The isolation and purification of endophyte DNA from Alnus glutinosa nodules

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  • The isolation and purification of endophyte DNA from Alnus glutinosa nodules

    BETH C. MULLIN, PRIYAVADAN A. JOSHI, A N D CHUNG SUN AN Botany and Plant and Soil Science Departments, The University of Tennessee, Kno.uville, TN, U.S.A. 37996-1100

    Received December 1, 1982

    MULLIN, B. C. , P. A. JOSHI, and C. S. AN. 1983. The isolation and purification of endophyte DNA from Alrz~~s gl~~tirzosa nodules. Can. J. Bot. 61: 2855-2858.

    Total DNA was extracted from leaves, roots, and nodules of Alrz~ts glutinosa seedlings. Leaf and root DNA was shown to have a buoyant density in CsCl of -1.68 with no satellite bands at a higher density. The endophyte DNA, which has a much greater buoyant density (p = - 1.72), was separated from the plant nodule DNA by centrifugation in CsCl. The yield of endophyte DNA was much greater when DNA was extracted directly from nodule tissue than when it was extracted from endophyte which had been isolated from nodules by sucrose density gradient centrifugation.

    MULLIN, B. C. , P. A. JOSHI et C. S. AN. 1983. The isolation and purification of endophyte DNA from Alr~usglutir~o.sa nodules. Can. J. Bot. 61: 2855-2858.

    L'ADN total a CtC extrait des feuilles, des racines et des nodules de plantules d'A1nu.s glutinosa. L'ADN des feuilles et des racines a une densite B 1'Cquilibre de sedimentation dans le CsCl de -1,68, sans bandes satellites j. une densite supCrieure. L'ADN de l'endophyte, qui a une densite i I'Cquilibre de skdimentation beaucoup plus ClevCe ( p = -1,72), a CtC sCparC de 1'ADN de la plante dans les nodules par centrifugation dans le CsC1. Le rendement en ADN de I'endophyte est beaucoup plus ClevC lorsque 1'ADN est extrait directement des tissus du nodule que lorsqu'il l'est de l'endophyte apres que celui-ci a CtC extrait des nodules par centrifugation dans un gradient de sucrose.

    [Traduit par le journal]

    Introduction We have been interested for some time in determining

    the degree of genetic relatedness among actinomycetes classified in the family Frankiaceae. Our studies to date have been limited to strains of frankiae that have been isolated and cultured in vitro. We now report a method that makes it possible to study the genome of any Frankia endophyte as long as nodule tissue is available. The method takes advantage of the difference in buoyant density that exists between the host-plant DNA and frankiae DNA.

    Materials and methods Leaves, roots, and nodules were collected from 1- to

    2-year-old greenhouse-grown Alnus glutinosa seedlings that had been inoculated 2 weeks after germination with a crushed nodule suspension. Leaf and root tissue was used within minutes of being collected; nodules were washed and used immediately or frozen in a dry ice - acetone bath and stored at -20C for no more than 2 days.

    DNA was extracted from leaf, root, and nodule tissue by a modified urea phosphate (MUP) procedure (Walbot and Goldberg 1979). Six grams of each tissue, chopped into small pieces, was ground in dry ice, with MUP buffer, extracted with phenol-chloroform-isoamy lalcohol (25:24: l ) , and chroma- tographed on hydroxylapatite. The interface from the phenol extraction of nodule tissue was added to MUP buffer and passed through a French pressure cell four times at 20 000 psi (1 psi = 6.894 757 kPa). DNA was extracted from the disrupted interface as described above.

    Endophyte filaments and vesicles were isolated from nodules by sucrose density gradient centrifugation (Baker et

    a/ . 1979). Six grams of nodule tissue was suspended in 10 mL of ice water and ground in an Omni-Mixer (Somall) for a total of 15 min. The extract was filtered through eight layers of cheesecloth and one layer of Miracloth. The resulting filtrate was layered on three discontinuous sucrose gradients com- posed of 6 mL of 60% sucrose, 6 mL of 45% sucrose, and 12 mL of 30% sucrose in cellulose nitrate centrifuge tubes. The gradients were spun at 23 000 rpm for 6 h in a Beckman SW 25.1 rotor at 4OC. Gradients were fractionated from the top with an ISCO density-gradient fractionator. Fifteen 2-mL fractions were collected and examined microscopically for the presence of endophyte filaments. Material that pelleted during centrifugation was examined microscopically and was subse- quently suspended in MUP buffer and passed through a French pressure cell four times at 20 000 psi. DNA was extracted from the disrupted pellet as described above.

    For preparative CsCl centrifugation, approximately 30 pg of purified DNA was mixed with CsCl and water to make a final volume of 5.3 mL and a refractive index of 1.3994 a 0.0002 at 20C. Samples were placed in nitrocellulose tubes and centrifuged at 30 000 rpm in a Beckman SW 50.1 rotor for 50 h. Gradients were fractionated from the top using an ISCO density-gradient fractionator. The absorbance of each gra- dient was monitored at 254nm, using an ISCO UA-5 absorbance monitor. The refractive index of each fraction was measured with a Bausch and Lomb refractometer and was converted to density, using standard tables (Sober 19680). Some samples were maintained in CsCl for recentrifugation; other samples were desalted on Sephadex G-25 or by dialysis before being ethanol precipitated.

    To determine the buoyant density of endophyte DNA, DNA was extracted from the endophyte Ar14 (Frankia sp. isolated from Alnus rubra by D. D. Baker) grown in pure culture as reported elsewhere in this volume (An et a/ . 1983). The

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  • CAN. J. BOT. VOL. 61, 1983

    5 10 15 20 25

    FRACTION

    FRACTION

    I TOP

    OF CELL BOTTOM OF CELL

    Fig. 1. (a) Equilibrium centrifugation in CsCl of DNA from alder leaves. (b) Equilibrium centrifugation in CsCl of DNA from alder nodules. ( c ) Analytical equilibrium centrifugation in CsCl of endophyte DNA.

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  • purified DNA was dialyzed overnight against 1 mM Tris pH7.0 containing 2 m M NaCl and 1 mM EDTA. Three micrograms of endophyte DNA and 3 pg of Escherichia coli DNA were mixed into 0.5 rnL of a CsCl solution containing 1 mM Tris pH 8.0. The refractive index of the solution was adjusted to 1.4000. Samples of 0.4 mL were centrifuged in double-sector quartz cells at 40 000 rpm on a Beckman Model E analytical ultracentrifuge at 25OC. The position of DNA in the cells was determined by direct photoelectric scanning at 265 nm. Cells were scanned at approximately 12-h intervals to monitor the development of equilibrium conditions as well as to check for spurious peaks. A final scan was recorded at 48 h and was used to determine the density of the DNA (Schildkraut et al. 1962), relative to E. coli at 1.7 1 g/cm3.

    MULLIN ET AL. 2857

    Results DNA was obtained in relatively high yields from

    nodule, leaf, and root tissue. By using the absorbance at 260 nm to quantitate DNA, it was estimated that 535 pg of DNA was purified from 6 g of nodule tissue, 350 pg of DNA from 6 g of leaf tissue, and 290 p.g DNA from 6 g of root tissue. The A260/A280 ratio was used as a measure of purity, and the following values were obtained for DNA from the three tissues: nodule, 1.82; leaf, 1.74; and root, 1.47. When spun to equilibrium in CsCl the root and leaf DNA (Fig. 1 a) banded as a single species of DNA with a density of 1.68, indicative of a very low mole percentage of guanine and cytosine. When nodule DNA was subjected to equilibrium centrifugation in CsC1, two distinct bands were evident (Fig. 1 b), one at the position of alder DNA (- 1.68) and the other at the position of authentic endophyte DNA (p = -1.72). The density of the cultured endophyte (ArI4) as determined by analytical ultracentrifugation was 1.728 (Fig. 1 c). The endophyte DNA can be resedi- mented in CsCl to obtain a sample free of host-plant DNA with a calculated yield of approximately 300 p.g of DNA per 6 g of nodule tissue.

    A large amount of cellular material remained in the interface during the phenol extraction of nodule tissue, suggesting that the initial grinding in dry ice might not have released all of the DNA. Indeed when this material was disrupted in a French pressure cell and extracted using the MUP procedure, an additional 100 p.g of DNA with an A260/A280 of 1.86 was recovered. This DNA also formed two bands when spun to equilibrium in CsC1, indicating the presence of both host and endophyte DNA. We have since found that initial grinding in liquid nitrogen greatly increases the amount

    endophyte. However, each centrifuge tube had a dark brown pellet which contained large numbers of fila- ments and vesicles. The combined pellets were passed through a French pressure cell and were subjected to the MUP extraction procedure. Only 75 pg of DNA was recovered, and this DNA was contaminated by host- plant DNA as evidenced by CsCl centrifugation.

    Discussion The urea phosphate extraction procedure in combina-

    tion with CsCl centrifugation provides a rapid method for the isolation of endophyte DNA. We recommend, however, that liquid nitrogen be substituted for dry ice in the initial grinding of tissue, because tissue is more easily ground to a fine powder in liquid nitrogen and the yield of nucleic acids is correspondingly higher.

    There are several explanations for the low yield of DNA from the endophyte purified by sucrose density gradient centrifugation. It is possible that filaments and vesicles were not totally disrupted by passage through the French pressure cell. We have used this method successfully to isolate DNA from cultured endophytes, but the pectinaceous layer surrounding the endophyte in vivo may provide extra resistance to shearing. It is also possible that during the initial tissue homogenization in water, DNA was released from the filaments and was separated from them during centrifugation. Finally it is possible that during homogenization and centrifuga- tion, in the absence of a denaturing agent, the DNA was degraded by nucleases. All three factors probably contribute to the poor yield of DNA from endophyte isolated from nodules.

    It is likely that the MUP-CsC1 method of DNA isolation described in this paper can be used to isolate endophyte DNA from a large number of host-plant nodules. The endophyte DNA has consistently demon- strated a relatively high mole percent of guanine plus cytosine (68.4-72.1) (An et al. 1983), and most higher plants observed to date have a much lower mole percent of guanine plus cytosine (

  • 2858 CAN. J. BOT. VOL. 61, 1983

    SCHILDKRAUT, C . L., J . MARMUR, and P. DOTY. 1962. Determination of the base composition of the deoxyribonu- cleic acid from its buoyant density in CsCl. J . Mol. Biol. 4: 430-443.

    SOBER, H. A. 1968a. Density at 25C of CsCl solution as a function of refractive index. In CRC handbook of bio- chemistry, selected data for molecular biology. Edited by H. A. Sober. CRC Press, Inc., Boca Raton, FL.

    19686. Distribution of purines and pyrimidines in DNAs. In CRC handbook of biochemistry, selected data for molecular biology. Edited by H. A. Sober. CRC Press, Inc., Boca Raton, FL.

    WALBOT, V., and R. GOLDBERG. 1979. Plant genome organization and its relationship to classical plant genetics. In Nucleic acids in plants. Edited by T. C. Hall and J. W. Davies. CRC Press, Inc., Boca Raton, FL. pp. 3-40.

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