In Vitro Transcription

Rohinton T. Kamakaka1, W. Lee Kraus2

1 NICHD, Bethesda, Maryland, 2 Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.6
DOI:  10.1002/0471143030.cb1106s02
Online Posting Date:  May, 2001
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Abstract

In vitro analysis of transcription and the factors that play a role in transcription require preparation of an extract that faithfully reproduces in vivo transcription. This unit describes protocols for generating extracts of mammalian cells and Drosophila embryos that are competent for in vitro transcription reactions using naked DNA or chromatin templates and for primer extension analysis of transcripts.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Transcription Reactions with Nuclear Extracts
  • Support Protocol 1: Preparation of a Nuclear Extract from HeLa Cells
  • Support Protocol 2: Preparation of High‐Salt Drosophila Extracts
  • Alternate Protocol 1: Preparation of the Soluble Nuclear Fraction from Isolated Drosophila Embryo Nuclei
  • Support Protocol 3: Primer Extension Analyses of In Vitro Transcription Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Transcription Reactions with Nuclear Extracts

  Materials
  • Nuclear extract (see protocol 2, protocol 3, or protocol 4)
  • 10% (w/v) polyvinyl alcohol (PvOH; mol. wt. 10,000; Sigma)
  • 10% (w/v) polyethylene glycol (PEG; mol. wt. 15,000 to 20,000)
  • 1 M HEPES (potassium salt), pH 7.6 and 8.0, adjusted with KOH
  • 200 ng/µl plasmid DNA template in TE buffer, pH 8.0 ( appendix 2A)
  • 5 mM ribonucleotide‐5′‐triphosphates (rNTPs; see recipe)
  • Transcription stop solution (see recipe)
  • 0.3 M sodium acetate
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (PCIAA), equilibrated with 10 mM Tris⋅Cl, pH 7.5
  • 75% and 100% ethanol
  • 30° and 37°C water baths

Support Protocol 1: Preparation of a Nuclear Extract from HeLa Cells

  Materials
  • ∼5–8 × 105 cell/ml suspension culture of HeLa cells grown in spinner flasks
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Freezing buffer (see recipe)
  • Glycerol
  • PBS ( appendix 2A containing 1 gm/litre MgCl 2
  • Hypotonic buffer (see recipe)
  • High‐salt buffer (see recipe)
  • Powdered ammonium sulfate
  • TM buffer (see recipe) with and without 0.1 M KCl
  • Beckman JS‐4.2 and SW‐28 and Sorvall GSA and SS‐34 rotors (or equivalents) and appropriate centrifuge bottles and tubes
  • 40‐ml Wheaton Dounce homogenizer with loose (B) pestle (or equivalent)
  • Insulated magnetic stir plate
  • Coffee bean grinder or mortar and pestle
  • Dialysis tubing (12,000 to 14,000 MWCO)
  • Conductivity meter (optional)
  • Additional reagents and equipment for dialysis ( appendix 3A)
NOTE: Perform all procedures at 4°C (i.e., on ice and in a cold room) using precooled solutions, glassware, and equipment. Perform all centrifugations at 4°C with precooled rotors. All low‐speed centrifugations are done in a low‐speed centrifuge (e.g., Sorvall RC‐5) while high‐speed centrifugations are done in an ultracentrifuge.

Support Protocol 2: Preparation of High‐Salt Drosophila Extracts

  Materials
  • Drosophila cultures
  • Molasses/agar plates (see recipe)
  • Bleach (5.25% sodium hypochlorite) diluted 1:1 in distilled H 2O (store at room temperature)
  • Embryo wash solution: 0.7% (w/v) NaCl/0.04% (v/v) Triton X‐100 (store at room temperature)
  • Disruption buffer (see recipe)
  • Resuspension buffer (see recipe)
  • 4 M ammonium sulfate, pH 7.0
  • Solid ammonium sulfate
  • HEMG containing 0.1 M KCl (see recipe)
  • Embryo collection apparatus (construct according to Fig. ) and paint brush
  • Nylon mesh (Tetko, #3‐70/43 and 3‐500/49) and interlocking rings for constructing embryo collection apparatus
  • Yamato LH‐21 homogenizer (Thomas)
  • 4 pieces of Miracloth (9 in. × 9 in.; Calbiochem)
  • GSA, 45‐Ti, and SS‐34 rotors (or equivalents) and appropriate centrifuge bottles and tubes
  • 40‐ml Wheaton Dounce homogenizer with a B pestle and 15‐ml homogenizer with A pestle
  • Rotating mixer
  • Coffee grinder
  • 5 × 22–cm G‐25 SF desalting column (430 ml; Pharmacia Biotech)
  • Conductivity meter
  • Additional reagents and equipment for growing Drosophila (Goldstein and Fyrberg, )
NOTE: Perform all procedures at 4°C (i.e., on ice and in a cold room) using precooled solutions, glassware, and equipment. Perform all centrifugations at 4°C with precooled rotors.NOTE: The use of frozen embryos or embryos stored >3 days results in transcription extracts of a very poor quality.

Alternate Protocol 1: Preparation of the Soluble Nuclear Fraction from Isolated Drosophila Embryo Nuclei

  • HEMG20 containing 0.1 M KCl (see recipe)
  • Ultracentrifuge with SW‐28 rotor

Support Protocol 3: Primer Extension Analyses of In Vitro Transcription Products

  Materials
  • ∼25‐base DNA oligonucleotide complimentary to the RNA products from the in vitro transcription reactions
  • T4 polynucleotide kinase
  • 150 µCi/µl [γ‐32P]ATP (7000 Ci/mmol; ICN Biomedicals)
  • 2.5 M ammonium acetate
  • 10 mg/ml glycogen
  • 100% ethanol
  • 1× TE buffer, pH 8.0 ( appendix 2A)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (PCIAA), equilibrated with Tris⋅Cl, pH 7.5
  • 3 M sodium acetate
  • 5× annealing buffer (see recipe)
  • RNA product (e.g., from protocol 1)
  • Primer extension mix (see recipe)
  • 50 U/µl Moloney murine leukemia virus (MMLV) reverse transcriptase
  • Formamide loading buffer (FLB; see recipe)
  • 8% urea‐polyacrylamide sequencing gel
  • 1× TBE buffer ( appendix 2A)
  • 37°, 58°, 70°C, and boiling water baths
  • Whatman 3MM filter paper
  • Additional reagents and equipment for primer synthesis, agarose gel electrophoresis, DNA elution, ethanol precipitation, DNA quantification, primer labeling ( appendix 3A), and phosphorimaging or autoradiography (unit 6.3)
NOTE: All reagents used for this protocol should be high‐quality molecular biology reagents. All procedures should be performed at room temperature unless otherwise noted. All microcentrifugations are done at maximum speed.CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out experiment and dispose of waste in appropriately designated areas, following the guidelines provided by the local radiation safety officer (also see appendix 1D).
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Figures

  •   FigureFigure 11.6.1 The embryo collection apparatus consists of three interlocking rings, which hold two nylon meshes of different porosity. The coarse mesh (Tetko 3‐500/49) is placed between the upper and middle cylinders to collect any dead flies and large particulate material. The fine mesh (Tetko 3‐70/43) is placed between the middle and lower cylinders to collect the embryos but allow the yeast to flow through. The dimensions of this apparatus can vary; 3‐in. high, 10‐in. diameter segments work well.

Videos

Literature Cited

Literature Cited
   Biggin, M.D. and Tjian, R. 1988. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell 53:699‐711.
   Croston, G.E., Kerrigan, L.A., Lira, L., Marshak, D.R. and Kadonaga, J.T. 1991. Sequence‐specific antirepression of histone H1‐mediated inhibition of basal RNA polymerase II transcription. Science 251:643‐649.
   Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 11:1475‐1489.
   Goldstein, L.S.B. and Fyrberg, E.A. 1994. Drosophila melanogaster: Practical uses in cell and molecular biology. Methods Cell Biol. Vol. 44.
   Heiermann, R. and Pongs, O. 1985. In vitro transcription with extracts of nuclei of Drosophila embryos. Nucl. Acids Res. 13:2709‐2730.
   Kamakaka, R.T., Tyree, C.M. and Kadonaga, J.T. 1991. Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos. Proc. Natl. Acad. Sci. U.S.A. 88:1024‐1028.
   Kamakaka, R.T., Bulger, M., and Kadonaga, J.T. 1993. Potentiation of RNA polymerase II transcription by Gal4‐VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 7:1779‐1795.
   Lue, N.F. and Kornberg, R.D. 1987. Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 84:8839‐8843.
   Manley, J.L., Fire, A., Cano, A., Sharp, P.A. and Gefter, M.L. 1980. DNA‐dependent transcription of adenovirus genes in a soluble whole‐cell extract. Proc. Natl. Acad. Sci. U.S.A. 77:3855‐3859.
   Parker, C.S. and Topol, J. 1984. A Drosophila RNA polymerase II transcription factor contains a promoter‐region‐specific DNA‐binding activity. Cell 36:357‐369.
   Soeller, W.C., Poole, S.J. and Kornberg, T. 1988. In vitro transcription of the Drosophila engrailed gene. Genes Dev. 2:68‐81.
   Woontner, M. and Jaehning, J.A. 1990. Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae. J. Biol. Chem. 265:8979‐8982.
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