SYNTHESIS AND PURIFICATION OF FLUORINATED BENZIMIDAZOLE AND BENZENE NUCLEOSIDE-5′-TRIPHOSPHATES

The expression “universal base” is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g., their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.     

"Portraying" of plant genomes using polymerase chain reaction amplification of ribosomal 5S genes.

Nontranscribed spacers of plant genes coding for ribosomal 5S RNA were amplified using the polymerase chain reaction. Primers were synthesized that were complementary to 3' (direct) and 5' (reverse) ends of the coding region and that are universal for higher plants. The patterns of polymerase chain reaction products are species and, sometimes, variety specific. The use of this approach for identification of barley 5S genes in chromosome-addition lines of wheat is discussed. This principle can be applied for the "portraying" of other tandem repetitive genes containing divergent regions.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

RNA polymerase--promoter recognition. Specific features of electrostatic potential of "early" T4 phage DNA promoters

Comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase alpha-subunit. The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase. A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase alpha-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.     

Processivity of ribozyme-catalyzed RNA polymerization

The "RNA world" hypothesis proposes that early in the evolution of life, before the appearance of DNA or protein, RNA was responsible both for encoding genetic information and for catalyzing biochemical reactions. Ribo-organisms living in the RNA world would have replicated their RNA genomes by using an RNA polymerase ribozyme. Efforts to provide experimental support for the RNA world hypothesis have focused on producing such a polymerase, and in vitro evolution methods have led to the isolation of a polymerase ribozyme that catalyzes primer extension which is accurate and general, but slow. To understand the reaction of this ribozyme, we developed a method of measuring polymerase processivity that is particularly useful in the case of an inefficient polymerase. This method allowed us to demonstrate that the polymerase ribozyme, despite its inefficiency, is partially processive. It is currently limited by a low affinity for the primer-template duplex, but once it successfully binds the primer-template duplex in the productive alignment, it catalyzes an extension reaction that is so rapid that it can occur multiple times during the short span of a single binding event. This finding contributes to the understanding of one of the more sophisticated activities yet to be generated de novo in the laboratory and sheds light on the parameters to be targeted for further optimization.     

The relationship between the activities of different pools of RNA polymerases I and II during PHA-stimulation of human lymphocytes.

Following PHA-stimulation of lymphocytes in culture, it is known that nuclear RNA synthesis and the amount of extractable RNA polymerase activity rise in these cells. The relationship between these two phenomena has been examined. Using an in vitro assay system which discriminates between polymerase activity which is "engaged" in nuclear RNA synthesis and a pool of "free" enzyme, the data suggest that the factors regulating the interaction between these two pools of enzyme activity are different for forms I and II RNA polymerases.     

Substitution of RNA-polymerase sigma-subunits and transcription regulation

Recent data on regulation of gene activity in bacteria by substitution of RNA polymerase sigma subunits are reviewed. The htpR gene which controls the switch-on of the Escherichia coli heat-shock protein synthesis codes for sigma 32 subunit. sigma 32-containing RNA polymerase transcribes the heat-shock genes in vitro from specific promoters of no use for RNA polymerase containing the major sigma 70 subunit. Several minor sigma subunits have been found in Bacillus subtilis vegetative cells, in addition to the major sigma 55 subunit, differing in the specificity of promoter recognition. Many B. subtilis genes are controlled by tandemly located promoters recognized by RNA polymerases carrying different sigma subunits. sigma 29 subunit is encoded by spoIIG gene and is probably involved in the regulation of sporulation. Specific sigma subunits for transcribing "middle" or "late" genes are encoded by a number of phages.