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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology
Purification And Properties Of A Hela Cell Enzyme Able To Remove The 5'-Terminal Protein From Poliovirus Rna, Victor Ambros, David Baltimore
Purification And Properties Of A Hela Cell Enzyme Able To Remove The 5'-Terminal Protein From Poliovirus Rna, Victor Ambros, David Baltimore
Victor R. Ambros
Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving …
An Enzymatic Activity In Uninfected Cells That Cleaves The Linkage Between Poliovirion Rna And The 5' Terminal Protein, Victor Ambros, Ralf Pettersson, David Baltimore
An Enzymatic Activity In Uninfected Cells That Cleaves The Linkage Between Poliovirion Rna And The 5' Terminal Protein, Victor Ambros, Ralf Pettersson, David Baltimore
Victor R. Ambros
The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of heLa cells and is activated by Mg++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5' terminal protein. The role of this enzyme …
Protein Is Linked To The 5' End Of Poliovirus Rna By A Phosphodiester Linkage To Tyrosine, Victor Ambros, David Baltimore
Protein Is Linked To The 5' End Of Poliovirus Rna By A Phosphodiester Linkage To Tyrosine, Victor Ambros, David Baltimore
Victor R. Ambros
Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either …
Identification Of A Protein Linked To Nascent Poliovirus Rna And To The Polyuridylic Acid Of Negative-Strand Rna, Ralf F. Pettersson, Victor R. Ambros, David Baltimore
Identification Of A Protein Linked To Nascent Poliovirus Rna And To The Polyuridylic Acid Of Negative-Strand Rna, Ralf F. Pettersson, Victor R. Ambros, David Baltimore
Victor R. Ambros
A protein similar to that previously demonstrated on poliovirus RNA and replicative intermediate RNA (VPg) was found on all sizes of nascent viral RNA molecules and on the polyuridylic acid isolated from negative-strand RNA. 32P-labeled nascent chains were released from their template RNA and fractionated by exclusion chromatography on agarose. Fingerprint analysis using two-dimensional polyacrylamide gels of RNase T1 oligonucleotides derived from nascent chains of different lengths showed that a size fractionation of nascent chains was achieved. VPg was recovered from nascent chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5' …
Separation Of Quantitation Of Intracellular Forms Of Poliovirus Rna By Agarose Gel Electrophoresis, Martinez Hewlett, Shmuel Rozenblatt, Victor Ambros, David Baltimore
Separation Of Quantitation Of Intracellular Forms Of Poliovirus Rna By Agarose Gel Electrophoresis, Martinez Hewlett, Shmuel Rozenblatt, Victor Ambros, David Baltimore
Victor R. Ambros
Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific …
Covalent Linkage Of A Protein To A Defined Nucleotide Sequence At The 5'-Terminus Of Virion And Replicative Intermediate Rnas Of Poliovirus, James B. Flanegan, Ralf F. Pettersson, Victor R. Ambros, Martinez J. Hewlett, David Baltimore
Covalent Linkage Of A Protein To A Defined Nucleotide Sequence At The 5'-Terminus Of Virion And Replicative Intermediate Rnas Of Poliovirus, James B. Flanegan, Ralf F. Pettersson, Victor R. Ambros, Martinez J. Hewlett, David Baltimore
Victor R. Ambros
The 5'-terminus of poliovirus polyribosomal RNA is pUp. A candidate for the 5'-terminus of poliovirion RNA was recovered as a compound migrating toward the cathode when 32P-labeled virion RNA was completely digested with ribonucleases T1, T2 and A and analyzed by paper ionophoresis at pH 3.5. Treatment with proteinase K reversed its direction of migration, indicating the presence of protein. Treatment with venom phosphodiesterase liberated all of the radioactivity as pUp, suggesting that poliovirion RNA has a protein-pUp 5'-terminus. Treatment of virion RNA with T1 ribonuclease alone generated a proteinase K-sensitive oligoribonucleotide. Analysis of the oligoribonucleotide using ribonucleases A and …