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The Laboratory of Jim Kadonaga at the University of California, San Diego
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| This protocol describes how to produce a soluble nuclear extract rich in basal pol II transcription factors from Drosophila embryos. This is a cell-free extract that contains all the necessary transcription factors and is capable of accurate initiation of transcription by RNA polymerase II but is deficient in core histones and histone H1. Thus, it can be used in a transcription assay with in vitro assembled chromatin (see Protocol on Transcription of Chromatin Reconstituted with Drosophila S-190 Chromatin Assembly Extract and Primer Extension Analysis). Transcribing RNA in vitro using reconstituted chromatin allows for the biochemical analysis of the molecular mechanisms controlling gene expression. This is also a simple and fast procedure; the extract preparation takes about 2.5 hr. |
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1. Grow Drosophila melanogaster in population cages (see Hint #1). Harvest the embryos from 0 to 12 hr after fertilization by washing the trays over a collection apparatus that consists of two Nitex nylon screens (Tetko). The top 500 μm screen retains the adult flies while the lower 70 μm finer screen retains the embryos and allows the yeast to flow through. Wash the embryos extensively with water to remove all traces of yeast. Store harvested embryos at 4°C before use. Harvest embryos for up to three days (72 hr) in successive collections (see Hint #2). Typically 100 to 150 grams of embryos will yield approximately 30 to 50 ml of Soluble Nuclear Extract (see Hint #3).
2. Dechorionate the embryos by immersion for 90 seconds in Bleach Solution at room temperature.
3. Quickly rinse the embryos with 1 liter of Embryo Wash Solution and then rinse thoroughly with running water at room temperature.
4. Dry the embryos with paper towels by placing the paper towels under the fine nylon mesh of the embryo collection apparatus to absorb the excess water.
NOTE: all subsequent steps should be performed as quickly as possible at 4°C.
5. Transfer the embryos into a chilled, preweighed 800 ml beaker and determine the mass of the embryos.
6. Combine 3 ml of cold (4°C) Buffer I per gram of embryos and disperse the embryos in the Buffer with a glass rod.
7. Disrupt the embryos with a single passage through the Yamato LH-21 homogenizer at 1,000 rpm (setting = 100). If a Yamato LH-21 homogenizer is not available, homogenize the embryos with 6 to 8 strokes in a motorized Potter-Elvehjem homogenizer with a serrated Teflon pestle and a glass vessel. This apparatus often does not yield complete disruption of the embryos, however, and it may be necessary to carry out further homogenization by using a 40 ml Wheaton Dounce homogenizer with a B pestle.
8. Filter the homogenate through a single layer of Miracloth (Calbiochem) into a Sorvall GSA centrifuge bottle.
9. Wash the Miracloth with additional 2 ml of Buffer I per gram embryos. Add that wash to the centrifuge bottle. The final volume of Buffer I should be about 5 ml/g embryos (see Hint #4).
10. Pellet nuclei by centrifugation in a Sorvall GSA rotor at 8,000 rpm (10,400 X g) at 4°C for 15 min.
11. Carefully decant the supernatant; the pellet is very loose. Wipe off the lipids from the walls of the bottle with laboratory tissue. Discard the supernatant.
12. The nuclei appear as a loose, tan-colored pellet. At the very bottom of the bottle is a small yellow yolk pellet. Suspend the nuclei gently by swirling in 3 ml of Buffer I per gram embryos. Do not suspend the yellow yolk pellet.
13. Use a 40 ml Wheaton Dounce homogenizer with a loose B pestle to disperse the nuclei with a single stroke of the pestle.
14. Transfer the homogenate to clean GSA centrifuge bottles.
15. Centrifuge in a Sorvall GSA rotor at 8,000 rpm (10,400 X g) at 4°C for 15 min to again pellet the nuclei.
16. Once again carefully decant the supernatant and wipe off the lipids from the walls of the bottle with Kimwipe tissues.
17. Resuspend the nuclei gently by swirling in 1 ml of Buffer AB per gram embryos. Use a 40 ml Wheaton Dounce homogenizer with a loose B pestle to disperse the nuclei with two to three strokes of the pestle.
18. Transfer the supernatant to one preweighed GSA centrifuge bottle.
19. Centrifuge in a Sorvall GSA rotor at 8,000 rpm (10,400 X g) at 4°C for 10 min.
20. Decant the supernatant, and weigh the bottle with the pellet. Determine the mass of the nuclei in the bottle.
21. Add 0.5 ml HEMG With 0.1 M KCl per g nuclei (see Hint #5). Suspend the nuclei by gentle swirling and shaking. Do not use the Dounce homogenizer.
22. Place the nuclear suspension on ice for at least 15 min and up to 60 min with occasional swirling and shaking.
23. Centrifuge in a Beckman SW28 rotor at 24,000 rpm (100,000 X g) for 1 hr at 4°C (see Hint #6).
24. After centrifugation is complete, there will be four distinct layers in the centrifuge tubes. From the top to bottom, they are as follows:
a. A thin grey-white lipid layer. Remove it with a spatula and discard.
b. A yellow liquid layer, which comprises about 50 to 60% of the total volume of the tube - this is the Soluble Nuclear Extract. Remove it with a pipet. Quick-freeze the Soluble Nuclear Extract in Liquid Nitrogen, and store at -80°C.
c. A grey layer. Avoid this material, because it will inhibit transcription.
d. At the bottom, a solid off-white layer composed of DNA and nuclear debris.
25. Determine the protein concentration of the extract with a Coomassie blue protein assay (Pierce) using Bovine Serum Albumin as the reference. The protein concentration of the Soluble Nuclear Extract prepared with KCl is usually about 7 to 10 mg/ml, whereas the protein concentration of the Soluble Nuclear Extract that is prepared with Potassium Glutamate is about 30 mg/ml.
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| HEMG With 0.4 M Potassium Glutamate |
| 0.1 mM EDTA
0.4 M Potassium Glutamate (L-glutamic acid, monopotassium salt; Sigma)
25 mM Hepes-KOH, pH 7.6
1 mM Benzamidine-HCl
Just before use, add the following:
1.5 mM DTT
12.5 mM MgCl2
20% (v/v) Glycerol
1 mM Sodium Metabisulfite
0.1 mM PMSF
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| HEMG With 0.1 M KCl |
| 0.1 mM EDTA
1 mM Benzamidine-HCl
25 mM Hepes-KOH, pH 7.6
Just before use, add the following:
0.1 M KCl
1.5 mM DTT
12.5 mM MgCl2
20% (v/v) Glycerol
0.1 mM PMSF
1 mM Sodium Metabisulfite
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| Buffer AB |
| 0.2 mM PMSF
0.1 mM EDTA
15 mM HEPES-KOH, pH 7.6
5 mM MgCl2
1 mM Benzamidine-HCl
Just before use, add the following:
110 mM KCl
2 mM DTT
1 mM Sodium Metabisulfite
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| Buffer I |
| 0.2 mM PMSF
1 mM DTT
0.1 mM EDTA
15 mM HEPES-KOH, pH 7.6
10 mM KCl
5 mM MgCl2
1 mM Benzamidine-HCl
Just before use, add the following:
350 mM Sucrose
0.5 mM EGTA
1 mM Sodium Metabisulfite
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| 0.5 M Benzamidine-HCl |
| Store at -20°C.
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| 0.2 M PMSF |
| Prepare in 100% Ethanol
Store at -20°C
0.2 M Phenylmethylsulfonyl Fluoride (PMSF; CAUTION! see Hint #7)
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| 0.5 M Sodium Metabisulfite |
| Prepare fresh, store at 4°C, and use within 10 hr.
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| 0.5 M DTT |
| Store at -20°C.
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| Embryo Wash Solution |
| 0.7% (w/v) NaCl
0.04% (v/v) Triton X-100
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| Bleach Solution |
| 50% (v/v) Household Bleach (CAUTION! see Hint #7)
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Potassium Chloride
Sucrose
Sodium Chloride
EGTA
Potassium Glutamate
Glycerol
Potassium Hydroxide
Phenylmethylsulfonyl Fluoride (PMSF)
Triton X-100
Bleach, Household
Magnesium Chloride
HEPES
Nitrogen, Liquid
Sodium Metabisulfite
DTT
Bovine Serum Albumin (BSA)
EDTA
Benzamidine-HCl
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1. The contributors of this protocol use wild-type Canton S flies.
2. The length of storage at 4°C, which is from 0 to 72 hr, does not appear to affect the properties of the Soluble Nuclear Extract.
3. This protocols works wells for amounts of less than 0.5 grams embryos to more than 150 grams.
4. Once the embryos are homogenized, work quickly. Proteins can leak out of the nuclei into the Buffer and be lost in the subsequent centrifugation step.
5. Alternatively, HEMG With 0.4 M Potassium Glutamate Buffer may be used, as described originally in Citation #2. At present, however, the contributors prefer to use 0.1 M KCl instead of 0.4 M Potassium Glutamate. The use of 0.1 M KCl instead of 0.4 M Potassium Glutamate results in less extraction of nonspecific DNA binding proteins, such as Histone H1.
6. A fixed-angle rotor is not recommended for this step.
7. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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1. Kamakaka RT and Kadonaga JT. The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos. Methods in Cell Biology. 1994; 44:225-235.
2. Kamakaka RT, Tree CM, Kadonaga JT. Accurate and efficient RNA polymerase II transcription with a soluble nuclear faction derived from Drosophila embryos. Proc. Natl. Acad. Sci. USA. 1991; 88:1024-1028.
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