Preparation of Nucleic Acids

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(1986) have published a quick, small-scale method for purifi- cation of DNA ... Lurquin et al. (1975) published a procedure for DNA preparation involv- ... dium iodide (Anet and Strayer, 1969) are superior to cesium chloride and are also more ...
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Phosphatases L o p p e s and Matagne (1973) gave the following methods for assay o f acid and neutral phosphatase activity in Chlamydomonas extracts and in colonies. A sample of 0.2 ml of cell suspension or crude extract (sonicated cells) is mixed with 1.1 ml o f 0.05 M acetate buffer, p H 4.8, and 0.2 ml o f a 2 mg/ml a-naphthylphosphate solution in the same acetate buffer. T h e mixture is incubated for 30 min at 37°C, after which 1 ml of a solution containing 4% SDS and 0.02% tetrazotized-O-dianisidine in acetate buffer is added. Absorption is read at 540 nm against a blank to which substrate is added after incubation. For colony assay, 6-7-day-old plates (2-mm colonies) are replicated onto sterile filter paper. T h e papers are allowed to dry at room temperature before being immersed in a solution containing 1 mg/ml a-naphthylphosphate and 2 mg/ml tetrazotized-O-dianisidine in 0.05 M acetate buffer, p H 4.8. T h e filter papers are then placed on wet paper and incubated for 30 min at 37°C. Colonies with acid phosphatase activity will produce a deep brown-red color on the filter paper. The same solutions can be used to assay for neutral phosphatase activity; in this case the filter papers are incubated for 5 min at room temperature, by which time colonies with neutral phosphatase activity will turn purple ( L o p p e s , 1976). T o assay alkaline phosphatase activity in cell extracts, L o p p e s (1976b) used the same reagents as for acid phosphatase but replaced the acetate buffer with a g l y c i n e - N a O H buffer at p H 9.5. A fluorometric assay for alkaline phosphatase activity using o-methyl fluorescein phosphate was published by Healey and Hendzel (1979).

Preparation of Nucleic Acids Preparation of Nuclear, Chloroplast and Mitochondrial D N A Fractions T h e following procedures are those currently used in our laboratory and are modified from the methods published by Grant et al. (1980). T h e extraction methods given here are satisfactory for wild-type and mutant strains o f C. reinhardtii. F o r an alternative procedure for preparation of chloroplast D N A from cell-wall deficient strains, see Rochaix (1982). W e e k s et al. (1986) have published a quick, small-scale method for purification o f D N A from a variety o f sources, including Chlamydomonas. Lurquin et al. (1975) published a procedure for D N A preparation involving agarose gel filtration at high ionic strength. Rochaix, Lurquin, and W e e k s all used centrifugation on CsCl density gradients to analyze the extracted D N A . F o r separation o f chloroplast and mitochondrial D N A bands from nuclear D N A , w e have found that density gradients o f sodium iodide (Anet and Strayer, 1969) are superior to cesium chloride and are also more economical to use.

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Solutions 1 . T E N buffer: 10 m M Tris-HCl, p H 8.0; 10 m M E D T A ; and 150 m M N a C l . A 10x stock solution should be prepared. 2. 20% ( w / v ) Sodium dodecyl sulfate ( S D S ) (Sequanal grade, Pierce Chemicals, Rockford, I L 61105) in water. This solution is stable at room temperature. 3. 20% ( w / v ) Sarkosyl N L 97 ( K & K Laboratories, I C N Biochemicals, Plainview, N Y 11803) in water. This solution is stable at room temperature. 4. Protease solution: 5 g protease (nonspecific, #165921 from Boehringer Mannheim Biochemicals, Indianapolis, I N 46250), 10 ml o f 1 M Tris-HCl, p H 7.5; 0.11 g o f C a C l 2 ; water to 100 ml. Heat to 80°C for 10 min to eliminate nucleases, then add 100 ml o f glycerol and store in freezer. A precipitate may form, but it can be ignored. 5. Phenol-CIA: U s e 35 ml o f phenol (redistilled and stored frozen until immediately before use) for each sample o f 6 liters o f Chlamydomonas culture. A d d to an equal volume o f T E N buffer in a separatory funnel, shake, and collect the lower (phenol) phase. Measure the volume o f this phase and add an equal volume o f C I A (chloroform : isoamyl alcohol 24:1 v / v ) . Swirl vigorously before use. 6. Ethidium bromide: 20 mg/ml in water. 7. Sodium iodide (Fisher Chemicals #S-324): prepare a saturated solution in T E N buffer, adding a pinch o f sodium bisulfite to decolorize and prevent further oxidation. This solution should have a den3 sity o f 1.9 g / c m at 25°C. It should be stored in a dark place. Isolation procedure Cells are harvested in logarithmic growth phase using large centrifuge bottles or a continuous flow apparatus (see p. 590). T h e procedure as written here assumes a starting culture volume o f 6 liters, at a cell 6 density in the range o f 2-4 x 10 /ml. Resuspend the pelleted cells in T E N buffer, wash by centrifugation, and resuspend again in T E N to a total volume o f 50 ml in a glass roller bottle in which a Teflon vane has been inserted ( F i g . 12.2). Slowly add 5 ml o f 20% S D S , 5 ml o f 20% sarkosyl, and 4 ml o f protease solution. W e find that leakage from these bottles can be prevented by placing a Teflon liner inside the cap and wrapping the bottle threads with plumber's Teflon tape before capping. T h e bottle is then put on a rotator (e.g., Bel-Art Vial Rotator, model 78, from TechniLab Instruments, Pequannock, Ν J 07440) for 24 hr at 4°C. A d d 65 ml phenol-CIA to the roller bottle. Rotate at room temperature for 1-2 hr. Transfer to a plastic 250-ml centrifuge bottle and centrifuge for 10 min at 15,000 g. Collect the aqueous (upper) phase with a plastic syringe and large bore (13-ga) needle and transfer it to a clean

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Figure 12.2. Roller bottles for detergent and phenol extraction of Chlamydomonas D N A . A vane is cut from 2-mm Teflon sheeting and inserted into a screw-cap bottle (Fisher #02892R, 250 ml). The screw threads on the bottle top are wrapped with 3.5-mil Teflon plumber's tape to minimize leakage.

plastic centrifuge bottle. T r y not to contaminate this with any o f the interphase material. W e find it is worthwhile to reextract the interface material with 10-20 ml more phenol-CIA for 30 min. After centrifugation in a 45-ml plastic tube, the upper phase from this second extraction is added to the previous upper phase. A d d t w o volumes o f cold 95% ethanol and put the sample in a - 2 0 ° C freezer overnight for precipitation. Collect the precipitated D N A by centrifugation for 20 min at 15,000 g. Invert the centrifuge bottle and drain the pellet for 5 min to r e m o v e ethanol; wipe off any excess E t O H around the shoulder o f the inverted bottle. Dissolve the pellet in 15 ml o f 10 m M T r i s - H C l , p H 7.5. A d d the following solutions: 2.0 ml o f 10x T E N ; 2.5 ml of 10 m M T r i s - H C l , p H 7.5, containing 0.050 ml ethidium bromide stock (20 m g / m l ) ; and 30.0 ml of saturated N a l in T E N . Invert the bottle several times to mix thoroughly. The refractive index o f the final mixture should be 1.4350. Transfer the dissolved D N A to the appropriate number o f ultracentrifuge tubes (Beckman #340382 polycarbonate " b o t t l e s , " 25 ml capacity)

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and balance the tubes with a T r i s - T E N - N a l - E B stock made according to the proportions given above. Centrifuge to equilibrium (44 hr at 44,000 rpm and 20°C in Beckman 50.2 T i , 60 T i , or 70 T i rotors; the 60 T i and 70 Ti can be run on an L8-60 or L8-70 centrifuge in slow acceleration mode with the brake on, whereas the 50.2 Ti can be run on an L2-65B centrifuge with the brake off). After centrifugation, the D N A bands can be visualized by long-wave U V light. T h e bottom and brightest band, which should be approximately in the center of the gradient, is nuclear D N A (density 1.723 3 g / c m ) . T h e second-brightest, topmost band is chloroplast D N A (1.696 3 g / c m ) . Between these bands t w o fainter bands may be seen. These are mitochondrial D N A and the nuclear r D N A satellite (densities 1.707 and 3 1.712 g / c m respectively). Collect the D N A fractions from the top o f the tube, using 13-ga needles and plastic syringes. R e m o v e the needle and transfer the D N A from the syringe to plastic tubes. A t this point the D N A can be extracted with isoamyl alcohol and precipitated with ethanol if desired (see b e l o w ) . H o w e v e r , w e routinely subject the D N A to a second round o f centrifugation by diluting each sample with T r i s - T E N N a l - E B stock solution to a volume of about 9.5 ml to fill a small centrifuge tube (Beckman #339573 polycarbonate " b o t t l e s , " to be run on Beckman 50 Ti or 70.1 Ti rotors). This second centrifugation is highly desirable for isolation o f chloroplast D N A bands and is essential for purification o f mitochondrial D N A . After the second round o f centrifugation, transfer the D N A samples to plastic tubes and extract them with one to t w o volumes o f isoamyl alcohol, three or more times, to remove the ethidium bromide. T h e top (pink-colored) layer should be discarded at each extraction. Then carefully remove the aqueous (lower) layer from the tube with a plastic syringe and 13-ga needle, leaving behind the interphase material. Transfer the D N A to a clean plastic tube and precipitate with t w o volumes o f cold 95% ethanol at - 2 0 ° C . Centrifuge the ethanol-precipitated D N A at 15,000 g for 15 min, discard the supernatant fluid, and wash the pelleted D N A once or twice with cold 95% ethanol to remove any residual N a l . Resuspend the D N A in sterile 10 m M Tris-HCl, p H 7.5. The volume to be used depends on the starting cell number; for chloroplast D N A , w e normally estimate this 8 at 1 μΐ Tris-HCl for 2 x 10 cells, or about 60 μΐ for a 6-liter culture at a 6 concentration o f 2 χ 10 cells/ml. Store the dissolved D N A at 4°C. D N A prepared in this manner usually can be cut without any problem by a wide variety o f restriction endonucleases.

Rapid Preparation of Whole-Cell D N A from Small Cultures of Chlamydomonas Harvest cells from a log phase 300-ml culture for 5 min at 10,000 g. Resuspend cells in 40 ml o f T E N buffer (see a b o v e ) and transfer to a 45-ml plastic centrifuge tube. Centrifuge 5 min at 10,000 g. Resuspend

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the cells in 5 ml o f T E N buffer and transfer to a plastic scintillation vial. A d d slowly, with stirring: 0.5 ml o f 20% S D S , 0.5 ml o f 20% sarkosyl, and 0.4 ml o f protease solution. Put the vial on a rotator for 24 hr at 4°C. A d d 13 ml o f phenol-CIA to each sample, and put on a rotator at room temperature for 30 min. Transfer to a 45-ml plastic centrifuge tube and centrifuge, preferably in a swinging bucket rotor, for 15 min at 15,000 g. Using a 5-ml plastic syringe and 13-ga needle, remove the upper phase from each sample. Dialyze this fraction overnight at 4°C against 10 m M Tris-HCl, p H 8.0; and 10 m M E D T A . Use 1-2 liters o f buffer per sample. Transfer the samples to graduated plastic tubes. A d d 0.1 volume o f 3 M ammonium acetate, 0.1 volume o f 0.5 M magnesium chloride, and 2 volumes o f cold ( - 2 0 ° C ) ethanol. Precipitate 30-60 min at - 2 0 ° C . Pellet the D N A by centrifugation in a plastic tube for 15 min at 15,000 g and 4°C. Decant the supernatant fluid and drain the pellet at room temperature for 10 min. Flush the tube with N 2 to remove residual ethanol. Resuspend the pellet in sterile 10 m M Tris-HCl, p H 7.5, to the 8 equivalent o f 7 x 10 cells/ml and transfer to plastic tubes or vials for storage. D N A prepared in this way will be cut by most, but not all, restriction endonucleases. I f partial digests are obtained with such enzymes as EcoRl or BamHl, the D N A should be reprecipitated with ethanol and dissolved again.

Preparation of R N A Total Cattolico (1978a) gave the following procedure for isolation o f total R N A from Chlamydomonas cells: After harvesting cells, resuspend at a 7 concentration o f 3.5 x 10 /ml in 25 m M Tris-HCl, p H 7.6; 25 m M M g C l 2 ; and 25 m M K C l at 5°C. Break cells by sonication for 3 x 15 sec and add sodium dodecyl sulfate to a final concentration of 0.8%. Extract for 20 min at 10°C with a 1.5x volume of redistilled phenol saturated with the above buffer by rapid shaking on a rotary shaker. R e c o v e r the supernatant fraction by centrifugation for 15 min at 7000 rpm in a Sorvall HB-4 swinging bucket rotor or equivalent. Reextract twice, using a 1: 1 ratio o f phenol : lysate. Extract three times with diethyl ether and precipitate R N A with t w o volumes o f 95% E t O H at - 2 0 ° C . Electrophoretic procedures for separation o f high molecular weight ( r R N A ) fractions were given by Cattolico and Jones (1972, 1975). +

Separation of Poly(A) and Poly(A)~ RNA Schmidt et al. (1984a) separated polyadenylated and nonpolyadenylated m R N A fractions by the following procedure, based on methods for higher plants published by Cashmore (1982). A l l steps should be carried out under sterile conditions. Cells from 6-liter log-phase cultures are harvested by centrifugation, 8 resuspended at a concentration o f 2 x 10 /ml in lysis buffer (50 m M TrisH C l , p H 7.5; 150 m M N a C l ; 15 m M E D T A ; 2% S D S ; 40 /xg/ml Protein-

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ase K ) , and gently shaken at room temperature for 20 min. R N A is extracted twice for 20 min at room temperature with an equal volume o f phenol (previously equilibrated with lysis buffer lacking Proteinase K ) : chloroform : isoamyl alcohol ( 5 0 : 5 0 : 1 ) and precipitated overnight at - 2 0 ° C with t w o volumes o f cold E t O H . T h e R N A pellet is resuspended in sterile H 2 0 and precipitated twice with t w o volumes o f E t O H at - 2 0 ° C , once with an equal volume o f 4 M L i C l at 0°C, and again with t w o volumes o f E t O H at - 2 0 ° C . Precipitated R N A is pelleted by centrifugation at 10,000 g for 10 min at 2°C, dried under vacuum, and resuspended in 10 mM Tris-HCl, p H 7.4; 5 mM E D T A ; 0.2% SDS to a concentration that shows absorbance o f 50 A 2 6 0/ m l or less. T o this + solution is added l/10th volume o f 4 M N a C l . T h e p o l y ( A ) R N A is separated from p o l y ( A ) ~ R N A by affinity chromatography on poly(U)Sepharose-4B in 10 mM Tris-HCl, p H 7.4; 5 mM E D T A ; 0.4 M N a C l ; 0.2% S D S . T h e p o l y ( A ) " R N A effluent is collected and precipi+ tated with t w o volumes o f E t O H at - 2 0 ° C . Bound p o l y ( A ) R N A is eluted with the column equilibration buffer without N a C l but containing 90% deionized formamide, diluted with an equal volume o f sterile H 2 0 , + brought to 0.4 M N a by addition o f 4 M N a C l and precipitated with ethanol. Determination

of DNA and RNA

Concentration

Reaction with diphenylamine is probably the most widely used method of determining D N A content o f Chlamydomonas cells, although many persons prefer other assays, notably the ones using diaminobenzoic acid (see b e l o w ) . T h e following procedure was adapted by J. Kates from that published by Burton (1956, 1968).

Diphenylamine Assay 8

A log-phase culture o f Chlamydomonas ( ~ 5 x 10 cells) is harvested by centrifugation and extracted with 80% acetone until all pigment has been removed. T h e pellet is suspended in 2.0 ml o f 0.3 M ice-cold perchloric acid and incubated on ice for 30 min, then extracted twice with 0.5 M perchloric acid at 70°C for 30 min, using 1.5 ml o f acid for the first extraction and 0.7 ml for the second. T h e supernatant fractions from these extractions are combined. Diphenylamine reagent is prepared from 1.5 g o f diphenylamine (which should be an off-white color, not pink; it can be recrystallized if necessary from hexane), 100 ml o f glacial acetic acid, and 1.5 ml o f concentrated sulfuric acid. This solution is stable for several months at 4°C. Just before use, 0.1 ml o f a solution o f 1.6% aqueous acetaldehyde is added per 20 ml o f reagent. A 2.0 ml aliquot o f the combined perchloric acid extracts is added to 4.0 ml o f diphenylamine reagent. Standards are prepared using 4.0 ml o f diphenylamine reagent and 2.0 ml o f 0.5 M perchloric acid containing 50 μg/m\ and 25 μg/ml deoxyadenosine, heated at 70°C for 25 min. Standard D N A solutions can also be used. Blank samples o f perchloric acid and diphe-

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nylamine reagent are also prepared. All samples are incubate( for 20 hr before reading absorption at 595 nm. A modification o f the diphenylamine assay that omits acet; and sulfuric acid and uses light activation (exposure to fluoresce for 4-6 hr at 40°C), followed by measurement o f the absorbant ence between 600 and 700 nm, has been published by Zachled*

3,5-Diaminobenzoic Acid Assay A n alternative D N A assay which is highly specific and very se fluorometric analysis using 3,5-diaminobenzoic acid ( D A B A (Cattolico and Gibbs, 1975; Lien and Knutsen, 1976a; Cattolicc A D A B A stock solution is prepared by dissolving 0.6 g in 2.0 r HCl at 5°C. This solution is transferred to a 12-ml conical centril containing 20 mg o f charcoal and is mixed with the charcoal by it into and out o f a Pasteur pipette. After centrifugation in a centrifuge (5 min at 2900 rpm), the solution is transferred t containing fresh charcoal and the extraction process repeated, as seven times, until a clear yellow solution is obtained (Catt( Gibbs, 1975). In the D N A assay method published by Cattolico (1978a), culture is pelleted in a conical test tube, rinsed with 0.5 ml ο medium, and transferred to a 6 x 50-mm Siliclad-coated centrifi The sample is extracted with 80% acetone to remove pigmei transferred to Metricel Alpha-6 cellulose filters (25 mm c Gelman Instrument C o . , Ann Arbor, M I 48106) and washed tv 0.15 ml o f 0.6 Ν trichloroacetic acid at 5°C, twice with 0. ethanol : water ( 2 : 1 v / v ) at 5°C, and twice with ethanol : water A vacuum flask system for washing the filters was illustrated t lico and Gibbs (1975). The filters are dried in polyethylene ca 20°C before being saturated with D A B A solution and incubate« for 30 min. After the samples have been cooled for 5 min temperature, 0.5 ml of 1 Ν H C l is added to each filter sample cence is read with excitation at 405 nm and emission at 520 ni

Ethidium Bromide Assay Valle et al. (1981) adapted a D N A - R N A determination based dium bromide fluorescence for use on Chlamydomonas cells, 5 7 samples o f cells at densities of 5 x 10 —1.5 x 10 cells/ml, 0.1 rr the following solutions is added: (1) sodium AMauryl sarcosine in distilled H 2 0 ; (2) 0.3 M E D T A , adjusted to p H 7.0 with N a O I N a O H . T h e mixture is stirred and incubated at room tempérât min (1 min is sufficient for cell wall-deficient cells). This pr which lyses cells without R N A breakdown, is used for determi total D N A plus R N A . T o digest R N A , similarly prepared sampl incubated at 60°C for 45 min. After incubation, both sets o f sar neutralized with 0.1 ml of 3 TV H C l , followed by 0.3 ml of 1.2 sium phosphate buffer, p H 6.8, and 2.1 ml of distilled water.

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Fluorescence is assayed by excitation at 530 nm and measurement o f emission at 590 nm. A blank solution o f 3.8 ml is prepared, to which 0.2 ml o f ethidium bromide (50 μ g / m l ) in water is added. Standard D N A solutions in the range o f 0.2-10 ^ g / m l and R N A at 2-50 μg/ml should also be prepared and tested. The 3.8-ml cell samples are measured for background fluorescence (from chlorophyll) before addition o f 0.2 ml ethidium bromide solution. After reading fluorescence o f these samples, standard D N A solutions (0.2 ml = 4 μ g ) should be added to the samples and fluorescence measured again to give an internal standard for quench correction. Valle et al. have provided equations for calculation o f the quench-corrected fluorescence and final D N A and R N A content.

Colony Hybridization The following procedure for colony hybridization o f Chlamydomonas cells was contributed by Stefan Surzycki. Cells should be replica-plated (Chapter 2) to Whatman #541 paper. The filter is washed with 60-70% methanol or acetone or with 100% chloroform to remove pigments and lipids and then dried. The filter should be layered on a wet plate with 10 μg/ml proteinase K , 50 m M E D T A , and incubated for 1.5 hr at 37°C. (Chloroplast D N A may require a longer incubation period.) From this point, the filter is treated as for bacterial colony hybridization (see Maniatis et al., 1982, or other manuals on techniques o f molecular biology).

Techniques for Studying Protein Synthesis Preparation of Cytoplasmic and Chloroplast Ribosomes from Chlamydomonas Cells Mets (1982) has published a detailed procedure for separation o f ribosomal subunits from both cytoplasmic and chloroplast ribosomes on a Beckman 15 Ti zonal rotor. Although this apparatus produces excellent separation o f ribosomal particles, w e have found that ribosome isolation on linear sucrose gradients in standard swinging bucket rotors is quite satisfactory for most purposes. A s shown in Table 12.3 and Figure 12.3, the basic protocol given below can be adapted for analytical profiles o f whole cell ribosome complements (Harris et al., 1974) and for preparation o f chloroplast or cytoplasmic monomers and subunits for protein synthesis assays (Bartlett et al., 1979). Cells are grown in liquid culture (Chapter 2) to a density o f about 5 x 6 10 cells/ml and are harvested by centrifugation. T h e y are rinsed by centrifugation and then resuspended in T K M buffer (25 m M Tris-HCl, 2+ p H 7.8; 25 m M M g acetate or M g C l 2 ; 25 m M K C l ; 5 m M glutathione) at an appropriate concentration (Table 12.3) and are broken by t w o passages through a French press at 5000 psi. The French press exudate is centrifuged for 30 min at 40,000 g at 4°C, and the supernatant fraction