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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 415 records · Page 23Linked to original sources
PubMed1977

Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A).

A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.

HeLa Cells↗
PubMed1977

Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration.

Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits. At limiting Mg2+ concentration, DNA is synthesized in good yield, up to a maximum size of about 2.4 X 10(6) daltons. DNA larger than 2 X 10(6) daltons, taken from alkaline sucrose gradients, has no detectable self-complementarity and was protected from digestion by S1 nuclease to an extent of 90% by annealing to 70S RNA. All size classes of DNA made in these reactions are primed with RNA, because all are initiated with a pApdAjunction. To produce such long molecules, it is necessary to keep the concentration of Mg2+ in the reaction mixture below the total concentration of deoxyribonucleoside triphosphates. Under these conditions, degradation of the RNA template is minimized. The rate of DNA synthesis is also slowed by 30 to 50%, but products longer than 5,000 nucleotides, which are not found otherwise, are completed between 3 and 6h of reaction.

Cell-Free System↗
PubMed1977

Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis.

The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro.

DNA, Viral↗
PubMed1977

Circular forms of Uukuniemi virion RNA: an electron microscopic study.

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.

Arboviruses↗
PubMed1977

Localization of two cellular forms of the vesicular stomatitis viral glycoprotein.

Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G1 and G2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. G1 has the higher electrophoretic mobility, but both forms migrate more slowly than G protein synthesized in a wheat germ cell-free system (G0), which presumably is the unglycosylated form. G1 is a kinetic precursor of the G2 form, and the apparent cause of the electrophoretic difference between the two species is the presence of N-acetylneuraminic acid on the G2 form. Conversion of G1 to G2 occurs 10 to 20 min prior to the appearance of the G2 form of the protein on the cell surface. This suggests that the G protein may be completely glycosylated several minutes prior to its migration to the cell surface and that glycosylation is not the limiting step in its maturation. No glycoprotein comigrating with G0 can be detected in the infected cells, even after 5-min labeling periods; this suggests that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.

Cell Line↗
PubMed1977

Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus.

Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.

Cell Fractionation↗
PubMed1977

Analysis of the defects of temperature-sensitive mutants of vesicular stomatitis virus: intracellular degradation of specific viral proteins.

The metabolism of viral RNA and proteins has been studied in cells infected with temperature-sensitive mutant strains of vesicular stomatitis virus. Certain viral proteins encoded by the mutant strains, usually the putative mutant protein for the assigned complementation group, were shown to be degraded more rapidly at the nonpermissive temperature than were the wild-type proteins. Group III mutants (tsG33, tsM301) encode M proteins which are degraded three- to fourfold faster than the wild-type protein. This defect cannot be fully rescued by coinfection with wild-type virus, and thus the defect appears to be in the M protein itself. Mutants tsM601 (VI) and tsG41(IV) encode N proteins which are degraded much faster than the wild-type protein and also share the property of being defective in replication of viral RNA, suggesting a correlation between these phenotypic properties. Furthermore, the L proteins of tsG11(I) and tsG13(I) are more labile than the wild-type protein at the nonpermissive temperature. The G protein of tsM501(V) did not undergo the change in electrophoretic mobility previously shown to be the result of sialylation, suggesting that it is defective in maturation or glycosylation at the nonpermissive temperature. Three of the mutants previously isolated in this laboratory, tsM502(V), tsM601(VI), and tsM602(VI), were shown to be defective in viral RNA synthesis at the nonpermissive temperature. Mutant tsM601(VI) was defective mainly in viral RNA replication, whereas tsM502(V) appeared to be totally defective for viral RNA transcription and replication at the nonpermissive temperature.

L Cells↗
PubMed1977

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.

Cell Fractionation↗
PubMed1977

Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses.

Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.

Cell Line↗
PubMed1977

Morphogenesis of poliovirus. IV. existence of particles sedimenting at 150S and having the properties of provirion.

An apparent precursor to the poliovirion that cosediments with the virion at 150S was identified by its content of VP-0. It has properties previously associated with the provirion, a structure that sedimented at 125S, and it may be an alternate form of provirion. Like virions, the 150S precursor binds to and elutes from cells, after which it sediments at about 125S.

HeLa Cells↗
PubMed1976

Terminal deoxynucleotidyl transferase is found in prothymocytes.

Terminal deoxynucleotidyl transferase is an enzyme which has the unique property of polymerizing polydeoxynucleotides onto a primer in the absence of a template (1,2). This enzyme is found both in the thymus and the bone marrow of birds, rodents, and humans (3-7). Whether the marrow cells that contain terminal transferase are related to thymocytes, or are on a separate pathway of differentiation, is not yet known (7,8). To determine the lineage of the murine bone marrow cells that have terminal transferase, we have investigated whether these cells have the antigen Thy-1 induced on the cells by treatment with thymopoietin (9). Thymopoietin is known to induce a set of characteristic T-cell markers including the Thy-1 alloantigen on the surface of a subpopulation of bone marrow cells committed to T-cell differentiation (prothymocytes) (10). Destruction of Thy- 1-positive cells after exposure to thymopoietin allows elimination of a substantial fraction of those bone marrow cells that can repopulate an irradiated thymus (11). We find that such an elimination after induction with the thymic polypeptide removes a substantial amount of terminal transferase from the bone marrow cell population, suggesting that at least one-half of the marrow cells bearing this enzyme are related to those found in the thymus.

Animals↗
PubMed1976

A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus.

A quantitative Abelson murine leukemia virus (A-MuLV) lymphoid cell transformation assay has been developed using a semisolid agarose culture system. Under these conditions lymphoid cell transformation was shown to vary linearly with the dose of A-MuLV used. The susceptibility of bone marrow cells from different strains of mice to A-MuLV-induced transformation can be estimated using the agarose assay. Strains with bone marrow cells of high, medium, and low susceptibility to A-MuLV can be identified. The assay has been used to study the susceptibility of cells from lymphoid organs of fetal and adult mice to A-MuLV. Cell suspensions from fetal liver, adult bone marrow, and adult spleen are susceptible to A-MuLV, while thymocytes are resistant to A-MuLV-induced transformation. Bovine serum albumin gradient fractionation of bone marrow cells before infection with A-MuLV demonstrates that the majority of A-MuLV-sensitive cells are recovered in a broad band partially overlapping the majority of the nucleated cells. The agarose assay system allows study of A-MuLV-lymphoid cell interaction at the level of single cell-single virus particle interaction.

Animals↗
PubMed1976

Terminal deoxynucleotidyltransferase. Serological studies and radioimmunoassay.

Mouse antisera against calf terminal deoxynucleotidyltransferase (terminal transferase) have been prepared. The sera have been used to characterize terminal transferase both by studying inhibition of enzyme activity and by developing a competition radioimmunoassay using highly purified 125I-labeled terminal transferase. By either assay, anti-terminal transferase serum did not cross-react significantly with calf DNA polymerases alpha and beta, Escherichia coli DNA polymerase I, or the reverse transcriptase of Moloney mouse leukemia virus. The calf terminal transferase did, however, share cross-reactive but not identical determinants with human and murine terminal transferase. The radioimmunoassay could detect as little as 2 ng of terminal transferase/mg of soluble protein in a tissue extract. Thymocytes were found to contain 280 ng of terminal transferase/mg of cell protein or about 1 X 10(5) molecules/cell; bone marrow had about 1% of the level of enzyme found in thymus. Extracts of spleen, peripheral white blood cells, lymph nodes, liver, muscle, and kidney all lacked detectable antigenicity of terminal transferase. These data indicate that terminal transferase is a tissue-specific enzyme and is not related to other DNA polymerases.

Animals↗