Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.     

In Vivo and In Vitro Synthesis of Human Rhinovirus Type 2 Ribonucleic Acid

HeLa cells infected with human rhinovirus type 2 synthesize a mixture of single-and double-stranded ribonucleic acid (RNA). The RNA synthesized by the membrane-bound RNA polymerase complex in vitro is also a mixture of single- and double-stranded RNA, whereas the deoxycholate-treated RNA polymerase complex synthesized only double-stranded RNA. Although twice as much cell-associated viral RNA is synthesized in vivo at 34 C than at 37 C, there is no difference in the rate of RNA synthesized in vitro at 34 C and 37 C by the polymerase complex. The RNA polymerase complex, after treatment with deoxycholate, sediments as a broad peak with an average sedimentation value of 120S.     

Nascent ribosomal and messenger RNA in DNA-membrane-complexes of Escherichia coli

Summary From Escherichia coli, DNA-membrane-complexes have been isolated which contain about 40% of the ribosomes, about 95% of the DNA and nearly all of the nascent RNA. The kinetic data on pulse labeled RNA show an average time of turnover of about 60 sec both for nascent messenger- and nascent ribosomal RNA. A proportion of the polysomes with nascent messenger RNA as well as most of the nascent ribosomal RNA is found in association with membranes, as has been shown by subfractionations of the DNA-membrane-complex involving treatment with DNase and desoxycholate. In this early transient stage, ribosomal precursor RNA already acquires some ribosomal proteins, as has been shown by arginine pulse label. Data on partial release of DNA from the DNA-membrane-complex by treatment with extremely low doses of DNase indicate that messenger RNA synthesis occurs in clusters on the DNA.The results support models in which, at any given time, RNA synthesis proceeds mainly in sections of the DNA close to the membrane. Thereby the DNA is linked to the membrane via nascent RNA contained in ribosomal precursors as well as via nascent messenger RNA on membrane-bound polysomes.     

Small-sample total RNA purification: laser capture microdissection and cultured cell applications.

Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.     

The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

Studies of newly synthesized ribosomal ribonucleic acid of Escherichia coli

1. RNA synthesized by Escherichia coli during one-hundredth of the generation time contains two fractions distinguishable by hybridization with homologous DNA. One fraction, approximately 30% of the newly synthesized RNA, did not compete with ribosomal RNA, being apparently messenger RNA. The other fraction, approximately 70% of the newly made RNA, hybridized as ribosomal RNA. These values are comparable with previous estimates (McCarthy & Bolton, 1964; Pigott & Midgley, 1968). 2. Hybridization-competition experiments showed that the newly made RNA associated with 70s ribosomes and larger ribosome aggregates was a mixture of ribosomal RNA and messenger RNA, whereas that associated with nascent ribosomal subunits consisted exclusively of ribosomal RNA. This observation provides means by which newly synthesized ribosomal RNA can be isolated free from messenger RNA. 3. Newly made ribosomal RNA in nascent ribosomal subunits was sensitive to shear under conditions where ribosomal RNA in mature ribosomes was shear-resistant. Thus, when RNA was extracted from cells of E. coli disrupted by mechanical means, newly made ribosomal RNA appeared heterogeneous in size, sedimenting as a broad peak extending from 8s to 16s. 4. Newly synthesized ribosomal RNA in nascent ribosomal subunits was rapidly degraded in the presence of actinomycin D and during glucose starvation. 5. Newly synthesized ribosomal RNA stimulated amino acid incorporation in a system synthesizing protein in vitro to the same extent as the RNA which contained the messenger RNA fraction.     

RD 114 Virus-Specific Sequences in Feline Cellular RNA: Detection and Characterization

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.     

Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.     

Secondary structure of the RNA component of a nuclear/mitochondrial ribonucleoprotein.

RNase mitochondrial RNA processing (MRP) is a site-specific endoribonuclease located in both the nucleus and mitochondria of vertebrate cells. The enzyme is a ribonucleoprotein whose RNA component has been shown to be encoded by a nuclear gene. Because RNase MRP is particular in its substrate requirement, RNA-RNA interaction has been proposed as important for the cleavage reaction. A secondary structure of this RNA from mouse cells has been derived by chemical modification of in vivo MRP RNA in ribonucleoprotein form, as isolated free RNA, and as RNA synthesized in vitro. Full-length MRP RNA appears to adopt a conformation containing a significant number of single-stranded residues and may form a pseudoknot. The data are consistent with both the RNA within the ribonucleoprotein and the free RNA possessing comparable secondary structures and suggest a possible site of interaction between enzyme and substrate. The human MRP RNA can be folded into a conformation very similar to that predicted for the mouse MRP RNA. A more limited analysis of human MRP RNA is consistent with the structure proposed for the mouse species.     

激光微切割与定量PCR技术分析肾脏病理切片RNA

采用激光微切割与定量PCR技术,分析使用不同提取方法从不同固定方法固定的病理切片中提取的RNA.用70%乙醇、丙酮、甲醇、4%多聚甲醛固定肾脏冰冻切片,使用激光微切割技术切取肾小球,用硫氰酸胍方法(guanidinethiocyanatemethods,GTC)和Trizol试剂方法提取RNA,使用Taqman定量PCR方法分析比较各组RNA的量;选取丙酮固定的石蜡切片,使用激光微切割技术切取肾小球,采用RNA裂解液提取RNA,使用Taqman定量PCR方法,比较石蜡切片和冰冻切片中RNA含量.结果显示:提取沉淀性固定剂如乙醇、丙酮、甲醇固定的冰冻切片的RNA时,2种提取方法和3种固定方法对RNA含量的影响都无明显差异;但在提取4%多聚甲醛固定冰冻切片时,使用Trizol提取RNA含量明显高于使用GTC方法,且其含量与沉淀性固定剂固定的切片RNA含量无明显差异.石蜡切片中经激光微切割肾小球的RNA含量与冰冻切片经激光微切割肾小球的RNA含量无明显差异.结果提示:切片的固定方法和RNA的提取方法是影响切片RNA提取量的主要原因.     

Effects of Potassium Nutrition on Ribonucleic Acid and Ribonuclease in Zea mays L

Potassium deficiency decreased the total RNA per shoot in corn (Zea mays L.) seedlings due to the reduced sizes of the plant, but increased the ratio of RNA to dry matter as much as 40%. Total base composition of RNA was unaffected by K⁺ deficiency. Thus K⁺ deficiency does not appear to alter RNA metabolism to such an extent that a lack of RNA becomes a factor limiting growth. The RNA contents of leaves were followed as deficiency developed or as the plant recovered from deficiency. Such time-course studies, although extremely useful in microbial work, did not yield as much information with higher plants. The ratio of RNA to dry matter in younger leaves was much higher than that in older leaves. The ribonuclease level in corn shoots was increased as much as threefold by K⁺ deficiency.