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Biomedical subjects

E Wimmer

Publications and source records attributed to E Wimmer.

At least 253 records · Page 14Linked to original sources
PubMed1978

Identification of specific fragments containing the 5' end of poliovirus RNA after ribonuclease III digestion.

The small protein (VPg) covalently linked to the 5' end of poliovirus Type 1 (PV-1) RNA has been labeled in vitro with 125I using the Bolton and Hunter reagent. The RNA is not degraded under the conditions used and nearly all the label enters VPg and not the poly-nucleotide chain. When this 125I-labeled RNA is cleaved with RNase III at low monovalent salt concentrations, one major 125I-labeled fragment, approximately 100 nucleotides long, is produced. The corresponding fragment from similar digests of 32P-labeled RNA has also been identified. The 32P-labeled fragment changes electrophoretic mobility after protease treatment indicating that it contains VPg. Furthermore, the RNase T1 oligonucleotide known to be at the 5' terminus of poliovirus RNA is found in T1 digests of the purified fragment. These results confirm that the fragment is derived from the 5' end of the RNA. This fragment will be useful in studies concerning the initiation of protein synthesis during poliovirus infection.

Oligoribonucleotides↗
PubMed1978

An electron microscope study of the proteins attached to polio virus RNA and its replicative form (RF).

A recently described method (Wu, M. and Davidson, N. (1978), Nucleic Acids Research 5, in press) for visualizing proteins attached to nucleic acids in the electron microscope has been applied to study proteins attached to poliovirion RNA and to the viral double-stranded intracellular RF form. A protein is found at the 5' end of the plus strand virion RNA, and protein components are found at both ends of the duplex RF. In the RF as usually extracted, there is frequently a larger or compound protein aggregate at the end which contains the 3' end of the plus strand and the 5' end of the minus strand. Banding in CsCl-guanidinium hydrochloride in the presence of sarkosyl causes dissociation of some components of this aggregate, leaving both ends labeled with the covalently bound VPg. These results confirm and extend previous biochemical studies of proteins bound to poliovirion RNA and to the RF form.

Cytochrome c Group↗
PubMed1978

Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells.

The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.

Cell Nucleus↗
PubMed1977

A protein covalently linked to poliovirus genome RNA.

Poliovirion [32P]RNA, after digestion with RNase T2, yields mononucleotides and a labeled compound "X," which is not negatively charged at pH 5. X contains, relative to the label in virion RNA, one to two phosphates and is partially acid insoluble. It can be labeled with tritiated amino acids 3 hr after infection, is insoluble in chloroform/methanol, and can be digested with Pronase. These observations suggest that X is a protein. The protein cannot be removed from the polio genome when the RNA is (i) sedimented through a sucrose gradient in 0.5 M NaCl, (ii) heated to 100 degrees in the presence of sodium dodecyl sulfate followed by sedimentation through a sucrose gradient in 80% dimethylsulfoxide, or (iii) banded in 4 M cesium trichloroacetate. Digestion of the 32P-labeled protein with Pronase yields one major 32P-labeled product, which contains pUp. The protein migrates faster than capsid protein VP4 in a polyacrylamide gel. Our data show that the genome of poliovirus, but not poliovirus mRNA [A. Nomoto, Y. F. Lee, and E. Wimmer (1976) Proc. Natl. Acad. Sci. USA 73, 375-380], is covalently attached to a small virus-coded protein (molecular weight less than 7000), which we call VPg. VPg is probably linked to the 5' end of the polio genome. Possible functions of VPg in viral replication are discussed.

Molecular Weight↗
PubMed1977

The 5'-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg.

The 5'-terminal, RNase T1-resistant oligonucleotide of poliovirus mRNA has been isolated. Its sequence is pU-U-A-A-A-A-C-A-Gp, which is identical to that of virion RNA except that the genome-linked protein VPg is absent [Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977) Nature 268, 208-213]. Because all newly synthesized viral RNAs are VPg-linked, we propose that VPg is cleaved from progeny RNA at the linkage between protein and nucleic acid prior to polyribosome formation. This may represent a new mode of processing of viral macromolecules. Virion RNA from which VPg has been cleaved proteolytically retains its specific infectivity, an observation suggesting that VPg is not involved in early steps (penetration and translation) of the infectious cycle initiated by RNA.

Base Sequence↗
PubMed1976

[Studies on the accuracy and precision of total serum cholesterol in regional interlaboratory trials (author's transl)].

The between-run precision of the Liebermann-Burchard reaction modified by Watson was, in our laboratory, 2-3%, the within-run coefficient of variation was 1-2%. The between-run precision of the enzymatic test was 3-4%, the within-run coefficient of variation was 3%. The regression analysis of 92 serum specimens from patients was y = -17.31 + 1.04 chi, the coefficient of regression was r = 0.996. Interlaboratory trials of serum cholesterol were studied in the normal and pathological range. Lyophilized samples of serum prepared commercially and from fresh specimens from patients were analysed by the method of Liebermann-Burchard as well as by the enzymatic procedure. Acceptable results estimated by Liebermann-Burchard were obtained in the different laboratories after using a common standard of cholesterol. The coefficient of variation of the enzymatic test in the interlaboratory trial was higher in comparison to the Liebermann-Burchard reaction. Methodological difficulties of the Liebermann-Burchard reaction are discussed and compared with the specific, enzymatic assay.

Cholesterol↗
PubMed1976

The 5' end of poliovirus mRNA is not capped with m7G(5')ppp(5')Np.

Poliovirus was grown in HeLa cells in the presence of phosphorus-32 and actinomycin D. Three to four hours after infection, viral mRNA was recovered from polyribosomes and its identity verified by two-dimensional gel electrophoresis of RNase T1 digests. Digestion of the viral [32P]mRNA with RNase T2 and separation of the products by ion exchange chromatography at pH 5 yielded pUp as possible 5' terminus but no "capping group" of the structure m7G(5')ppp(5')Np. Total cytoplasmic [32P]RNA of HeLa cells, on the other hand, was found to contain capping groups. Neither the capping group nor ppNp or pppNp was found in an RNase T2 digest of poliovirion [32P]RNA, in agreement with previous results [Wimmer, E. (1972) J. Mol. Biol. 68, 537-540]. The data indicate that 5'-terminal m7G(5')ppp(5')Np is absent from poliovirus RNAs and, therefore, is not involved in poliovirus protein synthesis.

Base Sequence↗
PubMed1976

"Fingerprinting" high molecular weight RNA by two-dimensional gel electrophoresis: application to poliovirus RNA.

Conditions are described under which complete RNase T1 digests of high molecular weight RNA can be separated into numerous components by two-dimensional gel electrophoresis. Small and large oligonucleotides (n = 1 - 2c0) can be resolved without losses. The procedure yields fingerprints which are diagnostic for a particular species of RNA and an index of its purity as will be shown for the genomes of poliovirus type 1 and 2.

Electrophoresis, Polyacrylamide Gel↗
PubMed1975

Replication of picornaviruses. I. Evidence from in vitro RNA synthesis that poly(A) of the poliovirus genome is genetically coded.

A crude replication complex has been isolated from poliovirus-infected HeLa cells and used for synthesis of poliovirus replicative intermediate (RI) RNA, replicative form (RF) RNA, and single-stranded (SS) RNA in vitro. All three classes of virus-specific RNA synthesized in vitro are shown to contain poly(A). Poly(A) of RF and of SS RNA [RF-poly(A) and SS-poly(A)] has a chain length (50 to 70 nucleotides) that is shorter than that of poly(A) of in vivo-synthesized RNAs. Poly(A) of RI [RI-poly(A),] however, is at least 200 nucleotides long and, therefore, larger than poly(A) of RI isolated from HeLa cells 4 h after infection. The crude membrane-bound replication complex contains a terminal adenylate transferase activity that is stimulated by Mn2+ and the addition of an (Ap)2AOH primer. This transferase activity is found also in extracts of mock-infected cells. Partial purificaiton of the replication complex in a stepwise sucrose gradient, in which the viral replicase is associated with the smooth cytoplasmic membrane fraction, does not remove the terminal transferase. However, when the partially purified replication complex is treated with deoxycholate and sedimented through a sucrose gradient, a soluble replication complex can be isolated that is free from terminal adenylate transferase. This soluble replication complex was found to synthesize viral RNA-linked poly(A) longer in chain length than that synthesized by the crude replication complex. Taking into account the 5'-terminal poly(U) in poliovirus minus strands, our data suggest that polyadenylation of poliovirus RNA occurs by transcription and not by end addition. When compared to other viral systems, poliovirus and, probably, all picornaviruses appear to be unique in that the poly(A) of their genome is genetically coded.

Adenosine Monophosphate↗