9
THE Joumar, OF BIOLOGICAL. CHE%USTRI Vol. 235, No. 4, April 1960 Printed in U.S.A. Nucleotides Derived from Enzymatic Digests of Nucleic Acids of T2, T4, and T6 Bacteriophages* JANET LICHTENSTEIN AND SEYMOUR S. COHEE From the Department of Biochemistry, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania (Received for publication, November 3, 1959) The compositions of the nucleic acids of the T even bacterio- phages, T2, T4, and T6 have been shown to be identical with respect to their purine and pyrimidine bases within the accuracy of existing analytical methods for these bases (1). However these viruses contain the unusual pyrimidine, 5-hydroxymethyl cytosine, which provides a unique opportunity for chemical variability in the composition of these nucleic acids. Cohen found (2) that enzymatic digestion of phage nucleic acids by pancreatic deoxyribonuclease and intestinal phosphatase released large amounts of the deoxyribonucleosides of adenine, guanine, and thymine but only a very small proportion of the HMCY deoxyribonucleoside, although a similar digestion of thymus deoxyribonucleic acid leads to a quantitative degradation to the deoxyribonucleosides. It was suggested that the structural relations of HMC in some way conferred resistance to the phos- phoesterase action of these enzymes. The HMC deoxyribo- nucleoside was released by phosphatase after a preliminary acid hydrolysis (2). It was reported in 1946 that purified preparations of T2r+ bacteriophage contained hexose which reacted like glucose (3). Jesaitis and Goebel (4) have reported on the presence of hexose in T4 preparations and Jesaitis (5) first demonstrated the asso- ciation of glucose with the viral nucleic acid. In 1954 Volkin (6) and Sinsheimer (7) showed independently that the DNA’s of T4r and T2r+ bacteriophages, respectively, could be hydrolyzed, albeit incompletely, by pancreatic DNase and by venom phosphodiesterase to liberate small amounts of a nucleotide which contained both HMC and glucose. The glu- cose was present as an 0-glycoside of the 5-hydroxymethyl sub- stituent on the pyrimidine. In the digests of T4r+ nucleic acid the ratio of glucose to HMC in various fractions was 1: 1 (6). However, two HMC nucleotides were isolated by ion exchange chromatography from digests of T2r+ DNA, one of which con- tained a mole of glucose per mole of HMC whereas the other was free of the hexose (7). It was reported that the monoglu- cosyl HMC nucleotide is dephosphorylated by a 5’-nucleotidase at a markedly slower rate than is the nonglucosylated dHMP (7). Jesaitis (8) has analyzed the DNA of wild type r+ strains of T2, T4, and T6 and has shown that the three strains, which contain essentially similar amounts of HMC, contain different amounts of glucose in the amount of approximately 0.8, 1.0, * This research was aided by a grant from the Commonwealth Fund. _ - 1 The abbreviations used are: HMC, 5-hydroxymethyl cytosine; d. used in combination with standard abbreviations, stands for dkoxy; and dHMP, 5-hydroxymethyl deoxycytidylate; and 1.6 moles of glucose per mole of HMC, respectively. He has isolated small amounts of a diglucosyl derivative of dHMP and of nonglucosylated dHMP from enzymatic digests of the DNA of T6r+ (9). The isolation of the diglucosyl derivative of HMC deoxyribonucleoside has recently been described by Loeb and Cohen (10). Biosynthetic routes to the formation of some of these unique viral compounds have been demonstrated. Flaks and Cohen (11) have described the biosynthesis of dHMP from dCMP by an enzyme produced only in Escherichia coli infected by T-even bacteriophages. The conversion of dHMP to the triphosphate and its enzymatic incorporation into DNA have been described by several laboratories (12-14). The monoglucosylated HMC nucleoside triphosphate does not seem to be active in this system (15). On the other hand Kornberg et al. (12) have described a monoglucosylation of DNA containing HMC by UDP-glucose in the presence of an enzyme derived from extracts of T2-in- fected E. coli. The glucose of T6r+ DNA is the sole constituent of virus DNA which is formed completely de lzouo after infection (10). This is in keeping with the requirement for UDP-glucose in formation of virus DNA, whereas other units, purines, py- rimidines, etc. are in part derived from preformed host con- stituents. The exact role of glucosyl moieties in determining the speci- ficity of the phage nucleic acids in heredity is not known. A relation has been observed between the extent of glucosylation of HMC nucleotides in phage DNA and the efficiency with which phage infection leads to virus multiplication (16). This is con- ceivably related to survival of phage polynucleotide sequences within the host, as postulated earlier (2, 17). This laboratory has long been interested in the nature of the r mutation affecting cell lysis and we have examined various properties of pairs of r+ and r mutants. No differences had been observed between the base compositions of the parent and mutant strains. However preparations of r strains and of the DNA derived from these strains were obtained which contained larger amounts of glucose (18) than did similar preparations of r+ phages and their DNAs, whose glucose contents were comparable to the values reported by other workers (&S). Variations in the glucose content of the r preparations and their isolated DNA’s suggested the possi- bility that the extra glucose might be derived from degradation products of bacteria lysed more extensively by r phages (18) and it was felt that to test this point it would be necessary to seek and possibly exclude the presence of HMC nucleotides contain- ing polyglucosyl units. Existing enzymatic methods of releasing HMC nucleotides gave low yields of such compounds and although considerable 1134 by guest on April 27, 2020 http://www.jbc.org/ Downloaded from

Nucleotides Derived from Enzymatic Digests of Nucleic ...Nucleotides Derived from Enzymatic Digests of Nucleic Acids of T2, T4, and T6 Bacteriophages* JANET LICHTENSTEIN AND SEYMOUR

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Page 1: Nucleotides Derived from Enzymatic Digests of Nucleic ...Nucleotides Derived from Enzymatic Digests of Nucleic Acids of T2, T4, and T6 Bacteriophages* JANET LICHTENSTEIN AND SEYMOUR

THE Joumar, OF BIOLOGICAL. CHE%USTRI Vol. 235, No. 4, April 1960

Printed in U.S.A.

Nucleotides Derived from Enzymatic Digests of Nucleic Acids of T2, T4, and T6 Bacteriophages*

JANET LICHTENSTEIN AND SEYMOUR S. COHEE

From the Department of Biochemistry, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania

(Received for publication, November 3, 1959)

The compositions of the nucleic acids of the T even bacterio- phages, T2, T4, and T6 have been shown to be identical with respect to their purine and pyrimidine bases within the accuracy of existing analytical methods for these bases (1). However these viruses contain the unusual pyrimidine, 5-hydroxymethyl cytosine, which provides a unique opportunity for chemical variability in the composition of these nucleic acids. Cohen found (2) that enzymatic digestion of phage nucleic acids by pancreatic deoxyribonuclease and intestinal phosphatase released large amounts of the deoxyribonucleosides of adenine, guanine, and thymine but only a very small proportion of the HMCY deoxyribonucleoside, although a similar digestion of thymus deoxyribonucleic acid leads to a quantitative degradation to the deoxyribonucleosides. It was suggested that the structural relations of HMC in some way conferred resistance to the phos- phoesterase action of these enzymes. The HMC deoxyribo- nucleoside was released by phosphatase after a preliminary acid hydrolysis (2).

It was reported in 1946 that purified preparations of T2r+ bacteriophage contained hexose which reacted like glucose (3). Jesaitis and Goebel (4) have reported on the presence of hexose in T4 preparations and Jesaitis (5) first demonstrated the asso- ciation of glucose with the viral nucleic acid.

In 1954 Volkin (6) and Sinsheimer (7) showed independently that the DNA’s of T4r and T2r+ bacteriophages, respectively, could be hydrolyzed, albeit incompletely, by pancreatic DNase and by venom phosphodiesterase to liberate small amounts of a nucleotide which contained both HMC and glucose. The glu- cose was present as an 0-glycoside of the 5-hydroxymethyl sub- stituent on the pyrimidine. In the digests of T4r+ nucleic acid the ratio of glucose to HMC in various fractions was 1: 1 (6). However, two HMC nucleotides were isolated by ion exchange chromatography from digests of T2r+ DNA, one of which con- tained a mole of glucose per mole of HMC whereas the other was free of the hexose (7). It was reported that the monoglu- cosyl HMC nucleotide is dephosphorylated by a 5’-nucleotidase at a markedly slower rate than is the nonglucosylated dHMP (7).

Jesaitis (8) has analyzed the DNA of wild type r+ strains of T2, T4, and T6 and has shown that the three strains, which contain essentially similar amounts of HMC, contain different amounts of glucose in the amount of approximately 0.8, 1.0,

* This research was aided by a grant from the Commonwealth Fund.

_ -

1 The abbreviations used are: HMC, 5-hydroxymethyl cytosine; d. used in combination with standard abbreviations, stands for dkoxy; and dHMP, 5-hydroxymethyl deoxycytidylate;

and 1.6 moles of glucose per mole of HMC, respectively. He has isolated small amounts of a diglucosyl derivative of dHMP and of nonglucosylated dHMP from enzymatic digests of the DNA of T6r+ (9). The isolation of the diglucosyl derivative of HMC deoxyribonucleoside has recently been described by Loeb and Cohen (10).

Biosynthetic routes to the formation of some of these unique viral compounds have been demonstrated. Flaks and Cohen (11) have described the biosynthesis of dHMP from dCMP by an enzyme produced only in Escherichia coli infected by T-even bacteriophages. The conversion of dHMP to the triphosphate and its enzymatic incorporation into DNA have been described by several laboratories (12-14). The monoglucosylated HMC nucleoside triphosphate does not seem to be active in this system (15). On the other hand Kornberg et al. (12) have described a monoglucosylation of DNA containing HMC by UDP-glucose in the presence of an enzyme derived from extracts of T2-in- fected E. coli. The glucose of T6r+ DNA is the sole constituent of virus DNA which is formed completely de lzouo after infection (10). This is in keeping with the requirement for UDP-glucose in formation of virus DNA, whereas other units, purines, py- rimidines, etc. are in part derived from preformed host con- stituents.

The exact role of glucosyl moieties in determining the speci- ficity of the phage nucleic acids in heredity is not known. A relation has been observed between the extent of glucosylation of HMC nucleotides in phage DNA and the efficiency with which phage infection leads to virus multiplication (16). This is con- ceivably related to survival of phage polynucleotide sequences within the host, as postulated earlier (2, 17). This laboratory has long been interested in the nature of the r mutation affecting cell lysis and we have examined various properties of pairs of r+ and r mutants. No differences had been observed between the base compositions of the parent and mutant strains. However preparations of r strains and of the DNA derived from these strains were obtained which contained larger amounts of glucose (18) than did similar preparations of r+ phages and their DNAs, whose glucose contents were comparable to the values reported by other workers (&S). Variations in the glucose content of the r preparations and their isolated DNA’s suggested the possi- bility that the extra glucose might be derived from degradation products of bacteria lysed more extensively by r phages (18) and it was felt that to test this point it would be necessary to seek and possibly exclude the presence of HMC nucleotides contain- ing polyglucosyl units.

Existing enzymatic methods of releasing HMC nucleotides gave low yields of such compounds and although considerable

1134

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April 1960 J. Lichtenstein and S. S. Cohen 1135

effort was expended in devising chemical techniques to increase the yield of HMC mononucleotides no such method was indeed devised. Accordingly in lieu of a better approach the DNA of pairs of r+ and r strains were degraded extensively by pancreatic DNase and venom phosphodiesterase and the incompletely hydrolyzed digests were analyzed by ion exchange chromatog- raphy. These analyses are presented in this paper, as are a number of properties of various HMC nucleotides.

EXPERIMENTAL

Materials and Methods

Biological Systems-The preparation of cultures of E. coli strain B in various media and their use in the production of various T-even bacteriophages have been described (19, 20), as have been the isolation and properties of r+ and r strains of the T2, T4, and T6 bacteriophages used in these studies. Dr. Seymour Benzer of Purdue University has analyzed our r strains and has informed us that of the strains used in the earlier studies (19,20) T2r was of the rI1 group, whereas T4r and T6r are of the r1 group. Starting with our T2r+ stock (T2C) Dr. Benzer kindly isolated a T2r+I (designated T2Cr8 in his files) and this strain was used in this analytical work.

Chemical Methods; Preparation and Degradation of DNA- Preparations of phage containing 100 to 200 pmoles of DNA phosphorus were degraded by urea (10) and after extensive deproteinization, fibrous preparations of sodium salts of DNA were precipitated by ethanol and dried in a vacuum over P,Os. The solids were soaked overnight at 4” in Hz0 (about to 2 mg per ml) and the very viscous solution was subsequently worked to a smooth consistency, adjusted to 0.02 M MgClz and to pH 6.5 with dilute NaOH.

Crystalline pancreatic DNase (Worthington) (1 mg) was added per 100 mg of the DNA preparation. The pH, deter- mined electrometrically with a glass electrode and pH meter, was followed continuously at 37” with COrfree Nt bubbling slowly through the digest. The pH was maintained at pH 6.5 by frequent additions of dilute standard alkali and the digestion was continued until no pH change occurred for 30 minutes. In each digestion another milligram of DNase was added, and the reaction was continued for another hour, although no additional pH change was ever observed. The digests were then adjusted to pH 9.2 with NaOH and purified phosphodiesterasez was added. This enzyme was isolated (21) from 100 to 200 mg quantities of the venom of Crotalus adawuznteus, obtained from the Ross Allen Institute, Silver Springs, Florida. The purified phosphodiesterase was demonstrated to be essentially free of phosphomonoesterase, as tested on DNase digests of thymus DNA. Phosphodiesterase in an amount equal to that derived from 40 mg of venom was added to an amount of digest equiva- lent to 100 mg of DNA, and the pH was maintained at pH 9.2 by addition of standardized alkali during digestion. Hydrolysis seemed to be complete in about 90 minutes. After another 30 minutes, added portions of diesterase did not increase hydroly- sis. Thus the limitation in enzymatic cleavage did not seem to arise from the development of products inhibitory to the en- zyme. However it was noted in one experiment with T2r+ DNA that repetitive treatment with DNase and phosphodi-

2 We are indebted to Dr. J. F. Koerner for telling us of an im- proved isolation method for this enzyme before its publication.

esterase did lead to a significantly higher yield of mononucleo- tides and particularly of the HMC nucleotides.

Analytical Methods-Analyses were made on the DNA solu- tions after digestion by DNase had reduced the viscosity of the solutions sufficiently to permit accurate pipetting. Phosphorus was determined by the method of King (22) and deosyribose by the diphenylamine reaction (23). The estimation of glucose by the anthrone method has been described (18). Since deoxy- ribose is reactive in this reaction, it is necessary to attempt suitable corrections for the color contributed by nucleotides containing this substance. The estimation of this correction is very difficult since not only does purine-bound deosyribose react, as in the diphenylamine reaction, but pyrimidine-bound deoxyribose does also to a slight extent. The estent of the correction may be made relatively accurately with mono- or dinucleotide fragments whose base compositions are known and whose nonglucosylated components may be assembled in a test mixture. In most estimations of the glucose content of the mononucleotides, dCMP was used as a blank. However the correction is necessarily less accurate with digests or larger polynucleotide fragments. With the latter types of material it is unlikely that the determination of glucose content by this calorimetric method is more accurate than &lo to 15%.

Ion Exchange Chromatography-The enzymatic digests were fractionated by the method of Sinsheimer and Koerner (24). Digests containing 100 to 200 pmoles of DNA phosphorus, ad- justed to pH 10, were applied to Dowex l-acetate columns (10 cm x 1 cm, 4% cross-linked) at concentrations of 0.01 to 0.02 M

with respect to total anion. The preparation of the column has been described (25). The column filtrate or eluate was collected at a flow rate of 0.2 to 0.4 ml per minute in 5 ml fractions on a Gilson fraction collector, equipped with an ultraviolet absorp- tion spectrophotometer, continuously recording percentage of transmission at 2537 A. The initial filtrate was washed through with 0.01 M NHbOH and the column was then washed with HzO. Elution was carried out with ammonium acetate buffers adjusted to pH 4.3. These were used successively at the follow- ing concentrations: 0.01 M, 0.03 M, 0.06 M, 0.1 M, 0.2 M, 0.3 M, 0.5 M, 1 M, and 2 M. With each eluent, a minimum of 100 ml of buffer was applied; and if a component was eluted, the eluent was not changed until the concentration of component in the eluate had fallen to essentially zero. After completing the elution of a component at the end of a day, the flow of eluent was frequently arrested until the following morning. On beginning the flow of buffer on the next morning a small amount of guanine usually came off quite rapidly, reflecting the small but significant la- bility of the deoxyribosyl linkage to guanine at pH 4.3 at room temperature.

Fractions containing the separated mononucleotides were pooled after a more extensive spectrophotometric examination and were concentrated by distillation under reduced pressure. Fractions lacking appreciable ultraviolet absorption were also pooled and concentrated. Phosphorus analyses were made of all fractions; phosphorus was present only in fractions charac- teristic of the known mononucleotides and larger components of the digest. After estimating the yields of the mononucleo- tides, dTMP, dAMP and dGMP, which were shown to have correct spectral properties, the pooled fractions were taken to dryness in a vacuum and finally lyophilized to minimize the ammonium acetate content of the preparations.

The yields of the mononucleotides of HMC were estimated

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1136 Nucleotides from Nucleic Acids of T2, T4, and T6 Bacteriophages Vol. 235, No. 4

TABLE I

Enzymatic Degradation of phage DNA

Effect of DNase

Sample ofiDNA

Moles H+ liberated per

mole DNA-P

T2r+ T2rI T4r+ T4rI T6rf T6rI Thymus E. coli

0.08 0.10 0.09 0.12 0.08, 0.09 0.11

1

al

% 34 30.9 34-37 33.7 29 32.3 36 36.3 35 42.1 36 50.2 34-38 15.4 34 22.8

.- :ncrement i;i;yey.-

sqw rate, 50%

xorption* Before After .-

Total moles [+ liberated per mole DNA-P

1.14 0.68 1.21 0.63 1.15 0.60 1.39 0.73 1.26 0.68, 0.65 1.36 0.68 1.26 0.98 1.12 0.98

Phosphodies- terase after

DNase

* These estimations were kindly made by Miss H. D. Barner.

from the sum of the ultraviolet absorption at 280 rnp of each fraction within each elution peak. The extinction coefficients of each HMC-containing mononucleotide were estimated by de- termining the P content and absorption at 280 rnp at pH 4.3 for aliquots taken from the maximum of each elution peak. Thus the 6~‘s at the pH of elution, pH 4.3, were estimated to be 8.5X 103,8.2 x 103, and 8.6 x lo3 for diglucosyl dHMP, monoglucosyl dHMP, and nonglucosylated dHMP, respectively.

RESULTS

Extent of Enzymatic Digestion-The electrometric back titra- tion of the digest to maintain digestion at a. constant pH pro- vided a measure of the extent of cleavage of the DNA chains. It can be seen in Table I that the extent of hydrolysis of phage DNA by DNase was of the order of 10% of the phosphodiester bonds, or about half that observed with DNA derived from other materials (26).

The subsequent degradation by phosphodiesterase cleaved an additional 50 to 60 y0 of the phosphodiester bonds as recorded in Table I. This amounts to a total of 60 to 70% of the total phos- phodiester bonds, as contrasted to an essentially quantitative cleavage of thymus DNA (27) or a greater than 93 y0 cleavage of T7 DNA (28) to mononucleotides by the same treatment. Thus the stepwise digestion of all the T-even phage nucleic acids by pancreatic DNase and Crotalus venom phosphodiesterase left 30 to 40% of the nucleotides in polynucleotide fragments. In con- trast to the results with phage DNA an identical digestion of E. coli DNA and fractionation by ion exchange separation resulted in a 940/, recovery in the form of mononucleotides as estimated on a phosphorus basis or in a 98% recovery estimated spectro- photometrically.

Analysis of the ultraviolet absorption at 2600 A at 37” during digestion of DNA at about 30 pg per ml by DNase at 0.45 pg of DNase per ml in 0.05 M Tris buffer pH 7.5 + 0.02 M MgClz re- vealed that the increase in this function for phage DNA was com- parable to that observed with samples of thymus DNA and E. coli DNA, as presented in Table I. At lower concentrations of the enzyme, which revealed the markedly slower rate of degrada- tion of the various phage DNA samples,3 as contrasted with

3 The rate of increase of ultraviolet absorption when solutions of phage DNA are degraded by pancreatic DNase was found by

thymus DNA, the increase in ultraviolet absorption finally at- tained was frequently less than 30% and appeared to reflect the inhibition of the enzyme by DNA and its degradation products. In any case, degradation of DNA by DNase carried to comple- tion appeared to eliminate essentially all structural integrity re- sponsible for the restriction of ultraviolet absorption in phage DNA.

Studies were also made of the sedimentation behavior of phage DNA and of DNase digests to determine whether the products of digestion of the phage DNA were markedly larger than those of thymus DNA. Sedimentation velocity studies of solutions of phage DNA and digests (3 to 4 pg of DNA phosphorus per ml) were carried out in a Spinco model E ultracentrifuge’ equipped with ultraviolet absorption optics (29). A Spinco Analytrol was used to read the films. The analysis of these densitometer read- ings to estimate sedimentation constants has been described (29). The s rates were corrected to that for water at 20”. Samples of intact DNA and phage digests having an optical density at 260 rnp of 0.7 to 0.9 were analyzed in 0.1 M Tris buffer at pH 7.6. The distribution of s rates of these samples was determined. The DNA’s were observed to be somewhat less heterogeneous and to possess somewhat higher s rates than are obtained for most sam- ples of thymus DNA. These samples also possessed a consid- erably higher gsp/c, extrapolated to infinite dilution and zero shear gradient, than had thymus DNA. The s rate recorded in Table I is that of material at the midpoint of the sedimenting boundary. As recorded earlier (30) a bimodal distribution of ~50% rates was ndt observed for phage DNA. In the absence of data on the concentration dependence of the s rate and distribution function for phage DNA, and in the absence of data on the ef- fect of the isolation procedure on the physical parameters of this material, the s rates for the DNA before digestion which are pre- sented in Table I will not be discussed further.

In order to analyze the sedimentation of the slowly sedimenting DNase digests, use was made of the synthetic boundary cell (29). As can be seen in Table I, the ~50% rates of the DNase digests of phage DNA and other DNA are not significantly different, and indeed the spread of the boundaries is also quite similar. After a digestion with DNase carried to completion the s rates at the 50 ‘% points of the various boundaries were 1.1 to 1.4, and at the 90% point were 6 to 7. Thus digestion of phage DNA by pan- creatic DNase did not leave a high molecular fraction comprising a significant portion of the initial DNA.

Analysis of Digests-The initial alkaline filtrate through the column usually contained a very small amount of deoxyribonu- cleosides and free bases. The ultraviolet absorption of this frac- tion, usually having a maximum at 255 to 260 rnp at pH 10 amounted to 0.5 to 1.5% of the total. This fraction, the Hz0 washes and the eluate made with 0.01 M acetate pH 4.3 did not contain phosphorus.

The first nucleotides to be eluted contained HMC and three such nucleotides were observed. These contained in the order

Miss H. D. Barner to be uniformly and markedly lower for T4 DNA than for T2 and T6 DNA under comparable conditions at low enzyme concentration. The T4r+ and T4r DNAs were de- graded at identical rates when so tested under identical conditions.

4 We are indebted to Dr. H. K. Schachman of the Virus Labora- tory of the University of California who kindly made his facilities available to us for these studies and in addition was very helpful in the interpretation of the data therein obtained. We are also grateful for the assistance of Miss Pearl Appel of his laboratory in the calculation of the distribution of s rates.

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April 1960 J. Lichtenstein and S. S. Cohen 1137

of their appearance from digests of T&DNA a diglucosyl moiety, a monoglucosyl moiety, and a final nucleotide lacking glucose. From such a digest (150 pmoles of P applied to the column) the diglucosyl dHMP could be eluted with 100 ml 0.03 M acetate (pH 4.3) and the monoglucosyl and nonglucosylated dHMP were eluted with 0.06 NI buffer. Occasionally a column was obtained with which the elution of the diglucosyl nucleotide with 0.03 M

acetate proceeded too slowly, and in this case 0.06 M acetate was used to elute the diglucosyl- and monoglucosyl-dHMP while 0.1 M acetate was used to elute dHh!IP. On this column dCMP is eluted with 0.1 M acetate; no trace of this compound was ever observed in these digests. Records of the elution patterns of the dHMP nucleotides for digests of DNA of T6r+ and T6r are pre- sented in Figure 1A and B, respectively. It can be seenthat each digest contains three comparable components; these are the dHMP nucleotides noted above, and the properties of these ma- terials will be considered in detail below.

As shown in Figure 1 and Table II, the monoglucosylated nu- _ cleotide is present only as a small fraction of the total dHMP nucleotides of which approximately 407, has been recovered in both cases. Jesaitis first discovered the existence of digluco- sylated dHMP and nonglucosylated dHMP in T6r+ digests (9) but in those studies did not detect the presence of the small amount of the monoglucosylated nucleotide. He has since ob- FIG. IA (upper). The ion exchange separation of various HMC-

served the three substances in T6 digests essentially in the pro- containing deoxynucleotides from an enzymatic digest of T6r+ DNA.

portions described above (31). B (lower). The ion exchange separation of various HMC-

containing deoxynucleotides from an enzymatic digest of T6r The similarity of the patterns in studies of digests of several DNA.

preparations of T6r+ DNA and in the T6r+ and T6r analyses sug- gest that the monoglucosyl dHMP is not an artifact arising from TABLE II the degradation of the diglucosyl derivative. The quantitative Yield of monon&eotides from DNA digest variation between the two preparations with respect to their content of the monoglucosyl component, as reported in Table II Percentage of phosphorus applied to column

may arise in several ways. The analysis of a great many prep- arations of phage DNA would be required to be certain that the observed differences are significant. Thus, although some care was taken to attempt to carry the digestion to completion, thereby accounting for higher yields of the dHMP mononu- cleotides than obtained earlier, it will be seen below that the hy- drolysis of the glucosylated dHMP nucleotides does occur very slowly. In the digestion of phage DNA, then, hydrolysis would proceed very slowly at the end and would yield disproportion- ately large amounts of the dHMP nucleotides at this stage of the digestion. Under such circumstances if digestion were not abso-

Phage DNA

T2rf A 0 T2r+ B 0 T2rI 0 T4r+ 0 T4rI 0 T6r+$ 3.1 T6rI 2.7

m& dHMP

-

B

g,

-

dono- ucosy IHMP

4.1 2.6 3.2 3.3 0.7 0.4

-

1 < 1HMP dTMP

6.9 5.5 2.6 0 0 2.4 3.4

24.1 28.0 22.0 13.6 22.2 27.7 21.7

- I -

.-

-

dAMP dGMP

--

22.9 14.7 26.8 16.5 20.8 10.7 17.4 15.3 21.0 12.9 21.8 11.8 20.9 12.8t

.- 1 I

.-

-

Recovery*

Mono- 1UClW.l. tides

-0ta1 P

% %

69 87 81 94 69 80 50 79 59 88 67 94 62 93

lutely completed one might expect to observe quantitative dif- * Including polynucleotides eluted from column. ferences with respect to particularly refractory parts of the t After correction for dinucleotide (1.2%).

polynucleotide chain. In this connection it can be noted that the total yield of dHMP nucleotides is lowest from T4 prepa- rations which appears to contain only the monoglucosylated nucleotide, affirming the high level of resistance to enzymatic cleavage conferred by the presence of this compound and by the absence of nonglucosylated dHMP.4

In Fig. 2A and B are presented the elution patterns of the dHMP nucleotides from digests of the DNA of T2rf and T4r+, respectively. As can be seen in Fig. 2A, no trace of the digluco- syl derivative was obtained from T2r+ and only the monogluco- syl- and nonglucosylated nucleotides were observed in similar amounts during elution with 0.06 M: buffer. In the analyses re- ported in this figure, in which the high yield of 60% of the total

5 For the purposes of estimating yields of recovery of dHMP nucleotides the total HMC content of T even phage DNA is as- sumed to be 0.16 mole HMC per mole phosphorus (1).

$ HMC nucleotides used for estimation of c, dephosphoryla- tion.

HMC of T2r+-B was obtained as mononucleotides in the digest, the digest had been subjected during one day to two successive treatments with DNase, one by phosphodiesterase, and on the next day another treatment by DNase followed by phosphodi- esterase. The second series of treatments liberated an additional 12 pmoles of acid per 149 pmoles of DNA phosphorus. Thus as noted above the somewhat lower yield of dHMP nucleotides from T2r1, although qualitatively reflecting the similar content of both nucleotides, may be interpreted as reflecting the resist- ance of the residual fragments which were not similarly subjected to such repetitive enzymatic assaults.

As can be seen in Fig. 2B, T4r+ was observed to liberate only the monoglucosylated dHMP which was eluted at 0.06 M buffer.

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Nucleotides from Nucleic Acids of T.2, T4, and T6 Bacteriophages Vol. 235, No. 4

FIG. 2A (upper). The ion exchange separation HMC-containing deoxynucleotides from an enzymatic digest of T2r+ DNA. B (lower). The ion exchange isolation of monoglucosyl dHMP from an enzymatic digest of T4r+ DNA.

As reported in Table II for both T4rf and T4r, only 20% of the total HMC was obtained in this form in each case.

The separation of dTMP and dAMP was never entirely com- plete in this system in a single run. The elution was effected

initially at 0.2 M acetate and a point was usually obtained where- in the elution of dTMP was almost complete but had not fallen to zero before dAMP began to be eluted. The degree of overlap did not involve more than 5% of either of the two nucleotides estimated spectrophotometrically. At the point where dTMP fell and dAMP appeared, the molarity of the buffer was increased to 0.3 M. The final mononucleotide, dGMP, was eluted at 0.5 M acetate buffer. Polynucleotide fragments were then eluted at 1.0 M, 1.5 M, and 2.0 M acetate. The columns were finally washed with M HCl; this final eluate rarely contained significant amounts of phosphorus.

No other mononucleotides were detected by this analytical pro- cedure. Digests of T6 DNA contained a dinucleotide of adenine and diglucosylated HMC which was eluted just before dGMP, as can be seen in Fig. 3. However it was not separated cleanly from dGMP in a single elution sequence. The properties of this fragment, comprising 7.5% of the total HMC, will be described below.

Properties of HMC Mononucleotides-The separated mononu- cleotides were lyophilized to minimize the ammonium acetate in the preparations and were stored at -20”. Further purification was effected by adsorption on and elution from charcoal as de- scribed by Pontis et al. (32). Yields were of the order of 50 to 70% of the nucleotide applied.

The dHMP nucleotides were characterized and differentiated by their composition, their ultraviolet absorption properties over a wide range of pH, chromatographic properties in two solvent systems, electrophoretic mobilities, and sensitivity to alkaline phosphatase.

The absorption maxima of the three dHMP nucleotides were found to be at 283 to 284 rnp in 0.1 N HCl andrtt 275 rnE.1 at neu- trality and in 0.1 N NaOH. The minima were at 245 rnp in acid and at 255 rnp at neutrality and in alkali. These properties and spectral changes in acid, neutral pH, and alkali afbrmed the iden- tity of ionizable groups in a common base in the three nucleotides. The pK of the amino groups of each nucleotide was estimated by the spectrophotometric method (33) ; in earlier studies by this method (10) the pK values of the HMC deoxyriboside and of the diglucosyl derivative of this deoxyriboside were found to be 3.5 to 3.6 and 3.4 to 3.5, respectively. In Figs. 4~4, B, and C are presented the effects of pH on the various extinction ratios of the mononucleotides. In these figures the diglucosylated and non-

FIG. 3. The ion exchange separation of a dinucleotide containing HMC from dGMP in the analysis of an enzymatic digest of T6r DNA.

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April 1960 J. Lichtenstein and S. S. Cohen 1139

2.5 t

4 I 0 2 4 6 a IO 12 14

PH

FIG. 4A FIG. 4. The effect of pH on the 290:260, 280:260, and 250:269

extinction ratios of A, dHMP; B, monoglucosyl dHMP; C, di- glucosyl dHMP.

glucosylated nucleotides were derived from the T6rI preparation while the monoglucosylated derivatives were obtained from the T4rf preparation. The diglucosyl derivative was found to con- tain 1.83 moles of glucose per mole of P, and the T4 derivative had 1.02 moles of glucose per mole of P. Identical pK values were obtained for the diglucosyl derivative derived from T6r+ (1.96 moles of glucose per mole of P) and from T6rI. It can be determined from the figures that the pK values of the amino group were 3.75 to 3.80 for the diglucosyl-dHMP, 3.6 to 3.75 for the monoglucosyl dHMP, and 4.0 to 4.2 for free dHMP. In free dHMP it may be inferred that the presence of the phosphate and its propinquity to the ammo group inhibit the loss of the proton from -NHs+, accounting for the higher pK of this group in dHMP as contrasted to the deoxyribonucleoside. Substitution at the -CH20H interposes a group between the phosphate and -NH3+ and reduces the pK of the latter. Thus the spectral properties of the glucosylated compounds were quite diierent from those of the nonglucosylated dHMP at the pH of elution (pH 4.3) from the ion exchange column as a function of the dif- ferences in extent of dissociation of the amino groups of these compounds.

The separated nucleotides were studied by descending paper chromatography in two solvent systems, the isobutyrate-NH40H system at pH 3.9 (34) and the ethanol-acetate-tetraborate sys- tem (35). The electrophoretic motilities of the compounds were also compared on paper in 0.2 M borate buffer at pH 9.2 in the E. C. 405 electrophoresis apparatus at 22.2 volts per cm for 2 hours. The behavior in these systems readily distinguished the compounds, as can be seen in Table III.

In the unfractionated digests of thymus DNA, chromatogra- phy in the isobutyrate system easily reveals the four nucleotides in the order of increasing RF: dGMP, dTMP, dCMP, and dAMP. Similar analyses of digests of phage DNA clearly demonstrates

2.5 t

2.5

2.c

0 F $ 1.5

MONOGLUCOSYL dHMP

J : : : : : : : : : : : : 0 2 4 6 a IO 12

PH FIG. 4B

DIGLUCOSYL dHMP

0 2 4 6 a IO 12 14 PH

FIG. 4C

the deficiency of dCMP, although a faint spot attributable to dHMP may be perceived in that area. In addition the gluco- sylated dHMP derivatives are easily detected in the area between polynucleotide fragments and the more mobile dGMP.

The sensitivities of the three HMC nucleotides and of dCMP to hydrolysis by calf intestinal alkaline phosphatase (35) were compared. Enzyme purified up to, but not including the acetone precipitation step (9), was used. Four units of phosphatase in 0.025 ml were added to 0.33 pmole of nucleotide in 1.1 ml of 0.05

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1140 Nucleotides from Nucleic Acids of T2, T4, and T6 Bacteriophages Vol. 235, No. 4

TABLE III

Chromatographic and electrophoretic properties of HMC nucleotides

dCMP Diglucosyl

dHMP.... Monoglucosy

dHMP. dHMP...... CMP

-.

(

( 1

( ( (

-

Paper chromatography

Isobutyrate E thanol-tetraborate

RF RF ___ RF dCMP RF

RF RF dCMP

I---- 1.39-0.45 1.0 0.18 1.0

).15-6.180.41-0.470.15 0.8

1.27-0.280.634.64 ).354.400.88-0.900.20 1.0 ).27-0.300.69-0.760.037-0.041 0.2

Paper elec- trophoresis

Borate pH 9.2

Mobility nucleotide

Ivffbilty

0

1.6-0.7

).84-0.86 ).93-0.98

dCMP .

MINUTES

FIG. 5. A comparison of the enzymatic dephosphorylation by alkaline phosphatase of dCMP and various glucosylated deriva- tives of dHMP.

Tris buffer pH 9.2 containing 0.03 M Mg++ and the mixture was incubated at 37”. Aliquots were removed, acidified, and inor- ganic P was determined. In Fig. 5, it can be seen that dHMP is dephosphorylated at a markedly slower rate than is dCMP. Glucosylation sharply reduces the rate of dephosphorylation of dHMP to 10% or less of the rate at which dCMP is cleaved. Of interest is the observation that the cleavage of the monogluco- syl derivative soon stopped completely.

Properties of Dinucleotide-The fraction present in T6 DNA digests which was eluted by 0.5 M acetate just before the elution of dGMP was concentrated and lyophilized. A solution of this material was subjected to various analyses. It did not chroma- tograph as a compact spot in the isobutyrate system. Spectra were measured at pH 1, 7, and 13 and absorption maxima were observed at 270 rnl.c, 266 rnp, and 265 mp, respectively. The absorbance ratios were those of a 1: 1 mixture of dAMP and dHMP. The material contained 0.5 mole of reactive deoxyri- bose per mole of P, suggesting the presence of equal amounts of

purine and pyrimidine nucleotides. Hydrolysis for 1 hour at 60” in N HCl, followed by chromatography in butanol-NHIOH (37), revealed adenine as the sole mobile base. The glucose con- tent of the material was estimated with a mixture of equimolar amounts of dAMP and dCMP as the blank. The substance con- tained 1.1 moles of glucose per mole of P. It was concluded that the fraction is a dinucleotide consisting of dAMP and the digluco- sylated dHMP.

Treatment with alkaline phosphatase converted slightly more than 50% of the total phosphorus to inorganic P. Hydrolysis at 100” with N HCl for 1 hour also cleaved half of the total P.

Note added in proof: Four structural possibilities exist for the dinucleotide. These are d-pApH, d-pHpA, d-ApHp, and d- HpAp, where H represents the diglucosyl hydroxymethyldeoxy- cytidine. The last two structures are excluded by the method of preparation. The second possibility would be expected on acid hydrolysis or treatment with Burton’s reagent to lead to the formation of the diphosphate of the pyrimidine nucleoside [Bur- ton, K. and Petersen, G. B., Biochim. et Biophys. Acta, 26, 667 (1957) and Potter, J. L. and Laskowski, J. L., J. Biol. Chem., 2S4, 1263 (1959)], whereas the first should lead to the formation of the 5’-monophosphate of the pyrimidine nucleoside. After treatment with Burton’s reagent, a compound was found whose RF in isobutyrate-NHIOH and electrophoretic mobility in borate were those of the diglucosyl dHMP. It is concluded that the nucleotide probably has the structure d-pApH.

DISCUSSION

No evidence could be obtained in these analyses to show that the DNA of a mutant r phage differed significantly in its content of glucosylated dHMP nucleotides from the DNA of the parent r+ phage. However these experiments revealed only a part of the total HMC of the DNA as mononucleotides and it might be imagined that the HMC attached to polynucleotide fragments contained other types of glucosylated nucleotides which were even more resistant to enzymatic degradation. It is of interest in this connection that Lehman has recently described (38) a nu- clease derived from E. coli which was capable of complete con- version of a partially digested phage DNA to mononucleotides and in a hydrolysate of T2r+ DNA was able to detect a small amount of the diglucosylated dHMP. From the yield of non- glucosylated dHMP from T2r+ DNA, in Analysis B, recorded in Table II, it may be calculated that the glucose content of T2 DNA is too high to be accounted for by monoglucosylated dHMP alone and leads one to postulate other polyglucosyl derivatives. The application of the new nuclease to rf and r DNA should be most helpful in settling the question originally posed.

Although the addition of glucosyl moieties to HMC deoxyribo- nucleoside did not affect the pK of the amino group to a marked extent, it is clear that the nucleotides present a different picture. The pK of the,amino group seems to be similar in dHMP and in dCMP, the former being 4.0 to 4.2 and the latter at 4.2. The addition of one or more glucosyl moieties to dHMP reduces the pK very significantly to 3.6 to 3.8, accounting in part for the ease of separation of the glucosyl derivatives from the nonglucosylated nucleotide. However the diglucosyl and monoglucosyl deriva- tives are also cleanly separated in elution from Dowex l-acetate despite the similarities of their pK values. This phenomenon of the effect of glucose in decreasing affinity for the resin may also be observed in the relatively early elution of the diglucosylated dinucleotide in T6 digests, when other dinucleotides from other

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April 1960 J. Lichtenstein and X. S. Cohen

digests are not detectable before release of all of the mononu- cleotides. The effects of such a substituent are also noteworthy in paper chromatography in the isobutyrate system in which af- finity for the aqueous phase is increased by addition of glucose. In paper electrophoresis in borate at pH 9.2 the increase in mo- lecular volume of the nucleotides resulting from addition of glu- cose decreases electrophoretic mobility and more than compen- sates for the effect of borate which increased net charge.

SUMMARY

The deoxyribonucleic acids of three pairs of parent and mutant phages, T2r+ and T2r, T4rf and T4r, T6r+ and T6r were de- graded by pancreatic deoxyribonuclease. Despite a slow and relatively low extent of cleavage of phosphodiester bonds by this enzyme, the sizes of the resulting fragments, estimated ultracen- trifugally, were of the same order as those from the deoxyri- bonucleic acid of thymus or Escherichia co&. The subsequent cleavage of these fragments by rattlesnake venom phosphodi- esterase released mononucleotides and a large proportion of poly- nucleotide fragments which were fractionated by ion exchange chromatography. Most of the viral 5-hydroxymethyl deoxycy- tidilic acid (dHMP) remained in the polynucleotide fragments. However of the dHMP mononucleotides released, similar distri- butions of the several types were observed in digests of parental r+ and mutant r strains.

The dHMP nucleotides isolated from T2 strains were of two types, monoglucosylated and nonglucosylated, in contrast to the monoglucosylated dHMP nucleotide isolated from T4 strains. Three types were isolated from T6 strains, including similar amounts of a diglucosylated and a nonglucosylated plus a small portion of the monoglucosylated derivative. In addition T6 strains yielded a dinucleotide of dAMP and of diglucosylated dHMP. Spectrophotometric, chromatographic, and electropho- retie properties of the three types of dHMP nucleotides are re- corded.

REFERENCES

1. WYATT, G. R., AND COHEN, S. S., &o&em. J., 66, 774 (1953). 2. COHEN, S. S., Cold Spring Harbor symposia on quantitative

biology, Vol. 18, Long Island Biological Association, Cold Spring Harbor, Long Island, New York, 1953, p. 221.

3. COHEN, S. S., AND ANDERSON, T. F., J. Exptl. Med., 64, 511 (1946).

4. JESAITIS, M. A., AND GOEBEL, W. F., Cold Spring Harbor symposia on quantitative biology, Vol. 18, Long Island Bio-

logical Association, Cold Spring Harbor, Long Island, New York, 1953, p. 205.

5. 6.

i: 9.

10. 11.

JESAITIS, M. A., Microbial Genetics Bull., 10, 16 (1954). VOLHIN, E. J., J. Am. Chem. Boc., 76, 5892 (1954). SINSHEIMER, R. L., Science, 120, 551 (1954). JESAITIS, M. A., Nature, 178, 637 (1956). JESAITIS, M. A., J. Exptl. Med., 106, 233 (1957). LOEB, M. R., AND COHEN, S. S., J. Biol. Chem., 234,364 (1959). FLAKS, J. G., AND COHEN, S. S., Biochim. et Biophys. Acta,

26, 665 (1957). 12.

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ZIMMERMAN, S. B., KORNBERG, S. R., JOSSE, J., AND KORN- BERG, A., Federation Proc., 18, 359 (1959).

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KOERNER, J. F., AND SMITH. M. S., Federation Proc.. 18, 264 (1959). ’

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KOERNER, J.F., SMITH, M.S., ANDBUCHANAN, J.M., J.Am. Chem. Sot., 81, 2594 (1959).

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COHEN, S. S., Advances in Virus Research, 3, 1 (1955). COHEN, S. S., Science, 123, 653 (1956). COHEN, S. S., AND ARBOGAST, R., J. Exptl. Med., 91,607 (1950).

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1049 (1957). 22. KING, E. J., Biochem. J., 26, 292 (1950). 23. DISCHE, Z., Mikrochemie, 8, 4 (1930). 24. SINSHEIMER, R. L., AND KOERNER, J. F., Science, 114, 42

(1951). 25. SINSHEIMER, R. L., J. Biol. Chem., 208, 445 (1954). 26. KUNITZ, M., J. Gen. Physiol., 33, 363 (1949). 27. SINSHEIMER, R. L., AND KOERNER,J. F., J. Biol. Chem., 198,

293 (1952). 28. LUNAN, K. D., AND SINSHEIMER, R. L., Virology, 2,455 (1956). 29. SCHACHMAN. H. K.. in S. P. COLOWICK AND N. 0. KAPLAN

30. COHEN, S. S., Cellular biology, nucleic acids and viruses, Vol. 6. New York Academy of Sciences. New York. 1957, v. 263.

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MAGASANIK, B., VISCHER, E., DONIGER, R., ELSON, D., AND CHARGAFF, E., J. Biol. Chem., 186, 37 (1950).

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(Editors); Methods in enzymology, Vol. IV, Academic Press, Inc., New York, 1957, p. 32.

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Janet Lichtenstein and Seymour S. CohenBacteriophages

Nucleotides Derived from Enzymatic Digests of Nucleic Acids of T2, T4, and T6

1960, 235:1134-1141.J. Biol. Chem. 

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