VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1209

9 Ramel, A., E. Stellwagen, and H. K. Schachman, Federation Proc., 20, 387 (1961).10 Markus, G., A. L. Grossberg, and D. Pressman, Arch. Biochem. Biophys., 96, 63 (1962)."1 For preparation of anti-Xp antisera, see Nisonoff, A., and D. Pressman, J. Immunol., 80,

417 (1958) and idem., 83, 138 (1959).12 For preparation of anti-Ap antisera, see Grossberg, A. L., and D. Pressman, J. Am. Chem.

Soc., 82, 5478 (1960).13 For preparation of anti-Rp antisera, see Pressman, D. and L. A. Sternberger, J. Immunol.,

66, 609 (1951), and Grossberg, A. L., G. Radzimski, and D. Pressman, Biochemistry, 1, 391 (1962).14 Smithies, O., Biochem. J., 71, 585 (1959).15 Poulik, M. D., Biochim. et Biophysica Acta., 44, 390 (1960).16 Edelman, G. M., and M. D. Poulik, J. Exp. Med., 113, 861 (1961).17 Breinl, F., and F. Haurowitz, Z. Physiol. Chem., 192, 45 (1930).18 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940).19 Pressman, D., and 0. Roholt, these PROCEEDINGS, 47, 1606 (1961).

THE ENZYMATIC SYNTHESIS OF GUANOSINE DIPHOSPHATECOLITOSE BY A MUTANT STRAIN OF ESCHERICHIA COLI*

BY EDWARD C. HEATHt AND ALAN D. ELBEINTRACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BACTERIOLOGY,

THE UNIVERSITY OF MICHIGAN

Communicated by J. L. Oncley, May 10, 1962

We have previously reported' the isolation of guanosine diphosphate colitose(GDP-colitose* GDP-3,6-dideoxy-L-galactose) from Escherichia coli 0111-B4;only 2.5 umoles of this sugar nucleotide were isolated from 1 kilogram of cells.Studies on the biosynthesis of colitose with extracts of this organism indicated thatGDP-mannose was a precursor;2 however, the enzymatically formed colitose wasisolated from a high-molecular weight substance and attempts to isolate the sus-pected intermediate, GDP-colitose, were unsuccessful.We now wish to report the enzymatic synthesis of GDP-colitose from GDP-man-

nose (Fig. 1) using extracts of a mutant strain derived from E. coli 0111-B4.This mutant (designated E. col; J-5) was isolated from aged cultures of E. coli

0111-B4, and appears to have properties similar to those of the mutant strains ofSalmonella typhi-murium and Salmonella enteritidis previously reported.3' 20 Thus.E. coli J-5 exhibits the following characteristics: (1) inability to ferment galactose,(2) galactose sensitivity, (3) accumulation of uridine diphosphate galactose (whengrowth media contain galactose), (4) accumulation of GDP-colitose. In addition,analysis of the cell-wall lipopolysaccharide isolated from the parent and mutantorganisms agreed with these findings. Thus, the parent organism produces lipo-polysaccharide containing glucose, galactose, and co'itose.4 When the mutant isgrown in the absence of galactose, the lipopolysaccharide contains no galactose andlittle or no colitose; supplementation of the growth medium with galactose yieldslipopolysaccharide that appears similar to the normal product.

Materials and Methods.-Tyvelose was prepared by mild acid hydrolysis of the cell-wall lipo-polysaccharide of Salmonella typhi4' followed by neutralization, deionization, and chromatog-raphy. 3-Deoxy-D-ribohexose was a generous gift of N. K. Richtmyer, National Institutes ofHealth, Bethesda, Md. Uniformly labeled L-fucose was kindly provided by H. S. Isbell of the

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CH2OH National Bureau of Standards. Allo o other chemicals were obtained from

CH3 commercial sources.--PPRG -- H o- PPRG Guanosine diphosphate D-glucose

H was a generous gift from D. M.Carlson of this laboratory. GDP-

GDP-MANNOSE GDP- COLI/OSE mannose and guanosine diphos-GUANOSINE DIPHOSPHATE GUANOSINE DIPHOSPHATE phate L-fucose (GDP-fucose) were

a- D- MANNOPYRANOSIDE P(?)- 3,6-DIDEOXY-L -GALACTOPYRANOSIDE chemically prepared by condensingFIG. 1.-The conversion of GDP-mannose to GDP- the corresponding hexose 1-phos-

colitose. phate with GMP morpholidate.6Mannose 1-phosphate was preparedby the method of Posternak and

Rosselet.7 The synthesis of L-fucose 1-phosphate has not been previously described. In thepresent studies, L-fucose was converted to L-fucose 1-phosphate by a series of procedures analo-gous to those used for the preparation of mannose 1-phosphate; thus, fucose - (crystalline) tetra-acetyl-fucose (crystalline) 1-chloro-triacetyl L-fucose L 1-diphenyl-phosphoryl-triacetyl-L-fucoside- L-fucose 1-phosphate. Although the anomeric configuration of L-fucose 1-phosphateand therefore of GDP-fucose is not known with certainty, it is assumed to be the ,B-L-pyrano-side. Thus, when 1-chlorotriacetyl-L-fucoside was converted to methyl-2,3,4-triacetyl-L-fuCopy -ranoside, only the ,8-glycoside (mp 96° could be isolated; this derivative is readily distinguish-able8 from the a-anomer (mp 670).

Chromatographic solvent systems used in these studies were as follows: I. Ethanol: 1 Aammonium acetate, pH 7.4 (7:3); II. Isobutyric acid: ammonium hydroxide: water (57:4:39);III. 0.1 M phosphate, pH 6.8: ammonium sulfate: n-propanol (100: 60:2); IV. Ethyl acetate:acetic acid:water (3:1:3); V. n-Butanol:pyridine:water (6:4:3); VI. n-Butanol:ethanol:water (10:4:3); VII. n-Butanol:pyridine:0.1 N hydrochloric acid (5:3:2); VIII. n-Butanol:acetic acid:water (4:1:5).For the determination of radioactivity on paper chromatograms, guide strips were scanned for

radioactivity in a windowless 47r scanner. For quantitation, appropriate areas of the paper werecut out, suspended in a toluene solvent,9 and counted in a Packard liquid scintillation spec-trometer.

Phosphate was determined by the method of Fiske and Subbarow;"° anthrone reagent' wasused for the estimation of hexose; diphenylamine reagent" was used for the estimation of deoxy-ribose; dideoxyhexose was determined by the thiobarbituric acid procedure."Due to the extreme acid-lability of GDP-colitose (see Fig. 3), the acid conditions employed in

the thiobarbituric acid test caused considerable hydrolysis of the nucleotide even at 37°. It wastherefore necessary to perform the periodate oxidation at pH 7. Under these conditions, freecolitose exhibited approximately one half the molar absorbancy index as that observed understandard conditions.

Conversion of GDP-Mannose to GDP-Colitose.-E. coli J-5 was grown in Trypti-case Soy broth at 370 for 12 hr with shaking. Cells were harvested by centrifuga-tion and washed with cold 0.15 M KC1. Extracts were prepared by suspendingcells in 3 volumes of water, sonicating for 5 to 10 min, and centrifuging at 25,000X g for 30 min. The incubation mixture contained the following (/umoles in a finalvolume of 36.5 ml): GDP-mannose-C"4 (34,100 cpm//Amole), 12; TPN, 50; glu-cose 6-phosphate, 50; potassium fluoride, 500; Tris buffer, pH 7.2, 5,000; and20 ml of crude extract. Disappearance of GDP-mannose and appearance of a prod-uct were followed by removing 0.4 ml aliquots at the indicated times (Fig. 2) andtransferring to 1 ml of warm ethanol to stop the reactions. After cooling and centri-fuging, the supernatant fluids were concentrated in vacuo to 0.2 ml, applied to What-man 3MM paper in one inch bands, and chromatographed in solvent I. Afterdeveloping the chromatograms for 17 hr, the areas of each strip corresponding to

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VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1211

GDP-mannose (Rf = 0.16) and 40 ' 'GDP-colitose (Rf = 0.27) were cut A

out and their radioactive content 32determined as described. These re- Nsuits (Fig. 2) indicated that GDP- ° 24

x 2

mannose was rapidly converted to E A GDP-Colifosea compound with chromatographic 6 GDP-Mannoseproperties similar to GDP-colitose.In addition, 85 to 100 per cent of / \the total radioactivitywasaccounted 8 1for in these two areas of the chro- u

matograms for each aliquot tested, 20 40 60 80 100 120indicating that GDP-colitose formed MINUTESby these extracts is not further FIG. 2.-The rate of enzymatic conversion of

conditions. GDP-mannose to GDP-colitose. The details of themetabolized under these conditions experiment are described in the text.After 120-min incubation, the re-mainder of the mixture was added to 100 ml of warm ethanol and centrifuged,and the supernatant fluids were chromatographed in the same manner as describedfor the aliquots. The radioactive band was eluted with water and rechromato-graphed on Schleicher and Schuell 589-Blue Ribbon paper first in solvent II andthen in solvent I. A single radioactive band was observed in each case which cor-responded in mobility to GDP-colitose (isolated from E. coli J-5). Using these pu-rification procedures, 6.6 umoles of GDP-colitose were isolated.Homogeneity of the Product.-The nucleotide appeared homogeneous (on What-

man 1 paper) in solvents I (Rf = 0.35), II (Rf = 0.37), and III (Rf = 0.36) andby paper electrophoresis in 0.05 M potassium phosphate buffer, pH 7.5, and in0.05 M citrate buffer, pH 4.6; in each case, the radioactive and ultraviolet-light-absorbing spots coincided. The chromatographic and electrophoretic mobilities ofthe enzymatically synthesized material were identical in all instances with those ofGDP-colitose which had been isolated from cells.

Characterization of GDP-Colitose.-The nucleotide exhibited ultraviolet-light-absorption spectra at pH 1, 7, and 11 which were indistinguishable from authenticGDP. Analysis of the nucleotide gave the following results (molar ratios): guano-sine, 1.00; phosphorus, 2.04; 3,6-dideoxyhexose, 1.04. Inorganic phosphorus wasnot detected in the preparation. Guanosine was estimated from the absorbancyof the sample at 252 mAt at pH 7, assuming a molar absorbancy index of 13.7 X103. The nucleotide gave negative reactions with diphenylamine and with anthroneindicating the absence of deoxyribose and of hexoses. The specific radioactivityof the nucleotide was found to be 30,200 cpm/,4mole agreeing with that of GDP-mannose (34,100 cpm/,jmole).The nucleotide is considerably more labile to acid hydrolysis than other nucleotide

diphosphate hexoses. As shown in Figure 3, both GDP-colitose (isolated from thecells) and the enzymatically prepared GDP-dideoxyhexose were completely hy-drolyzed at pH 2 in 1 min at 1000, as compared to about 6 minutes for GDP-mannose.The dideoxyhexose was shown to be glycosidically bound to the nucleotide since

(1) it gave a negative reducing sugar test, (2) the carbonyl group was resistant to

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1212 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S.

,,, reduction by sodium borohydride,and (3) it gave a negative thio-

100 - barbituric acid test.After mild acid hydrolysis, the

N 80 / nucleotide diphosphate moiety0 E / / was characterized as GDP by

60 chromatography in solvents IIand III. In both instances, the

U 40 g / / major ultraviolet-light-absorbing0: 40CL ^ / ;/ o GOP-Colilose(Enzymolic) components were indistinguish-

20 if / /f ^ GDP-Co/ltose (From cel/s) able from GDP (1, R. = 0.3; II,20/o GDP-Munnose Rf = 0.46), while traces of ma-

* E. Co/i 0-///

0Lpopo/ysocchoride terial were observed in both sol-5 10 15 vents which corresponded toMINUTES GMP. GDP is readily distin-

FIG. 3-Acid-lability of GDP-colitose. The rate of guishable from ADP, UDP, andhydrolysis of GI)P-colitose was compared to that of '(IDP-mannose and lipopolysaccharide (from E. coli CDP in these solvent systems.0111-B4) at pH 2 at 1000. In all cases, samples were In addition the nucleotide di-adjusted to pH 2 in an ice bath and placed in a boilingwater bath, and, at appropriate intervals, aliquots were phosphate was further shown toremoved and pipetted into 0.1 ml of ice cold 0.5 M be a pentose nucleotide as it con-phosphate buffer. In the case of GDP-colitose andlipopolysaccharide, the rate of hydrolysis was meas- sumed periodate when the chro-ured by the thiobarbituric acid test' (using neutral matograms were treated withperiodate as described). For GDP-mannose, the rateof hydrolysis was determined by the Park-Johnson periodic acid and benzidine.15reducing sugar method.14 The values presented for After mild acid hydrolysis ofthe rate of hydrolysis of lipopolysaccharide representonly colitose release; thus, these values do not reflect the sugar nucleotide, the radioac-hydrolysis of the other sugar components. tive dideoxyhexose was purified

by deionization with mixed-bedion-exchange resin (Dowex-1-CO3= and Dowex-50-H+) and was tentatively identi-fied as colitose by cochromatography with the authentic compound as indicated inTable L. Of the four possible pairs of enantiomorphs of the dideoxyhexoses, only

TABLE 1CHROMATOGRAPHY OF SUGAR FROM ENZYMATICALLY SYNTHESIZED NUCLEOTIDE

Solvent systems*Compound V VI VII VIII

Unknown 0.66 0.56 0.64 0.55Colitose 0.65 0.57 0.64 0.56Tyvelose 0.73 0.64 0.74 0.633-deoxy-D-ribo-hexose 0.51 0.39 0.47. 0.35i-fucose 0.44 0.33 0.39 0.312-deoxy-D-ribose 0.57 0.47Unknown (reduced) 0.58 - 0.54Colititol 0.58 0.55

* See text for description.

tyvelose (3,6-dideoxy-D-arabinohexose) and colitose (3,6-dideoxy-L-xylohexose)were available as standards. In all instances, the unknown sugar cochromato-graphed with authentic colitose, and the radioactivity corresponded precisely withthe colitose area of the chromatograms as determined with the ammoniacal silverreagent'6; no other reducing substances were detectable on the chromatograms.Further, after reduction of the sugar with sodium borohydride, a product was ob-

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VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1213

tained that was indistinguishable from authentic colititol by paper chromatographyin solvents V and VII. In addition, the sugar alcohol gave a positive thiobarbi-turic acid test with an absorption maximum at 532 mju; these results would not beobtained if the parent sugar were a 2-deoxyhexose since the corresponding alcoholcould not yield malondialdehyde upon periodate oxidation.The identity of the dideoxyhexose was definitely established as colitose by

preparation of a large quantity of the nucleotide and conversion of the sugar moietyto colititol. In this case, the incubation mixture contained the following (Amolesin a final volume of 32 ml): GDP-mannose-C 4 (2,900 cpm/,Lmole), 61; TPN,50; glucose 6-phosphate, 50; potassium fluoride, 500; Tris buffer, pH 7.2, 5,000;and 20 ml of crude extract from E. coli J-5. The mixture was incubated for 4hr at 370, treated with warm ethanol and streaked on Whatman 3MM filter paperas described. The GDP-colitose area of the chromatogram was eluted with water,heated in 0.01 N H2SO4 for 10 min at 1000, neutralized with solid BaCO3, and cen-trifuged. The supernatant fluid was treated with mixed-bed ion-exchange resin,concentrated in vacuo to a small volume, and streaked on Schleicher and Schuell589-Green Ribbon paper which was then developed with solvent IV for 3 hr.Guide strips were cut from the edge of the chromatograms and stained with theammoniacal silver reagent, revealing a band of material which corresponded tocolitose (Rf = 0.57; representing all of the radioactivity on the chromatogram)and only a trace of some other reducing substance near the origin (Rf = 0.10).The band corresponding to colitose was eluted with water and concentrated invacuo to about 5 ml. Analysis of the solution indicated the presence of approxi-mately 60 ,umoles of dideoxyhexose. The solution was treated with 75 mg of sodiumborohydride at room temperature for 30 min, adjusted to pH 1 with 2 N HCl andconcentrated in vacuo to dryness. The residue was dissolved in methanol and con-centrated in vacuo to dryness, and this process was repeated four times. Finally,the aqueous solution was deionized with mixed-bed ion-exchange resin and concen-trated to a clear, colorless syrup from which water was removed by dissolving thesyrup in ethanol and concentrating to dryness several times. The syrup was seededwith authentic colititol and stirred for several min. A few drops of acetone werethen added, and the suspension was placed in an ice bath and allowed to crystallizefor about 1 hr. The crystalline material was harvested by centrifugation, washedwith cold acetone and ether, and dried in vacuo. The mother liquor was again car-ried through the procedure and a second crop of crystals was obtained. The totalyield of crystalline material was approximately 6 mg. Thus, a total of 40 Amoles(2,240 cpm/,4mole) of crystalline colititol was obtained from approximately 61Mmoles of GDP-mannose (2,900 cpm/,4mole) which had been used as substrate, ora yield of about 66 per cent. The physical constants which have been reportedfor synthetic colititol'7 are: mp 92-940, [a]D -51 (c = 2.5 in methanol); andthe constants for authentic colititol prepared in our laboratory were, mp 89-910,[a]D23 -50.60 (c = 0.25 in methanol). The dideoxyhexitol derived from the en-zymatically synthesized GDP-colitose exhibited the following values: mp 89-910,[aCD23 -50.30 i 0.60 (c = 0.32 in methanol).'8 These results clearly establishthe identity of the dideoxy-hexitol as colititol, 3,6-dideoxy-L-xylo-hexitol and clearlydistinguish it from any of the other three possible pairs of enantiomorphs, 3,6-dideoxy-arabino-hexitol, 3,6-dideoxy-ribo-hexito, and 3,6-dideoxy-lyxo-hexitol.4

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1214 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S.

From these data, it was concluded that the sugar moiety of the isolated guanosinesugar nucleotide is colitose, or 3,6-dideoxy-L-galactose. In assigning the structureof the nucleotide as guanosine diphosphate colitose (Fig. 1), the following assump-tions have been made: (1) the colitose moiety is attached to the terminal phosphategroup of the nucleotide diphosphate; (2) the colitose moiety of the nucleotide isin the pyranose ring form; and (3) the anomeric configuration of the glycosidicbond is in the fl-form by analogy to the position of this bond in the substrate, GDP-mannose.

Specificity of the Reaction-To determine the specificity of the enzyme system forGDP-mannose, the extract was incubated with the other known guanosine hexosenucleotides, GDP-glucose and GDP-fucose. The results of these studies, shownin Table 2, clearly indicate that only GDP-mannose is capable of serving as a pre-

TABLE 2SPECIFICITY STUDIES

Total Radioactivity (Cpm) inSubstrate* Colitose Mannose Glucose Fucose

GDP-mannose, 3,600 2920 14GDP-mannose, t 3,600 4 3220GDP-glucose, 5,000 12 3153GDP-fucose, 2,500 5 - 2250

* Incubation mixtures contained the following (pmoles in final volumes of 0.35 ml): either GDP-mannose, 0.1, GDP-glucose, 0.03, or GDP-fucose, 0.25; TPN, 0.5; glucose 6-phosphate, 0.5; potas-sium fluoride, 5; Tris buffer, pH 7.2, 50; and 0.2 ml of crude extract of B. coli J-5. Incubations wereconducted at 370 for 1 hr. The mixtures were then treated with ethanol, hydrolyzed, deionized, andchromatographed on Whatman 1 paper with solvent VII. The strips were first scanned for radio-activity, and then the radioactive areas and the colitose areas of each strip were counted as described.

t The extract used in this experiment was heated at 1000 for 2 min before it was added to the incuba-tion mixture.

cursor of GDP-colitose. In addition, the data indicate that these extracts are in-capable of converting GDP-glucose to GDP-mannose and, further, that GDP-fucose cannot be an intermediate in the conversion of GDP-mannose to GDP-colitose.No definitive information is presently available concerning the cofactor require-

ments of this enzyme system. The ability of the extracts to convert GDP-mannoseto GDP-colitose was stimulated only 10 to 20 per cent by the addition of TPNHor a TPNH generating system. Exhaustive dialysis of the extracts against water,buffers, or EDTA resulted in substantial losses (50 per cent or more) of activity,although they could be reactivated only slightly by the addition of TPNH (or aTPNH-generating system) or by divalent cations.

Discussion.-Nikaido3 suggested from studies with mutants of S. typhi-muriumand S. enteritidis, which are similar in their properties to E. coli J-5, that in theabsence of a galactosyl donor (UDP-galactose), the organisms are able to synthesizeonly the glucan portions of their cell-wall lipopolysaccharides; when a galactosyldonor is available (cells grown in the presence of galactose), cell-wall lipopolysac-charides are present which contain a normal complement of sugars (glucose, galac-tose, mannose, rhamnose, and either abequose or tyvelose in S. typhi-murium andS. enteritidis, respectively). The present studies with E. coli J-5 support this con-tention in so far as glucose-grown cells possess essentially only glucose in theirlipopolysaccharides, while cells grown on galactose-supplemented media possessglucose, galactose, and colitose in amounts similar to the parent organism, E. coli

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VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1215

0111-B4. In addition, the studies with E. coli J-5 indicate that colitose is probablyattached terminally to the polysaccharide via galactose.The direct formation of GDP-colitose from GDP-mannose substantiates the

in vivo isotope experiments reported by Cynkin and Ashwell,'9 which indicatedthat colitose was formed from glucose without inversion or cleavage of the carbonchain. The conversion of GDP-mannose to GDP-colitose would appear to involvea complex series of enzymatic transformations; thus, this conversion requires re-duction at carbon atoms 3 and 6 in addition to epimerization at carbon atom 5.Although the isolation of CDP-abequose and CDP-tyvelose has been reported,20the present studies are the first report of the enzymatic synthesis of any of the dide-oxy-hexose nucleotides, and therefore no precedence has been established for a pos-sible mechanism for the biosynthesis of these unique sugar nucleotides. Gins-burg,2' in his study of the conversion of GDP-mannose to GDP-fucose and Glaserand Kornfeld22 and Okazaki et at.23 in their studies of the conversion of TDP-glucose to TDP-L-rhamnose, in each instance, concluded that one of the interme-diates in the biosynthesis of both of these 6-deoxyhexose nucleotides was a 4-keto-6-deoxyhexose nucleotide. It appears from the previous studies, therefore, that the6-deoxy group is formed without any net reduction of the molecule and that TPNHfunctions to reduce the compounds ultimately to the level of fucose or rhamnoseafter the formation of the 4-keto-6-deoxy intermediate. Whether or not analogousintermediates are involved in the biosynthesis of GDP-colitose remains to be estab-lished. In this regard, it is of interest to note in the present studies that GDP->-fucose does not serve as a precursor of GDP-colitose.

Further work is in progress in an attempt to elucidate the details of the mechanismof the biosynthesis of GDP-colitose from GDP-mannose.

* The Rackham Arthritis Research Unit is supported by a grant from the Horace H. RackhamSchool of Graduate Studies of The University of Michigan. This investigation was supported by agrant from the National Institute of Arthritis and Metabolic Diseases, National Institutes ofHealth.

t Research Career Development Awardee, U.S. Public Health Service.t Post-doctoral Fellow, National Institute of Allergy and Infectious Diseases, National Insti-

tutes of Health.I Heath, E. C., Biochim. Biophys. Acta, 39, 377-378 (1960).2Heath, E. C., Federation Proc., 19, 85 (1960).3Nikaido, H., Biochim. Biophys. Acta, 48, 460-469 (1961).4Westphal, O., and 0. Luderitz, Angew. Chem., 72, 881-891 (1960).5 This preparation was a generous gift from A. G. Johnson of this University.6Roseman, S., J. J. Distler, J. G. Moffatt, and H. G. Khorana, J. Am. Chem. Soc., 83, 659-

663 (1961).7Posternak, T., and J. P. Rosselet, Helv. Chim. Acta, 36, 1614-1623 (1953).8 Hockett, R. C., F. P. Phelps, and C. S. Hudson, J. Am. Chem. Soc., 61, 1658-1660 (1939).9 Hayes, F. N., D. G. Ott, V. N. Kerr, and B. S. Rogers, Nucleonics, 13, No. 12, 38-41 (1955).

10 Fiske, C. H., and Y. Subbarow, J. Biol. Chem., 66, 375-400 (1925).11 Loewus, F. A., Anal. Chem., 24, 219 (1952).2 Seibert, F. B., J. Biol. Chem., 133, 593-604 (1940).

13 Cynkin, M. A., and G. Ashwell, Nature, 186, 155-156 (1960).14 Park, J. T., and M. J. Johnson, J. Biol. Chem., 181, 149-151 (1949).6 Gordon, H. T., W. Thornburg, and L. N. Werum, Anal. Chem., 28, 849-855 (1956).16 Trevelyan, W. E., D. P. Procter, and J. S. Harrison, Nature, 166, 444 (1950).7 Luderitz, O., A. M. Staub, S. Stirm, and 0. Westphal, Biochem. Zeit., 330, 193-197 (1958).

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1216 BIOCHEMISTRY: HUANG AND BONNER PROC. N. A. S.

18 The authors are indebted to J. J. Distler of this laboratory for performing the polarimetrystudies.

19 Cynkin, M. A., and G. Ashwell, Bact. Proc., 161 (1960).20 Nikaido, H., and K. Jokura, Biochem. and Biophys. Res. Comm., 6, 304-309 (1961).21 Ginsburg, V., J. Biol. Chem., 236, 2389-2393 (1961).22 Glaser, L., and S. Kornfeld, J. Biol. Chem., 236, 1795-1799 (1961).23 Okazaki. R., T. Okazaki, and J. L. Strominger, Federation Proc., 20, 85 (1961).

HISTONE, A SUPPRESSOR OF CHROMOSOMAL RNA SYNTHESIS*

BY RU-CHIH C. HUANG AND JAMES BONNER

DIVISION OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY

Communicated May 22, 1962

We have previously reportedl1 2 that chromatin isolated from pea embryos pos-sesses the ability to carry out the DNA-dependent synthesis of RNA from the fourriboside triphosphates.3 The present paper concerns the roles in such synthesis ofthe several components of chromatin. It will be shown that the DNA of pea em-bryo chromatin is present in at least two forms, namely, as DNA itself and as DNAbound in nucleohistone complex. It will be further shown that DNA fully com-plexed with histone is inactive in the support of DNA-dependent RNA synthesis.

Materials and Methods.-Pea embryos: Pea seeds (var. Alaska) were germinated in 35-gallonbarrels in lots of 25 lb. The seeds were soaked for 5 hr in running water at 200C and then gentlysprayed with water for an additional 35 hr. The embryonic axes, approximately 1 cm in length,were next separated from the cotyledons in a semiautomatic 3-stage disassembly line. Fiftypound dry weight of seeds yield approximately 1 kg fresh weight of embryos.

Preparation of chromatin: The chilled, sterilized (with 10OX diluted Clorox) embryos wereground for approximately 1 min in a Blendor with an equal weight of grinding medium (sucrose0.25 M, tris pH 8.0,0.05 M, ,3-mercaptoethanol, 0.01 M, MgCl2, 0.001 M) and filtered successivelythrough cheesecloth and miracloth to remove cell wall debris. The filtrate was then centrifugedfor 30 min at 4,000 X g. Under these circumstances, mitochondria and smaller particles remainin suspension while starch and chromatin sediment. The gelatinous chromatin layer was scrapedfrom the underlying, firm starch layer and washed by successive recentrifugation (10,000 X g)in grinding medium (1X), sucrose, 0.25 M (2X), and tris, 0.05 M, pH8.0(2X). ,3-Mercapto-ethanol, 0.01 M, was included in all of the above media. The final pellet was resuspended in thetris buffer, 30 ml per kg initial embryos. The yield of such crude chromatin is approximately0.5 gm per kg embryos; the yield of DNA, 50 mg per kg embryos.

Purification of crude chromatin: The crude chromatin prepared as described above contains ca.95% of the DNA of the embryo but is contaminated by nonchromosomal protein, removable bysucrose gradient centrifugation. This was accomplished by layering 5 ml of crude chromatinsuspension on 25 ml of 2 M sucrose (0.01 M in j-mercaptoethanol). The upper third of the tubewas then gently stirred to form a rough gradient and the tubes centrifuged in the SW-25 swingingbucket head at 20 krpm for 3 hr. The resulting pellet of which the major constituents are DNAand histone will be referred to as purified chromatin. Approximately 70% of the DNA of crudechromatin is recovered in the purified chromatin.

Analyses: DNA and RNA were determined principally by the Schmidt-Tannhauser procedureaccording to Ts'o and Sato.4 The diphenylamine method of Burton6 was used for determinationof DNA in the presence of much protein. Total protein was determined by the Folin-phenolmethod as described by Lowry.8 Histone protein was separated from total protein on the basisof the solubility of the former in 0.2 N HC1 and determined on the acid extract by the Lowrymethod. Melting point determinations of DNA and nucleohistone were carried out

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on

Apr

il 18

, 202

1