Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.     

Footprint of the sigma protein: a re-examination

Escherichia coli RNA Polymerase is a multi-subunit enzyme that catalyzes RNA synthesis, using DNA as a template. The sigma subunit of this enzyme plays an important role in the recognition of promoter sites on DNA. Using DNase I footprinting, Utpala Ramesh and Claude F. Meares [(1989) Biochem. Biophys. Res. Comm. 160, 121-125] reported that in the absence of the other subunits, sigma binds specifically to the bacteriophage lambda PR promoter DNA sequence. We are unable to reproduce that result.     

大鼠老化过程大脑皮层细胞核、染色质转录研究

 本实验对不同鼠龄(4—,16—17—,33—34—和99—103周)大鼠老化动物模型进行脑细胞核、染色质体外转录研究,结果表明:(1)大脑皮层细胞核、染色质转录活性在老化过程中呈下降趋势,其中RNA聚合酶Ⅰ、Ⅱ活性与染色质模板效率变化一致,说明染色质模板活性降低是导致细胞核转录功能减退的原因之一。(2)幼年鼠染色质RNA和NHCP含量高于老年鼠,提示染色质结合蛋白及RNA可能参与不同生理时期脑神经元染色质结构和功能的调节。(3)老年鼠脑染色质DNA抗DN-aseⅠ酶解能力增强,提示衰老导致转录活性染色质区域减少。     

Action of pancreatic DNase: requirements for activation of DNA as a template-primer for DNA polymerase.

Pancreatic DNase requires both Ca2+ and Mg2+ for its activity as measured by formation of an activated DNA template for in vitro DNA polymerase alpha assay and by the hyperchromic shift. Mn2+ can partially satisfy the Mg2+ requirement of the DNase for activation of DNA but the resulting template is only 50% as active in the DNA polymerase assay. When precautions are taken to avoid divalent ion contamination, pancreatic DNase is not active in the presence of Ca2+ or Mg2+ alone. analysis of the DNA by sucrose gradient centrifugation shows that only in the presence of Ca2+ plus Mg2+ or Mn2+ does pancreatic DNase produce extensive strand breaks in the DNA. The activated DNA template that yields maximal DNA polymerase activity is low molecular weight material of 30,000 to 50,000 daltons.     

On the origin of DNA genomes: evolution of the division of labor between template and catalyst in model replicator systems

The division of labor between template and catalyst is a fundamental property of all living systems: DNA stores genetic information whereas proteins function as catalysts. The RNA world hypothesis, however, posits that, at the earlier stages of evolution, RNA acted as both template and catalyst. Why would such division of labor evolve in the RNA world? We investigated the evolution of DNA-like molecules, i.e. molecules that can function only as template, in minimal computational models of RNA replicator systems. In the models, RNA can function as both template-directed polymerase and template, whereas DNA can function only as template. Two classes of models were explored. In the surface models, replicators are attached to surfaces with finite diffusion. In the compartment models, replicators are compartmentalized by vesicle-like boundaries. Both models displayed the evolution of DNA and the ensuing division of labor between templates and catalysts. In the surface model, DNA provides the advantage of greater resistance against parasitic templates. However, this advantage is at least partially offset by the disadvantage of slower multiplication due to the increased complexity of the replication cycle. In the compartment model, DNA can significantly delay the intra-compartment evolution of RNA towards catalytic deterioration. These results are explained in terms of the trade-off between template and catalyst that is inherent in RNA-only replication cycles: DNA releases RNA from this trade-off by making it unnecessary for RNA to serve as template and so rendering the system more resistant against evolving parasitism. Our analysis of these simple models suggests that the lack of catalytic activity in DNA by itself can generate a sufficient selective advantage for RNA replicator systems to produce DNA. Given the widespread notion that DNA evolved owing to its superior chemical properties as a template, this study offers a novel insight into the evolutionary origin of DNA.     

应用RT-PCR检测基因的体外转录活性

体外转录分析已广泛应用于分子生物学领域．由于体外转录所产生的ＲＮＡ的量极少,需有灵敏的方法检测生成的ＲＮＡ．本研究发展了一种基于ＲＴ－ＰＣＲ技术鉴定体外转录产物的方法．将待研究基因的启动区与任何一段已知的含转录起始位点的编码序列相连,制备成体外转录的模板．体外转录后,用ＤＮａｓｅⅠ将ＤＮＡ模板完全降解;生成的ＲＮＡ经反转录合成ｃＤＮＡ;再以ｃＤＮＡ为模板,用相应的引物进行ＰＣＲ扩增,产物用琼脂糖凝胶电泳检测．该法灵敏度高,操作简便,无需同位素,且体外转录模板制备方便．还可结合其他技术,如Ｓｏｕｔｈｅｒｎ印迹分析,能获得更高的灵敏度．     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

Polymerase Activity of Pichinde Virus

Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg(2+) and Mn(2+) were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients.     

Long-term storage of DNA-free RNA for use in vaccine studies

RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved.