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The terminal nucleotides of 70 S RNA extracted from murine leukemia virus (AKR, L-1 strain MLV) have been characterized by the introduction at the 5′-terminus of 32 P and by chemical conversion of the 3-terminal sugar residue to a tri[ 3 H]alcohol. Identification of the 32 P- and 3 H-labeled products after alkaline digestion of the RNA established uridine and adenosine as the 3′- and 5′-terminal nucleosides, respectively.

Summary The virus proteins, reverse transcriptase (RT) and p30, were found to increase with time in the subcellular fractions of lymphocytic tissue from either the thymus or spleen of AKR mice with spontaneous lymphocytic leukaemia. Significant levels of RT activity were first detected in the microsomal fractions of the two tissues at 15 and 20 weeks old, respectively. Although low amounts of p30 could be found in both tissues within the first week of life, the overall increase in the amount of p30 within each tissue followed much the same course as that shown by RT. In addition, a protein complex consisting of p30 and RT was first found in thymus and spleen lymphocytes of 15 and 20 week-old animals, respectively. The complex increased in amount in both organs as the animals aged, reaching a maximum level in 30 week-old mice. Repeated attempts to detect other virus proteins such as gp70 in association with the complex by immunological means were unsuccessful. The complex could not be found in lymphocytic tissue taken from younger animals or in ‘non-target’ organs, such as liver or kidney, of animals with leukaemia. In animals treated with antiviral IgG, which delayed the development of spontaneous leukaemia, the complex did not appear until much later in life (45 weeks) and then in considerably smaller amounts.

Human spleen RNase was purified using an immunoabsorbant produced with anti-human liver RNase serum. The purification was more rapid than the procedure used to purify the liver RNase, yet the final specific activity was similar. Problems encountered previously using immunoabsorbants to purify enzymes were to a large degree avoided by injecting only microgram amounts of human liver RNase directly into the popliteal lymph nodes of rabbits, thereby producing a low avidity antibody. The low avidity antibody permitted elution from the immunoabsorbant with only dilute citrate buffer and without significant denaturation. An examination of crude spleen homogenates did not reveal any other RNase to be present except that which bound to the antibody. The enzyme was found to be antigenically unrelated to the human plasma RNase. A comparison of the physical properties of the human spleen enzyme with those of the human liver enzyme did not reveal any significant differences.

The Citrobacter ribonuclease, an enzyme that cleaves preferentially phosphodiester bonds of ribonucleic acid at uridylic acid residues, has been shown to bind the polyamines, putrescine, spermidine, and spermine. The binding of spermidine insures the maintenance of enzyme activity in the presence of the polynucleotide, polyguanylic acid, at a concentration which is ordinarily completely inhibitory. Enzyme activity, if inhibited by polyguanylic acid, could, however, be restored by spermidine, which appears to effect a removal of the polynucleotide from the enzyme surface. The substitution of the polyamine for the polynucleotide was used to obtain two spermidine-bound peptides from tryptic digests of a polyguanylic acid-enzyme complex. Amino acid analysis of the isolated peptides showed that one consisted of a chain having a minimum length of 17 residues but composed of 10 different amino acids. The other had a minimum length of 8 residues and contained only 6 different amino acids. In both cases, the ratio of peptide to polyamine was approximately 1 to 1.

The polyamines have been used to activate staphylococcal nuclease for degradation of both RNA and DNA in the absence of calcium ions. If low concentrations of calcium are added to the polyamine-activated enzyme, up to double the expected activity (based on additive values) is observed. The polyamines have also been shown to prevent inhibition of the nuclease by the ordered polynucleotides.

Serum RNase levels were measured in 34 patients with multiple myeloma and compared with 51 normal controls and 28 non-myeloma patients on chronic hemodialysis. Nineteen of the myeloma patinets with creatinine clearance (CCr) greater than 50 ml/minute had mean serum RNase levels that were statistically indistinguishable from those of the normal controls. The 15 myeloma patinets with CCr less than 50 ml/minute had mean RNase levels much higher than normal controls or myeloma patients with normal renal function. Patients without myeloma but on hemodialysis for chronic renal failure of varied etiologies had markedly elevated serum RNase levels. A strong correlation between RNase levels and renal insufficiency, as measured by CCr, has thus been demonstrated. In addition, case histories of 5 representative myeloma patients were analyzed in greater detail; they illustrated the rise and fall of RNase levels as a function of the status of their renal insufficiency, regardless of the extent of the underlying myeloma. We concluded that the serum RNase level was an indicator of renal function, and was not a biomarker either for the presence or extent of the plasma cell tumor.

A ribonuclease, purified some 2700-fold from human plasma, exhibited a strong predilection for the hydrolysis of internucleotide bonds containing cytidylic acid. Analysis of [3'-32P]- and [5'-32P]phosphoryl-terminal fragments obtained after enzymic digestion of rabbit liver and yeast RNA indicated that the nucleotide found at the 3' terminus of the fragments was invariably cytidylic acid. The nucleotide at the 5' terminus varied between cytidylic and uridylic acids in a ratio of 9:1. When characterized by DEAE-cellulose chromatography, approximately 70 per cent of the digest consisted of oligonucleotides from 4 to 8 nucleotides in length. Enzyme activity, when measured in low ionic strength buffer, could be increased severalfold above control levels by the addition of either of the polyamines, spermidine or spermine. These substances also restored nucleolytic activity to preparations inhibited by such ordered synthetic polyribonucleotides as polyguanylic acid. Estimations of the molecular weight of the enzyme, both by Sephadex gel filtration and sucrose density centrifugation, indicate that the weight may vary, depending on the presence or absence of certain cations. Of the cations examined, spermidine and spermine appear to have the greatest effect, causing an alteration in molecular weight from greater than 150,000 to approximately 32,000.

The effect of polyadenylic acid, a potent inhibitor of mammalian and bacterial RNAses, on the binding of human liver RNAse to its antibody was studied. To do this, a human liver RNAse antibody was immobilized on Sepharose 4B. Examination of the ability of the enzyme to bind to the immobilized anti-RNAse in the presence or absence of polyadenylic acid indicated that enzyme-antibody binding was more sensitive to the presence of polyadenylic acid than was enzyme activity. Furthermore, the effect of polyadenylic acid on enzyme-antibody binding was specific since neither polycytidylic acid nor polyuridylic acid had much effect on the antigenicity of the enzyme. The metal cation, Mg2+, and the polyamine, spermidine, but not putrescine, readily reversed the effects of polyadenylic acid on enzyme-antibody binding.

The RNase activity observed in the sera of leukemic guinea pigs was compared to that observed in white blood cell (WBC) lysates of the same animals. The WBC-associated RNase activity directed against polyuridylic acid decreased with the progression of neoplastic disease, though serum RNase activity remained unchanged. With certain forms of cancer, therefore, variations in cell RNase may be more sensitive markers than changes in serum RNase for the evaluation of the progression or regression of disease.

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.

The inhibitory properties of poly(A) on human spleen ribonuclease have been investigated. Hydrolytic activity has been shown to be strongly inhibited by poly(A) contained within RNAs isolated from a variety of natural sources. Furthermore, poly(A) segments of varying length have been covalently linked at the 3' terminus of Escherichia coli 5 S rRNA by polynucleotide phosphorylase in an attempt to construct an in vitro demonstration of the stabilization of RNA which contains poly(A). The extent to which these poly(A) tracts, varying from 4 to 132 nucleotides in length, could inhibit endonucleolytic attack on the 5 S rRNA to which they are linked was found to be dependent upon their length and upon small changes in spermidine concentration. The consequences of these findings are discussed in terms of a possible role for poly(A).

A heat-stable ribonuclease isolated from the organism Enterobacter sp. has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis, sucrose density centrifugation and Sephadex gel filtration. Comparison of the properties of the homogeneous enzyme with those of the ribonuclease partially purified from Enterobacter by another method indicates that the two enzymes are the same. Analysis of the hydrolytic products found after enzymatic degradation of synthetic ribonucleotide polymers and of yeast RNA suggests that those fragments terminating in 3′-adenylic acid as well as those terminating in 3′-cytidylic acid result from the activity of the same enzyme.

The enrichment culture technique has been used to isolate bacteria which contain enzymes for the hydrolysis of the terminal amino acid in various folate analogues. Each of the three purified enzymes is specific for the hydrolysis of only one of the following compounds : 4-amino-4-deoxypteroylalanine, 4-amino-4-deoxypteroyl-aspartate and 4-amino-4-deoxypteroylglutamate. The hydrolysis in each reaction is in accord with the equation: 4-amino-4-deoxypteroyl-N-(amino acid) + H2O → 4-amino-4-deoxypteroate + amino acid.

A partially purified extract from a soil pseudomonad catalyzes the reaction 4-amino-4-deoxypteroylaspartate + H2O → 4-amino-4-deoxypteroate + aspartate This activity is dependent on the presence of Co++ or Zn++ and can be resolved into two components by disc gel electrophoresis. The possible interrelationships between the two enzymes has not been clarified, but both enzymes exhibit absolute specificity for linkages of the form RCO—N—, in which nitrogen is contributed by l-aspartate. In these substrates RCOOH represents not only a pteroyl group but also a variety of acyl groups and all the amino acids tested. This absolute specificity for aspartate in the carboxyl-terminal position of peptides complements previous studies on carboxypeptidase G, in which an analogous specificity is exhibited for glutamate.

Elevated polyamine concentrations in human urine and serum appear to be associated with the presence of many types of cancer. The automated amino acid analyzer lends itself to the rapid separation and measurement of these substances with little sample handling and excellent reproducibility. The ion-exchange resin used was Beckman’s PA-35 (strong sulfonic acid cation exchanger, copolymerization product of styrene-divinylbenzene). Amino acids are bound less strongly to the resin than are the polyamines, and can be removed by first eluting with a buffer of lower ionic strength. If sample injections are overlapped, one sample can be analyzed every hour. We have corroborated that increased concentrations of polyamines appear in the urine of patients with a variety of cancers. Additionally, we have shown increased amounts of polyamines in the serum of patients having a diagnosis of cancer.

Purified Klebsiella ribonuclease (ribonucleate nucleotido-2′-transferase (cyclizing), EC 2.7.7.17) is able to hydrolyze poly(A), poly(U) and poly(C) more readily than yeast RNA, but cannot attack poly(G), poly(I), poly(X), poly(hU), poly(A) · poly(U) and poly(I) · poly(C). Hydrolysis of yeast RNA, poly(U), and poly(A) by this nuclease is inhibited markedly by poly(G) and to a much lesser extent by poly(I). Another compound, poly(X), is more potent than poly(I) as an inhibitor of RNA hydrolysis but less potent in inhibiting poly(U) hydrolysis. Relatively high concentrations of poly(hU) will inhibit poly(U) hydrolysis but have no effect when yeast RNA is the substrate. A number of mononucleotides were tested and have no effect on RNA hydrolysis by Klebsiella nuclease. Several other nucleases were found to be inhibited to a greater or lesser extent by poly(G) and poly(I). The data suggest that the nuclease prefers molecules of unordered secondary structure as substrates, and that ordered molecules are not attacked and may actually be inhibitors.

Melilotate hydroxylase partially purified from extracts of Arthrobacter species catalyzes the stoichiometric conversion of melilotic acid to 2,3-dihydroxyphenylpropionic acid. The hydroxylation is shown to require both reduced nicotinamide adenine dinucleotide and atmospheric oxygen. A partial resolution of the enzyme is achieved with ammonium sulfate. The preparation so treated is almost deviod of enzyme activity, but activity can be completely restored with flavin adenine dinucleotide. The role of melilotate hydroxylase in the general metabolism of coumarin is discussed.

A pseudomonad has been isolated from soil which can utilize glutamate derived from the hydrolysis of folate or methotrexate (4-amino-N10-methylpteroylglutamic acid) as a source of carbon and nitrogen. During growth of the organism on folate, large quantities of pteroic acid accumulate in the growth medium. The enzyme responsible for the deamidation has been partially purified. It has a pH optimum of 7.3 and a Km toward methotrexate of 2.4 x 10-4 m. The reactivity of the enzyme toward a number of folate analogues has been studied.

Ribonuclease A was found to behave in an unusual fashion on a Sephadex gel column. Though ribonuclease A produces a single, well-defined protein peak on elution, enzyme activity can be detected several void volumes after the protein peak. A second unrelated protein added to the column will displace further activity as will 0.5 M phosphate buffer. This additional activity, apparently due to ribonuclease A or an active fragment of the enzyme, would appear to make this enzyme unsuitable for use as a standard in molecular weight determinations of other nucleases.

A heat-stable ribonuclease, isolated from Citrobacter species, catalyzes the conversion of polyuridylic acid to cyclic 2',3'-uridylic acid but does not hydrolyze polycytidylic, polyguanylic, or polyadenylic acids. The enzyme, purified some 1500-fold, migrates as a single band after electrophoresis on polyacrylamide gels and exhibits an absorption maximum and minimum at 281 and 248 nm, respectively. Evidence from studies with synthetic polyribonucleotides and with yeast ribonucleic acid suggests that the nuclease has a distinct predilection for internucleotide bonds containing uridylic acid at either end of the phosphodiester linkage. Enzyme activity is affected profoundly by the polyamines, putrescine and spermidine. Both of these substances as well as the ammonium ion stimulate enzyme activity considerably. In addition, spermidine appears to alter the high degree of specificity exhibited by the enzyme to an activity much more random in nature. Enzyme activity is inhibited by polyguanylic acid and by the double- and triple-stranded complexes, polyadenylate + polyuridylate and polyadenylate + 2 polyuridylates, respectively. The inhibition of enzyme activity induced by these polynucleotides was found to be competitive, and activity could be restored completely by spermidine.

Metabolism of Coumarin by a Micro-Organism : O-Coumaric Acid as an Intermediate between Coumarin and Melilotic Acid

A NUMBER of laboratories have reported the isolation from eukaryotic cells of mRNAs linked covalently at their 3′-OH termini to polyadenylic add (poly(A))1–4. The chain of adenylic acid residues has been found also at the 3′ termini of RNAs isolated from several well characterised viruses5–7. Although considerable evidence exists to suggest that the insertion of poly(A) on the RNA chains destined to become mRNA is a post-transcriptional event8, there is at present little understanding of the part poly(A) plays in the subsequent stages of protein synthesis.

Our original interest in preparing this review lay in the fact that no one had presented a thorough examination of the topic, with particular attention to the several possible biological functions of polyadenylic acid [poly(A)]. However, as we scrutinized the literature, one point cropped up repeatedly: those engaged in research efforts aimed at clarifying the physiological significance of poly(A) did not make full use of the current body of knowledge concerning the chemical properties of the homopolymer. Similarly, results of experiments that clarify aspects of the physical nature of poly(A) were never interpreted in terms of intracellular functions. Thus, two vast bodies of literature exist in roughly equal proportions, one concerning the biochemistry of poly(A), the other dealing with more physically oriented considerations, and the amount that either group draws on the knowledge or experience gained by the other is small. It was therefore obvious to us that a deficit was present in the field of polyadenylic acid research: no source of knowledge concerning all facets of the biology and chemistry of poly(A) existed. Consequences of the lack of discussion between chemists and molecular biologists engaged in work involving poly(A) were manifest. Experimental results based on quantitation of poly(A) · polyuridylic acid (poly(U)] hybrids prepared under conditions wherein the triplex [poly(A) · 2 poly(U)] may exist is one example. Another is the attitude with which the structure of poly(A) is approached by most biologists. Few papers dealing with the molecular biology of poly(A) give consideration to the different structural forms that the polymer may assume. Despite overwhelming evidence from chemical and physical studies that this polymer is unique in many respects and that an alteration in experimental conditions may induce a radical change in polymeric structure, little consideration is given to this information. As a final example, the researchers attempting to define the nature of the poly(A)-binding proteins appear to be unaware of classes of enzymes that interact with poly(A) and are not cognizant of the consequences of the partially stacked structure of the polymer relevant to amino acid and protein binding. Thus, the knowledge regarding the many aspects of poly(A) chemistry and biochemistry are, in our opinion, in need of organization and presentation in one place. We feel that such an effort will be of importance to both the biochemist and the chemist, since no review of the chemical and physical properties of poly(A) has been published in more than a decade, and the last ten years have seen the most significant advances in knowledge concerning the structure of poly(A) and its interactions with cations, low-molecular-weight organic compounds, and macromolecules. Accordingly, we have divided our manuscript approximately in two; the first part deals with the biochemical and subcellular aspects of poly(A), and because of the biochemical importance of the structure of poly(A), the second half concentrates on this topic—but includes, as well, sections on metals, complementary monomers, and polymers that interact with poly(A).

The Enterobacter nuclease, which cleaves RNA between the 3′-phosphate group of cytidylic acid and the 5′-hydroxyl group of adenylic acid, has been shown to be affected by the polyamines, spermidine, spermine and putrescine. These substances enhance the hydrolytic activity of the enzyme against both poly(C) and yeast RNA. Spermidine and spermine also reverse the inhibition of the enzyme by the ordered polynucleotides, apparently by removing them from the surface of the enzyme. Treatment of poly(G)-bound peptides (obtained from tryptic digests of poly(G)-bound nuclease) with an excess of spermidine resulted in the isolation of spermidine-bound peptides. Purification of these peptides through ion-exchange chromatography resulted in the isolation of three spermidine-bound peptides which consisted of 17 residues (6 amino acids), 19 residues (10 amino acids), and 12 residues (9 amino acids). The binding ratio of spermidine to peptides varied from 1:1 to 3:1.

The thermal inactivation of a Citrobacter sp. ribonuclease (RNase) is subject to control by a number of factors. Low concentrations of naturally occurring polyamines such as spermidine and spermine, and certain analogs of these compounds, protect the enzyme from inactivation. Changes in ionic strength cause wide variations in the rate at which enzyme activity is lost. Additionally, depending on the type of ion added to the reaction mixture, the rate constant for enzyme inactivation-may either increase or decrease as the ionic strength is raised. Thermodynamic parameters were determined under a variety of experimental conditions for the thermal inactivation of this RNase. It was found in all of these cases that the entropy of activation is large and negative, implying that a gross change in enzyme conformation is not taking place. The concentration and identity of ions present and the amount of polyamine available to interact with this RNase determines the rate of loss, by thermal inactivation, of enzyme activity in this in vitro system. These factors therefore constitute a system whereby substrate hydrolysis may be controlled with time.

Polypeptides synthesized during productive infection of HSV-1 and HSV-2 were found to possess distinct characteristics in regard to localization within the cell, DNA-binding properties, and phosphorylation after synthesis. Continuous labeling for 14 hr or pulse-labeling at successive periods during the replicative cycle with radioactive precursors revealed two types of polypeptide localization: (a) selective accumulation or enhancement within the cytoplasm or nucleus with barely detectable concentrations elsewhere and (b) accumulation in significant concentrations within both cytoplasm and nucleus showing little selective enhancement. Of the polypeptides made during HSV-1 infection 22 were phosphorylated as compared with 16 phosphoproteins specified by HSV-2. Phosphorylation was also implicated in the generation of the four molecular forms comprising the ICP 5–8 complex. Twenty-three polypeptides with affinity for DNA were detected after either type of infection. Sufficient comparisons were made to provide a basis for the tentative listing of 20 polypeptides of HSV-1 with corresponding polypeptides of HSV-2.

Summary S-adenosyl- l -methionine decarboxylase has been purified more than 350-fold from rat liver by a new procedure. Some characteristics of the decarboxylase are described. Higher stages of purification did not abolish the stoichiometric relationship between CO2 formation and spermidine production.

A ribonuclease partially purified from Aerobacter sp. has been found to exhibit specificity towards polycytidylic acid. Polyuridylic, polyguanylic, and polyadenylic acids are not attacked. The enzyme catalyzes the hydrolysis of polycytidylic acid to 2′,3′-cyclic CMP and di- and trinucleotides of cytidylic acid. In its reactivity towards yeast RNA the ribonuclease exhibits a distinct proclivity for internucleotide bonds containing cytidylic acid. This proclivity disappears with increasing length of incubation. The molecular weight estimated by sucrose density centrifugation and Sephadex gel filtration gives an average molecular weight of 40 000.

A ribonuclease partially purified from Serratia sp. catalyzes the stoichiometric conversion of poly (A) to 5′-AMP, but attacks poly (C) and poly (U) very slowly. Poly (G) is not attacked at all. Studies with di- and trinucleotides indicate that the ribonuclease is inactiv against those oligonucleotides which have phosphoryl groups at their 3′-terminal positions. Oh the other hand, the corresponding dinucleoside monophosphates and trinucleoside diphosphates are readily degraded, although the larger molecule is hydrolyzed at a somewhat faster rate. The hydrolytic data obtained from the degradation of the trinucleoside diphosphates suggests that the 3′-termini of these compounds are the positions at which enzymatic attack is initiated. The enzyme also displays a marked proclivity for adenylic acid-containing heteropolymers, releasing, within the first two hours of incubation, 5′-AMP at a faster rate than other nucleotides. Yeast RNA was degraded in a similar manner. No detectable reaction was apparent, however, when calf thymus DNA was tested as a substrate.

The present study characterizes parameters of use of synthetic double stranded polynucleotides in the amplification of immunologically induced lymphocyte tritiated thymidine incorporation. Parameters for optimum use both in the one-way mixed lymphocyte reaction as well as in response to soluble antigen are presented. Each lymphocyte population has a range of poly AU which is effective for maximal amplification. Once determined, use of this concentration enables amplification of lymphocyte responsiveness over a broad range of antigen concentration. This optimum range of poly AU concentration appears more specific for the particular lymphocyte population than for the antigen inducing stimulation. When used above optimum concentration, poly AU has an inhibitory effect inversely proportional to the antigen concentration utilized. This study is of practical importance for those investigators studying situations in which minimal responsiveness is being evaluated. It is in this situation, that the relative stimulatory effect of poly AU appears to be greatest.

THE microbiological utilization of coumarin has not been examined extensively although coumarin is a natural constituent of many plants and, ultimately, therefore of soil. Bellis1, however, did isolate from soil two species of Penicillium moulds which grew slowly on coumarin ; but no derivatives could be identified in the external medium. In higher animals coumarin metabolism, prompted by reports of liver damage in the dog and rat2, has been investigated. Metabolic products identified in the urine when coumarin was fed to rabbits and rats have included 3-, 7-, and 8-hydroxycoumarins, melilotic, o-hydroxyphenyl-acetic and o-coumaric acids3,4. Similarly in the plant, Melilotus alba, degradation products of coumarin have been identified as melilotic acid and glucosides of melilotic, coumarinic and o-coumaric acids5,6.