Skip to main content
Genetics logoLink to Genetics
. 2002 Feb;160(2):727–740. doi: 10.1093/genetics/160.2.727

Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco.

Ludmila Mlynárová 1, Annelies Loonen 1, Elzbieta Mietkiewska 1, Ritsert C Jansen 1, Jan-Peter Nap 1
PMCID: PMC1461960  PMID: 11861574

Abstract

The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum). In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants. The A element thus creates an artificial chromatin domain that yields coordinated expression. Surprisingly, enzyme activities correlated poorly with their respective mRNA levels. We hypothesize that this indicates the occurrence of "error pipelines" in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations. In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked. Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.

Full Text

The Full Text of this article is available as a PDF (412.3 KB).

Selected References

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

  1. Bode J., Stengert-Iber M., Kay V., Schlake T., Dietz-Pfeilstetter A. Scaffold/matrix-attached regions: topological switches with multiple regulatory functions. Crit Rev Eukaryot Gene Expr. 1996;6(2-3):115–138. doi: 10.1615/critreveukargeneexpr.v6.i2-3.20. [DOI] [PubMed] [Google Scholar]
  2. Breyne P., van Montagu M., Depicker N., Gheysen G. Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell. 1992 Apr;4(4):463–471. doi: 10.1105/tpc.4.4.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen L., Marmey P., Taylor N. J., Brizard J. P., Espinoza C., D'Cruz P., Huet H., Zhang S., de Kochko A., Beachy R. N. Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol. 1998 Nov;16(11):1060–1064. doi: 10.1038/3511. [DOI] [PubMed] [Google Scholar]
  4. Cohen B. A., Mitra R. D., Hughes J. D., Church G. M. A computational analysis of whole-genome expression data reveals chromosomal domains of gene expression. Nat Genet. 2000 Oct;26(2):183–186. doi: 10.1038/79896. [DOI] [PubMed] [Google Scholar]
  5. Dasgupta S., Collins G. B., Hunt A. G. Co-ordinated expression of multiple enzymes in different subcellular compartments in plants. Plant J. 1998 Oct;16(1):107–116. doi: 10.1046/j.1365-313x.1998.00255.x. [DOI] [PubMed] [Google Scholar]
  6. Dean C., Favreau M., Tamaki S., Bond-Nutter D., Dunsmuir P., Bedbrook J. Expression of tandem gene fusions in transgenic tobacco plants. Nucleic Acids Res. 1988 Aug 11;16(15):7601–7617. doi: 10.1093/nar/16.15.7601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dean C., Jones J., Favreau M., Dunsmuir P., Bedbrook J. Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res. 1988 Oct 11;16(19):9267–9283. doi: 10.1093/nar/16.19.9267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elmayan T., Tepfer M. Synthesis of a bifunctional metallothionein/beta-glucuronidase fusion protein in transgenic tobacco plants as a means of reducing leaf cadmium levels. Plant J. 1994 Sep;6(3):433–440. doi: 10.1046/j.1365-313x.1994.06030433.x. [DOI] [PubMed] [Google Scholar]
  9. Forrester W. C., van Genderen C., Jenuwein T., Grosschedl R. Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions. Science. 1994 Aug 26;265(5176):1221–1225. doi: 10.1126/science.8066460. [DOI] [PubMed] [Google Scholar]
  10. Gygi S. P., Rochon Y., Franza B. R., Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 1999 Mar;19(3):1720–1730. doi: 10.1128/mcb.19.3.1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halpin C., Cooke S. E., Barakate A., El Amrani A., Ryan M. D. Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants. Plant J. 1999 Feb;17(4):453–459. doi: 10.1046/j.1365-313x.1999.00394.x. [DOI] [PubMed] [Google Scholar]
  12. Iida S., Mittelsten Scheid O., Saul M. W., Seipel K., Miyazaki C., Potrykus I. Expression of a downstream gene from a bicistronic transcription unit in transgenic tobacco plants. Gene. 1992 Oct 1;119(2):199–205. doi: 10.1016/0378-1119(92)90272-q. [DOI] [PubMed] [Google Scholar]
  13. Jach G., Görnhardt B., Mundy J., Logemann J., Pinsdorf E., Leah R., Schell J., Maas C. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J. 1995 Jul;8(1):97–109. doi: 10.1046/j.1365-313x.1995.08010097.x. [DOI] [PubMed] [Google Scholar]
  14. Jenuwein T., Forrester W. C., Fernández-Herrero L. A., Laible G., Dull M., Grosschedl R. Extension of chromatin accessibility by nuclear matrix attachment regions. Nature. 1997 Jan 16;385(6613):269–272. doi: 10.1038/385269a0. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K., Käs E., Poljak L., Adachi Y. Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev. 1992 Apr;2(2):275–285. doi: 10.1016/s0959-437x(05)80285-0. [DOI] [PubMed] [Google Scholar]
  16. Leech M. J., May K., Hallard D., Verpoorte R., De Luca V., Christou P. Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation. Plant Mol Biol. 1998 Nov;38(5):765–774. doi: 10.1023/a:1006000229229. [DOI] [PubMed] [Google Scholar]
  17. Ma J. K., Hiatt A., Hein M., Vine N. D., Wang F., Stabila P., van Dolleweerd C., Mostov K., Lehner T. Generation and assembly of secretory antibodies in plants. Science. 1995 May 5;268(5211):716–719. doi: 10.1126/science.7732380. [DOI] [PubMed] [Google Scholar]
  18. Marcos J. F., Beachy R. N. In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide. Plant Mol Biol. 1994 Feb;24(3):495–503. doi: 10.1007/BF00024117. [DOI] [PubMed] [Google Scholar]
  19. Marcos J. F., Beachy R. N. Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide. J Gen Virol. 1997 Jul;78(Pt 7):1771–1778. doi: 10.1099/0022-1317-78-7-1771. [DOI] [PubMed] [Google Scholar]
  20. Mlynarova L., Jansen R. C., Conner A. J., Stiekema W. J., Nap J. P. The MAR-Mediated Reduction in Position Effect Can Be Uncoupled from Copy Number-Dependent Expression in Transgenic Plants. Plant Cell. 1995 May;7(5):599–609. doi: 10.1105/tpc.7.5.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mlynarova L., Keizer LCP., Stiekema W. J., Nap J. P. Approaching the Lower Limits of Transgene Variability. Plant Cell. 1996 Sep;8(9):1589–1599. doi: 10.1105/tpc.8.9.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mlynarova L., Loonen A., Heldens J., Jansen R. C., Keizer P., Stiekema W. J., Nap J. P. Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region. Plant Cell. 1994 Mar;6(3):417–426. doi: 10.1105/tpc.6.3.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Muskens M. W., Vissers A. P., Mol J. N., Kooter J. M. Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing. Plant Mol Biol. 2000 Jun;43(2-3):243–260. doi: 10.1023/a:1006491613768. [DOI] [PubMed] [Google Scholar]
  24. Nap J. P., Conner A. J., Mlynárová L., Stiekema W. J., Jansen R. C. Dissection of a synthesized quantitative trait to characterize transgene interactions. Genetics. 1997 Sep;147(1):315–320. doi: 10.1093/genetics/147.1.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nap J. P., Dirkse W. G., Louwerse J., Onstenk J., Visser R., Loonen A., Heidekamp F., Stiekema W. J. Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf. Plant Mol Biol. 1992 Nov;20(4):683–694. doi: 10.1007/BF00046453. [DOI] [PubMed] [Google Scholar]
  26. Nap J. P., van Spanje M., Dirkse W. G., Baarda G., Mlynarova L., Loonen A., Grondhuis P., Stiekema W. J. Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of Photosystem I, in transgenic potato and tobacco plants. Plant Mol Biol. 1993 Nov;23(3):605–612. doi: 10.1007/BF00019307. [DOI] [PubMed] [Google Scholar]
  27. Nawrath C., Poirier Y., Somerville C. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12760–12764. doi: 10.1073/pnas.91.26.12760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nayler O., Strätling W., Bourquin J. P., Stagljar I., Lindemann L., Jasper H., Hartmann A. M., Fackelmayer F. O., Ullrich A., Stamm S. SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements. Nucleic Acids Res. 1998 Aug 1;26(15):3542–3549. doi: 10.1093/nar/26.15.3542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peach C., Velten J. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol. 1991 Jul;17(1):49–60. doi: 10.1007/BF00036805. [DOI] [PubMed] [Google Scholar]
  30. Poirier Y., Dennis D. E., Klomparens K., Somerville C. Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science. 1992 Apr 24;256(5056):520–523. doi: 10.1126/science.256.5056.520. [DOI] [PubMed] [Google Scholar]
  31. Poljak L., Seum C., Mattioni T., Laemmli U. K. SARs stimulate but do not confer position independent gene expression. Nucleic Acids Res. 1994 Oct 25;22(21):4386–4394. doi: 10.1093/nar/22.21.4386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Razin S. V., Gromova I. I., Iarovaia O. V. Specificity and functional significance of DNA interaction with the nuclear matrix: new approaches to clarify the old questions. Int Rev Cytol. 1995;162B:405–448. doi: 10.1016/s0074-7696(08)62623-6. [DOI] [PubMed] [Google Scholar]
  33. Scheres B. Non-linear signaling for pattern formation? Curr Opin Plant Biol. 2000 Oct;3(5):412–417. doi: 10.1016/s1369-5266(00)00105-9. [DOI] [PubMed] [Google Scholar]
  34. Strätling W. H., Yu F. Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP. Crit Rev Eukaryot Gene Expr. 1999;9(3-4):311–318. doi: 10.1615/critreveukargeneexpr.v9.i3-4.150. [DOI] [PubMed] [Google Scholar]
  35. Thompson J. F., Hayes L. S., Lloyd D. B. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene. 1991 Jul 22;103(2):171–177. doi: 10.1016/0378-1119(91)90270-l. [DOI] [PubMed] [Google Scholar]
  36. Vaucheret H., Elmayan T., Thierry D., van der Geest A., Hall T., Conner A. J., Mlynarova L., Nap J. P. Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants. Mol Gen Genet. 1998 Sep;259(4):388–392. doi: 10.1007/s004380050827. [DOI] [PubMed] [Google Scholar]
  37. Ye X., Al-Babili S., Klöti A., Zhang J., Lucca P., Beyer P., Potrykus I. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science. 2000 Jan 14;287(5451):303–305. doi: 10.1126/science.287.5451.303. [DOI] [PubMed] [Google Scholar]
  38. Zhao K., Käs E., Gonzalez E., Laemmli U. K. SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin. EMBO J. 1993 Aug;12(8):3237–3247. doi: 10.1002/j.1460-2075.1993.tb05993.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van Engelen F. A., Molthoff J. W., Conner A. J., Nap J. P., Pereira A., Stiekema W. J. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 1995 Jul;4(4):288–290. doi: 10.1007/BF01969123. [DOI] [PubMed] [Google Scholar]
  40. van Engelen F. A., Schouten A., Molthoff J. W., Roosien J., Salinas J., Dirkse W. G., Schots A., Bakker J., Gommers F. J., Jongsma M. A. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Mol Biol. 1994 Dec;26(6):1701–1710. doi: 10.1007/BF00019485. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES