Search PubMed⌕ Search

Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 199 records · Page 11Linked to original sources
PubMed1982

The double-stranded RNA genome segments of cytoplasmic polyhedrosis virus are independently transcribed.

The in vitro transcription of the genome segments of insect cytoplasmic polyhedrosis virus was examined for a possible mechanism of coordinate control. Initiation of the first transcription cycle, primed by preincubation in the absence of CTP, was synchronous for all 10 viral genome segments. The time required for the completion of each genome transcript was proportional to the chain length, and all 10 mRNA's were synthesized in molar amounts inversely proportional to their molecular weights. The rate of chain elongation for all mRNA's was approximately 6 nucleotides per s. The combined results indicate that each of the viral genome segments is transcribed independently and suggest that RNA chain elongation, not initiation, is rate limiting for cytoplasmic polyhedrosis virus mRNA synthesis in vitro.

Animals↗
PubMed1982

Homologous Terminal Sequences in the Double-Stranded RNA Genome Segments of Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori.

The 3'-terminal regions (20 to 32 residues) of the genome double-stranded RNA (dsRNA) segments of cytoplasmic polyhedrosis virus were sequenced. The dsRNAs, which were labeled at their 3' termini by incubation with [5'-(32)P]pCp and T4 RNA ligase, were denatured and resolved into the plus and minus strands by agarose-urea gel electrophoresis. Ten single-stranded RNAs thus obtained from the five dsRNA segments IV, V, VIII, IX, and X were sequenced by postlabeling methods. Common 3'-terminal sequences, -GUUAGCC and -UUACU, were found in the plus and minus strands, respectively, of all five dsRNA segments. However, adjacent sequences diverged and were considerably variable. The homologous sequences found in the 3' end may be important recognition signals for viral RNA polymerases and for assembly of the genome segments.

Journal Article↗
PubMed1982

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.

Cell Nucleus↗
PubMed1981

Separation of the plus and minus strands of cytoplasmic polyhedrosis virus and human reovirus double-stranded genome RNAs by gel electrophoresis.

The complementary strands of most of the genome double-stranded RNA segments of insect cytoplasmic polyhedrosis virus (CPV) and human reovirus are separated for the first time by agarose gel electrophoresis in in the presence of 7 M urea. CPV (+) strands and most reovirus (-) strands migrate faster than the corresponding strands of opposite polarity. Glyoxal treatment, which modifies guanine residues and prevents G-C basepairing, results in a loss of strand resolution and concomitantly a significant decrease in electrophoretic mobilities. Reovirus mRNAs synthesized in vitro with ITP substituted for GTP show similar decreased electrophoretic mobilities as the glyoxalated mRNAs. These results clearly indicate that the basis for (+) and (-) strand resolution is the presence of secondary structure formed mainly by G-C(U) base-pairs that are maintained during gel electrophoresis in the presence of 7 M urea. When the plus and minus strands of CPV genomes were separated and compared for protein synthesizing activity, it was found that only the plus strands were able to form stable 80S ribosome-RNA initiation complexes in wheat germ cell-free extracts.

Base Composition↗
PubMed1981

Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase.

Short oligonucleotides corresponding to the 5'-terminal sequence of reovirus mRNAs were produced in vitro by virion-associated transcriptase activity. Both capped and uncapped oligonucleotides were synthesized in molar excess relative to mRNA. Yields of uncapped oligomers including ppG-C and ppG-C-U were severalfold greater than the homologous capped structures. In partial reaction mixtures that were nonsupportive for mRNA chain elongation, capped oligomer synthesis was increased. Similarly, oligonucleotide formation was differentially resistant in viral preparations that were inactivated with respect to mRNA synthesis by modification of the genome RNA by dimethyl sulfate alkylation or psoralen photoreaction. The results suggest that reovirus mRNA synthesis involves excessive initiation by reiterative transcription of promoter sites by the reovirus polymerase. Only a small fraction of the resulting oligonucleotides are capped and extended to form full length mRNAs during a subsequent elongation step which is apparently mediated by transcriptase molecules that escape the reiterative phase of transcription.

Alkylating Agents↗
PubMed1981

Allosteric stimulatory effect of S-adenosylmethionine on the RNA polymerase in cytoplasmic polyhedrosis virus. A model for the positive control of eukaryotic transcription.

The stimulatory effect of S-adenosylmethionine (AdoMet) on cytoplasmic polyhedrosis virus transcription in vitro has been studied in detail. AdoMet affects the initiation of transcription but not chain elongation. It is required for each reinitiation step during multiple transcription cycles. Under limited conditions of synthesis in reaction mixtures depleted of UTP and CTP, a greater amount of initiator oligonucleotides was formed in the presence of AdoMet than in its absence. These small oligonucleotides were also formed along with the completed mRNA under conditions of mRNA formation. Although these capped (or uncapped) oligonucleotides corresponded to the 5'-terminal sequence of cytoplasmic polyhedrosis virus mRNA, they failed to substitute for AdoMet in promoting mRNA synthesis. Efficient synthesis of mRNA containing 5'-m7GpppAmpG was dependent on the concentrations of AdoMet and the initiating nucleotide, ATP. There was also an inverse relationship between AdoMet and ATP concentrations such that the apparent Km for ATP was decreased strikingly from 10 to 0.25 mM as the AdoMet concentration was increased from 5 to 500 microM. This effect was observed only for ATP. Double reciprocal plots for estimating the Km of ATP formed concave upward curves but became linear upon addition of a higher concentration of AdoMet, indicating a positive cooperativity of ATP and allosteric effect of AdoMet in the initiation process. Stimulation of transcription by lowering the Km for the initiating nucleotide at the promoter site is proposed as a model for positive regulation of eukaryotic gene expression.

Allosteric Regulation↗
PubMed1981

Chemical methylation of RNA and DNA viral genomes as a probe of in situ structure.

We used [methyl-(3)H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for phiX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [methyl-(3)H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, phiX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation.

Adenine↗
PubMed1979

Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap.

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.

DNA-Directed RNA Polymerases↗
PubMed1978

"Pretranscriptional capping" in the biosynthesis of cytoplasmic polyhedrosis virus mRNA.

The in vitro synthesis of cytoplasmic polyhedrosis virus (CPV) mRNA was previously shown to be dependent upon the presence of the methyl donor S-adenosylmethionine (AdoMet). We now find that the competitive inhibitor of methylation, S-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNAs containing 5'-terminal GpppA and ppA, rather than m(7)GpppAm as observed with Adomet. In addition to AdoHcy, other AdoMet analogues, including S-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. In order to study the relationship between cap formation and mRNA synthesis, nucleoside triphosphates were replaced in the RNA-synthesizing reaction mixture (containing AdoMet) by the corresponding beta,gamma-imido analogues, which are resistant to nucleotide phosphohydrolase, an enzyme involved in cap formation. Although mRNA synthesis occurred in the presence of UMP-pNHp or GMP-pNHp, none was observed when AMP-pNHp was substituted for ATP. Because the ATP molecule that becomes the 5'-terminal nucleotide of CPV mRNA must be cleaved at the beta-gamma position during cap formation, the results suggest that, in this viral transcription system, cap formation is prerequisite to mRNA synthesis-i.e., a "pretranscriptional" event.

Adenylyl Imidodiphosphate↗
PubMed1977

Characterization of the m7G(5')pppN-pyrophosphatase activity from HeLa cells.

The m7(G(5')pppN-pyrophosphatase activity previously detected in HeLa cells has been further characterized. Results from DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one enzyme activity in HeLa cell extracts which was capable of selectively hydrolyzing m7G(5')pppN to yield m7pG + ppN (where N = 2'-O-methylated or unmethylated ribonucleosides or oligonucleotides of up to 8 to 10 nucleosides in length). The majority (approximately 95%) of this activity was found in the cytoplasmic extract but appeared not to be associated with the lysosomal fraction. m7G(5')pppG was hydrolyzed by the partially purified enzyme in the absence of divalent cations at a pH optimum of 7.5 and a temperature optimum of 45 degrees, with a Michaelis constant (Km) of 1.7 micronM. Sedimentation analysis and gel filtration showed the molecular weight of the enzyme as approximately 81,000. Inhibition studies testing the effect of a number of prospective substrates on the rate of m7G(5')pppG hydrolysis have confirmed the importance of the methyl moiety at the N7 position of guanosine for enzyme-substrate interaction. Furthermore, the trimethylated guanosine-containing 5'-terminal structure derived from U-2 RNA was found not to serve as substrate, and 7-methylinosine, unlike 7-methylguanosine, was not an effective inhibitor of m7G(5')pppG hydrolysis. Thus, the 2-amino group of the 7-methylguanosine portion of m7G(5')pppN is also important for substrate interaction with this specific pyrophosphatase.

Cytoplasm↗
PubMed1977

5'-Terminal structure and mRNA stability.

Reovirus mRNAs with 5'terminal m7GpppGm or GpppG are more stable than mRNA containing unblocked ppG 5'-ends when injected into Xenopus laevis oocytes or incubated in cell-free protein synthesising extracts of wheat germ and mouse L cells. The greater stability of mRNA with blocked 5' termini is not dependent upon translation but seems to result from protection against 5'-exonucleolytic degradation.

Animals↗
PubMed1977

A simple method for the preparation of [beta-32P]purine nucleoside triphosphase.

A rapid, simple and inexpensive procedure is described for the preparation of purine ribo-and deoxyribonucleoside triphosphates specifically and highly radiolabeled with [32P]phosphate in the beta position. The method involves two successive enzymatic reactions: conversion of donor [gamma-32P]ATP in the presence of an excess of acceptor 5'-mononucleotide to the 5'-diphosphates by myokinase or guanosine 5'-monophosphate kinase followed by phosphorylation with pyruvate kinase to yield 5'-triphosphates.

Adenylate Kinase↗
PubMed1976

Mechanism of formation of reovirus mRNA 5'-terminal blocked and methylated sequence, m7GpppGmpC.

Blocked and methylated 5' termini of reovirus mRNA are formed by viral cores at an early stage of transcription. Cores incubated in a complete transcription reaction mixture for 30 s, or in a mixture lacking UTP and ATP for a longer time, synthesize the "cap" structure, m7GpppGmpC. The dinucleotide ppGpC functions as substrate for a core-associated guanylyltransferase and is converted to GpppGpC by addition of pG from GTP. For optimal conversion, both the diphosphate terminus and phosphodiester bond are required. pGpC is not a substrate, but pppGpC is utilized after removal of the gamma-phosphate by a core nucleotide phospohydrolase. Methyltransferases also present in cores transfer methyl groups sequentially from S-adenosylmethionine (AdoMet) to the N7-position of the 5'-terminal guanosine followed by the 2'-OH of the penultimate guanosine. GpppGpC is hydrolyzed by cores in the presence of pyrophosphate to ppGpC, the predominant 5'-terminal structure of reovirus mRNA made in the absence of S-adenosylmethionine. N7-methylation prevents pyrophosphorolysis of m7GpppGpC, which may explain the increased proportion of blocked, methylated 5' termini in viral mRNA synthesized in the presence of S-adenosylmethionine. On the basis of these findings, the following reaction series is proposed for the synthesis of reovirus mRNA caps. In the series, AdoHcy is the abbreviation for S-adenosylhomocysteine (see article)9

Edetic Acid↗