Inhibition of the reactions of DNA hydrolysis and RNA synthesis by the alkaloid sanguinarine

The increase of the contour length of the low molecular linear duplex DNA in the complex with an alkaloid sanguinarine has been evidenced by the viscometric method. The enzymatic hydrolysis of modified DNA by pancreatic deoxyribonuclease I and RNA synthesis of DNA by rat liver nuclear RNA polymerase were studied. Sanguinarine has been shown to inhibit the first stages of DNA hydrolysis. This alkaloid is a weaker inhibitor than ethidium bromide, a more potent inhibitor than actinomycin D and exerts an inhibiting effect similar to that of distamycin A. Sanguinarine also decreases the rate of the labelled precursor incorporation into the acid-insoluble fractions by nuclear RNA polymerase from rat liver. A 50% inhibition by sanguinarine was observed at the same alkaloid concentration as that of ethidium bromide.     

The role of sulfhydryl groups in the functioning of DNA-dependent RNA-polymerase

Sulfhydryl groups of Escherichia coli DNA-dependent RNA polymerase were chemically modified with alkylating and mercuric-containing compounds. Iodoacetic acid and iodoacetamide were shown not to affect the enzymatic activity, whereas N-ethylmaleimide and mercuric-containing compounds completely inhibit the RNA synthesis. RNA polymerase modified with mercuric ions looses the ability of binding with promoter--containing DNA fragments. Moreover, mercuric ions inhibit the RNA elongation stage. Suggestion is made the Cys residues of RNA polymerase play a key role in double-stranded DNA unwinding. It is shown that SH-groups of beta- and beta'-subunits participate in the binding with double-stranded fragments of DNA.     

Effect of Chemically Modified 70S RNA from Avian Myeloblastosis Virus (AMV) upon the Activity of AMV DNA Polymerase

Adenine residues of 70S avian myeloblastosis virus (AMV) RNA are modified when reacted with chloroacetaldehyde. This modification introduces characteristic fluorescent epsilon-adenosine (epsilonA) probes which were used to monitor the reaction. Under suitable conditions, modified 70S(epsilonA) RNA was maintained intact and was inactive as a template for the AMV DNA polymerase. Furthermore, it inhibited the reaction catalyzed by AMV polymerase when 70S RNA was used as template-primer and had no effect on the two tested bacterial polymerases. Protection against the 70S (epsilonA) RNA inhibition was observed when 70S RNA was primed with oligo(dT) indicating preference of the polymerase for the oligo(dT) primed regions.     

Modification of Escherichia coli RNA polymerase by diethylpyrocarbonate. II. Binding and unwinding of double-stranded DNA

E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Binding to a double-stranded DNA and unwinding of the DNA at the enzyme binding site by the modified enzyme were examined. It was found that RNA polymerase reversibly lost the ability to unwind DNA helix as well as the RNA synthetic activity when 9 to 11 histidyl residues of the enzyme were modified. In addition ot modification of the most reactive sulfhydryl or amino groups of the enzyme accompanying histidyl residues modification results in irreversible decrease of the salt concentration which is necessary to remove the enzyme from DNA cellulose column. Further modification of the less reactive sulfhydryl or amino groups leads to irreversible loss of the DNA binding ability and to the enzyme structure alteration.     

特异亲和活性蓝染料的小分子RNA的SELEX筛选

化学合成含有２０ 个核苷酸随机序列, 长度为７３ 个核苷酸的单链 Ｄ Ｎ Ａ 随机库; Ｐ Ｃ Ｒ 扩增和双链化后, Ｔ７ Ｒ Ｎ Ａ 聚合酶体外转录得到单链 Ｒ Ｎ Ａ 随机库。以活性蓝染料凝胶柱为筛选介质, 体外进化方法筛选特异亲和活性蓝染料的 Ｒ Ｎ Ａ 分子。经８ 轮循环筛选, Ｒ Ｎ Ａ 群体亲和染料的比例从小于０ ．０３ ％ 上升至２２ ．４ ％ 。克隆筛选出 Ｒ Ｎ Ａ 序列后, 测定了３０ 个克隆的序列, 得到三类 Ｒ Ｎ Ａ 分子。经二级结构分析和活性蓝染料特异亲和能力测试,发现 Ｒ Ｎ Ａ 特异亲和活性蓝染料的主要结构因素是 Ｒ Ｎ Ａ 形成的双链结构     

Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 × 10⁵ cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K_m) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.     

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

Multiple amino acid substitutions allow DNA polymerases to synthesize RNA

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10(3)-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.     

Complex Formation of E. coli RNA Polymerase with Bacteriophage T2 DNA: Long-Range Effects in DNA

Conformation behavior of phase T2 DNA in the process of its interaction with it E. coli RNA polymerase was studied using spin labeling technique. T2 DNA was modified by the spin-labeled imidazole at OH-groups of glucosylated cytidine residues. It was shown that the binding of RNA polymerase under the conditions favoring the formation of open promoter complexes induces specific conformational changes in the spin-labeled DNA. The observed conformational changes encompass not only the promoter regions of DNA which are involved in direct contacts with RNA polymerase molecules but extend over remote DNA sites (long-range effect). In relation to this effect, current theoretical models of DNA dynamics are discussed.     

A new eukaryotic RNA polymerase factor: a factor from chicken myeloblastosis cells which stimulates transcription of denatured DNA

A heat-stable protein factor, capable of stimulating RNA synthesis by nuclear RNA polymerase II, was found in isolated nuclei of chicken myeloblastosis cells. It is adsorbed to a DEAE-Sephadex column used for RNA polymerase purification and then is eluted with 0.1 M ammonium sulfate. This factor appears to differ from previously reported eukaryotic RNA polymerase factors in its property of stimulating the activity of denatured (or single-stranded) DNA template. When heated, this factor contains no detectable endonuclease or exonuclease activity. The degree of stimulation is greater with chicken myeloblastosis RNA polymerase IIb than IIa and is most efficient when homologous DNA is used as template. This factor causes no stimulation of $\text{E. coli}$ RNA polymerase.