The synthesis of polyribonucleotides by cytoplasmic enzymes

Wykes, J. R.; Smellie, R. M. S.

doi:10.1042/bj0990347

Cited by 22 publications

(13 citation statements)

References 27 publications

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“…The polymerase activity is a poly(U) polymerase that preferentially incorporates UTP ( Table 1). This activity has been previously described by Burdon and Smellie (5) and later by Wykes and Smellie (26). In agreement with their results we find BHK2I cells grown as a monolayer in 32-oz bottles were gently scraped off the glass into 0.15 M NaCI solution and pelleted in sterile test tubes by centrifuging for 5 min at 1,000 rpm in an International model PR-2 (small rotor).…”

Section: Resultssupporting

confidence: 92%

“…The gradients were fractionated and assayed for RNA polymerase activity at 28 C for 2 h as described in Materials and Methods. that the poly(U) polymerase does not require the other nucleoside triphosphates and preferentially utilizes UTP as a substrate in the presence of the other triphosphates (24)(25)(26). Table 1 demonstrates that the polymerase activity is almost undetectable prior to fractionating cytoplasm on a gradient.…”

Section: Resultsmentioning

confidence: 98%

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Transcribing Complexes in Cells Infected by Vesicular Stomatitis Virus and Rabies Virus

Villarreal

Holland

1974

J Virol

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We analyzed cell extracts from BHK21 cells infected with vesicular stomatitis virus (VSV) and rabies virus for in vitro RNA polymerase activity. Cells infected with VSV B virions exhibited several complexes with in vitro RNA polymerase activity in sucrose gradients. These complexes synthesize VSV transcriptase product (4 to 18S) polyadenylated in RNA complementary to virion RNA. Cells infected with a high multiplicity of B virions and T particles show only one RNA polymerase complex active in vitro. This complex sediments at 110S aud makes only small (2S) RNA. Carrier BHK21 cells persistently noncytopathically infected with VSV contain several complexes active in RNA polymerase, but both exhibit very low activity. Cytoplasms of cells noncytopathically infected with rabies virus also show very low levels of a complex containing RNA polymerase activity. No transcriptase nor any other in vitro polymerase activity could be found associated with purified rabies virions, although they do carry out primary transcription in cells treated with actinomycin D and cycloheximide before infection.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 98%

Transcribing Complexes in Cells Infected by Vesicular Stomatitis Virus and Rabies Virus

Villarreal

Holland

1974

J Virol

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“…TUTases and poly(U) polymerases have been described in a variety of tissues and organisms, ranging from plants to humans (12,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Association with ribosomes has been a consistent theme.…”

Section: Discussionmentioning

confidence: 99%

Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction.

Andrews¹,

Baltimore²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Poliovirus RNA polymerase requires a host factor to initiate RNA synthesis in vitro. The host factor was previously purified to near homogeneity from HeLa cells but was not assigned an enzymatic activity. This report describes the purification of a terminal uridylyltransferase that can act as host factor. By all criteria examined it is identical to the factor purified previously. It has the same molecular weight (68,000), chromatographic properties, and cellular localization. We present evidence that terminal uridylyltransferase can add uridine residues to the 3' poly(A) end of virion RNA and that these anneal back to the poly(A) and form a hairpin primer for polymerase.

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“…[ac-32P]UTP was prepared by the method described by Wykes & Smellie (1966). Iso-octane (2,2,4-trimethylpentane) was obtained from British Drug Houses Ltd. and Tween 80 from Koch-Light Laboratories Ltd. (Colnbrook, Bucks.).…”

Section: Methodsmentioning

confidence: 99%

“…Such reactions include the synthesis of the terminal pCpCpA sequence of transfer RNA by cytoplasmic enzymes, which is well documented (Heidelberger, Harbers, Leibman, Takagi & Potter, 1956;Daniel & Littauer, 1963), but the significance of other soluble fractions that catalyse the incorporation of ribonucleotides into oligoand poly-ribonucleotides remains obscure (Chung, Mahler & Enrione, 1960;Burdon & Smellie, 1962;Edmonds & Abrams, 1962;Klemperer, 1963aKlemperer, ,b, 1964Klemperer, , 1965Ventkataraman & Mahler, 1963). There are also reports ofthe RNA-dependent uptake of ribonucleotides catalysed by particulate fractions of both bacterial (August, Ortiz & Hurwitz, 1962;Smith & August, 1966) and animal cells (Baltimore & Franklin, 1963;Eason & Smellie, 1965;Dalgarno, Liu, Martin & Work, 1966;Naora, 1966;Wykes & Smellie, 1966).…”

mentioning

confidence: 99%

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

Wilkie

Smellie

1968

Biochemical Journal

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1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg(2+) ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2-3 uridine residues, and there is evidence that these may be added to the 3'-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg(2+) ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5'-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5'-triphosphate. The optimum pH is in the range 7.0-8.5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [(32)P]UTP in the absence of ATP, GTP and CTP, 80-90% of (32)P was recovered in UMP-2' or -3' after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the (32)P was recovered in nucleotides other than UMP-2' or -3'. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.

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The synthesis of polyribonucleotides by cytoplasmic enzymes

Cited by 22 publications

References 27 publications

Transcribing Complexes in Cells Infected by Vesicular Stomatitis Virus and Rabies Virus

Transcribing Complexes in Cells Infected by Vesicular Stomatitis Virus and Rabies Virus

Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction.

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

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