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Synthetic building stones of desoxyribonucleic acid

, : Synthetic building stones of desoxyribonucleic acid. Abhandl Deutsch Akad Wiss Berlin (1): 66-70

When Enterococcus stei or Escherichia coli B were grown in a medium containing 10 gamma thymine- [2-14C] per ml, 100 mg of the former consumed 200 gamma thymine and 100 mg of the latter consumed 15 gamma. After cleavage of both strains with HClO4 and separation of pyrimidine bases by paper chromatography, only thymine was radio-active. Under the same conditions, Enterococcus stei consumed 1250 gamma uracil-[2-14C]/100 mg bacteria and E. coli B, 750 gamma. HClO4 cleavage of bacteria grown with uracil- [2-14C] showed that uracil was partially changed into cytosine and thymine. After growth with 10 gamma cytosine- [2-14C]/ml, Enterococcus stei consumed 60 gamma/100 mg bacteria and E. coli B, 1250 gamma. Both strains partially change cytosine into uracil and thymine. Experiments with 5-bromocytosine-[82Br] and 5-bromocytosine-[2-14C] show that neither strain of bacteria utilizes these substances in the synthesis of DNA. The effect of bromouracil (BU) on sulfathiazole-inhibited cells of Proteus mirabilis was studied and the mutagenic action of BU was tested on Proteus mirabilis VI/Sb-3. The mutation step from streptomycin dependence to streptomycin independence was tested and compared with the action of the known mutagenic agent, MnCl2. Non-radioactive-14C and -82Br labelled BU were employed. The streptomycin-dependent strain P. mirabilis VI/Sb-3 was inhibited by 15 gamma sulfathiazol/ml agar. The inhibition was partially removed by 10 gamma thymine. BU and 5-chlorouracil had the same action as thymine on both liquid and solid media. This shows that strain P. mirabilis VI does not need streptomycin for growth. The number of spontaneously occurring streptomycin-independent mutants with BU under these experimental conditions was 20-40/108 plated cells. With MnCl2 the frequency of mutation was 500-1000/108. After addition of BU and simultaneous thymine inhibition by sulfathiazol, there was no significant increase in the frequency of mutation. No increase in mutation was noted when P. mirabilis was suspended in phosphate buffer with radioactive 5-bromouracil-[82Br], This mutation rate was not increased in P. mirabilis irrespective of whether inactive or radio-active BU was used. BU eliminated 5-aminouracil inhibition in bacteria. The consumption and metabolism of 5-nitrouracil and 5-aminouracil were studied with 14C-labelled compounds as well as the mechanism of competitive inhibition on: (1) Enterococcus stei, (2) Strep. faecalis R, (3) E. coli B, and (4) E. coli 1883 Co. All of these strains consumed 5-nitrouracil, 5-aminouracil and 2-thiothymine from the medium. However, the 2 E. coli strains consumed significantly less 5-aminouracil and 2-thiothymine than Enterococcus stei and Strep. faecalis R. If Enterococcus stei is grown with 5-nitrouracil-[214C], 5-aminouracil-[2-14C], or 2-thiothymine-[2-14C], radioactivity of the bacterial cells decreases if thymine or 5-bromouracil is present at the same time. 10 gamma thymine decreased the uptake of 10 gamma 5-nitrouracil by more than 80%, 10 gamma 5-bromouracil by about 70%. With 5-amino-uracil-[2-14C] and 2-thiothymine-[2-14C], thymine is more effective than 5-bromouracil in the suppression of synthetic pyrimidines. However, the consumption of radioactive thymine is decreased only by 5-bromouracil, but not by 5-nitrouracil, 5-aminouracil or 2-thiothymine. In bacteria grown with 5-nitrouracil-[2-14C], more than 90% of the radioactivity is not in the DNA. After HC1O4 cleavage of the DNA and paper chromatography, only one radioactive substance was found which is not identical with 5-nitrouracil. In bacteria grown with 5-amino-uracil-[2-14C], the largest part of the radioactivity is not in the DNA. After HClO4 cleavage of the DNA, 4 radioactive spots were found by paper chromatography. The Rf values of 3 correspond to 5-aminouracil, cytosine, and thymine. The structure of the 4th substance has not been elucidated. If the entire bacteria is cleaved with HClO4, uracil and another unidentified compound contain most of the radioactivity. Enterococcus stei takes up about equal portions of 2-thiothymine and 5-aminouracil. From cells grown with 2-thiothymine, the desoxyriboside was isolated from DNA by enzymatic hydrolysis, while in others DNA isolated with NaCl was cleaved with formic acid. In both cases only one radioactive substance was found which was identical with thymine desoxyriboside or thymine. After HClO4 cleavage of the entire bacteria, thymine was the only radioactive base found. As these experiments show, only very small quantities of 5-nitrouracil, 5-aminouracil and 2-thiothymine are consumed by these bacteria. In contrast to 5-bromouracil, which can largely replace thymine in DNA, only about 0.1% of 5-aminouracil is incorporated into the DNA in place of thymine. 5-nitrouracil and 2-thiothymine are not incorporated into the DNA as such. The extremely low amounts of 5-aminouracil in bacterial DNA and the complete transformation of 5-nitrouracil and 2-thiothymine in to other pyrimidine bases explains the negative results previously reported in the literature. (BENNETT, J.A.C.S. 74, 2432, 1952) on the consumption and incorporation of these synthetic pyrimidines. Bibliography includes 16 references.


Other references

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