Carl C. Levy

Parameters for effective use of synthetic double stranded polynucleotides in the amplification of immunologically induced lymphocyte tritiated thymidine incorporation

Abstract 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.

Polyamine activation of staphylococcal nuclease.

Abstract 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.

The enzymatic hydrolysis of folate analogues

Abstract 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.

Structures, Properties, and Possible Biologic Functions of Polyadenylic Acid

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).

Polyguanylic acid-inhibited ribonuclease of Klebsiella: II. Studies with synthetic polyribonucleotides

Abstract 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.

Polyguanylic acid-inhibited ribonuclease of Klebsiella: I. Purification and general properties

Abstract A ribonuclease (ribonucleate nucleotido-2′-transferase (cyclizing), EC 2.7.7.17) was purified 540-fold from Klebsiella sp. and its properties were investigated. The enzyme is an endonuclease able to hydrolyze yeast RNA completely to the 2′:3′-cyclic phosphate derivatives of AMP, CMP, UMP and GMP. The 3′-phosphates can be found as minor products of the hydrolysis. The enzyme has a pH optimum in the neutral range, is moderately heat stable, has a molecular weight of 24 000, and has a Km of 32 μg yeast RNA per ml. Polyguanylic acid was found to be a potent inhibitor of this enzyme.

Enterobacter ribonuclease: II. A reexamination of the enzyme's specificity toward internucleotide linkages☆

Abstract 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 properties of a ribonuclease from Serratia sp. with a proclivity for adenylic acid residues

Abstract 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.

Anomalous behavior or ribonuclease A on Sephadex G-100.

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.

Characterization of the 5′-terminus of murine leukemia virus RNA

Abstract 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 32P and by chemical conversion of the 3-terminal sugar residue to a tri[3H]alcohol. Identification of the 32P- and 3H-labeled products after alkaline digestion of the RNA established uridine and adenosine as the 3′- and 5′-terminal nucleosides, respectively.