Summary
Using an in vitro chromatin assembly system, we analyzed the influence of DNA superhelicity on the development of transcriptionally active minichromosomes. Plasmid DNA molecules containing either a Xenopus borealis 5S RNA gene or an X. laevis methionine tRNA gene were utilized as templates for the assembly of chromatin. Both plasmids were processed into active minichromosomes if introduced as supercoiled molecules into the extract (S150). The degree of superhelicity is a determining factor in the assembly of active chromatin. Molecules containing varying superhelical densities were processed into minichromosomes with different transcriptional activities. The absence of supercoils leads to the assembly of chromatin with substantially lower transcriptional activity. Assembled minichromosomes are stable enough to be isolated by sucrose gradient centrifugation while retaining their transcriptional phenotype. The formation of nucleosomes with a periodic spacing occurred with the same efficiency and to the same degree regardless of the initial DNA topology. Hence, a determining factor in the development of transcriptionally active chromatin may be the initial superhelicity of the DNA molecule to which activator (trans-acting factors) or repressor (histones) proteins bind. Once the chromatin assembly process has begun, the transcriptional activity of the resulting minichromosome may already have been determined.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ambrose C, McLaughlin R, Bina M (1987) The flexibility and topology of simian virus 40 DNA in minichromosomes. Nucleic Acids Res 15:3703–3721
Brown DD (1984) The role of stable complexes that repress and activate eukaryotic genes. Cell 37:359–365
Choder M, Aloni Y (1988) In vitro transcribed SV40 minichromosomes, as the bulk minichromosomes, have a low level of unconstrained negative supercoils. Nucleic Acids Res 16:895–905
Cozzarelli NR, Gerrard SP, Schlissel M, Brown DD, Bogenhagen DF (1985) Purified RNA polymerase III accurately and efficiently terminates transcription of 5S RNA genes. Cell 34:829–835
Dezelee S, Sentenac A, Fromageot P (1974) Role of deoxyribonucleic acid-ribonucleic acid hybrids in eukaryotes. J Biol Chem 249:5978–5983
Dunn T, Hahn S, Schleif R (1984) An operator at -280 that is required for repression of araBAD. Proc Natl Acad Sci USA 81:5017–5020
Engelke DR, Ng S-Y, Shastry BS, Roeder RG (1980) Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes. Cell 19:717–728
Garguilo G, Razvi F, Worcel A (1985) Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors. Cell 38: 511–521
Garner MM, Felsenfeld G, O'Dea MH, Gellert M (1986) Effects of DNA supercoiling on the topological properties of nucleosomes. Proc Natl Acad Sci USA 84:2620–2623
Glikin GC, Ruberti I, Worcel A (1984) Chromatin assembly in Xenopus oocytes: in vitro studies. Cell 37:33–41
Gottesfeld J, Bloomer LS (1982) Assembly of transcriptionally active 5S RNA gene chromatin in vitro. Cell 28:781–791
Han S, Udvardy A, Schedl P (1985) Novobiocin blocks the Drosophila heat shock response. J Mol Biol 183:13–29
Harland RM, Weintraub H, McKnight SL (1983) Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature 302:38–43
Huang D-H, Roeder RG (1988) Delineation of DNA sequences that are important for in vitro transcription from the adenovirus EIIa late promoter. Mol Cell Biol 8:1906–1914
Kmiec EB, Worcel A (1985) The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in Xenopus oocyte extracts. Cell 41:945–953
Kmiec EB, Razvi F, Worcel A (1986) The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes. Cell 45:209–218
Mizushima-Sugano J, Roeder RG (1986) Cell-specific transcription of an immunoglobulin K light chain gene in vitro. Proc Natl Acad Sci USA 83:8511–8515
Peterson DO, Beifuss KK, Morley KL (1987) Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes. Mol Cell Biol 7:1563–1567
Peterson RC, Doering JL, Brown DD (1980) Characterization of two Xenopus somatic 55 DNAs and one minor oocyte-specific 5S DNA. Cell 20:131–141
Petryniak B, Lutter LC (1987) Topological characterization of simian virus 40 transcription complex. Cell 48:289–295
Pruitt SC, Reeder RH (1984) Effect of topological constraint on transcription of ribosomal DNA in Xenopus oocytes. J Mol Biol 174:121–139
Razvi F, Garguilo G, Worcel A (1983) A simple procedure for parallel sequence analysis of both strands of 5′-labeled DNA. Gene 23:175–183
Reeves R (1984) Transcriptionally active chromatin. Biochem Biophys Acta 782:343–393
Ruberti I, Worcel A (1986) Mechanism of chromatin assembly in Xenopus oocytes. J Mol Biol 189:457–476
Ryoji M, Worcel A (1984) Chromatin assembly in Xenopus oocytes: in vivo studies. Cell 37:21–32
Sekiguchi JM, Swank RA, Kmiec EB (1989) Changes in DNA topology can modulate in vitro transcription of certain RNA polymerase III genes. Mol Cell Biochem 85:123–133
Simpson RT, Thoma F, Brubaker JM (1985) Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histories: a model system for the study of higher order structure. Cell 42:799–808
Sinden RR, Carlson JO, Pettijohn DE (1980) Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell 21:773–783
Singleton CK, Wells RD (1982) The facile generation of covalently closed, circular DNAs with defined negative superhelical densities. Anal Biochem 122:253–257
Tabuchi H, Hirose S (1988) DNA supercoiling facilitates the formation of the transcription initiation complex on the fibroin gene promoter. J Biol Chem 263:15282–15287
Wang JC (1985) DNA topoisomerases. Annu Rev Biochem 54:665–697
Weintraub H (1985) Assembly and propagation of repressed and derepressed chromosomal states. Cell 42:705–711
Weintraub H, Chang PF, Conrad K (1986) Expression of transfected DNA depends on DNA topology. Cell 46:115–122
Weischet WO, Glotov BO, Schnell H, Zachau HG (1982) Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei. Nucleic Acids Res 10:3627–3645
Workman JL, Roeder RG (1987) Binding of transcription factor TFIID to the major late promoter during in vitro nucleosome assembly potentiates subsequent initiation by RNA polymerase II. Cell 51:613–622
Author information
Authors and Affiliations
Additional information
Communicated by M. Sekiguchi
Rights and permissions
About this article
Cite this article
Sekiguchi, J.M., Kmiec, E.B. DNA superhelicity enhances the assembly of transcriptionally active chromatin in vitro. Molec. Gen. Genet. 220, 73–80 (1989). https://doi.org/10.1007/BF00260859
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00260859