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Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions
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Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells. 相似文献
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Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors. 总被引:42,自引:23,他引:19
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The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions. 相似文献
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Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1 总被引:6,自引:0,他引:6
By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements. 相似文献
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Identification of cis-acting elements that mediate the replication and maintenance of human papillomavirus type 16 genomes in Saccharomyces cerevisiae
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Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote, Saccharomyces cerevisiae. Specifically, papillomavirus DNA could substitute for the function of the autonomously replicating sequence (ARS) and centromere (CEN) elements that are normally both required for the stable replication of extrachromosomal DNAs in yeast. Furthermore, this form of replication in yeast was E1 independent. In this study, we map the elements in the human papillomavirus type 16 (HPV16) genome that can substitute for yeast ARS and CEN elements. A single element, termed rep, was identified that can substitute for ARS, and multiple elements, termed mtc, could substitute for CEN. The location of one of these mtc elements overlaps the location of rep, and this approximately 1,000-bp region of HPV16 was sufficient to support stable replication of a bacterial-yeast shuttle plasmid deleted of both ARS and CEN elements. 相似文献
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Several prophylactic human papillomavirus (HPV) vaccines have been developed based on virus-like particles (VLPs) made from viral L1 proteins. A substantial number of VLPs is necessary for biochemical characterization and diagnostic test development. To establish the optimum conditions for production and purification of HPV L1 in the yeast expression system we varied the amount and nature of the carbon source and evaluated HPV 16 L1 recovery by three purification methods. Maximally threefold more HPV 16 L1 was produced with a 4% carbon source than with a 2% carbon source. In addition, the productivity of HPV 16 L1 varied by 25% depending on the combination of glucose and galactose in the 4% carbon source. We introduced an ammonium sulfate precipitation step in place of the ultracentrifugation using a sucrose cushion routinely used for HPV L1 purification, and optimized the purification by cation-exchange chromatography. Overall L1 protein recovery using the ammonium sulfate precipitation method was 30%, the highest recovery achieved so far. The purified HPV 16 L1 protein successfully self-assembled into VLPs. Purification by ammonium sulfate precipitation was maximally 15 times greater than ultracentrifugation on a sucrose cushion. We anticipate that our procedures for production and purification will reduce the cost, time and labor involved in obtaining sufficient yields of VLPs. 相似文献
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The 23-kilodalton E1 phosphoprotein of bovine papillomavirus type 1 is nonessential for stable plasmid replication in murine C127 cells. 总被引:4,自引:2,他引:2
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The 23-kDa protein encoded by the 5' segment of the E1 open reading frame of bovine papillomavirus type 1 (BPV1) was previously ascribed a negative regulatory function for the replication of viral plasmid DNA. However, results from recent functional and biochemical studies do not readily support this genetic assignment. Therefore, we have reassessed the role of this protein in papillomavirus DNA replication by using a mutant of BPV1 which is unable to express this E1 protein. This mutant viral DNA was found to replicate extrachromosomally with stability and copy number per cell similar to those of wild-type plasmid DNA. Thus, the absence of expression of the 23-kDa E1 protein did not lead to deregulated viral plasmid replication. We conclude that the 23-kDa E1 protein is nonessential for stable plasmid replication. 相似文献
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A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state. 总被引:18,自引:12,他引:6
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A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids. 相似文献
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Transcriptional organization of bovine papillomavirus type 1 总被引:26,自引:17,他引:9
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Background
Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. 相似文献13.
Victoria Gavrias Alex Andrianopoulos Carlos J. Gimeno William E. Timberlake 《Molecular microbiology》1996,19(6):1255-1263
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Cell wall replication in Saccharomyces cerevisiae 总被引:5,自引:0,他引:5
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G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by noncanonical G-G base pairs. Genome-wide chromatin immunoprecipitation was used to determine the in?vivo binding sites of the multifunctional Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in?vitro. G4 motifs were a significant subset of the high-confidence Pif1-binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, whereas non-G4 Pif1-binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication-stressed Pif1-deficient cells, which was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication, and stimulate DNA breakage. These data suggest that G4 structures form in?vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage. 相似文献
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Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
Cook JC Joyce JG George HA Schultz LD Hurni WM Jansen KU Hepler RW Ip C Lowe RS Keller PM Lehman ED 《Protein expression and purification》1999,17(3):477-484
Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice. 相似文献
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To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. 相似文献