Vol. 16, No. 1
JOURNAL OF VIROLOGY, July 1975, p. 132-140 Copyright i 1975 American Society for Microbiology
Printed in U.SA.
lodination of Herpesvirus Nucleic Acids JAMES E. SHAW,* ENG-SHANG HUANG, AND JOSEPH S. PAGANO Cancer Center Program and Departments of Medicine and Bacteriology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 13 March 1975
A simple method is described for the iodination of herpes simplex virus (HSV) DNA. The procedure involved synthesis of 125I-labeled 5-iodo-dCTP which was subsequently used as a precursor for the in vitro repair synthesis of HSV DNA. Synthesis of 5-iodo-dCTP and purification from oxidation and reduction reagents, buffer salts, unreacted dCTP and Na 125I was accomplished in a single chromatographic step. It was possible to prepare 125I-labeled HSV DNA in vitro with specific activities exceeding 108 counts/min/,ug. The DNA prepared by this method reassociated with DNA extracted from HSV-infected HEp-2 cells but not with HEp-2 cell DNA. lodinated HSV DNA was susceptible to S ,-endonuclease digestion once denatured but was resistant to digestion in the native form. This method was used to synthesize 125I-labeled ribo-CTP (5-iodo-CTP) which was used to prepare cytomegalovirus-specific complementary RNA. The method should be of value in the preparation of viral probes and for use in autoradiography of viral nucleic acids.
The search for viral nucleic acids in transformed cells and malignant tissues has been greatly facilitated by nucleic acid hybridization techniques, the sensitivity of which depend in part on the specific activity of the viral DNA or RNA probe. To attain sufficiently high specific activities, the viral probe can be labeled in vitro, either chemically or by enzymic incorporation of radioactive precursors. Commerford (1) has described in detail a technique for direct iodination of DNA and RNA in vitro and has shown that essentially all the irreversibly bound iodine was covalently linked as 5-iodocytosine. The Commerford technique has been extensively applied (2, 5, 7, 12, 15-17). Iodination by the Commerford procedure involved treatment of the nucleic acid with oxidizing and reducing reagents at elevated temperatures and at high and low pH values which resulted in some single-stranded breakage. Also, native DNA was not an efficient acceptor of iodine and had to be denatured if high specific activities were to be obtained. We report here a simple procedure for obtaining herpes simplex virus DNA of high specific activity by iodination. This procedure avoided exposure of the DNA to high temperature, pH extremes, and oxidizing and reducing reagents, and the DNA could be recovered in the native state after iodination. The procedure involved synthesis of 12"-labeled 5-iodo-dCTP which was subsequently used as a precursor for
the in vitro repair synthesis of HSV DNA. A similar method was used to prepare 125_ labeled cytomegalovirus (CMV) complementary RNA (cRNA). MATERIALS AND METHODS Virus strains and purification of DNA. Strain 196 of herpes simplex type 2 (13) was propagated in human epidermoid cells (HEp-2) at a multiplicity of 0.1 PFU/cell. Infection was allowed to proceed at 37 C until cytopathic effect was evident in 80 to 90% of the population. HEp-2 cells were propagated in Eagle minimal essential medium containing 10% fetal calf serum and were changed to 5% fetal calf serum during infection. Human CMV (strain AD 169) was propagated in human fibroblasts (WI-38). HSV and CMV DNA was extracted and purified from extracellular virions as previously described (8).
Chemicals, buffers, and enzymes. Radioactive
iodine was purchased from New England Nuclear as carrier-free Na125I in aqueous solution at pH 8 to 10. Triethylamine (Eastman Kodak) was used without prior redistillation to prepare triethylammonium bicarbonate (TEABC). A I M working stock solution was usually prepared, adjusted to pH 8, and stored in the dark. Thallium chloride was purchased from ICN -K & K Laboratories, Inc., Jamaica, N. Y. Unlabeled ribo- and deoxyribonucleoside triphosphates, DNase I, RNase A, and calf thymus DNA
were purchased from Sigma. Polyethyleneimine (PEI)-cellulose thin-layer chromatographic sheets (Brinkman Instruments Inc.) were developed with water and allowed to dry at room temperature prior to use. DEAE-cellulose (DE 52, Whatman) was fined repeatedly in water and stored at 4 C as a 50%
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suspension. S,-endonuclease was purified by the procedure of Vogt (18) from ENZOPHARM, a concentrated fungal alpha amylase powder purchased from Enzyme Development Corp., New York. DNA polymerase I was purified as described previously (8). Escherichia coli RNA polymerase was purchased from Worthington. S,-digestion buffer contained 30 mM sodium acetate (pH 4.5), 0.1 M NaCl, and 1 mM ZnCl,. TBS contained 50 mM Tris (pH 7.4) and 0.15 M NaCl. The concentration of purified native, viral DNA was determined using the relation: 50Mgg of DNA per ml = 1 unit of absorbance at 260 nm when measured through a 1-cm light path. Iodination of dCTP. The reaction mixture was assembled at 0 C in the following order and contained in a total volume of 80 Ml: 0.1 M sodium acetate-0.04 M acetic acid buffer (pH 5), 4 x 10' pmol of dCTP, 17 x 10' pmol of Na"'2I (3.8 mCi), and 17 x 10' pmol of thallium chloride. A 0.02 M solution of thallium chloride was prepared just prior to use. Solutions of thallium chloride below 10-3 M are cloudy. The reaction mixture was heated to 60 C for 15 min, chilled to 0 C, then mixed at 0 C with 20 Ml of a freshly prepared solution of 80 mM sodium sulfite to reduce excess thallium chloride. After approximately 2 min in the presence of sodium sulfite, 100 M&l of 1 M ammonium acetate-0.5 M ammonium hydroxide solution (-pH 9) was added, and the mixture was heated to 40 C for 15 min to dissociate remaining unstable intermediates formed during the reaction (1). Iodination of CTP. The synthesis of 5-iodo-CTP from CTP was identical to that described for the synthesis of 5-iodo-dCTP. DEAE-ellulose chromatography. The iodination reaction mixture was diluted to a concentration of less than 0.01 M salt and loaded at 0.8 ml/min on a DE 52 cellulose column (0.9 by 22 cm) that had been washed with three column volumes of 1 M TEABC and equilibrated with 10 mM TEABC (pH 8). After loading, the column was rinsed with 10 ml of 10 mM TEABC, and then the sample was eluted with a 1-liter linear gradient of TEABC, pH 8 (0.01 to 0.5 M). The peak fractions containing 5-iodo-dCTP (see fractions 72-78, Fig. 1) were pooled and evaporated to dryness under vacuum at 40 C. If a residue of TEABC remained after evaporation, the sample was dissolved in a small volume of water and evaporated to dryness a second time. The final product, recovered as the triethylammonium salt of 5-iodo-dCTP, was dissolved in 0.5 ml of water and stored at - 20 C. Synthesis of "12-labeled HSV DNA. The reaction mixture was assembled at 0 C and contained in a total volume of 250 ;l: 70 mM K,HPO4, pH 7.4, 7 mM MgCl,, 1 mM B-mercaptoethanol, and 0.1 mM dATP, dGTP, and dTTP, 400 to 600 MCi of 5-iodo-dCTP, 0.5 1g of nicked HSV DNA, and 10 Ml of DNA polymerase I. (HSV DNA was nicked for 4 min at 37 C with 34 x 10-' ;Mg of DNase I in 225 gl of the above reaction mixture minus all deoxyribonucleoside triphosphates and DNA polymerase I. DNase was inactivated at 70 C for 10 min.) The details of this reaction were originally described by Kelly et al. (9). The reaction mixture was incubated at 18 C until 121I counts per minute approached maximum incorporation (approx-
IODINATION OF VIRAL DNA IN VITRO
133
imately 5 h) as assayed by trichloroacetic acidprecipitable counts per minute. The sample was then made 1% with Sarkosyl 97, 10 mM with EDTA, and filtered through G-50 Sephadex equilibrated with 10 mM Tris-hydrochloride (pH 7.4), 1 mM EDTA, and 0.1% Sarkosyl. The excluded volume was mixed with 1 mg of calf thymus DNA, made 0.5% with sodium dodecyl sulfate, and extracted once with an equal volume of water-saturated phenol. The DNA was concentrated from the aqueous phase by ethanol precipitation, dissolved in TBS containing neutralized 1 mM EDTA, and sonically treated with a microtip probe for 12 min at 0 C. The sheared DNA was dialyzed extensively against neutralized 2.5 mM EDTA and stored at - 20 C. Hybridization of iodinated HSV DNA and digestion with S-endonuelease. DNA was extracted from HSV-infected or mock-infected HEp-2 cells, sonically treated for 12 min at 0 C in TBS-1 mM EDTA, and dialyzed extensively against neutralized 2.5 mM EDTA. Two milligrams of each DNA preparation was mixed with iodinated HSV DNA (0.01 ,g, 1.7 x 10' counts per min/gg), denatured in a boiling water bath for 10 min, then chilled to 0 C. Each sample was adjusted to 1 M NaCl and 5 mM Tris-hydrochloride (pH 7.4) in a total volume of 1 ml. Aliquots (100 MAl) were sealed in glass micropipettes and incubated in a water bath at 66 C. At various times during incubation samples were removed and placed at -20 C. To determine the amount of double-stranded DNA, each 100-Ml sample was mixed with 1.9 ml of S,-nuclease buffer and divided into two equal volumes. Each received 20Mug of sonically treated, native, calf thymus DNA, and one tube of each pair received 50 MAl of S,-endonuclease. After digestion at 37 C the trichloroacetic acid-precipitable counts per minute were determined for each tube. A reconstruction experiment was used to determine the length of time necessary to digest single-stranded DNA under these conditions. This was performed with each new preparation of S 1-endonuclease. Synthesis of CMV cRNA. 'H-labeled CMV cRNA, 8 x 106 counts/min per jg, was synthesized as previously described (8). The synthesis of "2'I-labeled CMV cRNA followed the above procedure except 'H-labeled UTP was replaced with cold UTP (0.38 mM), and 40 MCi of "2'I-labeled 5-iodo-CTP replaced cold CTP. The specific activity of the cRNA was estimated to be 4 x 107 counts/min per Mg. CMV cRNA was purified by Sephadex gel filtration as described for the purification of iodinated HSV DNA. cRNA-DNA hybridization. 'H- or "'I-labeled virus-specific cRNA was hybridized to denatured, immobilized DNA by the procedure of Gillespie and Spigelman (6) and as previously described (8). In situ cytohybridization was carried out as described by Gall and Pardue (4) with modifications (8). In brief, frozen sections (6 to 10 gm) from a congenitally infected human kidney were fixed to a glass slide with ice-cold glacial acetic acid-ethanol (1:3) and then were covered with 0.4% agarose. DNA was denatured in situ by applying 0.07 N NaOH for 3 min. After washing and dehydration with alcohol, 0.1 ml of 6x SSC (0.15 M NaCl plus 0.015 M sodium citrate)
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containing 8 x 104 counts/min of I25I-labeled cRNA and 1 mg of yeast RNA was applied. The slides were incubated in a humidified atmosphere at 66 C for 20 h. For autoradiography, the slides were covered with NTB-2 emulsion (Kodak). At various times slides were developed and fixed with Kodak D-19 developer and rapid fixer, respectively. They were then stained with Giemsa for contrast. Radiospectrometry. 125I counts per minute were determined directly in a Nuclear Chicago gamma counter which recorded 60% of the 125I disintegrations per minute, or the samples were placed in a toluenebased scintillator [6.3 g of 2,5 diphenyloxazole (Fisher) and 60 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene (Packard) per liter of toluene] and were counted in a Packard-Tricarb spectrometer. The counting efficiency in the presence of scintillator was 30% at a 25% gain and window settings of 50 to 1,000
(14). RESULTS Purification of 5-iodo-dCTP on DEAEcellulose. 5-Iodo-dCTP was purified from the iodination reaction mixture by anion exchange on DEAE-cellulose. The elution profiles of the radioactive components of the reaction mixture after iodination are shown in Fig. 1. Three major peaks of radioactivity were resolved. Peak I, fractions 29 to 32, was unreacted iodide. Peak
J. VIROL.
II, fractions 56 to 60, was not identified but presumably was iodinated dCDP. Peak III, fractions 72 to 78, eluted just after dCTP and was 5-iodo-dCTP. From the data shown in Fig. 1 it was calculated that 51% of the 1251 added to the reaction mixture was converted to 1251_ labeled 5-iodo-dCTP. PEI-cellulose chromatography of 5-iododCTP. The purity of 5-iodo-dCTP was checked by PEI-cellulose thin-layer chromatography. As shown in Fig. 2, material from peak III (Fig. 1) did not comigrate with any of the four common deoxyribonucleoside triphosphates added as markers but as a single peak just ahead of the dATP marker. Material from peak I (Fig. 1) migrated ahead of dTTP as indicated by the arrow and comigrated with Na125I, thus confirming its identity as unreacted iodide. lodination of HSV DNA and determination of specific activity. The kinetics of synthesis of I25I-labeled HSV DNA are shown in Fig. 3. A zero time aliquot was removed just prior to the addition of DNA polymerase I to register the background counts per minute. The reaction dGTP dATP
dTlTP'
dUCT
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30
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14-
2 12 t
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6
8
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12
14
16
18
DISTANCE MIGRATED (CM)
O
10 20 30 40 50 60 70 80 90 100 110 FRACTION NO.
FIG. 1. DEAE-cellulose elution profile of the iodination reaction mixture. After iodination of dCTP the reaction mixture was diluted to reduce the salt concentration and loaded on a column of DEAE-cellulose. The components of the reaction mixture were eluted with a linear gradient of TEABC. The counts per minute of 2 gl of each fraction (6.4 ml) were determined. The arrow indicates the elution position of unreacted dCTP determined in a reconstruction experiment which employed trace quantities of labeled 5-iodo-dCTP and UW-absorbing quantities of dCTP.
FIG. 2. PEI cellulose chromatography of 5-iododCTP. An aliquot of the material from peak III (Fig. 1) was mixed with 25 nmol of each common deoxyribonucleoside triphosphate and spotted at the origin of a PEI-cellulose thin-layer plate. The chromatogram was developed in a closed container with 0.75 M potassium phosphate (pH 4.5) until the solvent had migrated to within a few centimeters from the top, and then it was dried and exposed to UV light to locate the marker nucleoside triphosphates. A copy of the chromatogram was made by Xerox, and then it was cut into 0.4-cm fractions including origin and solvent front and the counts per minute of each were determined. The marker triphosphates are shown above in their positions relative to the origin (0 distance migrated).
IODINATION OF VIRAL DNA IN VITRO
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30
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240
280
INCUBATION TIME (MIN)
FIG. 3. Kinetics of synthesis of 125I-labeled HSV DNA. 125I-labeled HSV DNA was synthesized in vitro FRACTION NO. with DNA polymerase I using 5-iodo-dCTP as the 4. FIG. Elution profile of '251-labeled HSV DNA only labeled precursor. The viral DNA template was Sephadex. When synthesis of '251-labeled HSV prepared for the DNA polymerase I by nicking it with from (Fig. 3) was terminated, the reaction mixture DNase as described. During the reaction, 2-Ml aliquots DNA was filtered through Sephadex G-50. The counts per were removed from the reaction mixture, and the of 2 Al of each fraction (0.6 ml) were recorded. acid-precipitable counts per minute of each was minute The excluded volume, fractions 8 to 10, contained the recorded. iodinated DNA. Fractions 18 to 29 contained the rucleoside triphosphates not incorporated into DNA. was allowed to proceed for approximately 5 h or until maximum incorporation of the labeled precursor had occurred. An estimate of the of HSV DNA had occurred as a result of specific activity of the DNA was made from iodination, an aliquot of the iodinated DNA knowledge of the DNA concentration before (tube 9, Fig. 4) was centrifuged to equilibrium addition of DNase I and from the acid-precipi- in CsCl, and the density of the DNA was table counts per minute of the last aliquot, determined. There was a single peak of radioacminus the background counts per minute. The tivity approximately two-thirds from the top of specific activities of two preparations of the tube (Fig. 5). The rather broad distribution iodinated DNA, estimated by the above proce- of counts per minute was due to fragmentation dure, were 1.2 x 108 and 1.7 x 108 counts/min of the DNA as a result of nicking with DNase per Ag. when it was prepared for the DNA polymerase I Purification of 1251-labeled HSV DNA by reaction. It was not due to insufficient time of Sephadex gel filtration. The elution profile of centrifugation since 32P-labeled HSV DNA iodinated HSV DNA from Sephadex is shown in which had not been nicked with DNase formed Fig. 4. Of the two peaks of radioactivity eluting a sharp band only a few fractions wide when from the column, only the excluded volume, centrifuged under identical conditions (data not fractions 8 to 10, contained acid-precipitable shown). The important feature to be pointed counts per minute. These fractions were pooled, out in Fig. 5 is that iodinated HSV DNA, mixed with 1 mg of carrier DNA, and extracted labeled as described here, had an average buoywith phenol to remove protein. The iodinated ant density (1.724 g/ml, fraction 14) in the DNA was to be used for hybridization experi- native form similar to that reported (10) for ments, and calf thymus DNA was used as the uniodinated viral DNA (1.728 g/ml). source of carrier because no sequence homology Susceptibility of iodinated HSV DNA to has been detected between these two nucleic S -endonuclease. The kinetics of digestion of acids. iodinated HSV DNA by S,-endonuclease are Buoyant density of iodinated HSV DNA. shown in Fig. 6. During the first 20 min of To determine if a change in the buoyant density incubation, native DNA was digested to 13%
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but was resistant to further digestion for the remainder of the incubation period. Heatdenatured DNA was rapidly hydrolyzed to 85% during the first 90 min and reached a maximum 1.85 of 94% at the end of the incubation period. These results showed that iodinated DNA was 1.80 resistant to S -endonuclease digestion in the 1.75 ID native form but was susceptible once denatured and confirmed that this enzyme could be used 1.70 to distinguish between these two iodinated
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SHAW, HUANG, AND PAGANO
5
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15 20 25 30 35 FRACTION NO. FIG. 5. Equilibrium density centrifugation of iodinated HSV DNA. An aliquot of fraction 9, Fig. 4, was mixed with 100 ,gg of calf thymus DNA, adjusted to 1.71 g/ml with CsCI, and centrifuged at 20 C in a fixed angle rotor (T40, Spinco) at 36,000 rpm (82,000 x g) for 60 h. Fractions of 0.4 ml were removed from the bottom of the tube, and the counts per minute of each were recorded. The density of the gradient was determined from the refractive index of every fifth fraction.
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40 80 120 160 200 INCUBATION TIME (HRS) FIG. 7. Kinetics of reassociation of iodinated HSV DNA. DNA which had been extracted from HSVinfected or mock-infected HEp-2 cells was allowed to reassociate in the presence of I 21-labeled HSV DNA as described. During reassociation aliquots were removed from the hybridization mixture and split into two equal volumes. One was incubated with S,endonuclease and then precipitated with trichloroacetic acid. The acid-insoluble counts per minute of the S,-treated sample were used as a measure of the amount of double-stranded DNA formed during reannealing. The amount of single-stranded DNA formed was then determined from the total acid-insoluble counts per minute of the other sample by subtraction. The results are plotted as the ratio (DJDt) of the fraction of single-stranded DNA present at time 0 (D.) to that present at any time t (D,). One sample (0) contained 2 mg of DNA from mock-infected HEp-2 cells and 0.01 Mg of iodinated HSV DNA. The second sample (0) contained 2 mg of DNA extracted from HSV-infected HEp-2 cells and 0.01 Mg of iodinated HSV DNA. 0
w
CD
5060
70~~~ 80 0
90
100
120 160 200 240 TIME (MIN) FIG. 6. S,-endonuclease digestion of iodinated HS V DNA. HS V DNA (10-2 Ug, 1. 7 106 counts per min/Mug) was mixed with 100 Mg of calf thymus DNA and split into two equal volumes. The DNA of one preparation was denatured at 100 C, and then both were adjusted to 0.5 ml with S1-digestion buffer and 50 Mtl of an S,-endonuclease preparation. At various times during incubation at 37 C 10-Mul aliquots were removed and the acid-insoluble counts per minute were determined. Symbols: 0, native DNA; 0, denatured DNA. 0
40
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IODINATION OF VIRAL DNA IN VITRO
DNA species in hybridization experiments. Extent of reassociation of 1251-labeled HSV DNA. The kinetics of reassociation of 125I1 labeled HSV DNA in the presence of DNA which had been extracted from HSV-infected or mock-infected HEp-2 cells was measured (Fig. 7). After 192 h iodinated DNA in the presence of mock-infected HEp-2 cell DNA reached a DdD, value of 1.25, whereas that in the presence of DNA extracted from HSV-infected cells reached a value of 8.6, indicating that 20 and 88% of the iodinated DNA had reassociated, respectively. Synthesis of CMV cRNA. 125I-labeled 5iodo-CTP was prepared as described above and was used to synthesize CMV cRNA. The synthesis of cRNA was essentially linear during the first 40 min of the reaction but slowed considerably during the next 50 min (Fig. 8). RNase sensitivity of cRNA. Since it was essential to be able to remove excess labeled cRNA during membrane hybridization and in situ cytohybridization experiments, the sensitivity of 125I-labeled cRNA to RNase was determined. As shown in Fig. 9, 90% of the cRNA was digested to acid-insoluble material in 10 min, and by 60 min 97% of the RNA had been digested. cRNA-DNA hybridization on nitrocellulose. The reassociation of 25I-labeled cRNA to CMV DNA was determined by membrane hybridization (Fig. 10). The experiment was duplicated using 3H-labeled cRNA for comparison. A direct relation was observed between
the amount of DNA present per filter and the number of counts per minute hybridized whether the cRNA was labeled with 3H or 125I. Also, the relative rates of hybridization of 3Hand 12"I-labeled cRNA were identical. In situ cytohybridization of CMV cRNA. Iodinated cRNA was used as a probe to localize CMV DNA in kidney tissue shown previously to harbor this virus (Fig. 11). In Fig. 11A an obvious area of grains can be detected, indi-
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FIG. 9. RNase sensitivity of cRNA. An aliquot of purified "2'I-labeled CMV cRNA was made 60 Ag/ml with yeast RNA and incubated at 37 C in the presence (broken line) or absence (solid line) of RNase (40 Ag/mi). The extent to which the RNA was digested was determined by measuring the acid-insoluble counts per minute of aliquots removed during the course of the reaction.
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FIG. 8. Synthesis of CMV cRNA. CMV cRNA was synthesized in vitro with RNA polymerase using "2'I-labeled 5-iodo-CTP as the only labeled precursor. The kinetics of synthesis of "2'I-labeled cRNA was determined by measuring the acid-precipitable counts per minute in 5-gil aliquots which were removed at various times during the reaction.
005 9gfiIter
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FIG. 10. Hybridization of virus-specific 'H- and "2'I-labeled cRNA to CMV DNA. Increasing quantities of CMV DNA were mixed with 50 gg of calf thymus DNA and denatured in 0.5 N NaOH for 2 h at 37 C. The DNA solution was neutralized, adjusted to 6x SSC, and immobilized on nitrocellulose filters. The input cRNA for each filter
was
8
x
104 counts/
min for both tritiated and iodinated cRNA. After 20 h at 66 C filters were treated with RNase and washed thoroughly. The amount of cRNA hybridized was determined by measuring the counts per minute of each filter. Solid lines, counts per minute by scintillation spectrometer; broken line, counts per minute measured by a gamma recorder.
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11. In situ cytohybridization of '2561-labeled CMV cRNA. Tissue from a CM V-infected human kidney prepared and exposed to iodinated CMV cRNA as described. The preparation was then exposed to a photosensitive emulsion, developed, and stained with Giemsa for contrast. A, 400x. B, 1000x.
FIG.
was
cating a cluster of CMV-positive cells. Figure liB is a higher magnification that emphasizes the grain size. The size of the grain produced by 1251 disintegration was approximately four times that produced by 3H decay under similar conditions. However, a much longer exposure time was necessary before the grains pro-
duced by 3H decay were visible. For comparison, 8 x 104 counts/min were added to the cells shown in Fig. 11 and grains were visible within 5 days, whereas 5 x 105 counts/min of 3H required 3 to 4 weeks of exposure before being visible. The relative amounts of cRNA used in each case were comparable.
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DISCUSSION Indirect iodination of viral nucleic acids has two major advantages over the direct method described by Commerford (1). It avoids exposure of the nucleic acid to oxidization and reduction reagents, pH extremes, and high temperature, and it provides a means of labeling native DNA with iodine to high specific activity. Although 32P-labeled precursors can be used for indirect labeling of viral nucleic acids they are expensive, more difficult to prepare and purify, and have a half-life only 1/4 that of
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of the assay if they are 20- to 100-fold higher in specific activity. We have used successfully iodinated cRNA to localize CMV DNA in diseased kidney tissue (Fig. 11). Although this procedure revealed the presence of grains considerably earlier than 3H-labeled cRNA, they were four times the size of those produced by 3H disintegration. Therefore, some loss in resolution must be considered when iodinated cRNA probes are used for in situ cytohybridization experiments. lodinated cRNA probes would be of significant advantage, however, in procedures where rapid screening of 125I. tissue is important. We applied the Commerford procedure to make 5-iodo-dCTP and ribo-CTP. However, to ACKNOWLEDGMENTS minimize the breakdown of precursor and derivWe wish to thank Carolyn Smith, Shu-Mei Huong, and ative, temperatures above 60 C, pH values Joel A. Kostyu for their excellent technical assistance. This below pH 5, and incubation periods longer than work was supported by a Public Health Service grant from 15 min were avoided. Also, to achieve high the Virus Cancer Program of the National Cancer Institute (NO1 CP33336), by the National Heart and Lung Institute specific activities of the derivative, cold carrier (NHLI-72-2911-B), and in part by a Public Health Service iodide was not added during synthesis. The use fellowship (1 F22 CA04032) from the National Cancer Instiof a volatile buffer during elution of the deriva- tute. tive from DEAE-cellulose enabled it to be LITERATURE CITED removed free from salt and thus reduced purification to a single step. As a result, the deriva- 1. Commerford, S. L. 1971. Iodination of nucleic acids in vitro. Biochemistry 10:1993-2000. tive could be prepared routinely without diffi- 2. Duke, S. K. 1973. Use of 125I in fingerprinting RNA. culty and long exposure to radioactive iodine. It Nature (London) 246:483. was possible to prepare the derivative with little 3. Frenkel, N., B. Roizman, E. Cassai, and A. Nahmias. 1972. A DNA fragment of herpes simplex type 2 and its contamination from its precursor by this procetranscription in human cervical cancer. Proc. Natl. dure. This was essential since contamination Acad. Sci. U.S.A. 69:3784-3789. would reduce the effective specific activity of 4. Gall, J. G., and M. L. Pardue. 1971. Nucleic acid hybridization in cytological preparations. Methods Enthe derivative during in vitro synthesis reaczymol. 24:470-480. tions. M. J., L. C. Altenberg, and G. F. Saunders. 1972. Although we have not determined the long- 5. Getz, The use of RNA labeled in vitro with iodine-125 in term stability of the iodinated derivative when molecular hybridization experiments. Biochim. Biolabeled to high specific activity, we have noted phys. Acta 287:485-497. that it does break down. This was first observed 6. Gillespie, D., and S. Spigelman. 1965. A quantitative assay for DNA-RNA hybridization with DNA immobiwhen an aliquot of purified 5-iodo-dCTP (peak lized on a membrane. J. Mol. Biol. 12:829-842. III, Fig. 1) was eluted from DEAE-cellulose a 7. Holmes, D. S., and J. Bonner. 1974. Sequence composisecond time several days after its synthesis. The tion of rat nuclear deoxyribonucleic acid and high molecular weight nuclear ribonucleic acid. Biochemispresence of peaks I and II (Fig. 1) during the try 13:841-848. second elution suggested that breakdown prod- 8. Huang, E.-S., S. Chen, and J. S. Pagano. 1973. Human ucts of 5-iodo-dCTP were indeed present. Howcytomegalovirus. I. Purification and characterization of viral DNA. J. Virol. 12:1473-1481. ever, this did not seem to affect the synthesis of iodinated DNA. Preparations as old as 1 month 9. Kelly, R. B., N. R. Cozzarelli, M. P. Deutscher, I. R. Lehman, and A. Kornberg. 1970. Enzymic synthesis of have been used to synthesize HSV DNA with a deoxyribonucleic acid. XXXII. Replication of duplex specific activity exceeding 108 counts/min per deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245:39-45. ,ug. We are testing HSV DNA labeled to high E. D., S. L. Bachenheimer, and B. Roizman. 1971. specific activity to determine if it can be used as 10. Kieff, Size, composition, and structure of the deoxyribonua probe to search for virus DNA in transformed cleic acid of herpes simplex virus subtypes 1 and 2. J. cells and in cervical carcinomas that might Virol. 8:125-132. carry the virus genome. Probes prepared previ- 11. Nonoyama, M., and J. S. Pagano. 1973. Homology between Epstein-Barr virus and viral DNA from Burously using 3H-labeled dTTP as the only labeled kitt's lymphoma and nasopharyngeal carcinoma deprecursor were in the range of 106 to 5 x 106 tected by DNA-DNA reassociation kinetics. Nature counts/min per ,ug (3, 11). Therefore, the use of (London) 242:44-47. iodinated probes should increase the sensitivity 12. Prensky, W., D. M. Steffensen, and W. L. Hughes. 1973.
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The use of iodinated RNA for gene localization. Proc. Natl. Acad. Sci. U.S.A. 70:1860-1864. 13. Rawls, W. E., D. Laurel, J. S. Melnick, J. M. Glickman, and R. H. Kaufman. 1968. A search for viruses in smegma, premalignant and early malignant cervical tissues. The isolation of herpesviruses with distinct antigenic properties. Am. J. Epidemiol. 87:647-655. 14. Rhodes, B. A. 1965. Liquid scintillation counting of radioiodine. Anal. Chem. 37:955-997. 15. Scherberg, N. H., and S. Refetoff. 1973. Hybridization of DNA labeled with 125I to high specific activity. Nature
J. VIROL. (London) 242:142-145. 16. Shoulder, A., G. Darby, and T. Minson. 1974. RNA-RNA
hybridization using 125I-labeled RNA from tobacco necrosis virus and its satellite. Nature (London) 251:733-735. 17. Tereba, A., and B. J. McCarthy. 1973. Hybridization of 1251-labeled ribonucleic acid. Biochemistry 12: 4675-4679. 18. Vogt, V. M. 1973. Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur. J. Biochem. 33:192-200.
JOURNAL OF VIROLOGY, July 1975, p. 132-140 Copyright i 1975 American Society for Microbiology
Printed in U.SA.
lodination of Herpesvirus Nucleic Acids JAMES E. SHAW,* ENG-SHANG HUANG, AND JOSEPH S. PAGANO Cancer Center Program and Departments of Medicine and Bacteriology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 13 March 1975
A simple method is described for the iodination of herpes simplex virus (HSV) DNA. The procedure involved synthesis of 125I-labeled 5-iodo-dCTP which was subsequently used as a precursor for the in vitro repair synthesis of HSV DNA. Synthesis of 5-iodo-dCTP and purification from oxidation and reduction reagents, buffer salts, unreacted dCTP and Na 125I was accomplished in a single chromatographic step. It was possible to prepare 125I-labeled HSV DNA in vitro with specific activities exceeding 108 counts/min/,ug. The DNA prepared by this method reassociated with DNA extracted from HSV-infected HEp-2 cells but not with HEp-2 cell DNA. lodinated HSV DNA was susceptible to S ,-endonuclease digestion once denatured but was resistant to digestion in the native form. This method was used to synthesize 125I-labeled ribo-CTP (5-iodo-CTP) which was used to prepare cytomegalovirus-specific complementary RNA. The method should be of value in the preparation of viral probes and for use in autoradiography of viral nucleic acids.
The search for viral nucleic acids in transformed cells and malignant tissues has been greatly facilitated by nucleic acid hybridization techniques, the sensitivity of which depend in part on the specific activity of the viral DNA or RNA probe. To attain sufficiently high specific activities, the viral probe can be labeled in vitro, either chemically or by enzymic incorporation of radioactive precursors. Commerford (1) has described in detail a technique for direct iodination of DNA and RNA in vitro and has shown that essentially all the irreversibly bound iodine was covalently linked as 5-iodocytosine. The Commerford technique has been extensively applied (2, 5, 7, 12, 15-17). Iodination by the Commerford procedure involved treatment of the nucleic acid with oxidizing and reducing reagents at elevated temperatures and at high and low pH values which resulted in some single-stranded breakage. Also, native DNA was not an efficient acceptor of iodine and had to be denatured if high specific activities were to be obtained. We report here a simple procedure for obtaining herpes simplex virus DNA of high specific activity by iodination. This procedure avoided exposure of the DNA to high temperature, pH extremes, and oxidizing and reducing reagents, and the DNA could be recovered in the native state after iodination. The procedure involved synthesis of 12"-labeled 5-iodo-dCTP which was subsequently used as a precursor for
the in vitro repair synthesis of HSV DNA. A similar method was used to prepare 125_ labeled cytomegalovirus (CMV) complementary RNA (cRNA). MATERIALS AND METHODS Virus strains and purification of DNA. Strain 196 of herpes simplex type 2 (13) was propagated in human epidermoid cells (HEp-2) at a multiplicity of 0.1 PFU/cell. Infection was allowed to proceed at 37 C until cytopathic effect was evident in 80 to 90% of the population. HEp-2 cells were propagated in Eagle minimal essential medium containing 10% fetal calf serum and were changed to 5% fetal calf serum during infection. Human CMV (strain AD 169) was propagated in human fibroblasts (WI-38). HSV and CMV DNA was extracted and purified from extracellular virions as previously described (8).
Chemicals, buffers, and enzymes. Radioactive
iodine was purchased from New England Nuclear as carrier-free Na125I in aqueous solution at pH 8 to 10. Triethylamine (Eastman Kodak) was used without prior redistillation to prepare triethylammonium bicarbonate (TEABC). A I M working stock solution was usually prepared, adjusted to pH 8, and stored in the dark. Thallium chloride was purchased from ICN -K & K Laboratories, Inc., Jamaica, N. Y. Unlabeled ribo- and deoxyribonucleoside triphosphates, DNase I, RNase A, and calf thymus DNA
were purchased from Sigma. Polyethyleneimine (PEI)-cellulose thin-layer chromatographic sheets (Brinkman Instruments Inc.) were developed with water and allowed to dry at room temperature prior to use. DEAE-cellulose (DE 52, Whatman) was fined repeatedly in water and stored at 4 C as a 50%
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suspension. S,-endonuclease was purified by the procedure of Vogt (18) from ENZOPHARM, a concentrated fungal alpha amylase powder purchased from Enzyme Development Corp., New York. DNA polymerase I was purified as described previously (8). Escherichia coli RNA polymerase was purchased from Worthington. S,-digestion buffer contained 30 mM sodium acetate (pH 4.5), 0.1 M NaCl, and 1 mM ZnCl,. TBS contained 50 mM Tris (pH 7.4) and 0.15 M NaCl. The concentration of purified native, viral DNA was determined using the relation: 50Mgg of DNA per ml = 1 unit of absorbance at 260 nm when measured through a 1-cm light path. Iodination of dCTP. The reaction mixture was assembled at 0 C in the following order and contained in a total volume of 80 Ml: 0.1 M sodium acetate-0.04 M acetic acid buffer (pH 5), 4 x 10' pmol of dCTP, 17 x 10' pmol of Na"'2I (3.8 mCi), and 17 x 10' pmol of thallium chloride. A 0.02 M solution of thallium chloride was prepared just prior to use. Solutions of thallium chloride below 10-3 M are cloudy. The reaction mixture was heated to 60 C for 15 min, chilled to 0 C, then mixed at 0 C with 20 Ml of a freshly prepared solution of 80 mM sodium sulfite to reduce excess thallium chloride. After approximately 2 min in the presence of sodium sulfite, 100 M&l of 1 M ammonium acetate-0.5 M ammonium hydroxide solution (-pH 9) was added, and the mixture was heated to 40 C for 15 min to dissociate remaining unstable intermediates formed during the reaction (1). Iodination of CTP. The synthesis of 5-iodo-CTP from CTP was identical to that described for the synthesis of 5-iodo-dCTP. DEAE-ellulose chromatography. The iodination reaction mixture was diluted to a concentration of less than 0.01 M salt and loaded at 0.8 ml/min on a DE 52 cellulose column (0.9 by 22 cm) that had been washed with three column volumes of 1 M TEABC and equilibrated with 10 mM TEABC (pH 8). After loading, the column was rinsed with 10 ml of 10 mM TEABC, and then the sample was eluted with a 1-liter linear gradient of TEABC, pH 8 (0.01 to 0.5 M). The peak fractions containing 5-iodo-dCTP (see fractions 72-78, Fig. 1) were pooled and evaporated to dryness under vacuum at 40 C. If a residue of TEABC remained after evaporation, the sample was dissolved in a small volume of water and evaporated to dryness a second time. The final product, recovered as the triethylammonium salt of 5-iodo-dCTP, was dissolved in 0.5 ml of water and stored at - 20 C. Synthesis of "12-labeled HSV DNA. The reaction mixture was assembled at 0 C and contained in a total volume of 250 ;l: 70 mM K,HPO4, pH 7.4, 7 mM MgCl,, 1 mM B-mercaptoethanol, and 0.1 mM dATP, dGTP, and dTTP, 400 to 600 MCi of 5-iodo-dCTP, 0.5 1g of nicked HSV DNA, and 10 Ml of DNA polymerase I. (HSV DNA was nicked for 4 min at 37 C with 34 x 10-' ;Mg of DNase I in 225 gl of the above reaction mixture minus all deoxyribonucleoside triphosphates and DNA polymerase I. DNase was inactivated at 70 C for 10 min.) The details of this reaction were originally described by Kelly et al. (9). The reaction mixture was incubated at 18 C until 121I counts per minute approached maximum incorporation (approx-
IODINATION OF VIRAL DNA IN VITRO
133
imately 5 h) as assayed by trichloroacetic acidprecipitable counts per minute. The sample was then made 1% with Sarkosyl 97, 10 mM with EDTA, and filtered through G-50 Sephadex equilibrated with 10 mM Tris-hydrochloride (pH 7.4), 1 mM EDTA, and 0.1% Sarkosyl. The excluded volume was mixed with 1 mg of calf thymus DNA, made 0.5% with sodium dodecyl sulfate, and extracted once with an equal volume of water-saturated phenol. The DNA was concentrated from the aqueous phase by ethanol precipitation, dissolved in TBS containing neutralized 1 mM EDTA, and sonically treated with a microtip probe for 12 min at 0 C. The sheared DNA was dialyzed extensively against neutralized 2.5 mM EDTA and stored at - 20 C. Hybridization of iodinated HSV DNA and digestion with S-endonuelease. DNA was extracted from HSV-infected or mock-infected HEp-2 cells, sonically treated for 12 min at 0 C in TBS-1 mM EDTA, and dialyzed extensively against neutralized 2.5 mM EDTA. Two milligrams of each DNA preparation was mixed with iodinated HSV DNA (0.01 ,g, 1.7 x 10' counts per min/gg), denatured in a boiling water bath for 10 min, then chilled to 0 C. Each sample was adjusted to 1 M NaCl and 5 mM Tris-hydrochloride (pH 7.4) in a total volume of 1 ml. Aliquots (100 MAl) were sealed in glass micropipettes and incubated in a water bath at 66 C. At various times during incubation samples were removed and placed at -20 C. To determine the amount of double-stranded DNA, each 100-Ml sample was mixed with 1.9 ml of S,-nuclease buffer and divided into two equal volumes. Each received 20Mug of sonically treated, native, calf thymus DNA, and one tube of each pair received 50 MAl of S,-endonuclease. After digestion at 37 C the trichloroacetic acid-precipitable counts per minute were determined for each tube. A reconstruction experiment was used to determine the length of time necessary to digest single-stranded DNA under these conditions. This was performed with each new preparation of S 1-endonuclease. Synthesis of CMV cRNA. 'H-labeled CMV cRNA, 8 x 106 counts/min per jg, was synthesized as previously described (8). The synthesis of "2'I-labeled CMV cRNA followed the above procedure except 'H-labeled UTP was replaced with cold UTP (0.38 mM), and 40 MCi of "2'I-labeled 5-iodo-CTP replaced cold CTP. The specific activity of the cRNA was estimated to be 4 x 107 counts/min per Mg. CMV cRNA was purified by Sephadex gel filtration as described for the purification of iodinated HSV DNA. cRNA-DNA hybridization. 'H- or "'I-labeled virus-specific cRNA was hybridized to denatured, immobilized DNA by the procedure of Gillespie and Spigelman (6) and as previously described (8). In situ cytohybridization was carried out as described by Gall and Pardue (4) with modifications (8). In brief, frozen sections (6 to 10 gm) from a congenitally infected human kidney were fixed to a glass slide with ice-cold glacial acetic acid-ethanol (1:3) and then were covered with 0.4% agarose. DNA was denatured in situ by applying 0.07 N NaOH for 3 min. After washing and dehydration with alcohol, 0.1 ml of 6x SSC (0.15 M NaCl plus 0.015 M sodium citrate)
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SHAW, HUANG, AND PAGANO
containing 8 x 104 counts/min of I25I-labeled cRNA and 1 mg of yeast RNA was applied. The slides were incubated in a humidified atmosphere at 66 C for 20 h. For autoradiography, the slides were covered with NTB-2 emulsion (Kodak). At various times slides were developed and fixed with Kodak D-19 developer and rapid fixer, respectively. They were then stained with Giemsa for contrast. Radiospectrometry. 125I counts per minute were determined directly in a Nuclear Chicago gamma counter which recorded 60% of the 125I disintegrations per minute, or the samples were placed in a toluenebased scintillator [6.3 g of 2,5 diphenyloxazole (Fisher) and 60 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene (Packard) per liter of toluene] and were counted in a Packard-Tricarb spectrometer. The counting efficiency in the presence of scintillator was 30% at a 25% gain and window settings of 50 to 1,000
(14). RESULTS Purification of 5-iodo-dCTP on DEAEcellulose. 5-Iodo-dCTP was purified from the iodination reaction mixture by anion exchange on DEAE-cellulose. The elution profiles of the radioactive components of the reaction mixture after iodination are shown in Fig. 1. Three major peaks of radioactivity were resolved. Peak I, fractions 29 to 32, was unreacted iodide. Peak
J. VIROL.
II, fractions 56 to 60, was not identified but presumably was iodinated dCDP. Peak III, fractions 72 to 78, eluted just after dCTP and was 5-iodo-dCTP. From the data shown in Fig. 1 it was calculated that 51% of the 1251 added to the reaction mixture was converted to 1251_ labeled 5-iodo-dCTP. PEI-cellulose chromatography of 5-iododCTP. The purity of 5-iodo-dCTP was checked by PEI-cellulose thin-layer chromatography. As shown in Fig. 2, material from peak III (Fig. 1) did not comigrate with any of the four common deoxyribonucleoside triphosphates added as markers but as a single peak just ahead of the dATP marker. Material from peak I (Fig. 1) migrated ahead of dTTP as indicated by the arrow and comigrated with Na125I, thus confirming its identity as unreacted iodide. lodination of HSV DNA and determination of specific activity. The kinetics of synthesis of I25I-labeled HSV DNA are shown in Fig. 3. A zero time aliquot was removed just prior to the addition of DNA polymerase I to register the background counts per minute. The reaction dGTP dATP
dTlTP'
dUCT
36 -
30
20 1816
24-
'n
24
L
14-
2 12 t
aI
18
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4
6
8
0
12
14
16
18
DISTANCE MIGRATED (CM)
O
10 20 30 40 50 60 70 80 90 100 110 FRACTION NO.
FIG. 1. DEAE-cellulose elution profile of the iodination reaction mixture. After iodination of dCTP the reaction mixture was diluted to reduce the salt concentration and loaded on a column of DEAE-cellulose. The components of the reaction mixture were eluted with a linear gradient of TEABC. The counts per minute of 2 gl of each fraction (6.4 ml) were determined. The arrow indicates the elution position of unreacted dCTP determined in a reconstruction experiment which employed trace quantities of labeled 5-iodo-dCTP and UW-absorbing quantities of dCTP.
FIG. 2. PEI cellulose chromatography of 5-iododCTP. An aliquot of the material from peak III (Fig. 1) was mixed with 25 nmol of each common deoxyribonucleoside triphosphate and spotted at the origin of a PEI-cellulose thin-layer plate. The chromatogram was developed in a closed container with 0.75 M potassium phosphate (pH 4.5) until the solvent had migrated to within a few centimeters from the top, and then it was dried and exposed to UV light to locate the marker nucleoside triphosphates. A copy of the chromatogram was made by Xerox, and then it was cut into 0.4-cm fractions including origin and solvent front and the counts per minute of each were determined. The marker triphosphates are shown above in their positions relative to the origin (0 distance migrated).
IODINATION OF VIRAL DNA IN VITRO
VOL. 16, 1975
135
30
25F
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20f
0 x;
0
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0
10
I
5
0
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80
120
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200
240
280
INCUBATION TIME (MIN)
FIG. 3. Kinetics of synthesis of 125I-labeled HSV DNA. 125I-labeled HSV DNA was synthesized in vitro FRACTION NO. with DNA polymerase I using 5-iodo-dCTP as the 4. FIG. Elution profile of '251-labeled HSV DNA only labeled precursor. The viral DNA template was Sephadex. When synthesis of '251-labeled HSV prepared for the DNA polymerase I by nicking it with from (Fig. 3) was terminated, the reaction mixture DNase as described. During the reaction, 2-Ml aliquots DNA was filtered through Sephadex G-50. The counts per were removed from the reaction mixture, and the of 2 Al of each fraction (0.6 ml) were recorded. acid-precipitable counts per minute of each was minute The excluded volume, fractions 8 to 10, contained the recorded. iodinated DNA. Fractions 18 to 29 contained the rucleoside triphosphates not incorporated into DNA. was allowed to proceed for approximately 5 h or until maximum incorporation of the labeled precursor had occurred. An estimate of the of HSV DNA had occurred as a result of specific activity of the DNA was made from iodination, an aliquot of the iodinated DNA knowledge of the DNA concentration before (tube 9, Fig. 4) was centrifuged to equilibrium addition of DNase I and from the acid-precipi- in CsCl, and the density of the DNA was table counts per minute of the last aliquot, determined. There was a single peak of radioacminus the background counts per minute. The tivity approximately two-thirds from the top of specific activities of two preparations of the tube (Fig. 5). The rather broad distribution iodinated DNA, estimated by the above proce- of counts per minute was due to fragmentation dure, were 1.2 x 108 and 1.7 x 108 counts/min of the DNA as a result of nicking with DNase per Ag. when it was prepared for the DNA polymerase I Purification of 1251-labeled HSV DNA by reaction. It was not due to insufficient time of Sephadex gel filtration. The elution profile of centrifugation since 32P-labeled HSV DNA iodinated HSV DNA from Sephadex is shown in which had not been nicked with DNase formed Fig. 4. Of the two peaks of radioactivity eluting a sharp band only a few fractions wide when from the column, only the excluded volume, centrifuged under identical conditions (data not fractions 8 to 10, contained acid-precipitable shown). The important feature to be pointed counts per minute. These fractions were pooled, out in Fig. 5 is that iodinated HSV DNA, mixed with 1 mg of carrier DNA, and extracted labeled as described here, had an average buoywith phenol to remove protein. The iodinated ant density (1.724 g/ml, fraction 14) in the DNA was to be used for hybridization experi- native form similar to that reported (10) for ments, and calf thymus DNA was used as the uniodinated viral DNA (1.728 g/ml). source of carrier because no sequence homology Susceptibility of iodinated HSV DNA to has been detected between these two nucleic S -endonuclease. The kinetics of digestion of acids. iodinated HSV DNA by S,-endonuclease are Buoyant density of iodinated HSV DNA. shown in Fig. 6. During the first 20 min of To determine if a change in the buoyant density incubation, native DNA was digested to 13%
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but was resistant to further digestion for the remainder of the incubation period. Heatdenatured DNA was rapidly hydrolyzed to 85% during the first 90 min and reached a maximum 1.85 of 94% at the end of the incubation period. These results showed that iodinated DNA was 1.80 resistant to S -endonuclease digestion in the 1.75 ID native form but was susceptible once denatured and confirmed that this enzyme could be used 1.70 to distinguish between these two iodinated
3
1.65
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SHAW, HUANG, AND PAGANO
5
8 -5
10
15 20 25 30 35 FRACTION NO. FIG. 5. Equilibrium density centrifugation of iodinated HSV DNA. An aliquot of fraction 9, Fig. 4, was mixed with 100 ,gg of calf thymus DNA, adjusted to 1.71 g/ml with CsCI, and centrifuged at 20 C in a fixed angle rotor (T40, Spinco) at 36,000 rpm (82,000 x g) for 60 h. Fractions of 0.4 ml were removed from the bottom of the tube, and the counts per minute of each were recorded. The density of the gradient was determined from the refractive index of every fifth fraction.
7
6 5 "I. 0
0
4
3
01 10
In
20 30
2
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°\ 0
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40 80 120 160 200 INCUBATION TIME (HRS) FIG. 7. Kinetics of reassociation of iodinated HSV DNA. DNA which had been extracted from HSVinfected or mock-infected HEp-2 cells was allowed to reassociate in the presence of I 21-labeled HSV DNA as described. During reassociation aliquots were removed from the hybridization mixture and split into two equal volumes. One was incubated with S,endonuclease and then precipitated with trichloroacetic acid. The acid-insoluble counts per minute of the S,-treated sample were used as a measure of the amount of double-stranded DNA formed during reannealing. The amount of single-stranded DNA formed was then determined from the total acid-insoluble counts per minute of the other sample by subtraction. The results are plotted as the ratio (DJDt) of the fraction of single-stranded DNA present at time 0 (D.) to that present at any time t (D,). One sample (0) contained 2 mg of DNA from mock-infected HEp-2 cells and 0.01 Mg of iodinated HSV DNA. The second sample (0) contained 2 mg of DNA extracted from HSV-infected HEp-2 cells and 0.01 Mg of iodinated HSV DNA. 0
w
CD
5060
70~~~ 80 0
90
100
120 160 200 240 TIME (MIN) FIG. 6. S,-endonuclease digestion of iodinated HS V DNA. HS V DNA (10-2 Ug, 1. 7 106 counts per min/Mug) was mixed with 100 Mg of calf thymus DNA and split into two equal volumes. The DNA of one preparation was denatured at 100 C, and then both were adjusted to 0.5 ml with S1-digestion buffer and 50 Mtl of an S,-endonuclease preparation. At various times during incubation at 37 C 10-Mul aliquots were removed and the acid-insoluble counts per minute were determined. Symbols: 0, native DNA; 0, denatured DNA. 0
40
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IODINATION OF VIRAL DNA IN VITRO
DNA species in hybridization experiments. Extent of reassociation of 1251-labeled HSV DNA. The kinetics of reassociation of 125I1 labeled HSV DNA in the presence of DNA which had been extracted from HSV-infected or mock-infected HEp-2 cells was measured (Fig. 7). After 192 h iodinated DNA in the presence of mock-infected HEp-2 cell DNA reached a DdD, value of 1.25, whereas that in the presence of DNA extracted from HSV-infected cells reached a value of 8.6, indicating that 20 and 88% of the iodinated DNA had reassociated, respectively. Synthesis of CMV cRNA. 125I-labeled 5iodo-CTP was prepared as described above and was used to synthesize CMV cRNA. The synthesis of cRNA was essentially linear during the first 40 min of the reaction but slowed considerably during the next 50 min (Fig. 8). RNase sensitivity of cRNA. Since it was essential to be able to remove excess labeled cRNA during membrane hybridization and in situ cytohybridization experiments, the sensitivity of 125I-labeled cRNA to RNase was determined. As shown in Fig. 9, 90% of the cRNA was digested to acid-insoluble material in 10 min, and by 60 min 97% of the RNA had been digested. cRNA-DNA hybridization on nitrocellulose. The reassociation of 25I-labeled cRNA to CMV DNA was determined by membrane hybridization (Fig. 10). The experiment was duplicated using 3H-labeled cRNA for comparison. A direct relation was observed between
the amount of DNA present per filter and the number of counts per minute hybridized whether the cRNA was labeled with 3H or 125I. Also, the relative rates of hybridization of 3Hand 12"I-labeled cRNA were identical. In situ cytohybridization of CMV cRNA. Iodinated cRNA was used as a probe to localize CMV DNA in kidney tissue shown previously to harbor this virus (Fig. 11). In Fig. 11A an obvious area of grains can be detected, indi-
C
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FIG. 9. RNase sensitivity of cRNA. An aliquot of purified "2'I-labeled CMV cRNA was made 60 Ag/ml with yeast RNA and incubated at 37 C in the presence (broken line) or absence (solid line) of RNase (40 Ag/mi). The extent to which the RNA was digested was determined by measuring the acid-insoluble counts per minute of aliquots removed during the course of the reaction.
100-
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cRNA
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60 (MIN) 40 REACTION TIME
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FIG. 8. Synthesis of CMV cRNA. CMV cRNA was synthesized in vitro with RNA polymerase using "2'I-labeled 5-iodo-CTP as the only labeled precursor. The kinetics of synthesis of "2'I-labeled cRNA was determined by measuring the acid-precipitable counts per minute in 5-gil aliquots which were removed at various times during the reaction.
005 9gfiIter
01
001
005
ug/fiIter
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FIG. 10. Hybridization of virus-specific 'H- and "2'I-labeled cRNA to CMV DNA. Increasing quantities of CMV DNA were mixed with 50 gg of calf thymus DNA and denatured in 0.5 N NaOH for 2 h at 37 C. The DNA solution was neutralized, adjusted to 6x SSC, and immobilized on nitrocellulose filters. The input cRNA for each filter
was
8
x
104 counts/
min for both tritiated and iodinated cRNA. After 20 h at 66 C filters were treated with RNase and washed thoroughly. The amount of cRNA hybridized was determined by measuring the counts per minute of each filter. Solid lines, counts per minute by scintillation spectrometer; broken line, counts per minute measured by a gamma recorder.
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11. In situ cytohybridization of '2561-labeled CMV cRNA. Tissue from a CM V-infected human kidney prepared and exposed to iodinated CMV cRNA as described. The preparation was then exposed to a photosensitive emulsion, developed, and stained with Giemsa for contrast. A, 400x. B, 1000x.
FIG.
was
cating a cluster of CMV-positive cells. Figure liB is a higher magnification that emphasizes the grain size. The size of the grain produced by 1251 disintegration was approximately four times that produced by 3H decay under similar conditions. However, a much longer exposure time was necessary before the grains pro-
duced by 3H decay were visible. For comparison, 8 x 104 counts/min were added to the cells shown in Fig. 11 and grains were visible within 5 days, whereas 5 x 105 counts/min of 3H required 3 to 4 weeks of exposure before being visible. The relative amounts of cRNA used in each case were comparable.
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DISCUSSION Indirect iodination of viral nucleic acids has two major advantages over the direct method described by Commerford (1). It avoids exposure of the nucleic acid to oxidization and reduction reagents, pH extremes, and high temperature, and it provides a means of labeling native DNA with iodine to high specific activity. Although 32P-labeled precursors can be used for indirect labeling of viral nucleic acids they are expensive, more difficult to prepare and purify, and have a half-life only 1/4 that of
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of the assay if they are 20- to 100-fold higher in specific activity. We have used successfully iodinated cRNA to localize CMV DNA in diseased kidney tissue (Fig. 11). Although this procedure revealed the presence of grains considerably earlier than 3H-labeled cRNA, they were four times the size of those produced by 3H disintegration. Therefore, some loss in resolution must be considered when iodinated cRNA probes are used for in situ cytohybridization experiments. lodinated cRNA probes would be of significant advantage, however, in procedures where rapid screening of 125I. tissue is important. We applied the Commerford procedure to make 5-iodo-dCTP and ribo-CTP. However, to ACKNOWLEDGMENTS minimize the breakdown of precursor and derivWe wish to thank Carolyn Smith, Shu-Mei Huong, and ative, temperatures above 60 C, pH values Joel A. Kostyu for their excellent technical assistance. This below pH 5, and incubation periods longer than work was supported by a Public Health Service grant from 15 min were avoided. Also, to achieve high the Virus Cancer Program of the National Cancer Institute (NO1 CP33336), by the National Heart and Lung Institute specific activities of the derivative, cold carrier (NHLI-72-2911-B), and in part by a Public Health Service iodide was not added during synthesis. The use fellowship (1 F22 CA04032) from the National Cancer Instiof a volatile buffer during elution of the deriva- tute. tive from DEAE-cellulose enabled it to be LITERATURE CITED removed free from salt and thus reduced purification to a single step. As a result, the deriva- 1. Commerford, S. L. 1971. Iodination of nucleic acids in vitro. Biochemistry 10:1993-2000. tive could be prepared routinely without diffi- 2. Duke, S. K. 1973. Use of 125I in fingerprinting RNA. culty and long exposure to radioactive iodine. It Nature (London) 246:483. was possible to prepare the derivative with little 3. Frenkel, N., B. Roizman, E. Cassai, and A. Nahmias. 1972. A DNA fragment of herpes simplex type 2 and its contamination from its precursor by this procetranscription in human cervical cancer. Proc. Natl. dure. This was essential since contamination Acad. Sci. U.S.A. 69:3784-3789. would reduce the effective specific activity of 4. Gall, J. G., and M. L. Pardue. 1971. Nucleic acid hybridization in cytological preparations. Methods Enthe derivative during in vitro synthesis reaczymol. 24:470-480. tions. M. J., L. C. Altenberg, and G. F. Saunders. 1972. Although we have not determined the long- 5. Getz, The use of RNA labeled in vitro with iodine-125 in term stability of the iodinated derivative when molecular hybridization experiments. Biochim. Biolabeled to high specific activity, we have noted phys. Acta 287:485-497. that it does break down. This was first observed 6. Gillespie, D., and S. Spigelman. 1965. A quantitative assay for DNA-RNA hybridization with DNA immobiwhen an aliquot of purified 5-iodo-dCTP (peak lized on a membrane. J. Mol. Biol. 12:829-842. III, Fig. 1) was eluted from DEAE-cellulose a 7. Holmes, D. S., and J. Bonner. 1974. Sequence composisecond time several days after its synthesis. The tion of rat nuclear deoxyribonucleic acid and high molecular weight nuclear ribonucleic acid. Biochemispresence of peaks I and II (Fig. 1) during the try 13:841-848. second elution suggested that breakdown prod- 8. Huang, E.-S., S. Chen, and J. S. Pagano. 1973. Human ucts of 5-iodo-dCTP were indeed present. Howcytomegalovirus. I. Purification and characterization of viral DNA. J. Virol. 12:1473-1481. ever, this did not seem to affect the synthesis of iodinated DNA. Preparations as old as 1 month 9. Kelly, R. B., N. R. Cozzarelli, M. P. Deutscher, I. R. Lehman, and A. Kornberg. 1970. Enzymic synthesis of have been used to synthesize HSV DNA with a deoxyribonucleic acid. XXXII. Replication of duplex specific activity exceeding 108 counts/min per deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245:39-45. ,ug. We are testing HSV DNA labeled to high E. D., S. L. Bachenheimer, and B. Roizman. 1971. specific activity to determine if it can be used as 10. Kieff, Size, composition, and structure of the deoxyribonua probe to search for virus DNA in transformed cleic acid of herpes simplex virus subtypes 1 and 2. J. cells and in cervical carcinomas that might Virol. 8:125-132. carry the virus genome. Probes prepared previ- 11. Nonoyama, M., and J. S. Pagano. 1973. Homology between Epstein-Barr virus and viral DNA from Burously using 3H-labeled dTTP as the only labeled kitt's lymphoma and nasopharyngeal carcinoma deprecursor were in the range of 106 to 5 x 106 tected by DNA-DNA reassociation kinetics. Nature counts/min per ,ug (3, 11). Therefore, the use of (London) 242:44-47. iodinated probes should increase the sensitivity 12. Prensky, W., D. M. Steffensen, and W. L. Hughes. 1973.
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The use of iodinated RNA for gene localization. Proc. Natl. Acad. Sci. U.S.A. 70:1860-1864. 13. Rawls, W. E., D. Laurel, J. S. Melnick, J. M. Glickman, and R. H. Kaufman. 1968. A search for viruses in smegma, premalignant and early malignant cervical tissues. The isolation of herpesviruses with distinct antigenic properties. Am. J. Epidemiol. 87:647-655. 14. Rhodes, B. A. 1965. Liquid scintillation counting of radioiodine. Anal. Chem. 37:955-997. 15. Scherberg, N. H., and S. Refetoff. 1973. Hybridization of DNA labeled with 125I to high specific activity. Nature
J. VIROL. (London) 242:142-145. 16. Shoulder, A., G. Darby, and T. Minson. 1974. RNA-RNA
hybridization using 125I-labeled RNA from tobacco necrosis virus and its satellite. Nature (London) 251:733-735. 17. Tereba, A., and B. J. McCarthy. 1973. Hybridization of 1251-labeled ribonucleic acid. Biochemistry 12: 4675-4679. 18. Vogt, V. M. 1973. Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur. J. Biochem. 33:192-200.
