| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a B.M. Sutherland, Biology Dep., Brookhaven National Laboratory, Upton, NY 11973-5000
b Kyoto Univ., Gokanoshi, Uji, Kyoto 611-0011, Japan
c Institute of Genetic Ecology, Tohoku Univ., Sendai, Japan
d Dep. of Pathology, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115
e Dep. of Natural Resource Science and Landscape Architecture, Univ. of Maryland, College Park MD 20742
f Argonne National Laboratory, Argonne, IL 60439
g Dep. of Biology, Toho Univ. School of Medicine, Tokyo, Japan
Corresponding author (bms{at}bnl.gov)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Abbreviations: CPD, cyclobutyl pyrimidine dimers • kb, kilobase • LN2, liquid nitrogen • Mb, megabase • UV, ultraviolet radiation • UVA, 320–400 nm • UVB, 290–320 nm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
In this method, the number of lesions per DNA molecule is measured, and thus use of DNA molecules with high single strand lengths allows higher sensitivity in terms of damages per base. For damages affecting only one DNA strand, such as UV-induced cyclobutyl pyrimidine dimers (CPD), the frequency of lesions per single stranded DNA molecule is determined. Therefore, high sensitivity measurements of CPD require DNAs with high single strand molecular lengths. Most methods for extraction of plant DNAs, especially chemical extractions, yield DNA that contains sufficient levels of single strand breaks (not revealed by electrophoresis of double-stranded DNAs on neutral gels) to limit severely its use in measuring low damage levels.
To facilitate damage analysis in a variety of economically significant plants, we have developed a plant DNA isolation method that minimizes induction of single strand breaks (as assessed by the appearance of high single strand molecular length DNAs on denaturing gels). We show that such DNA is readily digestible by a lesion-recognizing endonuclease. This procedure can be readily scaled up to large numbers of samples, allows rapid harvest by immersion in liquid nitrogen (LN2), is amenable to storage between harvest and further processing, and avoids the use of toxic chemicals. Although optimized principally for production of enzymatically digestible, high (single-strand) molecular length DNA for studies of damage and repair in plants, yields are sufficient (
1–2 µg per sample, e.g., 16 first true leaves of alfalfa) to make it useful for other genomic studies.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Plant Material
Unless otherwise described, all plants were grown in an environmental chamber [20°C, 16-h photoperiod; cool white fluorescent bulbs (Sylvania/GTE, Danvers, MA) filtered by UF4 plexiglas (Rohm and Haas, Philadelphia, PA) to exclude wavelengths <400 nm]. The intensity of photosynthetically active radiation in the chamber, measured with an LI-250 light meter (LI-COR Inc., Lincoln, NE), was 140 to 230 µmol m-2 s-1 at the level of the leaves of the various plants.
Alfalfa seed was germinated on Whatman #1 filter paper, and grown for 2 wk in the environmental chamber described above. After irradiation of the seedlings, 16 first leaves were processed per sample.
Soybean (cv. Forrest) seed was germinated on Whatman #1 filter paper in petri dishes with sterile double-distilled water in the dark at room temperature for 3 d until the roots were 2 to 3 cm long, then transplanted into pots with a 5:1:0.5 mixture of Custom blend 90-1 (Grace Sierra Co., Millipilas, CA): sand: vermiculite (Schundler Co., Metuchen, NJ). Pots were placed in a shallow dish of water, providing a constant water supply; seedlings were grown for 10 to14 d in the chamber. Six 1-cm punches from the fully expanded first true leaves were harvested per sample.
Rice seedlings (cv. Sasanishiki) were grown on plastic net floating on tap water (pH 5.0–5.5) in the chamber. Three fully expanded third leaves of 12-d-old seedlings were harvested per sample.
Sorghum seed (cv. Acme Broom corn) was soaked for 48 h in 20°C running water, and sown in pots containing a 5:1:1:0.25 mixture of Pro-Mix BX (Premier Horticulture Inc., Red Hill, PA): sand: Perlite (Schundler Co., Metuchen NJ): Sierra 17-6-12 (Grace Sierra Co., Millipilas, CA). Dark seedlings were grown for 3 d at 25°C in the dark to a height of 7 cm; seven (3-cm) first internodes were used per sample. Seedlings were grown under light in the environmental chamber for 3 to 7 d; two leaves per plant were harvested (first leaves of 3-d-old seedlings, the first and second leaves of 4-d and the first, second, and third leaves of 7-d plants).
Spinach seedlings (cv. 242) were grown in the chamber for 2 wk in pots containing the same mixture as sorghum. Two cotyledons or one first leaf were harvested per sample.
Pea seedlings (cv. Alaska) were grown in a greenhouse for 2 wk in the same mixture as sorghum, and 1 g of first-fourth leaves was used in initial development of DNA isolation methodology.
For field growth, soybean seed (cv. Essex), provided by Dr. William Kenworthy, Department of Natural Resource Sciences and Landscape Architecture, University of Maryland, was sown in late May in rows spaced 0.4 m apart at a density of 30 seeds/m of row in 4.5- by 2.5-m plots. One week after germination, plants were thinned for uniformity in growth to 15 plants/m of row. The third true leaves of 5- to 6-wk-old seedlings were harvested (four 1-cm-diam punches per sample).
UV Irradiation of Rice Seedlings
Laboratory-grown rice seedlings were irradiated with Westinghouse FS20 lamps (primarily UVB radiation, 290–320 nm) to produce initial CPD levels of 10 to 60 sites/Mb (Mb, megabase, 1 million bases). UVB was monitored with a Jagger meter (Jagger, 1961) calibrated vs. a YSI radiometer (Yellow Springs Instruments, Yellow Springs, OH). Subsequent manipulations were carried out in dim yellow light (General Electric, 25W; 
> 500 nm) to minimize uncontrolled photoreactivation. The top 4 cm of each leaf was harvested immediately after irradiation by immersion in LN2, then stored in LN2 until processed as described above. At each dose point, at least three independent gels were used to determine the dimer frequencies; the results from all gels at each dose point were averaged and the standard error of the mean computed.
Field Sunlight and UV Exposure
Field-grown soybean was exposed to natural (College Park, Maryland, early July) sunlight supplemented by UVA radiation from Q-Panel UVB-313 sunlamps (Sullivan and Teramura, 1992) suspended above and perpendicular to the plant rows, filtered with 0.13-mm polyester that absorbs almost all radiation below 316 nm. Spectral irradiances were determined with a Model 742 spectroradiometer (Optronic Laboratories Inc., Orlando, FL). Plants received ambient levels of UV-B radiation, reduced by 10% by the suspended lamps and filters. The height of lamps was adjusted weekly to maintain a distance of 75 cm to the tops of the plants.
Processing Plant Material
Harvesting and processing is described in detail for alfalfa, and variations optimized for other plants as noted. DNA was obtained from plants that were not exposed to UV, with the following exceptions: pea seedlings from the greenhouse, soybean leaves from field-grown plants, and rice seedlings exposed to UV in the laboratory for measurement of pyrimidine dimer induction. Unirradiated or UV-exposed tissues (immersed in LN2 immediately after irradiation) were harvested identically: Sixteen first true leaves were placed in aluminum foil envelopes prechilled in LN2. The tissue was then kept immersed in LN2 until processed further. Plant material was poured into a 5-cm (ID), LN2-chilled ceramic mortar; after the LN2 evaporated, it was ground lightly in the dry mortar 45 s (alfalfa, rice, soybean, 45 s; sorghum and spinach, 10 s) reducing the sample to a fine powder. Samples were concentrated by adding LN2 and swirling. Immediately after the LN2 evaporated, the powdered plant material was transferred by means of an LN2-chilled spatula to a droplet of Lysis buffer 1 [10 mM Tris-HCl, pH 8, 0.83 M EDTA, 2% (w/v) sarcosyl, 13% (w/v) mannitol, 1 mg/mL proteinase K (Boehringer Mannheim, Indianapolis, IN); rice, 150 µL; all others, 300 µL] in a plastic dish (model 171099, Nalge Nunc, Naperville IL), kept tilted to minimize buffer spreading. [Modifications to Lysis buffer tested individually for increasing DNA size included 100 mM Tris; 5% sarcosyl; 10% (v/v) dimethyl sulfoxide (DMSO); 10% DMSO plus 0.1% (v/v) 8-hydroxyquinoline; 10% DMSO plus 0.1% (v/v) 
-tocopherol; and 0.1 M ethyleneglycol-bis-(ß-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA).] The dish was then placed immediately in a desiccator, and vacuum applied for 1 min.
Agarose Plug Preparation
The vacuum-infused plant slurry was mixed with an equal volume of agarose solution [2% (w/v) InCert or SeaPlaque agarose (FMC, Rockland ME) in 10 mM Tris-HCl, pH 8, 1 mM EDTA, boiled for 10 min and held at 95°C], and the suspension was pipetted into prechilled molds [(LADD Industries, Burlington VT, cat. no 21775) or GeNunc Modules (Nalge Nunc, cat. no. 2-32549)] and placed on ice. After plugs solidified, they were placed in 2 mL Lysis Buffer 2 [10 mM Tris-HCl, pH 8, 0.5 M EDTA, 2% sarcosyl, 1 mg/mL proteinase K (Boehringer Mannheim)] and digested at 42°C for 24 h without shaking. The solution was replaced with 2 mL of the same solution (rice, sorghum, spinach), or, for alfalfa and soybean, freshly prepared Lysis Buffer 3 (10 mM Tris, pH 7.2, 0.1 M EDTA, 20 mM NaCl, 1% sarcosyl, 1 mg/mL proteinase K), and digestion continued for 24 to 48 hr. This solution was replaced with 2 mL fresh Lysis Buffer 3, and digestion continued for 48 h. Plugs were rinsed 3x with TE (10 mM Tris, pH 7.5, 1 mM EDTA), soaked in 4 mL phenylmethylsulfonate (PMSF), (for alfalfa and soybean, 0.23 mM, all others, 2.5 mM; 2x, 30 min each), then rinsed 2 to 3x with TE.
Determination of Pyrimidine Dimer Frequencies
Dimer levels were determined as previously described (Freeman et al., 1986; Quaite et al., 1992b; Quaite et al., 1994a). In brief, plugs in UV endonuclease buffer (30 mM Tris-HCl, pH 7.6, 1 mM EDTA, 40 mM NaCl), containing 1 µg/mL bovine serum albumin were digested with sufficient dimer-specific UV endonuclease to cleave at all dimers while a companion plug was incubated in buffer alone. UV endonuclease from Micrococcus luteus was purified by the method of Carrier and Setlow (Carrier and Setlow, 1970). Enzyme activity toward CPD and specificity were determined using supercoiled DNA containing and lacking CPDs; typical activities were 4 x 1015 CPD cleaved µL-1 h-1. The quantity of endonuclease required was determined for each species and plant growth conditions by adding increasing quantities of UV endonuclease to sample plugs (containing more DNA than is used on a typical gel) from UV-irradiated plants (containing a higher dimer frequency than expected in the experimental samples), and determining the quantity of enzyme required for cleavage at all dimer sites; the absence of nonspecific cleavage at that enzyme level was ascertained by similar treatment of sample plugs containing DNA from unirradiated plants. Plugs were rinsed 2x with 2 mM EDTA, 30 mM NaOH, then incubated in 0.5 M NaOH, 50% (v/v) glycerol, 0.25% (w/v) bromocresol green for 30 min at 37°C. Samples, along with molecular length standards [bacteriophage G, 750 kb; T4, 170 kb; , 48.5 kb; and a HindIII digest of (23.1, 9.4, 6.6, 4.4, 2.3, 2, 0.56 kb); in some cases, chromosomes of Hansenula wingei Wickerham, the smallest of which is 1.05 Mb, were used as a high molecular length marker] were electrophoresed in 0.4% alkaline gels by means of unidirectional pulsed field gel electrophoresis [15 V/cm; 0.3 s pulse, 10 s interpulse period; 10°C with buffer recirculation (Sutherland et al., 1987b)] for CPD levels < 20 CPD/Mb, or static field electrophoresis for higher CPD levels. Gels were neutralized (500 mL of 0.1 M Tris-Cl, pH 8, 2x, 30 min), stained with ethidium bromide (1 µg/mL in H2O), and a quantitative electronic image was obtained with a charged-coupled device-based camera-based system (Sutherland et al., 1987a). A DNA dispersion curve was calculated from the migration distance and molecular lengths of the DNA size standards; from the dispersion curve, and lane profiles of sample DNAs, the number average molecular lengths of each + and - endonuclease companion pair were calculated, and from them, the lesion frequency in that sample (Freeman et al., 1986).
Other DNA Extraction/Isolation Trial Procedures
Trial Procedure I
Pea leaves (1.5 g) were ground in a mortar and pestle chilled with LN2; resuspended in 6 mL 0.1 M Tris-HCl, pH 8, 0.1 M EDTA (Tris-EDTA) containing 2% (w/v) SDS(sodium dodecyl sulfate), and chilled on dry ice. Redistilled phenol (5 mL, freshly equilibrated with 3x SSC; 1x SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) was added, mixed with a vortex mixer, and incubated on water ice for 15 min. The mixture was placed at room temperature for 15 min, then centrifuged (100 x g, 5 min). The upper phase was reextracted with phenol as before. The resulting top phase was brought to 1 M NaClO4, extracted with an equal volume of freshly mixed chloroform:phenol (1:1 v/v) and centrifuged as before. The lower phase was reextracted with 0.15 volumes of Tris-EDTA; the aqueous phases were combined and extracted with equal volumes of chloroform/isoamyl (24:1 v/v) until a clean interface was obtained. To the aqueous phase, Na acetate was added to 0.3 M final concentration, 2 volumes cold ethanol added, and the DNA was precipitated at -20°C overnight. The samples were centrifuged at 11 000 x g in the same rotor, the liquid removed, and the sample drained to remove traces of ethanol. DNA was taken up in 400 µL of 9 mM Tris, pH 8, 11 mM EDTA.
Trial Procedure II. (McCouch, 1988)
Leaves (1 g), ground as above, were resuspended in 10 mL extraction buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA containing, per mL, 4.8 g urea, 0.2 g. NaCl, 0.074 g EDTA and 0.2 g sarcosyl), then 1 mL phenol was added, mixed gently and 0.06 g SDS added. The mixture was extracted with 24:1 chloroform: isoamyl alcohol, and centrifuged at 1500 x g for 15 min. The supernatant was extracted with an equal volume of the same chloroform:isoamyl mixture, and centrifuged as before. The top layer was removed, and the DNA precipitated by adding 2 volumes cold ethanol, and centrifuging at 11 000 x g in the same rotor; the precipitate was rinsed gently with cold 70% (v/v) ethanol, and drained, then resuspended in 1 mL 10 mM Tris-HCl, pH 8, 1 mM EDTA.
Trial Procedure III
Leaves (1 g) were ground as above, and 15 mL Extraction Buffer II (100 mM Tris-HCl, pH 8, 50 mM EDTA, 500 mM NaCl, 10 mM ß-mercaptoethanol) plus 1 mL 20% SDS were added, and incubated at 65°C for 10 min. Then 5 mL of 5 M potassium acetate was added, the tube shaken vigorously, and incubated at 0°C for 20 min. The tubes were centrifuged at 25 000 x g for 20 min. The supernatant solution was mixed with 10 mL isopropanol, mixed, and stored 30 min at -20°C. The solution was centrifuged as before, the supernatant removed and tube drained, and the DNA redissolved in 0.7 mL 50 mM Tris, 10 mM EDTA, pH 8.
Trial Procedure IV and V (Molnar, 1989)
Leaves (1 g) were ground as above, and 10 mL of 30 mM Tris-HCl, pH 8, 30 mM EDTA containing 6% SDS was added. The mixture was extracted with phenol: chloroform:isoamyl (25:24:1), then centrifuged 10 min at 700 x g. The aqueous layer was extracted three times with chloroform:isoamyl alcohol (24:1), and precipitated with (Trial Procedure IV) an equal volume of isopropanol or (Trial Procedure V) with 2 volumes of ice cold ethanol. The samples were centrifuged as above, drained, and the DNA redissolved as in Procedure III.
Protoplast Production
Leaves were chopped, then added to Solution A [0.7 M mannitol, 5 mM DTT (dithiothreitol), 0.4% (v/v) PEG 6000, 5 mM KCl, 2 mM CaCl, 25 mM 2-(N-morpholino)ethanesulfonic acid-KOH, pH 5.8] to which cellulysin, macerase, pectolyase, and hemicellulase had been added to 1% (w/v) each just before use. The leaves were digested overnight at 10°C, filtered using cheesecloth, centrifuged (100 x g), and the pelleted protoplasts purified on a Percoll gradient [3 1-mL steps of 70, 50, and 25% (v/v) Percoll in 0.7 M mannitol, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH, pH 7.5) centrifuged at 200 x g for 15 min. The protoplasts were removed, mixed with an equal volume of 1.4% low melting point agarose (which had been melted and cooled to 38°C), formed into plugs, and allowed to solidify. They were incubated overnight at 50°C in 10 mM Tris, 0.5 M EDTA, pH 8, containing 1% SDS and 1 mg/mL proteinase K.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
|
100 kb. Unlike the homogeneous viral DNA size markers, genomic DNAs contain endogenous single strand breaks and breaks induced in DNA isolation and handling that result in single strand size heterogeneity. Similarly, preparations from spinach (Lane S in Panel B), rice (Lane R in Panel C), and sorghum (Lane Sgh in Panel D) contain DNAs with single strand molecular lengths principally greater than 100 kb. Soybean seedlings from the growth chamber yielded somewhat smaller DNAs (principally in the 100–150 kb range, but including molecules as small as 20 kb). Soybean DNA from plants in the field (subjected to UVA as well as sunlight) were distinctly smaller, with most molecules in the 25- to 50-kb range.
|
4–5, we increased the buffer concentration to 100 mM, which produced higher molecular length DNA. Increasing the sarcosyl content to 5%, adding 10% DMSO or 10% DMSO plus 0.1% 8-hydroxyquinoline produced modest increases in the size of the isolated DNA, while adding 10% DMSO plus 0.1% -tocopherol or 0.1 M EGTA yielded principally high single stranded molecular length DNA. High molecular length DNA from spinach leaves (S in Panel B) was obtained by including 10% DMSO and 0.1% -tocopherol in the buffer. These modifications should also be useful for isolating DNA from other recalcitrant samples.
DNA Damage Quantitation: UV-Induced Cyclobutyl Pyrimidine Dimers
DNA obtained by this method can be digested by a damage-specific endonuclease, allowing quantitation of DNA lesions such as CPD. Figure 3A
shows an electronic image of an alkaline electrophoretic gel containing DNAs from seedlings that received progressively increasing UV doses as well as unirradiated controls. Adjacent lanes contain companion pairs of DNA, treated without (left member of pair) and with (right) UV endonuclease. The samples incubated without endonuclease show no systematic variation in size with increasing dose; however, endonuclease treatment, which produces a single strand nick at each pyrimidine dimer site, clearly reduces the size of the DNA. Further, with increasing dose, the high molecular length DNA decreases, and the fraction of smaller molecules increases. Figure 3B shows the induction of CPD in DNA in rice seedlings by a broad spectrum FS20 lamp. The slope of the line for dimer induction by the broad spectrum UVB lamp is somewhat higher than that obtained with 302-nm radiation from a monochromator (Hidema et al., 1996), probably resulting from shorter wavelength UV radiation in the broad spectrum FS20 lamp (Lepre et al., 1998).
|
The method we have developed provides enzymatically digestible DNA that contains low frequencies of single strand breaks (thus producing high single strand molecular length DNA in denaturing gels). It provides for instantaneous harvest of plant material, avoiding protoplast isolation (Bancroft et al., 1992; Guzman and Ecker, 1988); this is important because DNA repair could occur during the production of protoplasts from plant tissue. Further, the method of isolation of protoplasts, and subsequent irradiation of the isolated protoplasts (Hall et al., 1992) for studying DNA damage is not applicable to the quantitation of steady-state damage levels induced by environmental agents levels in field-grown crop plants, or in plant in natural communities. The method described in this manuscript is ideal for accurate DNA damage assessment, allows extended storage of tissue before further processing, does not require use of toxic chemicals, and can readily be expanded to large numbers of samples. DNA can be obtained from many crop plants with only minor variations. We have also developed methods for successfully dealing with tissues that yield low molecular length DNA.
|
ACKNOWLEDGMENTS |
|---|
Received for publication December 30, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Vadose Zone Journal | |||
| Journal of Natural Resources and Life Sciences Education |
Soil Science Society of America Journal | ||||
| Journal of Plant Registrations | Journal of Environmental Quality |
The Plant Genome | |||
