Studies on the polypeptides of poxvirus. II. Comparison of virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses.

Virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses were examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. At least 42 vaccinia virus-induced polypeptides were identified among the polypeptides of cells pulse-labeled with [35S]-methionine and/or of fractionated cells labeled with [14C]-leucine for 24 hr. They consisted of 15 polypeptides (early polypeptides) which were synthesized even in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl, and 27 polypeptides (late polypeptides) which were synthesized only in the absence of cytosine-1-beta-D-arabinofuranosyl-HCl. By the same procedure at least 40 cowpox virus-induced polypeptides (14 early polypeptides and 26 late polypeptides) and at least 31 Shope fibroma virus-induced polypeptides (13 early polypeptides and 18 late polypeptides) were identified. Comparative studies of virus-induced polypeptides on the basis of migration in SDS-polyacrylamide gel electrophoresis revealed that 11 polypeptides were early polypeptides common to both vaccinia and cowpox viruses; 21 were late polypeptides common to both vaccinia and cowpox viruses; 4 were early polypeptides common to both vaccinia and Shope fibroma viruses; 7 were late polypeptides common to both vaccinia and Shope fibroma viruses; 5 were early polypeptides common to both cowpox and Shope fibroma viruses; 9 were late polypeptides common to both cowpox and Shope fibroma viruses; 4 were early polypeptides common to all three viruses; and 7 were late polypeptides common to all three viruses.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Effect of 5-bromodeoxyuridine on heterogeneous nuclear RNA in rat hepatoma cells.

Heterogeneous nuclear RNA HnRNA) was isolated from untreated and 5-bromodeoxyuridine (BrdUrd) treated hepatoma tissue culture (HTC) cells. analysis of this RNA by either electrophoresis on polyacrylamide-agarose gels or centrifugation in sucrose gradients demonstrated that BrdUrd caused a shift in the labeled HnRNA population toward a smaller size distribution. This effect was produced by concentrations of BrdUrd which specifically lower the level of the differentiated enzyme tyrosine aminotransferase, but do not greatly affect cell growth. Differential binding to oligo(dT) cellulose was used to fractionate HnRNA further into classes containing poly(A) (alpha), oligo(A) (beta) or neither category of A-rich sequences (gamma). BrdUrd did not alter the relative rates of uridine incorporation into the three classes. The shift in the labeled HnRNA population due to BrdUrd was observed in all three subclasses of HnRNA.     

Induction of prolactin synthesis in rat pituitary tumor cells by 5-bromodeoxyuridine.

The clonal strain of pituitary tumor cells GH₁2C₁ does not produce detectable amounts of prolactin (<5 ng/mg cell protein per 24 hr), although it does synthesize growth hormone. When GH₁2C₁ cells were grown in the presence of 5-bromodeoxyuridine (BrdU, 3 μg/ml), the cells did produce prolactin as determined by quantitative microcomplement fixation and incorporation of ³H-leucine into ³H-prolactin. BGH₁2C₁ and F₁BGH₁2C₁, two BrdU-resistant (r) substrains derived from GH₁2C₁ which grow in the presence of 30 μg/ml BrdU, also synthesized prolactin (100–500 ng/mg cell protein per 24 hr). Growth of BrdU^r strains was not dependent upon on the presence of the drug in the medium; however, the continued production of prolactin by F₁BGH₁2C₁ cells was dependent upon the presence of BrdU. Growth hormone production in both BrdU^s and BrdU^r strains was not affected by BrdU. Resistance of F₁BGH₁2C₁ cells to BrdU was not due to a defect in BrdU uptake. Thymidine inhibited the incorporation of ³H-BrdU into DNA in both sensitive and resistant strains, and also reduced BrdU-induced prolactin synthesis in F₁BGH₁2C₁. We postulate that induction of prolactin synthesis by BrdU in GH₁2C₁ and F₁BGH₁2C₁ cells is mediated by the incorporation of the drug into cellular DNA. Furthermore, the lack of measurable prolactin synthesis by the parent strain GH₁2C₁ is not due to deletion of the gene for prolactin, but is probably the result of regulatory mechanisms which do not permit expression of this gene.     

Effect of 5-bromodeoxyuridine on the appearance of cytoplasmic poly-A containing RNA

Cytoplasmic poly-A containing RNA, synthesized by cultured chick embryo cells, was examined during growth in 5-bromodeoxyuridine. The kinetics of ³H-adenosine incorporation into this species of RNA, when compared to the rest of the cytoplasmic RNA, and to control cells, indicates that the rate of synthesis of this RNA is slower in BrdU treated cells. An examination of the rate at which a steady state distribution of radioactivity, between the poly-A segment and non-poly-A portion of poly-A containing RNA is reached also indicated that this species is synthesized at a lower rate in BrdU treated cells.     

5-Azacytidine inhibits the induction of transient TK-deficient cells by 5-bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine.

This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.     

Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1.

Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity.     

Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.     

Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells.

The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.