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5 Dische, Z., "Color reaction of nucleic acid components," in The Nucleic Acids, ed. E. Chargaff and J. Davidson (New York: Academic Press, 1955), Vol. 1, pp. 258-303. 6 Burton, K., Biochem. J., 62, 315 (1956). 7Oyama, W. I., and H. Eagle, Proc. Soc. Exptl. Biol. Med., 91, 305 (1956). 8 Marmur, J., J. Mol. Biol., 3, 208 (1961). 9 Tamm, C., M. E. Hoder, and E. Chargaff, J. Bio. Chem., 195, 49 (1952). '0Szybalski, W., Anal. Biochem., 3, 267 (1962). 11 Sueoka, N., J. Mol. Biol., 3, 31 (1961). 12Trowne, P. W., and B. R. Rabin, these PROCEEDINGS, 52, 88 (1964). 13Goldberg, I. H., M. Rabinowitz, and E. Reich, these PROCEEDINGS, 48, 2094 (1962). 14 Siminoff, P., Appl. Microbiol., 9, 66 (1961). 15 Crowther, D., and J. Melnick, Virology, 15, 65 (1964). I6 Smith, C. G., W. L. Lummis, and J. E. Grady, Cancer Res., 19, 847 (1959). 17 Reich, E., Science, 143, 684 (1964). 18 These experiments were performed by Drs. J. J. Vavra and H. E. Renis of The Upjohn Company, and by Dr. J. J. Holland, University of California, Redwood, California. 19 Liersch, M., and G. Hartman, Biochem. Z., 340, 390 (1964). 20 DiMarco, A., et al., Nature, 201, 707 (1964). 21 Ward, D., and E. Reich, Federation Proc., 24, 603 (1965). 22 Chamberlin, M., and P. Berg, these PROCEEDINGS, 48, 81 (1962). 23 Goldberg, I. H., Biochim. Biophys. Acta, 51, 201 (1961). 24 Magee, W. E., Virology, 17, 604 (1962).

A BASIS FOR UTILIZATION OF UNNATURAL AND PENTJTOLS BY AEROBACTER AEROGENES* BY R. P. MORTLOCK,t D. D. Fossirr, AND W. A. WOOD DEPARTMENT OF BIOCHEMISTRY, MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN Communicated by H. G. Wood, June 18, 1966 Although not rigorously studied, it has long appeared that bacteria are unique in their ability to metabolize those organic materials which are rarely, if ever, found in nature. Aerobacter aerogenes, PRL R3, particularly illustrates this phenomenon with its ability to utilize as a source of energy seven of the eight aldopentoses and all four of the pentitols.' 2 Of this group of 11 structures, D-, D-, L- , D-, and ribitol are found in nature, whereas D-arabinose, D- , L-xylose, L-lyxose, , and L-arabitol3 seem to be rarely, if ever, en- countered in the natural environment.4 The routes of utilization of all of these C, compounds have been documented (Fig. 1).2. 5-18 Referring to Figure 1, it can be seen that metabolism of the unnatural compounds, D-lyxose and xylitol, for example, involves only one new or unique reaction for each, i.e., isomerization of D-lyxose or oxidation of xylitol to D-. The phosphorylation of D-xylulose is common to the route for utilization of D-xylose and D-arabitol, both naturally occurring substrates readily attacked by A. aero- genes. Similarly, the conversions of L-xylose, L-lyxose, and L-arabitol involve either isomerizations or an oxidation to L-xylulose.2 8 The subsequent reactions of the pathway are common to those for L-xylulose and L-ribulose2 19 which appear in nature as metabolic intermediates in utilization of L-ascorbate20 and L-arabi- Downloaded by guest on September 26, 2021 VOL. 54, 1965 BIOCHEMISTRY: MORTLOCK ET AL. 573

D-RIBOSE kinose D-RIBOSE-5-PHOSPHATE

isomerose D-ARABID-ARABINOSENOSE - isomeroseIsmrs -D-RIBULOSED- kinose D-RIBULOSE-5- PHOSPHATE RIBITOL dehydrogenose i D-XYLOSE isomerose 3-epimerose D-LYXOSE isomerose D-XYLULOSE kino, ID-XYLULOSE-5-PHOSPHATEI D-ARABITOL dehydrogenose XY LITOL dehydrogenose 4-epimerase

-ARABINOSE 4r- L-RIBULOSE k L-RIBULOSE-5-PHOSPHATE

isomerose L-XYLOSE - 3-epimerose L-LYXOSE isomerose L-XYLULOSE L-XYLULOSE-5-PHOSPHATE L-ARAB TOL dehydrogenose I FIG. 1.-Pathways of and pentitol dissimilation in A. aerogenes. nose,'4 16, 18 respectively. Thus, only one unique reaction for each rare compound is necessary for its dissimilation. The remainder of the sequence involves inducible which function in dissimilation of naturally occurring pentoses and penti- tols and for which the necessary genes are pre-existent.2" 22 Thus, the central problem concerns the means by which the organism acquires an ability to catalyze the unique isomerizations and dehydrogenations which convert the rare structures to ketopentoses. From growth and induction experiments, it has been established that the necessary enzymes for conversion of the naturally occurring structures to D-xylulose-5-phosphate are formed in response to inducer in the usual way." 22 In addition, it is assumed that preservation of the genetic in- formation for these enzymes involves the usual selective forces. The same situation would not be predicted for the unnatural substrates. The existence of genes direct- ing the synthesis of unique enzymes seems unlikely since an environment for their selection presumably does not exist, and their eventual loss, if indeed they once existed, would result from mutations. Data indicating that a mutational event precedes the initiation of growth on the unnatural substrates have been obtained" 22 and, hence, it is possible that these mutations lead to the acquisition of a new . Yet this is unlikely because several specific acquisitions of a unique enzyme would be indicated from the ability to grow on several of the unnatural substrates. Further, because of the relatively short period required to select a mutant population for many of these substrates, each mutation leading to a new enzyme would have to occur at a very high fre- quency. Thus, these acquisitions of a unique activity must be viewed as short-term Downloaded by guest on September 26, 2021 574 BIOCHEMISTRY: MORTLOCK ET AL. PROC. N. A. S.

events and not as evolutionary gain processes. Hence, the mutational acquisition of unique enzymes seems unlikely. It is the purpose of this report to present data which show, when considered with already published observations, that a-fortuitous combination of circumstances can act as a mechanism which will allow growth on several unnatural pentoses and pentitols without necessitating the acquisition of a new enzyme. Methods.-Aerobacter aerogenes was cultured at 300C on a salts-medium supple- mented with either 0.5 per cent , 1 per cent peptone, or casein hydroly- sate (Difco).' The method of Lin et al.23 was used to determine the proportion of constitutive cells and to isolate mutant strains, 4R1 and 5R1, after growth of the wild type on xylitol or L-arabitol, respectively. Preparation of extracts and anti- sera and methods of purification and determination of S20 values were described pre- viously.24 Pentitol dehydrogenases were assayed as the reduction of NAD by pentitol in the presence of semicarbazide buffer, pH 8.5,25 or by the oxidation of NADH by ketopentose in tris buffer, pH 7.5.24 One unit of dehydrogenase cata- lyzed an absorbancy change of 1.0 per minute at 340 mu in a reaction volume of 0.15 ml (1 = 1 cm). activity was measured as the rate of ketopentose forma- tion by the method of Anderson and Wood.8 One unit of isomerase in 2.0 ml cata- lyzed the formation of 1 umole of ketopentose per hour at 370C. Protein was de- termined by the method of Lowry et al.,26 and ketopentose was estimated by the cysteine-carbozole test of Dische and Borenfreund.2Y Results.-Enzymes for xylitol and L-arabitol utilization: The data of Mortlock et al.24 28 show that the xylitol dehydrogenase activity of cells grown on xylitol is attributable to a ribitol dehydrogenase which is present in high levels. Both dehy- drogenase activities fractionate identically on DEAE and in classical pro- cedures; each has the same S20 value (6.0-6.2), and antibodies elicited by ribitol dehydrogenase purified from D-arabinose-grown cells inhibited xylitol dehydro- genase and ribitol dehydrogenase from xylitol-grown cells to the same extent (91- 96%). Careful examination of extracts by DEAE-cellulose chromatography, density centrifugation, sucrose density electrophoresis, and by disk electro- phoresis failed to reveal the existence of another xylitol dehydrogenase. Similarly, the L-arabitol dehydrogenase activity of cells grown on L-arabitol ap- pears to be attributable solely to ribitol dehydrogenase. The ratios of ribitol dehy- drogenase to L-arabitol dehydrogenase in the crude extract and various purified fractions were identical. Also, no other L-arabitol dehydrogenase was found in effluents from DEAE cellulose columns or by disk electrophoresis. We have now crystallized (a) the inducible ribitol dehydrogenase from ribitol- grown cells, (b) the ribitol dehydrogenase from a constitutive mutant isolated after growth on xylitol, and (c) the ribitol dehydrogenase isolated from a constitutive mutant isolated after growth on L-arabitol.29 Each catalyzes the following reac- tions: Ribitol + DPN+ >r± D-ribulose + DPNH + H+ Xylitol + DPN+ T± D-xylulose + DPNH + H+ L-arabitol + DPN+ 2± L-xylulose + DPNH + H+ As shown in Table 1, the specific activities of the crystalline material were identical in each case (32,000). The relative maximum velocities for xylitol and L-arabitol Downloaded by guest on September 26, 2021 VOL. 54,1965 BIOCHEMISTRY: MORTLOCK ET AL. 575

TABLE I COMPARISON OF RIBITOL D)EHYDROGENASE FROM DIFFERENT SOURCES Dehydrogenasec Inducible, Constitutive mutant, Constitutive mutant, ribitol culture xylitol culture L-arabitol culture KM, ribitol 2.6 X 10-' 3.5 X 10-3 2.6 X 10-3 L-arabitol 2.9 X 10-1 2.5 X 10-1 4.0 X 10-1 Xylitol 2.9 X 10-1 2.7 X 10-1 4.2 X 10-1 Ratio-V.., ribitol 100 100 100 L-arabitol 35 39 46 Xylitol 34 23 44 Heat sensitivity 4.5-4.8 5.0 5.0 S20,. 6.0 5.8-6.2 Gel electrophoresis Protein (Rf) 0.40 0.4-0.41 0.39 Activity (Rf) 0.40 0.40 0.40 Specific activity 32,000 30,000-32,000 32,000 determined from 1/s versus 1/v, s/v versus s, and v/s versus v calculations30 were 23-46 per cent of the value for ribitol. The Km values for ribitol were essentially equal in each case at 3 X 10-3 M. Also, the values for xylitol and L-arabitol were essentially identical, but approximately 100-fold greater than that of ribitol. The heat sensitivities, determined at 56°C, were also virtually identical. Each dehy- drogenase migrated identically in polyacrylamide gel with a single protein band which coincided with the band produced in a dye-coupled test for activity.31 Selection of constitutive mutants by growth on xylitol and L-arabitol: After growth for 8 generations on xylitol, dehydrogenases for both ribitol and D-arabitol were present, as noted previously.2 Following transfer to peptone medium, the levels of ribitol dehydrogenase and D-arabitol dehydrogenase were followed in subsequent generations. As shown in Figure 2, ribitol dehydrogenase persists much longer than expected from an inducible enzyme deprived of inducer, whereas D-arabitol dehy- drogenase approximately follows the curve expected for dilution of a constant amount of enzyme during exponential growth in the absence of inducer. Thus, there appears to be a partial selection of constitutive mutants for ribitol dehydro- genase which are, at the same time, inducible for D-arabitol dehydrogenase. Induc- tion of D-arabitol dehydrogenase is considered to result from the presence of D- xylulose which arises from xylitol oxidation.2 A mutant constitutive for ribitol dehydrogenase was then isolated after growth on xylitol and subcultured sequentially on media containing (a) casein hydrolysate, (b)

100 FIG. 2.-Persistence of ribitol dehydro- >_ | \genase in xylitol-grown cells after transfer to peptone medium. A. P 80 t \ grown for 8 generationsaerogenes,in aPRLxylitol-saltsR3, was < t \ medium and used as inoculum for a peptone- O IRIBITOL DEHYDROGENASE salts medium. Samples were removed at time E 60 intervals, cells harvested by centrifugation, o ^ washed, broken by sonic oscillation, and the cell-free extract was assayed for ribitol dehy- 40 drogenase. After growth on peptone, the per Z cent of the original activity remaining was o 2 \ITHEORETICAL DILUTION plotted against the number of doublings of cell o 20 \ 7 mass which had occurred. Ribitol dehydro- D-ARABITOL DEHYDROGEN genase activity was assayed as the reduction of ribulose by NADH. Under the conditions I employed (low D-xylulose level), D-xylulose 0 4 8 12 16 20 24 reduction measured almost exclusively D- GENERATIONS (FOLD DRY WEIGHT) arabitol dehydrogenase activity. Downloaded by guest on September 26, 2021 576 BIOCHEMISTRY: MORTLOCK ET AL. PROC. N. A. S.

TABLE 2 RIBITOL DEHYDROGENASE CONTENT OF A CONSTITUTIVE MUTANT AFTER GROWTH ON SEVERAL ENERGY SOURCES Cumulative Generations by: Ribitol dehydrogenase* Energy source Dry weight Viable count (units/mg protein) Casein hydrolysate 0 0 507 2.1 0.2 242 4.2 1.86 15( 7.0 4.23 175 9.0 188 9.7 19.8 230 16.5 29.9 234 2)5.5 39.6 276 34.9 339 1)- 44.1 45 Casein hydrolysate 53.3 464 Xylitol 60.1 490 * Measured as D-ribulose reduction. A mutant strain 4R1 was isolated by plating a xylitol-grown culture and spraying the colonies as de- scribed by Lin et al.23 A pink colony was picked and grown for 9 generations on xylitol-salts medium. The first casein hydrolysate medium was then inoculated. Then glucose-salts medium, casein hydroly- sate medium, and xylitol-salts medium were inoculated at the generation noted in the table. The cells were then harvested, washed, ruptured by sonic oscillation, and assayed for ribitol dehydrogenase. Another portion was centrifuged, washed, and weighed, whereas a third portion was appropriately di- luted and plated on glucose-salts agar. glucose, (c) casein hydrolysate, and (d) xylitol as energy sources (Table 2). After 54 generations in the absence of inducer during which 9 generations were reared on glucose, the ribitol dehydrogenase level was 87 per cent of the initial value. There was, however, a slow decline in activity during growth on casein hydrolysate which was followed by a gradual increase. This constantly observed phenomenon may be due to the presence of a small amount of catabolite repressor in the casein hydroly- sate. In other experiments, after growth on xylitol or L-arabitol, cultures were plated on peptone agar and the number of mutants constitutive for ribitol dehydrogenase determined according to Lin et al.23 (Table 3). Over a period of 69 hr on xylitol, there was a rapid rise in the number of constitutive mutants until the population was almost entirely composed of constitutive mutants. Similarly, after eight gen- erations on L-arabitol, 332 of the 819 colonies observed, or about 41 per cent, were constitutive for ribitol dehydrogenase. After serial transfer on L-arabitol medium, the proportion of constitutive mutants approached 100 per cent. The isolated constitutive mutants, when inoculated into the standard xylitol or L-arabitol medium, did not exhibit the lag of 2-4 days which characteristically pre- cedes growth of the wild type on L-arabitol or xylitol,' but grew immediately, as is typical of growth on a readily utilized substrate. Mutants isolated after growth on L-arabitol grew immediately on xylitol, and vice versa. Examination of cultures grown under conditions of ribitol dehydrogenase induc- TABLE 3 SELE(CTlrIoN OF CONsrrIITT1vE MUTANTS DURING GROWTH ON XYIITOL Hours after --Cells per Ml inoculation Viable total Constitutive % Constitutive 0) 1.2 X 1() 0 0 20 1.7 X 1i)7 5.0( X 10 0:.3 42 9.5 X 1)7 1.9 X 1(7 19.5 69 3.7 X 10O' 3.7 X 109 100.0 Wild type A. aerogenes, PRL R3, was grown on D-glucose-salts medium and then transferred to xylitol-salts medium and incubated anaerobically at 30°C. Aliquots were plated at intervals on glucose-salts agar (total viable count) and on the constitutive assay medium of Lin et al.23 Downloaded by guest on September 26, 2021 VOL. 54, 1965 BIOCHEMISTRY: MORTLOCK ET AL. 577

TABLE 4 RIBITOL DEHYDROGENASE ACTIVITIES OF CELL-FREE EXTRACTS OF Aerobacter aerogenes Ribitol dehydrogenase Strain Energy source (units/mg protein) * Wild type Peptone 0-1 Wild type Ribitol 46-154 Wild type Xylitol 270-80() Wild type L-Arabitol 47-200 Constitutive mutant, xylitol-grown (4R1) Peptone 750-100() Constitutive mutant, L-arabitol-grown (5R1) Peptone 650-850 * The spectrophotometric assay followed the oxidation of NADH in the presence of D-ribulose.' One unit of ribitol dehydrogenase catalyzes an absorbancy change of 1.0 per minute at 340 my in an assay volume of 0.2 ml. tion (ribitol-salts medium) failed to reveal the production of any constitutive mutants. Mutants isolated from typical pink colonies (Lin et al.23) and the wild type were grown in the presence and absence of inducer (ribitol), and extracts were prepared by sonic treatment of washed suspensions. Table 4 summarizes the specific activi- ties of ribitol dehydrogenase in the wild type and these mutants. It can be seen that the wild type was devoid of ribitol dehydrogenase when grown on peptone and that ribitol dehydrogenase of moderate specific activity was induced during growth on ribitol. A considerably higher level of ribitol dehydrogenase was present after growth on xylitol. In contrast, the constitutive mutants isolated after growth on xylitol or L-arabitol and then grown on peptone (without ribitol) had about 10-fold the activity of the wild type grown on ribitol. L-Xylose utilization: L-Xylose utilization has been shown to start with the isomerization of L-xylose to L-xylulose.8 The isomerase appears only after a lag of about 400 hr22 and is not elaborated under nonproliferating conditions. After growth on L-xylose, however, extracts contained high levels of D-arabinose iso- merase. As shown in Table 5, D- was present after growth on D-arabinose, but at least 10-fold more was present after growth on L-xylose. L- activity was barely detectable after growth on D-arabinose, but was more than 10-fold higher after growth on L-xylose. The ratios of D-arabinose isomerase to L-xylose isomerase activities remained constant whether D-arabinose or L-xylose was used as an energy source. After growth on L-xylose, the organism possessed specific activities for D-ara- binose isomerase of 239 and for L-xylose isomerase of 21. After 6.3 generations on peptone-salts, the specific activities were 139 for D-arabinose isomerase and 6.6 for L-xylose isomerase. After 18 generations, a specific activity of 5.3 was still detected TABLE 5 for D-arabinose isomerase, and after 25 ISOMERASE ACTIVITIES OF CELL-FREE generations, the value was 2.1. These EXTRACTS OF Aerobacter aerogenes values are considerably above those ex- D-Arabinoseisomerase isomeraseL-Xylose Growth (units/mg (units/mg pected for an inducible enzyme undergoing substrate protein) protein) dilution Peptone 0 0 due to continued growth after re- D-Arabinose 17-39 moval of inducer and indicate the presence L-Xylose 239-584 0.5-18-15.0 of a high proportion of D-arabinose iso- After aerobic growth on a salts-medium con- of merase-constitutivemerase-constitutive mutants. ~~~~~1taining% peptone,either the0.5%cells D-arabinose,were harvested,L-xylose,washed,or Partial of D-arabinose and extracts prepared with the French pressure ms3purificationldofD-arabinoseratioiso-of cell. Isomerase activity was determined by measurement of ketopentose formed after incu- merase (30-fold) did not change the ratio of btoofaldopentose and enzyme at 370C. Downloaded by guest on September 26, 2021 578 BIOCHEMISTRY: MORTLOCK ET AL. PROC. N. A. S. D-arabinose isomerase to L-xylose isomerase, and the configurational similarity of these two about 2 and 3 lends support to the idea that both isomer- izations are catalyzed by the same enzyme. Discussion.-For at least two and probably three of the rarely encountered C6 substrates (L-arabitol, xylitol, and L-xylose), growth is facilitated by a combination of circumstances which circumvent a requirement for pre-existent genetic informa- tion (in the usual sense) for the production of enzymes specifically required for the dissimilation of these . The mechanism also circumvents the necessity of acquiring de novo a unique enzyme by mutation. The organism does possess genetic information for synthesis of a dehydrogenase and an isomerase which can utilize unnatural pentitols and pentoses related to the normal substrates. How- ever, this capacity at enzyme levels normally induced is insufficient to permit growth on the unnatural substrate. When mutation results in derepression and constitu- tive synthesis of high levels of these enzymes, utilization of the uncommon C5 com- pounds occurs at a sufficient rate to permit growth, and the mutant predominates in the culture which emerges. An apparent inconsistency in this conclusion with respect to L-xylose utilization is the fact that D-arabinose is rare in nature and that growth on D-arabinose is characteristic of the process of selection of mutants (i.e., growth is delayed in the first transfer and rapid thereafter).' Camyre and Mortlock32 have recently clarified this situation by showing that both the L-xylose isomerase and D-arabinose iso- merase activities are due to an L- isomerase. All of the activities are induci- ble by L-fuculose, and the affinity for L-fuculose is higher than for D-arabinose or L-xylose. Since growth pccurs on L- without a lag and L-fucose is found in nature, growth on both L-xylose and D-arabinose may now be explained by the phenomenon just described. Following our preliminary report of this phenomenon,28 Lerner et al.,3 published similar information for xylitol utilization by another strain of A. aerogenes and pointed to a possible extension of this phenomenon to evolutionary changes. How- ever widely applicable this phenomenon may be, it is certain that such circumstances do not govern growth on the unnatural pentose, D-lyxose, and the production of D- lyxose isomerase. Anderson and Allison34 have shown that D-lyxose isomerase is a distinct isomerase induced only by D-lyxose. Serial transfer on D-lyxose did not select mutants constitutiye for D-lyxose isomerase. D-, a secondary sub- strate, did not induce the formation of D-lyxose isomerase, and the Km for D-man- nose was much higher than that for D-lyxose. * Supported by a grant from the National Science Foundation. Contribution no. 3635 of the Michigan Agricultural Experiment Station. t Postdoctoral fellow, National Institutes of Health. Present address: Department of Micro- biology, University of Massachusetts, Amherst, Mass. 1 Mortlock, R. P., and W. A. Wood, J. Bacteriol., 88, 838 (1964). 2 Fossitt, D., R. P. Mortlock, R. L. Anderson, and W. A. Wood, J. Biol. Chem., 239, 2110 (1964). 8 McCormick, D. B., and 0. Touster, Biochim. Biophys. Acts, 54, 598 (1961). 4 Sowden, J. C., in The Carbohydrates, ed. Ward Pigman (1957), p. 77. 6 Cohen, S. S., J. Biol. Chem., 201, 71 (1953). 6 Stumpf, P. K., and B. L. Horecker, J. Biol. Chem., 218, 753 (1956). 7 Burma, D. R., and B. L. Horecker, J. Biol. Chem., 231, 1053 (1958). 8 Anderson, R. L., and W. A. Wood, J. Biol. Chem., 237, 296 (1962). 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9Ibid., 1029. '0Bhuyan, B. K., and F. J. Simpson, Can. J. Microbiol., 8, 737 (1962). " Fromm, H. J., J. Biol. Chem., 233, 1049 (1958). 12Wood, W. A., M. J. McDonough, and L. B. Jacobs, J. Biol. Chem., 236, 2190 (1963). 13 Hulley, S. B., S. E. Jorgensen, and E. C. C. Lin, Biochim. Biophys. Acta, 67, 219 (1963). 4 Lampen, J. O., Abstracts, National Meeting of American Chemical Society, 1954, p. 44c. 1Simpson, F. J., M. J. Wolin, and W. A. Wood, J. Biol. Chem., 230, 457 (1958). 16 Simpson, F. J., and W. A. Wood, J. Biol. Chem., 230, 473 (1958). 7 Neish, A. C., and F. J. Simpson, Can. J. Biochem. Physiol., 32, 147 (1954). 18 Altermatt, H. A., F. J. Simpson, and A. C. Neish, Can. J. Biochem. Physiol., 33, 615 (1955). 19 Wolin, M. J., F. J. Simpson, and W. A. Wood, J. Biol. Chem., 232, 559 (1958). 20 Cabib, E., in Annual Review of Biochemistry, ed. J. M. Luck (1963), p. 346. 21 Mortlock, R. P., and W. A. Wood, Bacteriol. Proc., 110 (1963). 22 Mortlock, R. P., and W. A. Wood, J. Bacterial., 88, 845 (1964). 23 Lin, E. C. C., S. A. Lerner, and S. E. Jorgensen, Biochim. Biophys. Acta, 60, 422 (1962). 24 Mortlock, R. P., D. Fossitt, D. H. Petering, and W. A. Wood, J. Bacterial., 89, 129 (1965). n Bonnichsen, R. K., and H. Theorell, Scand. J. Clin. Lab. Invest., 3, 58 (1951). 26Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 27 Dische, Z., and E. Borenfreund, J. Biol. Chem., 192, 583 (1951). 28 Mortlock, R. P., D. Fossitt, and W. A. Wood, Bacteriol. Proc., 95 (1964). 29 Fossitt, D., R. P. Mortlock, and W. A. Wood, Bacteriol. Proc., 82 (1965). 30 Dowd, J. E., and D. S. Riggs, J. Biol. Chem., 240, 863 (1965). 31 Moore, R. O., and Claud A. Villee, Science, 142, 389 (1963). 32 Camyre, K. P., and R. P. Mortlock, J. Bacteriol., in press. 33 Lerner, S. A., T. T. Wu, and E. C. C. Lin, Science, 146, 1313 (1964). 34 Anderson, R. L., and D. P. Allison, J. Biol. Chem., 240, 2367 (1965).

SPECIFIC TEMPLATE REQUIREMENTS OF RNA REPLICASES* By I. HARUNA AND S. SPIEGELMAN

DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA Communicated June 21, 1965 We have previously1 reported the isolation of an RNA-dependent RNA polym- erase (termed a "replicase" for brevity) from E. coli infected with the RNA bac- teriophage MS-2. The purified enzyme showed a mandatory requirement for added RNA and, furthermore, exhibited a unique preference for its homologous RNA. Ribosomal and sRNA of the host could not substitute as a template and neither of these cellular RNA types showed any ability to interfere with the tem- plate function of the viral RNA. We pointed out1 that the ability of the replicase to discriminate solved a crucial problem for an RNA virus attempting to direct its own duplication in an environ- ment replete with other RNA molecules. By producing a polymerase which ignores the mass of pre-existent cellular RNA, a guarantee is provided that replication is focused on the single strand of incoming viral RNA, the ultimate origin of progeny. It seems worth noting that sequence recognition by the enzyme can be of value not only to the virus but also to the investigator. The search for viral RNA rep- licases must perforce be carried out in the midst of a variety of highly active cellular Downloaded by guest on September 26, 2021