Seymour S. Cohen

Introduction to the Biochemistry of D-Arabinosyl Nucleosides

Publisher Summary The chapter discusses the biochemistry of D-arabinosyl nucleosides. Arabinosyl compounds can be thought of as analogs of both ribo and deoxy ribo compounds. They do participate actively in many enzymatic reactions, e.g., deamination of ara-C and ara-A, by cytidine deaminase and adenosine deaminases, respectively, phosphorylation of ara-C, by wheat phosphotransferase, phosphorylation of cytosine arabinoside monophosphate (ara-CMP), by an appropriate kinase, the activity of adenine arabinoside triphosphate (ara-ATP), with yeast hexokinase, etc. The epimerization of the 2-OH in the sugar does not so distort the structure, as to prevent these compounds from approaching a variety of catalytic sites in several enzymes. The apparent lack of phosphorylation of ara-M and its apparent inhibitory properties, as a nucleoside, in the conversion of cytosine monophosphate (CMP) to deoxycytidine monophosphate (dCMP) are also consistent, with the possibility that some arabinosyl compounds may not inhibit as nucleotides, but may be active solely as nucleosides. At present, there is little experimental evidence to suggest why a deficiency in deoxyribonucleotide should be lethal. The hypothesis, that in some way these deficiencies provoke chromosome breaks, is supported by the observation of these breaks in animal cells. Some slight evidence concerning deoxyribonucleic acid (DNA) degradation in bacteria, provoked by these treatments, is becoming available, of which induction of lysogenic bacteria, by such methods, may be considered to be an example. It is evident then that the chemistry and biochemistry of the D-arabinonuckosides have been launched, but that the biological activities of these compounds present numerous mysteries. The practical and esthetic aspects of the mysteries and its relation to the molecular mechanism and cellular physiology is discussed in this chapter.

THE SYNTHESIS OF MESSENGER RNA WITHOUT PROTEIN SYNTHESIS. II. SYNTHESIS OF PHAGE-INDUCED RNA AND SEQUENTIAL ENZYME PRODUCTION.

A polyauxotrophic mutant of Escherichia coli which is deficient in the ability to synthesize thymine, uracil and histidine has been used to analyse virus-induced processes of making specific nucleic acids and enzymes. The RNA made in this T6 r + -infected mutant strain in the absence of required amino acid or in the presence of chloramphenicol has the characteristic base ratios and electrophoretic mobility of normal phage-induced RNA, and is mainly associated with ribosomes. Incubation of the isolated RNA-charged ribosomes with inorganic phosphate leads to the selective degradation of the phage-induced RNA. This RNA in infected cells is degraded actively either in the presence or absence of histidine. In the presence of histidine, the degradation products of this RNA then appear in DNA. Thus, phage-infected cells appear to produce normal phage-induced RNA in the absence of protein synthesis. In T6 r + -infected cells deoxycytidylate hydroxymethylase, thymidylate synthetase and lysozyme appear in that order at different times after infection. The syntheses of these enzymes are completely inhibited in the absence of histidine. The appearance of these enzymes is considerably delayed in the absence of uracil, that of lysozyme never attaining a high level. After charging ribosomes with phage-induced RNA in the absence of protein synthesis, addition of the essential amino acid to infected cells produces a stimulated rate of synthesis of early phage-induced enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase. However, such prior synthesis of RNA does not significantly affect the rate of synthesis, nor even the time of appearance, of a late enzyme, lysozyme. From these results we suggest that the early RNA determines early proteins alone, and that the sequential production of phage proteins is determined by the sequential production of phage-induced RNA. On infection by phage inactivated by u.v. light, the synthesis of hydroxymethylase continues for a considerable time, whereas the syntheses of DNA and of lysozyme are strongly inhibited. Our results are consistent with the hypothesis that the cessation of synthesis of early enzymes and inception of synthesis of late proteins require DNA synthesis.

ON THE NATURE OF THYMINELESS DEATH

It seems appropriate among discussions of the chemotherapeutic efficacy of folic acid antagonists to include a discussion of the phenomenon of thymineless death. The latter appears to be a major response to treatment with antifolates under certain conditions, and there is some point in indicating the interrelations of the two, as well as in considering the mechanism of thymineless death as an approach to the problem of increasing the efficacy of antifolate therapy. Thymineless death was discovered and so named in our laboratory in 1953. This occurred during studies directed toward clarifying the origin of the unusual pyrimidine, 5-hydroxymethylcytosine, a compound found uniquely (at that time) in T-even bacteriophages.1 A thymine-requiring strain of Escherichia coli strain 15Twas obtained and tested in nutritional experiments. Its growth was not supported by 5-hydroxymethylcytosine or by 5-hydroxymethyluracil. However, the properties of the bacterium were explored further when it was found that infection by T2 in the absence of thymine induced the ability to synthesize thymine, DNA, and phage. This surprising result marked the discovery of the phenomenon of the virus-induced acquisition of function.2 On closer scrutiny it was observed that, under conditions of thymine deficiency in an otherwise complete medium, the turbidity of an aerated culture increased, but that cell viability, measured as the ability to form colonies on agar plates, decreased (FIGURE l ) . s We said that the cells died under conditions in which they grew, i.e. made RNA and protein. It was subsequently shown that a carbon and energy source was essential for the killing of the cells,’

Protein synthesis and RNA turnover in a pyrimidine-deficient bacterium.

a phenomenon ascribed to unbalanced g r ~ w t h . ~ By this was meant that a specific inhibition in DNA synthesis under conditions in which other normal processes continued led in some manner to the irreversible lesions connoted by “thymineless death.” Cells dying in this way were nevertheless capable of synthesis of RNA In subsequent studies, it was shown that a concomitant inhibition of RNA synthesis, provoked by a deficiency of uracil in addition to that of thymine, markedly retarded thymineless death.6 Initially the effects of amino acid deficiency appeared more complicated. The test of thymineless auxotrophs having various amino acid requirements revealed that amino acid deficiency decreased the percentage of cells killed, although the cells frequently began to die at the same time as in the presence of the amino acid.’ These studies led to the isolation and characterization of the much used multiple auxotroph, strain 15 TAU, which is unable to synthesize arginine and uracil as the result of the single block in carbamyl phosphate synthetase.Gs and inducible enzymes.’,

The synthesis of messenger RNA without protein synthesis: I. Studies with thymineless strains of Escherichia coli*

Abstract An auxotrophic mutant of E. coli strain 15 has been isolated possessing thymine and uracil requirements. Lack of oxogenous thymine prevents DNA synthesis; lack of oxogenous uracil inhibits net RNA synthesis. Lack of both pyrimidines prevents net nucleic acid synthesis, but does not prevent protein synthesis. The protein synthesized under such conditions may be directed by induction and be a specific enzyme such as xylose isomerase. Despite the absence of net nucleic acid synthesis in pyrimidine deficiency, a fraction of the RNA was found to have a marked turnover in which preformed uracil of RNA was released and reincorporated with newly formed ribose generated in the presence of glucose-14C. The very low isotope content of thymidine under comparable conditions indicates a negligible turnover of DNA. The possible significance of the RNA turnover has been discussed.

Some physiological and genetic properties of a strain of Escherichia coli requiring thymine, arginine and uracil

Escherichia coli , deficient in the ability to synthesize thymine, uracil and an amino acid, synthesize a small fraction of their normal RNA from uracil in the absence of thymine and the amino acid. The synthesis of even this fraction of RNA is inhibited by concomitant DNA synthesis. Most (75%) of the RNA made appears on the ribosomes and can be degraded on these structures in the presence of inorganic phosphate. When synthesized in the presence of an inducer of β -galactosidase, the RNA appears to permit the rapid synthesis of a small amount of this enzyme in the apparent absence of the inducer. These observations are consistent with the hypothesis that the RNA made in the absence of an essential amino acid and of protein synthesis is largely messenger RNA.

The lethality of streptomycin and the stimulation of RNA synthesis in the absence of protein synthesis.

A polyauxotrophic mutant of E. coli strain 15 has been studied. The organism requires thymine, uracil and arganine for normal growth and multiplication. The formation of citrulline and ureidosuccinate is blocked almost completely. Nevertheless, significant although depressed amounts of both ornithine and aspartate transcarbamylases are readily detectable in cell-free extracts of the organism. n nThe bacterium can be induced to synthesize xybose isomerase in the presence of exogenous arginine and in the absence of exogenous uracil, as in strain 15T−U−. Relatively extensive peptide synthesis is obtained under these conditions. Analysis of the observed RNA metabolism suggests a turnover of part of the RNA and a greater conservation of the bases than of the ribose. Most of the turnover appears to depend on an exogenous supply of arginine, as does the function of enzyme formation. n nReversion to arginine and uracil independence occurs in a single genetic step. Thymine starvation increase the percentage of revertants in the culture before the bacteria die as a result of unbalanced growth.

Arteriovenous Fistula after Percutaneous Needle Biopsy — Surgical Repair with Preservation of Renal Function

The action of streptomycin and related drugs has been studied with thymineless, arginineless and uracilless strains of Escherichia coli. Many experiments were performed in the absence of arginine, a condition shown to reduce 35SO42−incorporation (mainly protein synthesis) to 0·5% and less of the rate of incorporation during growth. In the absence of arginine and in the presence of thymine, under conditions in which the rate of RNA synthesis is 10 to 15% of that during normal growth, streptomycin initially inhibits and then markedly stimulates RNA synthesis. In a wide variety of conditions, the inception of stimulation coincides closely with the inception of lethality. Also the rate of stimulated synthesis of RNA correlates well with the rate of loss of capacity to form colonies. Such a stimulation of RNA synthesis and correlation with lethality also occurs in the presence of neomycin and kanamycin. The stimulation does not occur in a derived streptomycin-resistant strain in which streptomycin does inhibit slightly the basal level of RNA synthesis occurring in the absence of protein synthesis. Anaerobiosis, which protects against the lethal action of streptomycin, also prevents the stimulation of RNA synthesis, although the internal concentrations of phosphates and of streptomycin during anaerobiosis were not significantly changed as compared to the concentrations of these substances during aerobiosis. Reduction of the phosphate concentration in a medium containing streptomycin reduced the lag before both the beginning of killing and the stimulation of RNA synthesis, and increased the degree of the latter function. Indeed under these conditions, the stimulation produced by streptomycin approached the stimulated rate of synthesis of RNA produced on addition of chloramphenicol. The RNA made under the influence of streptomycin was partially associated with the ribosomal fraction and had a rapid but incomplete turnover; however, the partial degradation of this RNA in the absence of the antibiotic did not permit the restoration of apparently killed bacteria to viability. The incorporation of fluorouracil in the presence or absence of uracil was not lethal in the absence of streptomycin. In the presence of the antibiotic, lethality was increased by the analog, concomitant with the stimulated incorporation of the analog into the new type of RNA. At the beginning of stimulation of RNA synthesis, the capacity to incorporate arginine is reduced about 50%. The formation of the RNA stimulated by the antibiotic in the absence of protein synthesis (measured by uptake of 35SO42−) is coincident with a rapid reduction of the capacity to incorporate arginine to less than 5% of the normal rate. Thus physiological lethality, i.e. the irreversible loss of the ability to make proteins in the absence of streptomycin, occurs without protein synthesis.

Comparative biochemistry and virology.

PERCUTANEOUS renal biopsy has become an established diagnostic technic in the evaluation of both adults and children with parenchymal renal disease. Although complications are few,1 the increasing use of renal arteriography has demonstrated an appreciable incidence of arteriovenous fistula after biopsy.2 , 3 Most fistulas are small and disappear spontaneously,3 4 5 but there are several reports of hemorrhage or hypertension requiring operative intervention.6 7 8 9 10 11 Renal exploration has most often resulted in nephrectomy,7 8 9 although there are occasional reports of partial nephrectomy with preservation of the uninvolved portion of the kidney.10 , 11 The case reported below illustrates the extent of hemorrhage that can occur after renal biopsy. .xa0.xa0.

On Biochemical Variability and Innovation

Publisher Summary Within the past decade, the questions posed concerning the bacterial viruses and their interactions with their host bacteria have shifted from the biological to the chemical level. Many of the most important biological questions have been rephrased as chemical problems. Questions are now being posed concerning the nature of the building blocks and the pathways of their biosynthesis. The time course of infection, duplication, and virus liberation is being dissected minute by minute in terms of the molecular transformations occurring in these systems. This chapter shows that the problems of genetic duplication and virus synthesis are being posed and tested at molecular and intermolecular levels. Many of the biologists working on the phage are now engaged in chemical work, in learning the elements of chemical manipulation, or in hiring the chemically trained personnel for their research projects. Since the biological prerequisites for chemical work in the field of the animal viruses are essentially solved, that is, the systems of infected cells can be made readily available, it can be anticipated that animal virology will soon see a burst of activity in chemical directions as well. It therefore seems appropriate to scan the present status of chemical virology in the light of the impending growth of this specialization. It also seems desirable to assess some current trends in biochemistry for the purposes of the orienting approaches to the biochemical work in virology.