Skip to main content
NCBI home page
The Plant Cell logoLink to The Plant Cell
. 1996 May;8(5):899–913. doi: 10.1105/tpc.8.5.899

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

G C Allen 1, G Hall Jr 1, S Michalowski 1, W Newman 1, S Spiker 1, A K Weissinger 1, W F Thompson 1
PMCID: PMC161147  PMID: 8672887

Abstract

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Allen G. C., Hall G. E., Jr, Childs L. C., Weissinger A. K., Spiker S., Thompson W. F. Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell. 1993 Jun;5(6):603–613. doi: 10.1105/tpc.5.6.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. An G. Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol. 1986 May;81(1):86–91. doi: 10.1104/pp.81.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Archer T. K., Lefebvre P., Wolford R. G., Hager G. L. Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science. 1992 Mar 20;255(5051):1573–1576. doi: 10.1126/science.1347958. [DOI] [PubMed] [Google Scholar]
  4. Barry C., Faugeron G., Rossignol J. L. Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4557–4561. doi: 10.1073/pnas.90.10.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benfey P. N., Chua N. H. Regulated genes in transgenic plants. Science. 1989 Apr 14;244(4901):174–181. doi: 10.1126/science.244.4901.174. [DOI] [PubMed] [Google Scholar]
  6. Bonifer C., Hecht A., Saueressig H., Winter D. M., Sippel A. E. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci. J Cell Biochem. 1991 Oct;47(2):99–108. doi: 10.1002/jcb.240470203. [DOI] [PubMed] [Google Scholar]
  7. Boulikas T. Nature of DNA sequences at the attachment regions of genes to the nuclear matrix. J Cell Biochem. 1993 May;52(1):14–22. doi: 10.1002/jcb.240520104. [DOI] [PubMed] [Google Scholar]
  8. Breyne P., Van Montagu M., Gheysen G. The role of scaffold attachment regions in the structural and functional organization of plant chromatin. Transgenic Res. 1994 May;3(3):195–202. doi: 10.1007/BF01973987. [DOI] [PubMed] [Google Scholar]
  9. Butler P. J., Thomas J. O. Changes in chromatin folding in solution. J Mol Biol. 1980 Jul 15;140(4):505–529. doi: 10.1016/0022-2836(80)90268-5. [DOI] [PubMed] [Google Scholar]
  10. Camerini-Otero R. D., Hsieh P. Parallel DNA triplexes, homologous recombination, and other homology-dependent DNA interactions. Cell. 1993 Apr 23;73(2):217–223. doi: 10.1016/0092-8674(93)90224-e. [DOI] [PubMed] [Google Scholar]
  11. Conkling M. A., Cheng C. L., Yamamoto Y. T., Goodman H. M. Isolation of transcriptionally regulated root-specific genes from tobacco. Plant Physiol. 1990 Jul;93(3):1203–1211. doi: 10.1104/pp.93.3.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cook P. R. The nucleoskeleton and the topology of replication. Cell. 1991 Aug 23;66(4):627–635. doi: 10.1016/0092-8674(91)90109-c. [DOI] [PubMed] [Google Scholar]
  13. Davey M. R., Rech E. L., Mulligan B. J. Direct DNA transfer to plant cells. Plant Mol Biol. 1989 Sep;13(3):273–285. doi: 10.1007/BF00025315. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Dickinson L. A., Joh T., Kohwi Y., Kohwi-Shigematsu T. A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition. Cell. 1992 Aug 21;70(4):631–645. doi: 10.1016/0092-8674(92)90432-c. [DOI] [PubMed] [Google Scholar]
  16. Dorer D. R., Henikoff S. Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell. 1994 Jul 1;77(7):993–1002. doi: 10.1016/0092-8674(94)90439-1. [DOI] [PubMed] [Google Scholar]
  17. Eissenberg J. C., Elgin S. C. Boundary functions in the control of gene expression. Trends Genet. 1991 Oct;7(10):335–340. doi: 10.1016/0168-9525(91)90424-o. [DOI] [PubMed] [Google Scholar]
  18. Elgin S. C. On the importance of taking a firm position. Chromatin structure and gene expression sponsored by Fundación Juan March, Madrid, Spain September 24-26, 1990. New Biol. 1991 Jan;3(1):37–41. [PubMed] [Google Scholar]
  19. Elton T. S., Reeves R. Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I. Anal Biochem. 1986 Aug 15;157(1):53–62. doi: 10.1016/0003-2697(86)90195-8. [DOI] [PubMed] [Google Scholar]
  20. Gasser S. M., Amati B. B., Cardenas M. E., Hofmann J. F. Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol. 1989;119:57–96. doi: 10.1016/s0074-7696(08)60649-x. [DOI] [PubMed] [Google Scholar]
  21. Gasser S. M., Laemmli U. K. The organisation of chromatin loops: characterization of a scaffold attachment site. EMBO J. 1986 Mar;5(3):511–518. doi: 10.1002/j.1460-2075.1986.tb04240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Getzenberg R. H., Pienta K. J., Ward W. S., Coffey D. S. Nuclear structure and the three-dimensional organization of DNA. J Cell Biochem. 1991 Dec;47(4):289–299. doi: 10.1002/jcb.240470402. [DOI] [PubMed] [Google Scholar]
  23. Grosveld F., van Assendelft G. B., Greaves D. R., Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987 Dec 24;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
  24. Haber J. E., Leung W. Y., Borts R. H., Lichten M. The frequency of meiotic recombination in yeast is independent of the number and position of homologous donor sequences: implications for chromosome pairing. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1120–1124. doi: 10.1073/pnas.88.4.1120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hall G., Jr, Allen G. C., Loer D. S., Thompson W. F., Spiker S. Nuclear scaffolds and scaffold-attachment regions in higher plants. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9320–9324. doi: 10.1073/pnas.88.20.9320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hobbs S. L., Kpodar P., DeLong C. M. The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol. 1990 Dec;15(6):851–864. doi: 10.1007/BF00039425. [DOI] [PubMed] [Google Scholar]
  27. Hobbs S. L., Warkentin T. D., DeLong C. M. Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol. 1993 Jan;21(1):17–26. doi: 10.1007/BF00039614. [DOI] [PubMed] [Google Scholar]
  28. Jackson D. A. Structure-function relationships in eukaryotic nuclei. Bioessays. 1991 Jan;13(1):1–10. doi: 10.1002/bies.950130102. [DOI] [PubMed] [Google Scholar]
  29. Jefferson R. A., Kavanagh T. A., Bevan M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987 Dec 20;6(13):3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kalos M., Fournier R. E. Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol Cell Biol. 1995 Jan;15(1):198–207. doi: 10.1128/mcb.15.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Matzke M. A., Matzke A. J. Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol. 1991 May;16(5):821–830. doi: 10.1007/BF00015074. [DOI] [PubMed] [Google Scholar]
  32. Matzke M. A., Matzke A. J. Homology-dependent gene silencing in transgenic plants: what does it really tell us? Trends Genet. 1995 Jan;11(1):1–3. doi: 10.1016/s0168-9525(00)88973-8. [DOI] [PubMed] [Google Scholar]
  33. Matzke M. A., Neuhuber F., Matzke A. J. A variety of epistatic interactions can occur between partially homologous transgene loci brought together by sexual crossing. Mol Gen Genet. 1993 Jan;236(2-3):379–386. doi: 10.1007/BF00277137. [DOI] [PubMed] [Google Scholar]
  34. Matzke M. A., Primig M., Trnovsky J., Matzke A. J. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 1989 Mar;8(3):643–649. doi: 10.1002/j.1460-2075.1989.tb03421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McKnight R. A., Shamay A., Sankaran L., Wall R. J., Hennighausen L. Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6943–6947. doi: 10.1073/pnas.89.15.6943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mielke C., Kohwi Y., Kohwi-Shigematsu T., Bode J. Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo. Biochemistry. 1990 Aug 14;29(32):7475–7485. doi: 10.1021/bi00484a017. [DOI] [PubMed] [Google Scholar]
  37. Mittelsten Scheid O., Paszkowski J., Potrykus I. Reversible inactivation of a transgene in Arabidopsis thaliana. Mol Gen Genet. 1991 Aug;228(1-2):104–112. doi: 10.1007/BF00282454. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Peng J., Wen F., Lister R. L., Hodges T. K. Inheritance of gusA and neo genes in transgenic rice. Plant Mol Biol. 1995 Jan;27(1):91–104. doi: 10.1007/BF00019181. [DOI] [PubMed] [Google Scholar]
  40. Phi-Van L., von Kries J. P., Ostertag W., Strätling W. H. The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. 1990 May;10(5):2302–2307. doi: 10.1128/mcb.10.5.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pienta K. J., Getzenberg R. H., Coffey D. S. Cell structure and DNA organization. Crit Rev Eukaryot Gene Expr. 1991;1(4):355–385. [PubMed] [Google Scholar]
  42. 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]
  43. Schöffl F., Schröder G., Kliem M., Rieping M. An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants. Transgenic Res. 1993 Mar;2(2):93–100. doi: 10.1007/BF01969382. [DOI] [PubMed] [Google Scholar]
  44. Selker E. U. DNA methylation and chromatin structure: a view from below. Trends Biochem Sci. 1990 Mar;15(3):103–107. doi: 10.1016/0968-0004(90)90193-f. [DOI] [PubMed] [Google Scholar]
  45. Selker E. U., Fritz D. Y., Singer M. J. Dense nonsymmetrical DNA methylation resulting from repeat-induced point mutation in Neurospora. Science. 1993 Dec 10;262(5140):1724–1728. doi: 10.1126/science.8259516. [DOI] [PubMed] [Google Scholar]
  46. Selker E. U. Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet. 1990;24:579–613. doi: 10.1146/annurev.ge.24.120190.003051. [DOI] [PubMed] [Google Scholar]
  47. Spiker S., Thompson W. F. Nuclear Matrix Attachment Regions and Transgene Expression in Plants. Plant Physiol. 1996 Jan;110(1):15–21. doi: 10.1104/pp.110.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stief A., Winter D. M., Strätling W. H., Sippel A. E. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature. 1989 Sep 28;341(6240):343–345. doi: 10.1038/341343a0. [DOI] [PubMed] [Google Scholar]
  49. Thompson E. M., Christians E., Stinnakre M. G., Renard J. P. Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues. Mol Cell Biol. 1994 Jul;14(7):4694–4703. doi: 10.1128/mcb.14.7.4694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tomes D. T., Weissinger A. K., Ross M., Higgins R., Drummond B. J., Schaaf S., Malone-Schoneberg J., Staebell M., Flynn P., Anderson J. Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves. Plant Mol Biol. 1990 Feb;14(2):261–268. doi: 10.1007/BF00018566. [DOI] [PubMed] [Google Scholar]
  51. Wan Y., Lemaux P. G. Generation of Large Numbers of Independently Transformed Fertile Barley Plants. Plant Physiol. 1994 Jan;104(1):37–48. doi: 10.1104/pp.104.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Zlatanova J., Van Holde K. Histone H1 and transcription: still an enigma? J Cell Sci. 1992 Dec;103(Pt 4):889–895. doi: 10.1242/jcs.103.4.889. [DOI] [PubMed] [Google Scholar]
  54. van der Krol A. R., Mur L. A., Beld M., Mol J. N., Stuitje A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell. 1990 Apr;2(4):291–299. doi: 10.1105/tpc.2.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES