Clease (Sulbsequenit Eiizymic Actioln Beinig Inhihited by the Addition of 20 J4tii of Tri- Sodium Citrate) Led to a 40 Per Cent, Reduction in C14 Uptake

A'l.0 41IOCHEMISTR?19)54 Y: COHEN AND BARNER88885 clease (sulbsequenit eiizymic actioln beinig inhihited by the addition of 20 j4TiI of tri- sodium citrate) led to a 40 per cent, reduction in C14 uptake. When desoxyrihoull- clease was allowed to act throughout the whole of the incubation period (75 minutes total), the C14 uptake was only 15 per cent of that observed in control experiments. a. The addition of supplementary calf thymus desoxyribonucleic acid (DNA) to nuclei previously treated with desoxyribonuelease results in higher amino acid incorporations than are observed in the absence of added DNA. This result is reminiscent of the recent experiments of Gale8 on amino acid incorporation by broken bacterial cells, in which it was found that added DNA (as well as RNA and a purine-pyrimidine mixture) could restore the protein synthetic capacity of nu- clease-treated cells. In summary, the experiments described above have shown that isolated calf thymus nuclei can incorporate C14-alanine into nuclear protein and that this incorporation requires that the desoxyribonucleic acid of the nucleus remain intact. H. Stern and A. E. Mirsky, J. Gen. Physiol., 37, 177, 1953. 2 V. Alifrey, H. Stern, A. E. AMirsk, and H. Saetren, J. Gen. Physiol., 35, 529, 1952. 3V. Allfrey, AI. .4. Daly, and A. E. Mirsky, J. Gen. Physiol., 37, 157, 1953. 4 R. M. Schneider and M. L. Petermann, Cancer Research, 10, 751, 1950. 5 P. Siekevitz, J. Riot. Chem., 195, 549, 1952. 6 G. K. Schweitzer and B. R. Stein, Nucleonics, 7, 65, 1950. 7K. Lang, H. Lang, G. Siebert, and S. Lucius, Biochem. Z., 324, 217, 1953 8 E. F. Gale and J. P. Folkes, Nature, 173, 1223, 1954. STUDIES ON UNBALANCED GROWTH IN ESCHERICHIA COLI* BY SEYMOUR S. COHEN AND HAZEL D. BARNER CHILD)REN S HOSPITAL OF PHILADELPHIA (DEPARTMENT OF PEDIATRICS) AND DEPARTMENT OF PHYS- IOLOGICAI CHEMISTRY OF THE UNIVERSITY OF PENNSYLVANIA SCHOOL OF MEDICINE Communicated by David Goddard, August 11, 1954 The discovery of a unique viral constituent, 5-hydroxymethylcytosine,l has led this laboratory to metabolic studies on pyrimidines. Among the approaches em- ployed have been nutritional investigations with mutants of Escherichia coli requiring specific pyrimidines. One of these organisms was a thymine-requiring strain, designated 1T-. It was found that, on infection with T2 bacteriophage in the absence of thymine, the infected 15T - produced as much virus and DNA as if thymine had been added to the medium.2 More detailed investigation revealed that the infected cell synthesized thymine in considerable quantity as a component essential to virus deoxyribonucleic acid (DNA).' Indeed, the only nucleic acid pyrimidines synthesized after infection were thymine and 5-hydroxymethylcytosine, neither of which appeared to be synthesized in appreciable amounts before infection. The existence of this phenomenon led to a study of other properties of strain 15T-- Consequences of Thymine Deficiency.-It has been observed that, in a synthetic medium lacking thymine, the organism irreversibly loses the power to form colonies ("dies") at the rate of about 90 per cent per division time. This type of steriliza- 886 BIOCHEMISTRY: COHEN AND BARNER PROC. N. A. S. tion or "death" is accompanied by a significant increase in the turbidity of the culture, a marked increase in bacterial length and girth, doubling of ribose nucleic acid (RNA) content, and a very slight increment in DNA.3 The respiration of the culture increases considerably on incubation in glucose and a nitrogen source, and the assimilation of limited amounts of glucose is only slightly impaired.4 The RQ is essentially unchanged. Thus death appears to be a concomitant of growth which is vigorous with respect to cytoplasmic function and deficient with respect to DNA and nuclear synthesis. This unbalanced growth would appear to produce a struc- tural change, since it can no longer be reversed even when thymine is added to the medium at a later time, although DNA synthesis may still occur on addition of the pyrimidine. Recent studies with 5-bromouracil have revealed that this compound can partially replace the thymine requirement of 15T-, as can be seen in Figure 1. Bromo- uracil permits a five- to sixfold increase of turbidity of the culture and a temporary increase in viable cell number, 8 which eventually falls more slowly than in complete thymine defi- 120 \ \Br U ciency. In the presence of bro- \ mouracil there is a very extensive 100. / \ \ 7 : synthesis of RNA and DNA which 80 *BrU / is many times that seen in the ab- _ / \ < sence of thymine. X. / \ .6 >3 The phenomenon of thymineless death was also observed in E. coli, 40/ strain B, rendered nutritionally deficient by growth in sulfanila- 20\5 mide. The resulting folic acid requirement may be partially re- C 3 2 3 placed a of HOURS containingby essentialgroup compoundsone-carbon FIG. 1-The effect of 5-bromouracil on turbidity and fragments. These compounds in- viability of E. coli strain 15T, incubated in the absence of thymine. dlude methionine, serine, xanthine, pantothenate, etc., and thymine. Elimination of thymine from the fortified medium results in a rapid loss of cells capable of forming colonies. Elimination of the other compounds in the presence of thymine inhibits growth but does not produce death. A uracil-less strain, WC-, did not die in the absence of uracil. In such a medium the cell appears to be incapable of unbalanced growth, since uracil is essential to both RNA and DNA synthesis. It would appear reasonable to suppose that death similar to thymineless death may be provoked in many organisms only by a specific block in DNA synthesis. Quantitative Aspects of the Inabilety to Synthesize Thymine.-The following experiments were made to test the ability of 15T- to a synthesize thymine: The bacteria were grown in a synthetic medium containing uniformly labeled glucose-C14 (1 mg/ml) and exogenous thymine at 2 ,ug/ml. The bases of the nuclteic acids were isolated by paper chromatography in two solvent mixtures.3 The thymine was further purified by paper electrophoresis in 0.1 N NaOH and by ion exchange on a Voi,. 40, 19z5-1 BIOCHEMISTRY: COHEN AND BARNER 887 Dowex-l-acetate column. The radioactivities of the compounds are given in Table 1 and show a slight activity in the thymine, equal to 2 per cent of the carbon of the glucose used. Thus at least 98 per cent of the thymine of the organism is derived from exogenous thymine. Bacteria were also grown in unlabeled glucose and thymine, washed, and resus- pended in the mineral medium containing uniformly labeled glucose-C'4 (1 mg/ml) as sole carbon source. The culture was incubated for 2 hours, in which time the glucose was exhausted and the number of colony-formers fell from 6.2 X 108 to 9.4 X 106 per milliliter. The bacteria were harvested, and the bases were isolated, the thymine being purified by the three types of procedures described above. The activities of the compounds are given in Table 2. TABLE 1 RADIOACTIVITIES OF THE BASES OF STRAIN 15T- GROWN IN UNIFORMLY LABELED GLUCOSE-C'4 AND UNLABELED EXOGENOUS THYMINE ,AM. Carbon Counts/Min/ Substance Plated Counts/Min* juM Carbon Glucose 1.5-6.2 ... 259 Uracil 1.70 440 258 Cytosine 1.81 423 235 Guanine 1.90 477 251 Adenine 1.55 340 220 Thymine: Chromatography 4.90 67.2 13.8 Electrophoresis 9. 4 62.7 6.7 6.5 50.2 7.7 Ion exchange 10.6 45.9 4.4 All planchets were counted to 3,000 counts. TABLE 2 RADIOACTIVITIES OF THE BASES OF STRAIN 15T-INCUBATED IN UNIFORMLY LABELED GLUCOSE- C14 IN THE ABSENCE OF EXOGENOUS THYMINE Per Cent EL\I. Carbon Counts/Min/ Activity Substance Plated Counts/Min AM Carbon of Glucose Glucose ... ... 259 100 Uracil 2. 90 368 127 49 Cytosine 2.29 335 114 44 Adenine 4.20 538 128 49 Guanine 3.98 386 97a :37 Thymine: Electrophoresis 5.50 78.4 12.6 4.9 Ion exchange 4.45 46.7 10.6 4.1 The activity of the uracil, a specific RNA constituent, indicates a synthesis of this substance to the extent of 96 per cent of that of the RNA originally present. The activity of the thymine, a specific DNA constituent, indicates a maximal synthesis of thymine and DNA of about 4 per cent of that of the DNA originally present. These data show that under conditions of normal growth or of death by unbalanced growth there appears to be a very low level of thymine synthesis. However, this is insufficient to permit balanced growth in the absence of exogenous thymine. During unbalanced growth on radioactive glucose the bacteria excreted ultra- violet-absorbing materials into the medium. These were isolated by adsorption on, and elution from, charcoal-celite,' followed by paper chromatography of the major fractions in n-butanol-H20. Six white crystalline solids were isolated, of which 888 BIOCHEMISTRY: COHEN AND BARNER PROC. N. A. S. three have beeni positively identified as uracil, orotic acid, and hypoxanithine. The probable identities of the three substances were established by spectra in acid, neu- tral, and alkaline solutions and by Rf values in a variety of solvent mixtures. The radioactivities of the compounds permitted estimations of the carbon contents of the compounds, which were compared with those calculated from the extinction coefficients of known substances. These data are given in Table 3. Since the substances isolated from the media possessed activities equivalent per carbon atom to that of the glucose fed, it is clear that they were synthesized and excreted during unbalanced growth and are not autolytic products derived from the unlabeled cell.

Load more