Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

Synthesis of 5'(3')-O-amino nucleosides

Synthetic methods leading to 5'(3')-O-amino nucleosides have been developed in an effort to prepare derivatives that may have antitumor or antiviral activities. They are based on ring opening of O2,5'-cyclonucleosides with the N-protected hydroxylamines and dehydrative coupling of 5'(3')-O-unprotected nucleosides with N-hydroxyphthalimide.     

Rapid amplification of 5' complementary DNA ends (5' RACE)

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.     

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.     

A convenient synthesis of adenylyl-(2'----5')-adenylyl-(2'----5')-adenosine (2-5A core)

A simple synthesis of adenylyl-(2'----5')-adenylyl (2'----5')-adenosine (2-5A core) has been achieved on the basis of selective 3'-O-silylation of 5'-O-p-monomethoxytrityladenosine and chemo-selective formation of the 2'-5' internucleotide linkage using N-unprotected nucleosides.     

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.