Proc. Nat. Acad. Sci. USAVol. 70, No. 11, pp. 3260-3264, November 1973

Application of Fingerprinting Techniques to lodinated Nucleic Acids(iodine-125/ribonuclease/55 RNA/messenger RNA/RNA chemical labeling)

HUGH D. ROBERTSON*, ELIZABETH DICKSON*, PETER MODEL*, AND WOLF PRENSKYt* The Rockefeller University, New York, N.Y. 10021; and t Sloan-Kettering Institute for Cancer Research, New York, N.Y. 10021

Communicated by Nortoh D. Zinder, July 10, 1973

ABSTRACT Several techniques of RNAand DNA finger-printing and determination of sequence have been appliedto nucleic acids labeled with 125I. Fingerprints of human5S RNA and bacteriophage f2 RNA resemble those of theirnoniodinated counterparts both in complexity and in spe-cific pattern. Iodination as used here is thus a generallabeling procedure, and appears principally to labelcytidine residues. This iodination method shows littlesensitivity to potential structure in single-strandedRNA molecules, yields stable oligonucleotide productsin a reproducible manner, and does not change the speci-ficity of several ribonucleases and deoxyribonucleases.

Recent advances in the techniques of nucleic acid finger-printing and determination of sequence have yielded bio-logically interesting RNA and DNA sequences (1-5) andhave fostered comparative studies of closely related nucleicacid species (6, 7). Previous experiments concentrated uponnucleic acids that can be labeled to high specific activity with32p and isolated in high yield, usually from microorganisms.With increasing interest in applications of these methods toeukaryotes, indirect approaches to obtain radioactive nucleicacids are being studied. For example, one can attempt tocopy isolated nucleic acids with various polymerases, obtain-ing complementary copies of high specific radioactivity (8-10).Alternatively, nonradioactive nucleic acids are labeled afterisolation from the organism (11).

Several reports have appeared describing methods foriodination of nucleic acids with 125I (12, 13). Prensky et al. (13)have described a modification of Commerford's iodinationprocedure (12) by which they obtain RNA of very high spe-cific radioactivity (106-101 dpm/,g). These authors showed thespecific in situ hybridization of Drosophila 5S RNA with band56F of Drosophila polytene chromosomes.

In the studies reported here, we examined several prokary-otic and eukaryotic RNAs in order to compare fingerprints ofspecies previously studied by conventional techniques. Wealso applied iodination to molecules that have not beenavailable as radioactive species. Our results indicate that,although the iodinated oligonucleotides show altered electro-phoretic behavior in comparison to their noniodinated coun-terparts, this approach is useful for comparative fingerprintingstudies, and may eventually be applied to sequence deter-mination as well.

MATERIALS AND METHODS

Nucleic Acids forIodination. RNA from bacteriophage f2 wasobtained and purified as in reference 14. Bacteriophage fimessenger RNA (mRNA) was synthesized in vitro with double-stranded replicative form DNA isolated from infected Escheri-

chia coli as template, by the method of Model (manuscript inpreparation). Single-stranded DNA from phage fi was thegift of K. Jakes, The Rockefeller University. We thank Dr.T. Borun, Fells Research Institute, Philadelphia, Pa., forhis gifts of purified HeLa cell 5S ribosomal RNA and twofractions containing histone mRNA. We received duck globinmRNA and rabbit 5S RNA from Dr. D. Housman, Massachu-setts Institute of Technology. Fractions of Drosophila tRNAand 5S RNA were obtained from Dr. D. M. Steffensen,University of Illinois, and maize 5S RNA from Dr. D. E.Wimber, University of Oregon. Polyribocytidylic acid andpolyuridylic acid were purchased from Sigma.

Isotopes. Carrier-free 125I as Na121I was purchased fromNew England Nuclear Corp. Carrier-free 32p was obtainedfrom Schwarz-Mann. Ribonucleoside triphosphate precur-sors for in vitro RNA synthesis ([14C]CTP, specific activity500 Ci/mol, and [a-32P]ATP, [a-32P]GTP, [a.-32P]UTP, and[ca-32P]CTP, average specific activity 100,000 Ci/mol) wereobtained from New England Nuclear Corp.

Other Materials. Ribonucleoside triphosphates were ob-tained from P-L Biochemicals Inc. Yeast nucleic acid forhomochromatography was purchased from CalBiochem.RNases T1, T2, and U2 (Sankyo Co., Ltd., Tokyo, Japan)were purchased from CalBiochem. Pancreatic RNase (5Xrecrystallized), electrophoretically purified pancreatic DNase(DPFF), and snake venom phosphodiesterase (VPH) wereobtained from Worthington Biochemical Corp.

Iodination of Nucleic Acids. All carrier-free iodinationswith 125I were done by the methods of Commerford (12) andPrensky et al. (13). Concentrations and volumes of all reagentswere the same as given by Prensky et al., except that sometypes of RNA were used in concentrations smaller than 1mg/ml. Also, some iodination products were separated not onhydroxyapatite but by use of CFi1 cellulose (15). The re-action mixture was brought to 1 ml with 0.1 M NaCl-50mM Tris - HCl (pH 7.0)-i mM ethylene diamine tetraaceticacid (EDTA). 0.65 ml of absolute ethanol was added, and themixture was loaded on CF11 cellulose and washed. The [121].-RNA was eluted in 2 ml of the same buffer without ethanol.All samples of [125I]RNA were purified at least twice beforeuse in further steps; their specific activities were determinedas before (13).

Digestion and Fingerprinting of Nucleic Acids. IodinatedRNA or DNA samples were digested and fingerprinted bystandard techniques (1, 2, 6, 16-19). Oligonucleotides were

eluted after two-dimensional fractionation by conventional

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Fingerprints of Jodinated Nucleic Acids 3261

techniques (18). 30% triethylamine carbonate was used foroligonucleotides from thin-layer plates, while a 22.5% solu-tion was used for oligonucleotides on DEAE paper to obtainmore rapid elution. Further base composition and enzymaticanalyses were carried out using the procedures reviewed byBarrell (18); any changes are noted in legends to figures describ-ing individual experiments. Radioactive oligonucleotides weredetected by autoradiography on Dupont Cronex 2 x-ray film.

RESULTS

RNase Ti Fingerprints of Previously Characterized RNAs.Fig. 1 shows RNase T1 fingerprints of 5S ribosomal [125II-RNA prepared from HeLa cells. These patterns were ob-tained by either electrophoresis at pH 1.9 or thin-layer homo-chromatography in the second dimension. Both proceduresyield simple patterns with this 120-nucleotide RNA species.

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FIG. 1. Ribonuclease T1 fingerprints of '2I-labeled HeLacell 5S RNA. About 2 X 106 dpm of '25I-labeled HeLa cell 5SRNA (specific activity 1.1 X 107 dpm/,ug) were mixed with10i g of f2 phage RNA and digested with 2 ug of RNase Ti in2 Mul of 0.01 M Tris * HCl (pH 7.5)-i mM EDTA for 30 min at 37°.Reaction mixtures were spotted directly onto 2.5 X 57-cm cellu-lose acetate strips (Schleicher & Schuell, Keene, N.H.) soakedwith 5% glacial acetic acid, containing 5 mM EDTA in 7 M urea,

and subjected to high-voltage electrophoresis (25 min, 6 kV).The oligonucleotides were transferred either to Whatman DE81DEAE paper or Machery-Nagel 20 X 40-cm DEAE-cellulosethin-layer chromatography plates by standard procedures (18).Second dimensions consisted either of high-voltage electrophoresisat pH 1.9 or thin-layer homochromatography. (A) Two-di-mensional fingerprint of 1251-labeled HeLa 5S RNA with electro-phoretic second dimension. The origin is at the upper right;the first dimension was from right to left, and the second [high-voltage electrophoresis at pH 1.9 on Whatman DE81 paper(18, 20)] from the top of the picture to the bottom. (B) Two-dimensional pattern of 125I-labeled HeLa 5S RNA with homo-chromatographic second dimension. The origin is at the lowerright; the first dimension-was from right to left, and the second[thin-layer homochromatography with homochromatographymixture "b" of Brownlee and Sanger (17)] from the bottom ofthe picture to the top. Further analyses of oligonucleotides a, b,and c are presented in Fig. 4.

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FIG. 2. Ribonuclease T1 fingerprints of 32p- and 1251-labeledf2 phage RNA. About 5 X 106 dpm of either 32P-labeled (specificactivity 2 X 106 dpm/,.g) or "12-labeled (specific activity be-tween 0.3 and 1.1 X 107 dpm/,ug) f2 RNA was mixed with 10 ,gof unlabeled f2 phage RNA and digested and fingerprintedas described in the legend to Fig. 1. (A) RNase T1 fingerprintof f2 [32P]RNA. (B) RNase Ti fingerprint of f2 [125I]RNA.In each case the origin is at the lower right, and the bracketedregion near the bottom of each picture represents that populationof unique oligonucleotides in each fingerprint which correspondsto those characterized previously (6). The different size of the'twobrackets is explained by the somewhat different spread of com-parably sized oligonucleotides obtained in the two fingerprints,which were chromatographed at different times.

We next analyzed an RNA molecule of much larger size.Fig. 2 shows fingerprints obtained after RNase T1 digestionof bacteriophage f2 RNA, which is about 3500 nucleotideslong. Fig. 2A shows the well-characterized fingerprint off2 [32P]RNA isolated from phage particles (6). The bracketedspots represent a unique group of Tl-resistant oligonucleo-tides which occur only once each in the RNA molecule. Fig.2B shows that f2 [1251]RNA yields a very similar fingerprintto that in Fig. 2A, and that, in particular, the unique spotsare arrayed in a similar pattern.

The Composition of Iodinated Nucleic Acids. Commerford(12) has shown that the most efficiently labeled product ofiodination of homopolymers is 5-iodocytosine, while uraciland guanine are labeled at 3-4% and 0.2-0.3% of the levelof cytosine, respectively. Poly(C) and poly(U) were iodinated,reduced to monomers by enzymatic digestion with RNaseT2 (containing RNase Ti and pancreatic RNase), and char-acterized by high-voltage electrophoresis at pH 3.5 on What-man 3MM paper (Fig. 3a, lanes 3 and 5). Under these con-ditions, [1lII]CMP runs slightly slower than [32P]GMP(Fig. 3a, lane 1), while [1251]UMP has a mobility midwaybetween 132P]GMP and [32P]UMP. Similar digestion ofiodinated HeLa 5S RNA yields a major component withmobility identical to that of [125J]CMP, while a trace spot isobserved at the mobility of [251I]UMP (Fig. 3b, lane 1). Thesame result was obtained in base composition determinationsof RNAs from diverse sources except for transfer RNA.T2 RNase digestion of tRNA yields a complicated patternof up to nine 125I-labeled components (Fig. 3c). Perhaps invitro labeling with 125I will be a valuable technique for identi-fying modified bases in such RNAs.

Proc. Nat. Acad. Sci. USA 70 (1973)

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FIG. 3. Base composition analysis of iodinated nucleicacids. Alkaline hydrolysis was for 18 hr at 370 in 0.4 M NaOH in10 jl (Alk). RNase T2 (2 units/ml) was used, together withRNase T1 (0.05 mg/ml) and pancreatic RNase (0.05 mg/ml).Incubation was for 2 hr at 370 in 10 of buffer containing 0.05 Mammonium acetate (pH 4.5) (T2). Various samples were spottedonto Whatman 3MM paper and exposed to descending high-voltage electrophoresis at pH 3.5 in 0.5% pyridine-5% glacialacetic acid-5 mM EDTA. (a) Lane 1-RNase T2 digestion offl [32P]RNA (104 dpm; specific activity 2 X 108 dpm/,ug) pre-

pared with all four [a-32P]ribonucleoside triphosphates; lane2-alkaline hydrolysis of [251I]poly(C) (104 dpm; specific activity2.5 X 106 dpm/,ug); lane 3-RNase T2 digestion of [12511]poly(C)(104 dpm as in lane 2); lane 4-alkaline hydrolysis of [125I]poly(U)(5 X 103 dpm; specific activity 105 dpm/,ug); lane 5-RNaseT2 digestion of [12nllpoly(U) (5 X 103 dpm as in lane 4). (b)Lane 1-RNase T2 digestion of HeLa 5S [125I]RNA (104 dpm;specific activity 1.1 X 107 dpm/,ug); lane 2-alkaline hydrolysisof fl[32P]RNA (104 dpm as in a, lane 1). (c) RNase T2 digestionof 2 X 104 dpm of Drosophila['25I]tRNA (specific activity >106dpm/uAg). Bracketed spots represent additional 125I-labeled moi-eties which we have observed only in RNase T2 digests of severaliodinated RNA samples that contained tRNA.

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FIG. 4. Enzymatic analysis of RNase Tl-resistant oligo-nucleotides from HeLa 5S [12I]RNA. Oligonucleotides a, b, and c

(Fig. 1B) were eluted after two-dimensional fractionation byconventional techniques (18). Each eluted oligonucleotide was

split into four equal aliquots and treated as follows: lane 1-notreatment; lane 2-sample was incubated with 2 units/ml ofRNase U2 in 10 1A for 2 hr at 37° in 0.05 M sodium acetate(pH 4.5), 2 mM EDTA, and 0.1 mg/ml of crystallized bovineserum albumin (Pentex Inc., Kankakee, Ill.); lane 3-incubationwith 1 mg/ml of pancreatic RNase in 10 M1 of 0.01 M Tris * HCO(pH 7.4) and 1 mM EDTA; lane 4-treatment with RNase T2in the enzymatic mixture described in the legend to Fig. 3.-The three groups of four analyses are labeled a, b, and c andrepresent spots a, b, and c from Fig. 1B, respectively. The sampleswere spotted onto Whatman DE81 DEAE paper and subjectedto high-voltage electrophoresis in pH 1.9 buffer (2.5% formicacid-8.7% acetic acid). The various positions for mono- andoligonucleotides refer to the positions tentatively assigned tosuch moieties containing one [15I]C. For [l5I]CMP, (C in thefigure), this position was confirmed directly by chromatographing[1251]CMP obtained by RNase T2 digestion of ['25I]poly(C) asin Fig. 3a, lane 3.

A second method commonly used for determination ofbase composition of RNA is alkaline hydrolysis (incubationin 0.4 N NaOH, at 37° for 18 hr). This treatment is unsuitablefor iodinated species for two reasons. First, a significantportion of the 125I is lost from [1251]poly(C) during this treat-ment. Second, upon electrophoresis of iodinated RNAs afteralkali treatment, there is a shift of most of the radioactivityfrom the position of [11-I]CMP to that of [1251]UMP (Fig.3a, lane 2). Similar treatment of [1251]poly(U) results in a

slight loss of radioactivity, but no change in mobility of theresulting [1251I]UMP (Fig. 3a, lane 4). These observationscould be explained if either deamination or deiodination of[12IS]CMP occurs under extreme alkaline conditions leadingto the production of either [12511 UMP or CMP, respectively.This has not been confirmed.Whatever the exact chemistry of these changes involving

[1251]CMP, these phenomena have been used to confirm inde-pendently that CMP is the predominant iodinated nucleotide.Bacteriophage fl RNA containing ['4C]CMP, synthesizedin vitro with RNA polymerase from a double-stranded rep-licative form DNA template, was iodinated and digestedwith RNase T2 under conditions described in the legend to

Fig. 3. The nucleotide spot with the mobility of [1251]CMP waseluted and treated with alkali as in Fig. 3. These conditionsremoved 125I from some of the previously iodinated residues,allowing the resultant [14C]CMP to migrate once again atits characteristic mobility, as in Fig. 3a, lane 1, or in Fig. 3b,lane 2 (data not shown).

Enzymatic Digestion of lodinated Oligonucleotides. RNaseTi-resistant fragments from HeLa 5S RNA (Fig. 1) have beencharacterized further by enzymatic digestion. RNA frag-ments eluted from DEAE-cellulose thin-layer plates were

divided into four equal parts and treated with pancreaticRNase, RNase U2, and RNase T2. The products were sepa-rated by high-voltage electrophoresis on Whatman DE81paper at pH 1.9, and an untreated sample of each oligonucleo-tide was included as marker. Such characterizations of threeof the RNase Ti-resistant oligonucleotides shown in Fig. 1Bare illustrated in Fig. 4. Spot a has been assigned as CG sinceRNase U2, which cleaves after purines, does not alter itsmobility (Fig. 4a, lanes 1 and 2), while pancreatic RNaseand RNase T2 shift the mobility of the 1251I-labeled product

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Proc. Nat. Acad. Sci. USA 70 (1973)

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Fingerprints of Jodinated Nucleic Acids 3263

to that of [1251]CMP (Fig. 4a, lanes 3 and 4). Similar datafor spots b and c (Fig. 4b and c) are consistent with the se-quences ACCG and UACUUG reported in human 5S RNA(20). Tentative correlations of the remaining RNase T1-resistant fragments shown in Fig. 1B with those whose se-quences have been determined by Forget and Weissman (20)can also be made.

Fingerprints of Previously Unavailable Radioactive RNAs.One of the most promising applications of this approachshould be in studying nucleic acids isolated from cells notreadily grown at high specific radioactivity. Fig. 5 shows anexample of this approach in which rabbit 5S ribosomal RNAwas iodinated and fingerprinted after RNase T1 digestion,while Fig. 6 illustrates such a pattern obtained with duckglobin mRNA.

Finally, the single-stranded DNA of bacteriophage fi hasbeen iodinated. Base composition analysis by digestionwith a mixture of pancreatic DNase and venom phospho-diesterase and electrophoresis as in Fig. 3 reveals one majorcomponent migrating towards the anode, with a mobilityslightly slower than the major component of RNA afterRNase T2 digestion (Fig. 3a, lane 3). Depurination analysisof this DNA species by the method of Ling (19) revealedseveral spots which could represent the C-containing poly-pyrimidine tracts.

DISCUSSION

The results reported here allow a preliminary evaluation ofthe potential usefulness of 125J-labeled nucleic acids in com-parative fingerprinting and sequencing studies. Most im-portant, the iodination reaction used here is at least qualita-tively a general labeling procedure, giving simple RNaseT1 fingerprints for small RNAs (Figs. 1 and 5) and morecomplex fingerprints of expected pattern for the larger RNA

FIG. 5. Ribonuclease T1 fingerprint of rabbit 5S RNARabbit 5S RNA was iodinated to give a specific activity of be-tween 106 and 107 dpm/,og. About 1 X 105 dpm was mixed with10 1Ag of f2 phage RNA and digested as in Fig. 1 and 2. Finger-printing analysis with thin-layer homochromatography on DEAE-cellulose as a second dimension was as in Fig. lB.

molecules (Figs. 2 and 6). We estimate that the specific activ-ities of the nucleic acids used here (106-108 dpm of 1251 perMgg) reflect a range of from one [121I]cytidine residue per15,000 bases to one in every 150 bases (assuming 25% cyti-dine). Thus, the proportion of RNase Ti-resistant oligonu-cleotides containing more than one [125f]cytidine residueshould be extremely small.Apparently the sort of hairpin loops that may occur in

5S RNA (20) or f2 phage RNA (21) do not hinder iodinationsufficiently to alter the expected patterns. This had been acause for concern since Commerford (12) reports that double-stranded DNA is a poor substrate for iodination. In an at-tempt to detect influence of structure on the iodination re-action, D. Housman, A. Jacobson, and W. Prensky (personalcommunication) fingerprinted HeLa 5S RNA iodinated ateither 400, 600, or 700 and observed qualitatively similarpatterns. We confirmed these observations using HeLa 5SRNA iodinated at the same temperatures. These observationsalso provide good evidence for the reproducibility of the iodina-tion procedure in several reactions.

Iodination causes a systematic shift of iodinated mono-and oligonucleotides to a faster electrophoretic mobility inthe direction of the anode at pH 3.5 (Fig. 3). This shift ismost dramatic with the shortest oligonucleotides, and be-comes increasingly less apparent in the larger ones (Fig. 2and unpublished data). It is also evident that the acquisitionof a single 1251 is insufficient to alter grossly the mobility ofthe longer oligonucleotides on the thin-layer fingerprintingsystem with homochromatography (Fig. 2). We can alsoconclude that the majority of the 1251 bound to RNA is stableto the conditions of the standard fingerprinting and sequenc-ing techniques used here. Furthermore, iodinated RNA isnot subject to breakdown during storage at -200 for severalmonths, as judged by fingerprinting assays. Specifically, weobtained a fingerprint similar to that shown in Fig. 6 afterthe duck globin mRNA had been stored at -200 for 4 months.Our preliminary characterization of the labeled base in

iodinated RNA (Fig. 3) allows us to form the hypothesis

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FIG. 6. Ribonuclease Ti fingerprint of duck globin mRNA.About 1 X 105 dpm of iodinated duck globin mRNA (estimatedspecific activity 1.2 X 107 dpm//ug) was mixed with 10 ,ug of f2phage RNA, digested with RNase Ti, and fingerprinted as inFig. lB.

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3264 Cell Biology: Robertson et al.

that at least 95% of the 1251 taken up into RNA is incorpo-rated into cytidine residues. Two lines of evidence supportthis idea. First, [1251]poly(C) yields a single nucleotide spotupon digestion with either pancreatic RNase or RNase T2,while various iodinated biological RNAs yield a spot withidentical mobility to that from [1251]poly(C) on several elec-trophoretic systems after RNase T2 digestion (Figs. 3 and4). Second, our base composition studies of [14C]CMP-con-taining iodinated fi RNA demonstrate that a single compo-nent obtained after RNase T2 digestion contains both the125I and the 14C label. Since the 14C label originally was derivedfrom [14C]CTP, we conclude that CMP is the iodinatedspecies.Base composition studies with [1251]poly(U) confirm Com-

merford's observation (12) that it is much less efficientlyiodinated than is poly(C). However, a faint spot with themobility of [1251]UMP is observed in RNase T2 digests ofiodinated biological RNA samples. Since iodinated poly(C),handled in the same way as the biological samples in allrespects, does not yield such a spot, we conclude that naturalRNAs iodinated in vitro contain a low level of [1251 ]uridine.

Nuclease specificities previously shown for unmodifiedRNAs (18) appear to be preserved for iodinated species. Inparticular, RNase T2, in conjunction with RNase T1 andpancreatic RNase, reduces iodinated poly(C) and poly(U)and various biological species to mononucleotides (Figs. 3and 4). This is of particular importance because the alkalinehydrolysis method conventionally used to determine basecomposition of RNA is unsuitable (Fig. 3a, lane 2). RNaseT1 appears to retain its specificity for guanosine when itdigests iodinated RNA. Although free [1251 ]CMP is sometimesliberated by this enzyme, we found that this correlates withRNA breakdown. Intact species yield no free ['25I]CMPupon RNase T1 digestion. Furthermore, as shown in Figs.1 and 4a, CG sequences are released from HeLa 5S RNA byRNase T1 treatment, indicating that this enzyme splitsRNA chains between guanosine and [1251]cytidine. Since thedata in Fig. 4 suggest that pancreatic RNase and RNaseU2 also give the expected digestion products, we concludethat the presence of [1251]cytidine neither prevents the ex-pected cleavages by these four enzymes nor induces unex-pected ones.The fingerprints in Figs. 5 and 6 demonstrate that we can

compare various related RNA species by use of the iodina-tion reaction followed by RNase T1 fingerprinting. For ex-ample, in addition to HeLa and rabbit 5S RNA (Figs. 1 and5) we have fingerprinted 5S [1251 ] RNAs from maize and Dro-sophila (not shown). The high degree of similarity observedamong all of the patterns obtained suggests that these 5SRNA molecules are highly conserved. This approach shouldalso be useful in judging the purity and complexity of RNAs.In addition to the patterns shown in Figs. 1, 2, 5, and 6, wehave fingerprinted two fractions containing histone mRNAand a partially purified sample of rabbit globin mRNA.

We therefore conclude that, in addition to its demonstratedusefulness in in situ hybridization experiments, iodination ofnucleic acids should also prove valuable in nucleic acid finger-printing and sequencing studies. For example, messengerRNAs or viral nucleic acids could be compared without thenecessity for preparing [32P]RNA or [32P]DNA. The ap-proach applied by Robertson and Jeppesen (6) in comparingthree closely related bacteriophage RNAs can be used. RNaseTi-resistant oligonucleotides from closely related species suchas globin mRNAs or tumor virus nucleic acids could be com-pared both by their patterns (as in Fig. 2) or by further en-zymatic analysis (as in Fig. 4). In particular, we have char-acterized the 10 largest oligonucleotides from the duckglobin mRNA fingerprint shown in Fig. 6 in this way. Theirspecific breakdown products, when fully analyzed, shouldallow us to obtain useful information about the compositionand evolution of such eukaryotic nucleic acid molecules.

We thank Mr. E. Pelle for crucial technical assistance. Weare grateful for gifts of nucleic acids from Drs. D. Housman,T. Borun, D. M. Steffensen, and D. E. Wimber. Part of this workwas supported by a grant to Dr. N. D. Zinder, The RockefellerUniversity, from the National Science Foundation.

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