurralde, E., Ferreira, J., Daneholt, B., and Mattaj, I.W. (1996) A nuclear cap-binding complex binds Balbiani ring pre-mRNA co-transcriptionally and accompanies the ribonucleoprotein particle during nuclear export. J. Cell Biol. 133, 5-14PubMedCrossRefGoogle Scholar
  33. 33.
    Zhao, J., Jin, S.B., Björkroth, B., Wieslander, L., and Daneholt, B. (2002) The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm. EMBO J. 21, 1177-1187PubMedCrossRefGoogle Scholar
  34. 34.
    Alzhanova-Ericsson, A.T., Sun, X., Visa, N., Kiseleva, E., Wurtz, T., and Daneholt, B. (1996) A protein of the SR family of splicing factors binds extensively to exonic Balbiani ring premRNA and accompanies the RNA from the gene to the nuclear pore. Genes Dev. 10, 2881-2893PubMedCrossRefGoogle Scholar
  35. 35.
    Kiseleva, E., Wurtz, T., Visa, N., and Daneholt, B. (1994) Assembly and disassembly of spliceosomes along a specific pre-messenger RNP fiber. EMBO J. 13, 6052-6061PubMedGoogle Scholar
  36. 36.
    Skoglund, U., öfverstedt, L.-G., and Daneholt, B. (1998) Procedures for three-dimensional reconstruction from thin sections with electron tomography. In: RNP particles, splicing and autoimmune diseases, Springer lab manual, J. Schenkel ed., Springer Verlag Berlin, Heidelberg, New York, pp 72-94CrossRefGoogle Scholar
  37. 37.
    Sun, X., Alzhanova-Ericsson, A.T., Visa, N., Aissouni, Y., Zhao, J., and Daneholt, B. (1998) The hrp23 protein in the Balbiani ring pre-mRNP particles is released just before or at the binding to the nuclear pore complex. J. Cell Biol. 142, 1167-1180CrossRefGoogle Scholar
  38. 38.
    Sabri, N. and Visa, N. (2000) The Ct-RAE1 protein interacts with Balbiani ring RNP particles at the nuclear pore. RNA 6, 1597-1609PubMedCrossRefGoogle Scholar
  39. 39.
    Gournemann, J., Kotovic, K.M., Hujer, K., and Neugebauer, K.M. (2005) Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex. Mol. Cell 19, 53-63CrossRefGoogle Scholar
  40. 40.
    Cheng, H., Dufu, K., Lee, C.-S., Hsu, J.L., Dias, A., and Reed, R. (2006) Human mRNA export machinery recruited to the 5 end of mRNA. Cell 127, 1389-1400PubMedCrossRefGoogle Scholar
  41. 41.
    Baurén, G., Jiang, W.O., Bernholm, K., Gu, F., and Wieslander, L. (1996) Demonstration of a dynamic transcription-dependent organization of pre-mRNA splicing factors in polytene nuclei. J. Cell Biol. 135, 929-941CrossRefGoogle Scholar
  42. 42.
    Baurén, G. and Wieslander, L. (1994) Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription. Cell 76, 183-192PubMedCrossRefGoogle Scholar
  43. 43.
    Lambert, B. and Daneholt, B. (1975) Microanalysis of RNA from defined cellular components. Methods Cell Biol. 10, 17-47PubMedCrossRefGoogle Scholar
  44. 44.
    Wetterberg, I., Baurén, G., and Wieslander, L. (1996) The intranuclear site of excision of each intron in the Balbiani ring 3 pre-mRNA is influenced by the time remaining to transcription termination and different excision efficiencies for the various introns. RNA 2, 641-651PubMedGoogle Scholar
  45. 45.
    Bentley, D. (2005) Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr. Opin. Cell Biol. 17, 251-256PubMedCrossRefGoogle Scholar
  46. 46.
    Baurén, G., Belikov, S., and Wieslander, L. (1998) Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3-end formation and excision of the 3-terminal intron. Genes Dev. 12, 2759-2769PubMedCrossRefGoogle Scholar
  47. 47.
    Aguilera, A. (2005) Co-transcriptional mRNP assembly: from the DNA to the nuclear pore. Curr. Opin. Cell Biol. 17, 242-250PubMedCrossRefGoogle Scholar
  48. 48.
    Rodriguez-Navarro, S., Fischer, T., Luo, M.J., Antunez, O., Brettschneider, S., Lechner, J., Perez-Ortin, J.E., Reed, R., and Hurt, E. (2004) Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75-86PubMedCrossRefGoogle Scholar
  49. 49.
    Casolari, J.M., Brown, C.R., Drubin, D.A., Rando, O.J., and Silver, P.A. (2005) Developmentally induced changes in transcriptional program alter spatial organization across chromosomes. Genes Dev. 19, 1188-1198PubMedCrossRefGoogle Scholar
  50. 50.
    Schmid, M., Arib, G., Laemmli, C., Nishikawa, J., Durussel, T., and Laemmli, U. K. (2006) Nup-PI: The nucleopore-promoter interaction of genes in yeast. Mol. Cell 21, 379-391PubMedCrossRefGoogle Scholar
  51. 51.
    Zachar, Z., Kramer, J., Mims, I.P., and Bingham, P.M. (1993) Evidence for channelled diffusion of pre-mRNAs during nuclear RNA transport in metazoans. J. Cell Biol. 121, 729-742PubMedCrossRefGoogle Scholar
  52. 52.
    Singh, O.P., Björkroth, B., Masich, S., Wieslander, L., and Daneholt, B. (1999) The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles. Exp. Cell Res. 25, 135-146CrossRefGoogle Scholar
  53. 53.
    Politz, J.C., Tuft, R.A., Pederson, T., and Singer, R.H. (1999) Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr. Biol. 9, 285-291PubMedCrossRefGoogle Scholar
  54. 54.
    Shav-Tal, Y., Darzacq, X., Shenoy, S.M., Fusco, D., Janicki, M., Spector, D.L., and Singer, R.H. (2004) Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797-1800PubMedCrossRefGoogle Scholar
  55. 55.
    Miralles, F., öfverstedt, L.G., Sabri, N., Aissouni, Y., Hellman, U., Skoglund, U., and Visa, N. (2000) Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm. J. Cell Biol. 148, 271-282PubMedCrossRefGoogle Scholar
  56. 56.
    Vinciguerra, P. and Stutz, F. (2004) mRNA export: an assembly line from genes to nuclear pores. Curr. Opin. Cell Biol. 16, 285-292PubMedCrossRefGoogle Scholar
  57. 57.
    Mehlin, H., Daneholt, B., and Skoglund, U. (1992) Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell 69, 605-613PubMedCrossRefGoogle Scholar
  58. 58.
    Cole, C.N. and Scarcelli, J.J. (2006) Transport of messenger RNA from the nucleus to the cytoplasm. Curr. Opin. Cell Biol. 18, 299-306PubMedCrossRefGoogle Scholar
  59. 59.
    Björk, P., Baurén, G., Gelius, B., Wrange, ö., and Wieslander, L. (2003) The Chironomus tentans translation initiation factor eIF4H is present in the nucleus but does not bind to mRNA until the mRNA reaches the cytoplasm. J. Cell Sci. 116, 4521-4532PubMedCrossRefGoogle Scholar
  60. 60.
    Meyer, B., Mähr, R., Eppenberger, H.M., and Lezzi, M. (1983). The activity of Balbiani rings 1 and 2 in salivary glands of Chironomus tentans larvae under different modes of development and after pilocarpine treatment. Develop. Biol. 98, 265-277PubMedCrossRefGoogle Scholar
  61. 61.
    Wyss, C. (1982) Chironomus tentans epithelial cell line sensitive to ecdysteroids, juvenile hormone, insulin and heat shock. Exp. Cell Res. 139, 309-319PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Petra Björk
    • 1
  • Lars Wieslander
    • 1
  1. 1.Department of Molecular Biology and Functional GenomicsStockholm UniversityStockholmSweden

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