RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template

DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.     

Halogenation of tubercidin by N-halosuccinimides. A direct route to 5-bromotubercidin, a reversible inhibitor of RNA synthesis in eukaryotic cells

Tubercidin may be directly brominated by reaction with N-bromosuccinimide in DMF to give 5-bromotubercidin, a reversible inhibitor of RNA synthesis. When buffered with potassium acetate the major product is 6-bromotubercidin. 5,6-Dibromotubercidin is formed in minor amounts under both conditions. N-Chlorosuccinimide and tubercidin give 5-chlorotubercidin and 5,6-dichlorotubercidin.     

Completion of RNA synthesis by viral RNA replicases

How the 5′-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA–DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5′-terminus and the C2′-hydroxyl of a ribose at the third nucleotide from the 5′-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.     

Bacteriophage M13 DNA-directed in vitro synthesis of gene 5 protein

A method is presented which allows one to follow in a relative rapid and accurate way the synthesis of gene 5 protein of the bacteriophage M13 in a DNA-dependent cell-free system. Due to its unique property of binding selectively to single-stranded but not to double-stranded DNA, gene 5 protein can readily be separated, by means of sucrose densitygradient centrifugation, from the other polypeptides synthesized in the cell-free system. The meaning of this technique for the elucidation of the mechanism(s) which regulates gene 5 protein synthesis is discussed.     

Biochemical and cellular mechanisms of low-dose effects

Low-dose irradiation is usually considered to be rather ineffective in producing biologically relevant effects. Yet, individual radiation absorption events within cell nuclei or whole cells interact stochastically with subcellular structures due to the multiple ionizations along primary or secondary particle tracks, depending on ionization density. Whereas radiation effects are usually seen in the context of structure and function of DNA, other cellular effects, perhaps influencing DNA by secondary biochemical mechanisms, also warrant attention. Thus, previous work from this laboratory with bone marrow that was obtained from whole-body exposed mice, has shown that single or few instantaneous radiation absorption events per cell from gamma-rays produce an acute and temporary partial inhibition of the enzyme thymidine kinase; the effect appears within about 1 h after the event, reaches its maximum at approximately 4 h and disappears completely within another 6 h. This pattern of enzyme inhibition is fully concordant with the pattern of inhibition of uptake of tritiated thymidine or 125I-labelled deoxyuridine into the DNA; also concordant is a temporary increase in the concentration of free thymidine in the blood serum of the exposed mice. The particular response of thymidine kinase was considered to relate to some, thus far unknown, repair systems and/or to a defence mechanism of the hit cells. In order to further elucidate the role of the acute and temporary partial inhibition of thymidine kinase in cellular metabolism, experiments were carried out in which mice were acutely exposed to 0.01 or 0.1 Gy and again exposed to the same dose at different times up to 12 h after the first exposure. At regular time intervals after the second exposure bone marrow cells were obtained and thymidine kinase activity was studied by various assays. The results indicate that the first acute irradiation conditioned the cells in such a way that the second acute irradiation produced either an enhanced inhibition and recovery of thymidine kinase activity, or no effect at all was seen, when the second irradiation was given between about 3 and 8 h after the first irradiation. From 8 to 12 h after the first irradiation the cells apparently resumed their original state, so that the second irradiation produced effects quite similar to those seen after a single irradiation in unconditioned cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid synthesis of double-stranded cucumber mosaic virus-associated RNA 5: Mechanism controlling viral pathogenesis?

In cucumber mosaic virus infections of tobacco where disease attenuation is observed, viral RNA synthesis is quickly overtaken by the synthesis of cucumber mosaic virus-associated RNA 5, a satellite-like RNA dependent upon the virus for its replication, and that of its double-stranded form. A disease regulatory mechanism is proposed in which the sequestration of rapidly synthesized cucumber mosaic virus-associated RNA 5 molecules of complementary nucleotide sequence enables their successful competition with and suppression of, viral RNA synthesis.     

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.     

Biochemical properties and cellular localisation of STIM proteins

Human and murine STIM1 were originally discovered as candidate growth regulators in tumours and in the bone marrow stroma, and the structurally related vertebrate family members, STIM2 and the Drosophila homologue D-Stim, were subsequently identified. STIM proteins are ubiquitously expressed type I single-pass transmembrane proteins which have a unique combination of structural motifs within their polypeptide sequences. The extracellular regions contain an N-terminal unpaired EF-hand Ca(2+) binding motif adjacent to an unconventional glycosylated SAM domain, while the cytoplasmic regions contain alpha-helical coiled-coil domains within a region having homology to ERM domains adjacent to the transmembrane region, and phosphorylated proline-rich domains near the C-terminus. STIM1, STIM2 and D-Stim diverge significantly only in their structure C-terminal to the coiled-coil/ERM domains. The STIM structural domains were predicted to function in Ca(2+) binding as well as in mediating interactions between STIM proteins and other proteins, and homotypic STIM1-STIM1 and heterotypic STIM1-STIM2 interactions were demonstrated biochemically. However, the functional significance of the cellular localisation of STIM1 and its domain structure only became evident after recent breakthrough research identified STIM1 as a key regulator of store-operated calcium (SOC) entry into cells. It is now clear that STIM1 is both a sensor of Ca(2+) depletion in the endoplasmic reticulum (ER) lumen and an activator of Orai1-containing SOC channels in the plasma membrane. On the basis of recent functional studies a model can be proposed to explain how the biochemical properties of STIM1 contribute to its precise membrane localisation and its function in regulating SOC entry.     

The synthesis and biological properties of some 5-substituted-2'-deoxyuridines

The reaction of various nucleophiles with 5-vinyl-2'-deoxyuridine has been studied in an attempt to explain the in vitro toxicity of the compound. Attempts to synthesise pro-drugs of 5-vinyl- and 5-ethynyl-2'-deoxyuridine are described.