Publications
| Title | Abstract | Year Filter | PMID(sorted descending) Filter |
|---|
| analysis of a restriction endonuclease map for bovine papillomavirus type 2 dna. | a physical map was constructed for bovine papillomavirus type 2 dna by the use of restriction endonucleases. a comparison between the genomes of bovine papillomavirus types 1 and 2 in regard to location and number of cleavage sites of seven enzymes is also presented. this comparison revealed similarities between the two genomes. | 1981 | 6271276 |
| persistence of non-integrated viral dna in bovine cells transformed in vitro by bovine papillomavirus type 2. | 1981 | 6270574 | |
| unintegrated viral dna sequences in a hamster tumor induced by bovine papilloma virus. | a fibrosarcoma was induced in a hamster by bovine papilloma virus type 2 (bpv2). the content of bpv2 dna sequences was measured by dna-dna and crna-dna hybridizations. the tumor contained approximately 300 bpv2 genome equivalents per cell. southern blot hybridization indicated that the viral dna was in free form, the entire genome most likely being present. in situ hybridization with bpv2 crna showed that multiple genome copies were present in each cell. neither virus particles nor virus coat an ... | 1981 | 6270360 |
| detection and characterization of viral genomes and search for tumoral antigens in two hamster cell lines derived from tumors induced by bovine papillomavirus type 1. | 1981 | 6270023 | |
| molecular heterogeneity and lesion site specificity of cutaneous bovine papillomaviruses. | 1981 | 6267786 | |
| isolation and characterization of the major capsid protein of bovine papilloma virus type 1. | the major capsid protein of bovine papilloma virus type 1 (bpv-1) was isolated by gel filtration following disruption of purified virus particles with guanidine hydrochloride. the capsid protein, vp1, has a mol. wt. of about 53 500. amino acid composition studies of vp1 showed that it is a highly acidic protein containing almost twice the average number of acidic residues than basic residues. relatedness was observed between vp1 and the major capsid proteins of simian virus 40 (sv40) and polyoma ... | 1981 | 6267171 |
| bracken fern and papilloma virus in bovine alimentary cancer. | 1980 | 6266569 | |
| characterization of a papilloma virus from the european elk (eepv). | 1981 | 6266142 | |
| mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral dna sequences. | the viral dna sequences in mouse c127 cells transformed by bovine papillomavirus type 1 (bpv-1) virions, by full-length linear bpv-1 dna, or by a defined transforming subgenomic dna segment of bpv-1 were examined by reassociation kinetics and blot hybridization. in all cases, the transformed cells contained multiple copies of bpv-1 dna, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral dna molecules. in the transfo ... | 1981 | 6265905 |
| a new phenomenon (smog) associated with solid phase immune electron microscopy. | solid phase immune electron microscopy (spiem) has been used to study the interaction of virus and antibody both quickly and with economy of reagents. unexpectedly, the present study shows that when virus-coated grids are floated on drops of specific antibody the virus particles migrate to form complexes. this has been termed specific migration on the grid (smog) and it is suggested that it can be used both to assess the properties of an antiserum and to make virus more readily detectable. the m ... | 1980 | 6262338 |
| apparent lack of integration of bovine papillomavirus dna in virus-induced equine and bovine tumor cells and virus-transformed mouse cells. | 1981 | 6258289 | |
| identification of papillomaviruses in butchers' warts. | we have studied the papillomaviruses found in the hand warts of 60 butchers, most of them from 2 distant slaughterhouses. warts differing in morphology and location were studied separately. the viruses were identified by molecular hybridization, restriction enzyme analysis and immunofluorescence. four known human papillomaviruses (hpv-1, hpv-2, hpv-3, hpv-4) were detected and one hitherto unknown papillomavirus was identified in 9 butchers. the dna of the latter virus did not anneal with any of ... | 1981 | 6257792 |
| equine connective tissue tumors contain unintegrated bovine papilloma virus dna. | bovine papilloma virus (bpv) appears to be the etiological agent of common equine connective tissue tumors. we investigated the physical state of the viral dna within such tumors and found no indication for integration into the host genome. the bpv genomes were present as free circular episomes. two equine sarcoids were shown to contain multiple copies of free circular bpv type 1 (bpv-1) dna. when the tumors were digested with several single-cut restriction enzymes, there were only form iii bpv- ... | 1980 | 6252350 |
| in vitro tumorigenic transformation by a defined sub-genomic fragment of bovine papilloma virus dna. | 1980 | 6251381 | |
| a quantitative in vitro focus assay for bovine papilloma virus. | 1980 | 6247821 | |
| synthesis and glycosylation of the common alpha subunit of human glycoprotein hormones in mouse cells. | the synthesis and the post-translational modification of the alpha subunit of human glycoprotein hormones have been studied in a mouse cell. a full-length cdna coding for the human alpha subunit has been expressed in mouse c127 cells under the control of mouse metallothionein regulatory sequences, using a bovine papilloma virus vector. stable clones secreting the alpha subunit into the medium have been obtained. two intracellular forms of 22,000 da and 21,000 da have been detected. pulse-chase e ... | 1984 | 6208554 |
| selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites. | we have constructed a recombinant ( pmop ) which is based on the bacterial plasmid pml2 and contains bovine papilloma virus type 1 (bpv 1) sequences linked to an artificial mouse dihydrofolate reductase (dhfr) transcription unit. this unit consists of the sv40 early gene promoter, a complete dhfr coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. intact pmop or a fragment thereof devoid of pml2 sequences will transform mouse cells to methotrexate resistance. the ... | 1984 | 6202514 |
| current concepts in the management of laryngeal papillomatosis. | a wide variety of methods have been described for the treatment of laryngeal papillomatosis. this attests to the difficulties encountered in controlling this often refractory disease. recent trends have been directed toward immunotherapy and improved techniques of endoscopic excision. the various treatment modalities are reviewed to provide the reader with updated information and a perspective of the current management of this perplexing disease. | 1984 | 6200454 |
| interferon induces morphologic reversion with elimination of extrachromosomal viral genomes in bovine papillomavirus-transformed mouse cells. | the effect of mouse l-cell interferon on bovine papillomavirus type 1 (bpv-1) transformation of murine cells was examined. mouse interferon reduced the level of bpv-1-induced transformation of mouse c127 cells by 95%. long-term treatment of established bpv-1-transformed mouse cell clones with mouse l-cell interferon led to a decrease in the average number of the plasmid viral genomes present in these cells to 1/3 to 1/8. although revertant lines could not be isolated from these lines in the abse ... | 1982 | 6185955 |
| bovine papilloma virus transcription: polyadenylated rna species and assessment of the direction of transcription. | the bovine papilloma virus type 1 (bpv-1)-specific rna species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. in each case two major species were present (1.1 and 1.3 kilobases [kb]). also two species of 1.6 and 1.8 kb appearing in variable amounts were found. only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb rna species. in this tissue, however, the 1.6-kb spe ... | 1982 | 6180175 |
| transcriptional organization of bovine papillomavirus type 1. | multiple bovine papillomavirus type 1 (bpv-1)-specific polyadenylated rna species in a bpv-1-infected bovine fibropapilloma were identified and mapped. all of the rna species were transcribed from the same dna strand of the bpv-1 genome. five rna species previously identified in bpv-1-transformed mouse cells were also present in the bovine fibropapilloma. these five species measured 1,050, 1,150, 1,700, 3,800, and 4,050 bases, mapped within the 69% transforming segment of the bpv-1 genome, and s ... | 1983 | 6137574 |
| comparative analysis of human and bovine papillomaviruses. | a method is presented for the analysis and comparison of nucleic acid and protein sequences utilizing all identity blocks (the term "identity block" refers to a set of consecutive matches between two sequences) above a prescribed length. moreover, such identity blocks are determined for various groupings of amino acids according to chemical, functional, charge, and hydrophobic classifications. alignment maps based on these classifications and containing all statistically significant identity blo ... | 1984 | 6100988 |
| bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector. | a novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. the foreign deoxyribonucleic acid (dna) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. we have constructed a dna hybrid molecule, bpv69t-ri1, containing the transforming region of bovine papilloma virus dna and the rat preproinsulin ... | 1981 | 6100967 |
| expression of hepatitis b virus surface and e antigen genes cloned in bovine papillomavirus vectors. | sequences of the hepatitis b virus (hbv) genome coding for the surface antigen (hbsag), but lacking the regulatory (pres) sequences, were cloned into a bovine papillomavirus (bpv) vector consisting of the transforming 69% of the bpv genome (bpv 69t), and transformed into mouse c127 cells. clones carrying hbv and bpv sequences in the same transcriptional orientation did not produce immunologically active hbsag, while those with the opposite orientation produced and secreted hbsag. rna species of ... | 1984 | 6099321 |
| papillomas of the teats and udder of cattle and their causal viruses. | an abattoir survey was carried out on 1657 cattle to determine the incidence and parameters of teat and udder papillomas and their causal viruses. recent research has characterised six different bovine papillomaviruses of which three have been found to be particularly associated with teat tumours. of the population studied 37.3 per cent had teat warts and 86.2 per cent of these had multiple infections; 28.4 per cent had papillomas attributed to bovine papillomavirus 1, 88.5 per cent to bovine pa ... | 1984 | 6098066 |
| identification of a second transforming region in bovine papillomavirus dna. | bovine papillomavirus type 1 (bpv-1) has been used as a model for studying papillomavirus genetics because bpv-1 virions or bpv-1 genomic viral dna efficiently induce morphologic transformation of certain cultured cells. previous studies of bpv-1-induced transformation have found that a cloned 5.4-kilobase (kb) fragment (69t) of the genome is transforming and that a 2.3-kb segment from the 3' end of this fragment is also transforming if activated by a retroviral regulatory element (the long term ... | 1984 | 6096867 |
| localization and analysis of bovine papillomavirus type 1 transforming functions. | bovine papillomavirus type 1 (bpv-1) or cloned bpv-1 dna can transform susceptible rodent cells, and the viral dna remains as a stable extrachromosomal plasmid in the transformed cells. the transforming region of the bpv-1 genome has previously been localized to a specific fragment comprising 69% of the genome, which also contains the elements sufficient for extrachromosomal plasmid maintenance. to define more precisely the viral dna sequences which are involved in cellular transformation, we ha ... | 1984 | 6092667 |
| expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera. | recombinant plasmids containing the genomes of both bovine papillomavirus type i and minute virus of mice (mvm) were constructed and used to transform mouse c127 cells. transformed lines that express mvm gene products with high efficiency were isolated and characterized. these transformants synthesize large amounts of mvm structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. these lines were, however, genetically unstable; ... | 1984 | 6092662 |
| origin of replication in episomal bovine papilloma virus type 1 dna isolated from transformed cells. | the origin of replication of bovine papilloma virus type 1 (bpv-1) has been determined by isolating replicative intermediates (ri) of bpv-transformed hamster embryo fibroblasts (hef-bpv). these ri were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'cairns'-type ri molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. the position of this eye ... | 1984 | 6092063 |
| the mouse mammary tumor virus model in studies of glucocorticoid regulation. | 1984 | 6091192 | |
| a common function for polyoma virus large-t and papillomavirus e1 proteins? | nucleotide sequencing has revealed a common genetic organization for three papillomaviruses: bpv-1 (bovine papillomavirus type 1), hpv-1 (human papillomavirus type 1a) and hpv-6 (human papillomavirus type 6b). several open reading frames, corresponding to as yet uncharacterized proteins, were observed in these genomes in the region that is required for oncogenic transformation by bpv-1 and for plasmidial maintenance of its genome. the longest of these frames, e1, is also the most conserved betwe ... | 1984 | 6090931 |
| efficient production of hepatitis b surface antigen using a bovine papilloma virus-metallothionein vector. | we have developed a highly efficient system for producing hepatitis b virus surface antigen in cultured mammalian cells. this system utilizes a recombinant bovine papilloma virus in which the hepatitis surface antigen coding sequences are inserted into the 5' untranslated region of the mouse metallothionein-i gene. mouse fibroblasts stably transformed with this molecule produce surface antigen at levels as high as 10 mg/l/24 h and can be maintained in continuous culture for up to 85 days. | 1984 | 6090567 |
| constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector. | mouse c127i cells were transformed with a chimeric plasmid consisting of bovine papillomavirus and human interferon (huifn) gene (either ifn-gamma or ifn-alpha 5) placed under the control of simian virus 40 early promoter. the transformed cells retained 30-50 copies each of the hybrid plasmid extrachromosomally and secreted a high level of huifns constitutively up to 3-4 x 10(5) international units/ml. the secreted huifn-gamma had no detectable activity on mouse cells, whereas huifn-alpha 5 was ... | 1984 | 6089176 |
| genome of an avian papillomavirus. | a papillomavirus which we designate fpv was isolated from chaffinches (fringilla coelebs). a physical map of the fpv genome was constructed, and selected regions of this genome were studied by nucleotide sequence analysis. the results make it possible to align the fpv genome with the genome of bovine papillomavirus type 1 and to show, moreover, that avian and mammalian papillomaviruses have a similar genome organization. | 1984 | 6088809 |
| a novel bovine papillomavirus (bpv-6) causing true epithelial papillomas of the mammary gland skin: a member of a proposed new bpv subgroup. | a papillomavirus has been isolated from frond epithelial papillomas of the bovine udder. it is clearly distinguishable from all other bovine papillomaviruses (bpvs) based on dna sequence homology and antigenic properties and is thus characterised as a new entity, designated bpv-6. bpv-6 does not possess the interspecific papillomavirus antigen, its genomic dna (7.2 kb) is smaller than, and does not show any sequence homology to bpv-1, bpv-2, or bpv-5, whereas it is approximately the same length ... | 1984 | 6087545 |
| alimentary fibropapilloma in cattle: a spontaneous tumor, nonpermissive for papillomavirus replication. | fibropapillomatosis of the upper alimentary canal of cattle is described. the tumors, found in the esophagus, esophageal groove, and rumen, showed involvement of the subepithelial fibroblasts as well as of the squamous epithelial layer. although the fibropapilloma cells harbored multiple episomal copies of the genome of bovine papillomavirus type 2 (bpv-2) easily detected by hybridization techniques, no mature virus could be isolated from these lesions or seen by electron microscopy, and no vira ... | 1984 | 6087008 |
| the minute virus of mice p39 transcription unit can encode both capsid proteins. | the right-hand 80% of the genome of minute virus of mice (mvm) was cloned into the bovine papillomavirus type i shuttle vector and used to transfect mouse c127 cells. transformed lines were isolated that efficiently produce both authentic mvm capsid proteins at a ratio similar to that seen in a normal viral infection, and these proteins assemble into intact empty virions. the only transcription of mvm sequences detected in these lines was representative of the viral p39 transcription unit, which ... | 1986 | 3951017 |
| demonstration that a chemically synthesized bpv1 oncoprotein and its c-terminal domain function to induce cellular dna synthesis. | bovine papillomavirus type 1 contains the smallest known oncogene (orf e5), encoding a hydrophobic 44 amino acid protein. to study the biochemical functions of the e5 oncoprotein, we have chemically synthesized it and several deletion mutant peptides. we demonstrate induction of cellular dna synthesis in growth-arrested cells by microinjection of e5 oncoprotein. this activity can be broken down into two functionally distinguishable domains. remarkably, the first domain, which alone is sufficient ... | 1987 | 3677173 |
| cell cycle-dependent expression of a stable episomal human histone gene in a mouse cell. | we have constructed a recombinant plasmid that includes a cell cycle-dependent human h4 histone gene with 650 base pairs of 5' and 900 base pairs of 3' flanking sequences and the 69% transforming fragment of bovine papilloma virus. when transfected into c127 mouse cells, this plasmid is maintained as a stable episome with approximately 20 copies per cell. micrococcal nuclease digestion indicates that the episomal human histone gene is packaged as chromatin. the human h4 histone transcript is ini ... | 1986 | 3458197 |
| human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. | stably transfected cell lines containing the normal human growth hormone (hgh-n) and human growth hormone-variant (hgh-v) genes have been established in order to study the expression of these two highly homologous genes. each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse c127 cells. the transfected cells containing the hgh-n gene secrete two hgh ... | 1988 | 3392209 |
| expression and processing of a recombinant human macrophage colony-stimulating factor in mouse cells. | a human macrophage colony-stimulating factor encoded by a 4-kilobase cdna was expressed with bovine papillomavirus vectors in mouse cells. pulse-chase analyses revealed that the 62-kilodalton (kda) translation product was glycosylated, cleaved, and efficiently secreted within 1 h of synthesis. the secreted product contained both n-linked and o-linked oligosaccharide chains. macrophage colony-stimulating factor was present extracellularly as an 80-kda homodimer and as a multimeric species of grea ... | 1988 | 3264878 |
| expression of human epidermal growth factor precursor cdna in transfected mouse nih 3t3 cells. | stable cell lines expressing the human epidermal growth factor (egf) precursor have been prepared by transfection of mouse nih 3t3 cells with a bovine papillomavirus-based vector in which the human kidney egf precursor cdna has been placed under the control of the inducible mouse metallothionein i promoter. synthesis of the egf precursor can be induced by culturing the cells in 5 mm butyric acid or 100 microm zncl2. the egf precursor synthesized by these cells appears to be membrane associated; ... | 1988 | 3257563 |
| contingent replication assay (cra) procedure for rapid isolation of enhancers. | a rapid procedure for the isolation of functional enhancer sequences consists of the construction of a shotgun dna library in sv40-based plasmid shuttle vectors which depend on an enhancer for replication, the replication in monkey (cvi) cells of those vectors into which an enhancer sequence was inserted, the selective cleavage of unreplicated vectors by dpni and the recovery of the replicated vectors by transfection into escherichia coli. we describe conditions for the fusion of protoplasts to ... | 1987 | 3040530 |
| levels of bovine papillomavirus rna and protein expression correlate with variations in the tumorigenic phenotype of hamster cells. | three independent cell lines were established from primary cultures of lsh hamster embryo cells infected with bovine papillomavirus type 1 (bpv-1). although these cell lines differed in their in vitro saturation densities, none was capable of colony formation in soft agar. interestingly, two cell lines (bpv-he1 and bpv-he3) were tumorigenic in nude mice, syngeneic hamsters, and allogeneic hamsters, whereas bpv-he2 was not. all three cell lines contained similar numbers of the bpv-1 genome (appro ... | 1987 | 3039179 |
| identification of proteins encoded by the l1 and l2 open reading frames of human papillomavirus 1a. | the human papillomavirus 1 (hpv-1) virion is composed of two virally encoded proteins: a 57,000-molecular-weight polypeptide (57k polypeptide), which is the product of the l1 open reading frame (orf), and a 78k polypeptide, which is derived from the l2 orf. the 57k (l1) product, which represents the major structural component, appears to be disulfide cross-linked in virus particles. the 78k (l2) protein is a minor component of the virion and does not appear to be disulfide linked either to the l ... | 1987 | 3039170 |
| the nucleotide sequence and genome organization of bovine papillomavirus type 4. | the nucleotide sequence of bovine papillomavirus type 4 (bpv-4) was determined. the viral genome is 7261 base pairs long. several overlapping open reading frames (orfs) have been identified both on the basis of amino acid comparison with other papillomaviruses and on their transcriptional pattern. eight early orfs (e1 to 8) were recognized, coding for dna replication and cell transformation functions, and three late orfs (l1 to 3), coding for structural proteins. like the e5 orf of human papillo ... | 1987 | 3039043 |
| a promoter with an internal regulatory domain is part of the origin of replication in bpv-1. | extrachromosomal elements that are stably maintained at a constant copy number through cell doublings are a good model system for the study of the regulation of dna replication in higher eukaryotes. previous studies have defined both cis and trans functions required for the regulated plasmid replication of the bovine papilloma virus in stably transformed cells. here, a sequence known to be a cis-dominant element of the replication origin of the plasmid is shown to contain a promoter for transcri ... | 1987 | 3037693 |
| nucleotides in the polyomavirus enhancer that control viral transcription and dna replication. | the polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. we introduced multiple mutations in the enhancer which reduced transcription and dna replication. polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. revertants of the viral mutants were isolated and characterized. reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 ... | 1987 | 3037332 |
| nucleosomal organization of a bpv minichromosome containing a human h4 histone gene. | to address the relationship between chromatin structure and histone gene expression, the nucleosomal organization of a cell cycle-dependent human h4 histone gene in a bovine papilloma virus (bpv) minichromosome was examined. the nucleosome repeat length of the human h4 histone gene, maintained as a stable episome in a c127 mouse cell line designated i-8, was compared with that of the chromosomal copy of the h4 gene in human (hela) cells. in both cell lines, the h4 histone gene is predominantly e ... | 1987 | 3037307 |
| enhancers and trans-acting e2 transcriptional factors of papillomaviruses. | the upstream regulatory regions of human papillomavirus (hpv) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (ec 2.3.1.28) (cat). enhancer activity, reflected by cat gene expression, was detected in all of the upstream regulatory regions tested only whe ... | 1987 | 3037119 |
| human papillomavirus types 6 and 11 mrnas from genital condylomata acuminata. | we have identified and mapped a number of rna species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic r-loop technique. each of the early (e)- and late (l)-region open reading frames (orfs) deduced from the dna sequences was represented in one or more transcripts. in addition, rna species that could encode the modulator of dna replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomaviru ... | 1987 | 3037118 |
| expression of human papillomavirus types 6b and 16 l1 open reading frames in escherichia coli: detection of a 56,000-dalton polypeptide containing genus-specific (common) antigens. | the human papillomavirus (hpv) genome contains two large open reading frames (orfs), designated l1 and l2. to characterize the antigenic properties of the l1 orf-encoded proteins, we cloned the l1 orfs of hpv6b and hpv16 in plasmids, and these were expressed in escherichia coli. first, the hpv6b dna, representing 85.2% of the l1 orf, was cloned in puc19 and expressed in e. coli jm83 and rb791 as a 160,000-molecular-weight (160k) fusion protein with e. coli beta-galactosidase (6bl1/beta-gal). sec ... | 1987 | 3037103 |
| purification of papillomavirus structural polypeptides from papillomas by immunoaffinity chromatography. | a broadly cross-reactive monoclonal antibody directed against papillomavirus, coupled to immunoaffinity columns, was used to isolate bovine papillomavirus type 1 (bpv-1) and human papillomavirus type 1 (hpv-1) structural polypeptides from homogenates of productively infected cells. one of the polypeptides isolated from bovine fibropapillomas appeared to be the bpv-1 major capsid protein since it had a mol. wt. of 54k and was reactive by western blots with papillomavirus genus- and bpv-1 type-spe ... | 1987 | 3037012 |
| measurement of cell-mediated immunity against bovine papilloma virus by lymphoproliferative reactions. | to study cellular immunity towards bovine papillomavirus (bpv), calves were infected intradermally with bpv-1, and the cellular immune response was measured by the lymphocyte proliferation assay. peripheral blood lymphocytes were obtained which in control experiments were highly reactive towards mitogen stimulation. different batches of bpv-1 were prepared by the use of density gradients. in the first set of experiments, a nonspecific mitogenic effect of the virus preparation was observed. this ... | 1987 | 3036693 |
| identification of the hpv-16 e6 protein from transformed mouse cells and human cervical carcinoma cell lines. | human cervical carcinoma cell lines that harbor human papillomavirus (hpv) have been reported to retain selectively and express hpv sequences which could encode viral e6 and e7 proteins. the potential importance of hpv e6 to tumors is suggested further by the observation that bovine papillomavirus (bpv) e6 can induce morphologic transformation of mouse cells in vitro. to identify hpv e6 protein, a polypeptide encoded by hpv-16 e6 was produced in a bacterial expression vector and used to raise an ... | 1987 | 3036495 |
| differential promoter utilization by the bovine papillomavirus in transformed cells and productively infected wart tissues. | expression of the 'late' genes of bovine papillomavirus type 1 (bpv-1) occurs only in the differentiated keratinocytes of the productively infected fibropapilloma. a detailed analysis of viral transcription in the fibropapilloma was performed and compared to bpv-1 specific transcription in transformed c127 cells. a cdna library was constructed from bovine fibropapilloma mrna using the method of okayama and berg. analysis of full length cdnas showed that the majority of viral transcripts in the f ... | 1987 | 3036488 |
| a transcriptional repressor encoded by bpv-1 shares a common carboxy-terminal domain with the e2 transactivator. | a negative-acting transcriptional regulatory factor encoded by bovine papillomavirus type 1 (bpv-1) was identified. this factor inhibits bpv-1-mediated transformation of mouse c127 cells; inhibition is bpv-1-specific and occurs only when the bpv-1 transforming genes are regulated by authentic transcriptional control elements. plasmids expressing the inhibition function also repress e2 transactivation of the bpv-1 e2-dependent enhancer, and this repression is mediated by the same cis-acting eleme ... | 1987 | 3036366 |
| bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout s phase of the cell cycle. | bovine papilloma virus (bpv) replicates as a multicopy nuclear plasmid in mouse fibroblasts. using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of bpv occurs throughout s phase of the cell cycle and that replication is confined to s phase. after one round of chromosomal dna replication, almost one quarter of bpv plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. while multiple forms of bpv ... | 1987 | 3036365 |
| [genetic transformation of somatic cells. xi. the autonomic replication of the cloned dna of the bovine papilloma virus in nih/3t3 mouse fibroblasts and the changes in the growth characteristics of the transformed cells]. | mouse fibroblasts nih 3t3 were transfected with the plasmid pbpv (142-6) containing full genome of bovine papilloma virus 1, and focuses of morphological transformation were selected 2-3 weeks later. dna molecules, containing bpv-1 sequences, were isolated from extrachromosomal fraction of transformed clones suggesting stable autonomous replication of bpv in 3t3 nih cells. in some rescued plasmids deletions spanning e6, 7 genes of bpv were found. it is suggested that these genes are not essentia ... | 1987 | 3035762 |
| assessment of precancerous lesions of the uterine cervix for evidence of human papillomavirus infection: a histological and immunohistochemical study. | cervical biopsies obtained by colposcopic direction from 358 women were histologically examined for squamous dysplasia (cervical intra-epithelial neoplasia; cin) and human papillomavirus (hpv) infection. of the 358 biopsies, 136 were stained by an immunoperoxidase method using an antiserum against genus-specific (common) antigen of bovine papillomavirus. hpv antigens were detected in 40% of biopsies showing definite histological evidence of hpv effect, and in 7.9% and 2.6% of those with possible ... | 1987 | 3035470 |
| methods for analyzing bovine papilloma virus-based calmodulin expression vectors. | 1987 | 3035329 | |
| bovine papillomavirus transcriptional regulation: localization of the e2-responsive elements of the long control region. | the long control region (lcr) of the bovine papillomavirus type 1 genome can function as a conditional transcriptional enhancer which can be specifically trans-activated by the viral e2 gene product. to precisely map the target(s) of this trans-activation, bal 31 exonuclease was used to generate two overlapping series of deleted dna segments through the lcr. these fragments were assayed for their ability to activate transcription from the enhancer-deleted simian virus 40 early promoter of pa10ca ... | 1987 | 3035214 |
| organization and expression of the transforming region from the european elk papillomavirus (eepv). | the nucleotide sequence of the early (transforming) region from the european elk papillomavirus (eepv) double-stranded dna has been determined together with flanking regions. the established sequence, which is 5732 bp long, shows that the genome of eepv is closely related to the previously sequenced bovine papillomavirus type 1 (bpv-1) and deer papillomavirus (dpv) genomes. seven open reading frames (orfs), designated e1-e7, were identified in similar positions as in the bpv-1 genome. the e1 and ... | 1986 | 3034730 |
| characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells. | mouse c127i cells were transformed with a chimeric plasmid consisting of bovine papillomavirus dna and human granulocyte-colony-stimulating factor (g-csf) cdna placed under the control of the sv40 early promoter. the transformed cells secreted constitutively a high level of human g-csf, 10-20 micrograms/ml in a low-serum medium. the secreted g-csf has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. the purified recombinant g-cs ... | 1987 | 3034599 |
| trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral e2 gene. | the approximately 1000 nucleotide long upstream regulatory region (urr) of bovine papilloma virus-1 (bpv-1) contains a cis element which responds to trans-activation by a diffusible factor encoded in the viral e2 open reading frame (orf). a series of urr dna fragments have been linked to two heterologous genes, bacterial chloramphenicol acetyl transferase (cat) or herpes simplex virus-1 thymidine kinase (tk), and tested in transient transfection assays for transcription initiating at the authent ... | 1987 | 3034572 |
| dna-binding activity of papillomavirus proteins. | we demonstrate dna binding by papillomavirus (pv) open reading frame (orf) proteins that correspond to the early transforming and trans-activating (e6 and e2) and late structural regions (l2 and l1) from bovine pv type 1 and human pv types 6b and 16. all pv proteins were synthesized in escherichia coli and had a common 13-amino-acid leader sequence from the expression vector pra10. antibodies have been generated in rabbits against these pv proteins. the pv orf proteins bind double-stranded dna, ... | 1987 | 3033292 |
| transcriptional trans-activation by the human papillomavirus type 16 e2 gene product. | we identified a conditional transcriptional enhancer in the long control region (lcr) of human papillomavirus type 16 (hpv-16). this conditional enhancer requires activation in trans by a product of the viral early-region open reading frames (orfs). primer extension analysis of chloramphenicol acetyltransferase rna isolated from transiently transfected cv-1 cells demonstrated that trans-activation of the hpv-16 lcr enhancer operated at the transcriptional level. mutational analysis of the early ... | 1987 | 3033289 |
| human papillomavirus type 17 transcripts expressed in skin carcinoma tissue of a patient with epidermodysplasia verruciformis. | certain types of human papillomavirus (hpv) are involved in skin carcinogenesis in epidermodysplasia verruciformis. however, no gene or gene product of hpv associated with skin carcinogenesis has yet been identified. here, we report hpv-17 transcripts expressed in skin carcinoma tissue of an epidermodysplasia verruciformis patient infected with hpv-17. further, we show that one of these transcripts was localized to a portion of the genome which contains the 3' open reading frames of the early re ... | 1987 | 3032809 |
| episomal maintenance of a bovine papilloma virus vector in transgenic mice. | we have used a bovine papillomavirus-based vector to generate transgenic mice. transgenic mice result from either pronuclear or cytoplasmic injections of the vector into fertilized eggs. of 30 mice generated by microinjection, 27 (90%) contained the vector in its episomal form, at less than one copy per cell. this represents a highly efficient means of gene transfer in which the transgene is in a controlled genetic environment. | 1987 | 3031486 |
| the e2 "gene" of bovine papillomavirus encodes an enhancer-binding protein. | the e2 early open reading frame (presumably gene) of bovine papillomavirus-1 was fused in frame with the collagen-beta-galactosidase-encoding region of the vector pjg200 and was expressed in and partially purified from escherichia coli. the hybrid protein specifically bound to the enhancer region of bovine papillomavirus at several sites. dnase i-cleavage protection analysis of one such site revealed the protected sequence. a comparison of the protected sequence with the remainder of the dna seq ... | 1987 | 3029771 |
| comparison of peroxidase-antiperoxidase and avidin-biotin complex methods for the detection of papillomavirus in histological sections of the cervix uteri. | a study was undertaken to determine the relative sensitivities of the peroxidase-antiperoxidase (pap) and avidin-biotin complex (abc) methods for the detection of human papillomavirus (hpv) antigens in acetic acid-ethanol fixed paraffin-embedded cervical tissue. tissue sections prepared from 14 women suspected to have hpv infections with either atypia or dysplasia were stained immunohistochemically using an antiserum against genus-specific (common) antigen of bovine papillomavirus. detection of ... | 1986 | 3029653 |
| mutational analysis of open reading frame e4 of bovine papillomavirus type 1. | open reading frame (orf) e4 is a 353-base-pair orf of bovine papillomavirus type 1. to determine the biological activities of this orf in mouse c127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of orf e4 proteins while leaving the amino acid sequence encoded by the overlapping orf e2 unchanged. neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate ... | 1987 | 3029420 |
| stability of a bacterial gene in a bovine papillomavirus-based shuttle vector maintained extrachromosomally in mammalian cells. | in order to analyse the stability of cloned genes in a viral vector we have constructed a shuttle vector based on bovine papillomavirus and the escherichia coli gene lacz. propagation of this vector in mouse c127 cells and analysis of vector sequences in bacteria produced no detectable mutations in the lacz gene in over 6137 clones analysed. this is 100-fold less than the mutation frequency observed when the same and similar target genes are replicated in monkey cos cells using a simian virus 40 ... | 1987 | 3027246 |
| duplication of a viral enhancer sequence improves the stability of a vector based on bpv-1 dna. | various recombinant constructions involving bovine papillomavirus type 1 (bpv-1) dna and bacterial plasmids have been tested for their ability to transform mouse c127 cells and replicate as intact extrachromosomal monomeric structures. when bpv-1 dna was linked to pbr328, pat153 or derivatives of these plasmids lacking the 344 bp hindiii-bamhi fragment or another small segment, the resulting vectors replicated in c127 cells as high molecular weight structures and, in some cases, deleted extrachr ... | 1986 | 3026114 |
| bovine papillomavirus e2 trans-activating gene product binds to specific sites in papillomavirus dna. | enhancers are cis-acting elements that activate transcription in higher eukaryotes independently of their position or orientation relative to the promoter that they activate. the mechanisms by which enhancers activate transcription are poorly understood, in part because, with the exception of the glucocorticoid receptor, the proteins that directly interact with enhancers have not been purified, nor have the genes encoding them been cloned. the upstream regulatory region (urr) that immediately pr ... | 1987 | 3025749 |
| the noncoding region of hpv-6vc contains two distinct transcriptional enhancing elements. | hpv-6vc subgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (cat) and assayed for the presence of transcriptional enhancing elements. a transcriptional enhancing element was detected in the noncoding region (ncr) of the hpv-6vc viral genome when the cat assays were performed in viral transformed human kidney cell lines (293 and 324k), in human cervical carcinoma cell lines (hela and siha), and in bovine papillomavirus type 1 (bpv- ... | 1986 | 3024399 |
| glucocorticoid inhibition of transcription from episomal proopiomelanocortin gene promoter. | glucocorticoid hormones alter transcription of specific genes. glucocorticoid-stimulated genes have been especially useful in unraveling molecular events responsible for positive gene regulation in mammals. the gene encoding proopiomelanocortin (pomc), which is under feedback inhibition by glucocorticoids, provides a model system to study negative gene regulation. using an episomal bovine papilloma virus vector, we now demonstrate that a 769-base-pair fragment containing the rat pomc promoter is ... | 1986 | 3024155 |
| efficient transcription of a caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally. | a divergently transcribed pair of caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. the hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. in a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each ce ... | 1986 | 3023964 |
| complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene. | we identified a bovine papilloma virus function encoded by the e6/e7 gene, which is required for both bpv high-copy-number replication and maintenance of transformation of cultured cells. a cdna copy of this gene was isolated and expressed from a retrovirus vector. we found that complete complementation of a bpv low-copy-number mutant (dl576) by the cdna encoding the e6/e7 gene was temporally dependent. when both the e6/e7 cdna and dl576 were introduced together into cells, wild-type replication ... | 1986 | 3022134 |
| a bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state. | 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 ... | 1986 | 3022005 |
| bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions. | we examined bovine papillomavirus type 1 (bpv-1) dnas mutated in the e2 open reading frame (orf) to determine their ability (i) to transform c127 cells and (ii) to remain extrachromosomal in transfected cells. results obtained with deletion mutants and insertion mutants containing a linker with translational termination codons in all possible reading frames indicated that an e2 orf gene product(s) is necessary for efficient transformation, as well as viral plasmid replication and maintenance in ... | 1986 | 3021996 |
| differential regulation of papilloma virus early gene expression in transformed fibroblasts and carcinoma cell lines. | treatment of bovine papilloma virus (bpv) 1-transformed mouse fibroblasts with cycloheximide led to a 10-fold increase in the amount of viral transcripts, after as little as 1 h of protein synthesis inhibition. northern blots revealed no qualitative changes in the rna pattern. nuclear run-on experiments showed about a 7-fold increase in specific transcriptional activity after cycloheximide treatment. the half-life of bpv1 mrna was twice as long as in untreated controls. these results indicate th ... | 1986 | 3019673 |
| characterization of fischer rat embryo (cref) cells transformed by bovine papillomavirus type 1. | transformation of an established fischer rat embryo (cref) cell line by bovine papillomavirus type 1 (bpv-1), in contrast to transformation by type 5 adenovirus or the t24 (ha-ras) oncogene, resulted in transformants which did not exhibit a major increase in saturation density or a decrease in 125i-epidermal growth factor binding. bpv-1-transformed cref clones did, however, grow in agar suspension culture and were tumorigenic in both nude mice and fischer rats. the majority of transformed clones ... | 1986 | 3019001 |
| bovine papillomavirus genome elicits skin tumours in transgenic mice. | transmission of the bovine papillomavirus-1 (bpv-1) genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue-specific phenotype analogous to that observed in natural bpv-1 infection of cattle. oncogenesis is slow, with tumours first arising at 8-9 months of age, usually in areas prone to wounding. extrachromosomal bpv-1 dna is detected in all tumours, whereas normal tissues show only integrated dna. | 1986 | 3018579 |
| enhancer-dependent expression of the rat preproinsulin gene in bovine papillomavirus type 1 vectors. | the effect of position in a bovine papillomavirus type 1 (bpv-1) vector on foreign gene expression was assessed with the rat preproinsulin (ri1) gene. the ri1 gene was inserted at each of the bpv-1/pml2d junctions in either transcriptional orientation in derivatives of the pdbpv-1(142-6) vector which consists of the bamhi linear genome of bpv-1 dna cloned into pml2d. transformed lines of c127 cells were established and assayed for ri1 gene expression. cells containing the ri1 gene at the 3' end ... | 1985 | 3018523 |
| type 1 human t-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector. | in an attempt to express the small (transmembrane) envelope protein p21e of type 1 human t-cell leukemia (lymphotrophic) virus (htlv-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pmbe-1) in a bovine papillomavirus-derived mammalian expression vector. mouse c127 cells transfected with the pmbe-1 plasmid expressed the introduced htlv-1 viral gene(s) as demonstrated by northern blot and indirect immunofluorescence with natural human antisera. the transf ... | 1985 | 3018517 |
| the bovine papillomavirus distal "enhancer" is not cis essential for transformation or for plasmid maintenance. | we constructed a mutant of bovine papillomavirus type 1 (bpv-1) dna that lacked a transcriptional enhancer located 3' to the polyadenylation site of the early viral rnas expressed in transformed cells. this mutant dna, when separated from the procaryotic sequences, transforms mouse cells with an efficiency comparable to that of the full bpv-1 genome, and it exists as a stable multicopy plasmid in transformed cells. the bpv-1 distal enhancer suppresses the effects of a cis-inhibitory element in p ... | 1985 | 3018516 |
| the expression of human papillomavirus type 18 e6 protein in bacteria and the production of anti-e6 antibodies. | human papillomavirus type 18 (hpv-18) has recently been closely linked with human malignant cervical lesions. the early region of the genome of the related bovine papillomavirus (bpv) has been shown to be important for the production of the transformed phenotype. bpv e6 has been shown to be a transforming protein. we report the primary structure of the hpv-18 e6 open reading frame and its predicted amino acid sequence. both e6 protein and e6-beta-galactosidase fusion protein were synthesized in ... | 1986 | 3018129 |
| cleavage of vimentin in dense cell cultures. inhibition upon transformation by type 5 adenovirus. | the analysis on two-dimensional isoelectric focusing and sds polyacrylamide gels (2d gels) of the triton x-100 and high salt-insoluble fraction of fibroblast cell lines, certain epithelial cell lines and granulosa cells revealed various amounts of a vimetin cleavage product, with a more basic pi and with a mw (1,500-2,000) lower than that of intact vimentin. this cleavage product of vimentin which constituted as much as 30% of the total vimentin in an established rat embryo fibroblast cell line ... | 1986 | 3017738 |
| repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor. | cells transformed with bovine papilloma virus type 1 mutants in the e6 or e6/7 genes are resistant to high-copy-number amplification of wild-type dna after supertransfection. transient and stable replication assays demonstrate this effect. if the supertransfected dna has a mutation in a newly defined gene (m), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input dna. in contrast, the resident plasmid does not participate in amplifica ... | 1986 | 3017567 |
| negative control of dna replication in composite sv40-bovine papilloma virus plasmids. | to identify dna sequences that function in the control of dna replication, we designed a hybrid replicon consisting of linked sv40 and bpv dna sequences. in the composite sv40-bpv plasmid negative control encoded by bpv is dominant over the uncontrolled replication encoded by the positive factor, sv40 t antigen. using a transient replication assay, we show that replication control requires three bpv elements. two cis-acting sequences are closely linked to bpv replication origins. a third trans-a ... | 1986 | 3017566 |
| aetiology of enzootic haematuria. | the precise aetiology of enzootic haematuria in cattle remains unknown. the involvement of bracken fern (pteridium aquilinium) appears certain because of the close association between bracken fern infested farms and enzootic haematuria. several toxic principles have been identified but the main carcinogenic element remains to be conclusively demonstrated. more recently, bovine papilloma virus has been implicated in the aetiology of enzootic haematuria. its possible interaction with bracken fern ... | 1986 | 3016971 |
| comparison of methods for introducing vectors based on bovine papillomavirus-1 dna into mammalian cells. | the intracellular structure of several vectors based on bpv-1 dna has been analyzed following transfection into mouse c127 cells by the calcium phosphate method or, for the first time, by microinjection directly into the nucleus. it is shown that the method of introduction markedly affects the fate of a bpv-1 based vector. in general, microinjection appears to do little damage to dna and is more likely to result in a vector replicating extrachromosomally as a monomeric structure of the same size ... | 1986 | 3016916 |
| the e5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide. | bovine papillomavirus (bpv-1) contains two independent transforming genes that have been mapped to the e5 and e6 open reading frames (orf's). the e5 transforming protein was identified by means of an antiserum against a synthetic peptide corresponding to the 20 cooh-terminal amino acids of the e5 orf. the e5 polypeptide is the smallest viral transforming protein yet characterized; it had an apparent size of 7 kilodaltons. the transforming polypeptide is encoded entirely within the second half of ... | 1986 | 3014660 |
| the bovine papillomavirus replicon. | the bovine papillomavirus genome contains two cis-acting sequences which can serve as signals for replication. at least three virally encoded genes seem to be involved in plasmid replication: e6, e6/7 and e1. mutations in either the e6 or the e7 open reading frame create plasmids that are maintained at a low copy number per cell. mutations in the e1 open reading frame are absolutely lethal to replication. complementation experiments show that these mutations define separate genes. experiments ar ... | 1986 | 3013526 |
| papillomavirus transforming functions. | the bovine papillomavirus type 1 (bpv-1) has served as a model for unravelling the molecular genetics of the papillomaviruses. bpv-1 transformation of rodent cells in tissue culture has provided a means to study the viral functions involved in latent infection of cells and in the induction of cellular proliferation functions. bpv-1 has been shown to encode two independent transforming genes, each of which can induce cellular transformation in susceptible rodent cells. these two genes apparently ... | 1986 | 3013525 |
| classification of the papillomaviruses--mapping the genome. | papillomaviruses form one genus of the papovaviridae family. they share common antigenic determinants and their dnas cross-hybridize under conditions of low stringency. the classification of papillomaviruses is at present based on the host range and the relatedness of the nucleic acids. isolates are considered independent types if there is less than 50% cross-hybridization in the liquid phase according to a standard protocol. at least 31 human and six bovine papillomavirus types can be different ... | 1986 | 3013524 |
| organization and expression of the genome of bovine papillomavirus type 1. | the viral mrnas present in c127 cells transformed by bovine papillomavirus type 1 (bpv-1) have been mapped by a variety of techniques, including s1 nuclease analysis, northern blot analysis, primer extension and electron microscopic heteroduplex analysis. the results reveal a very complex mrna pattern, comprising at least five types of spliced cytoplasmic mrnas. both unspliced and partially processed nuclear rna species have also been identified. the transforming region of bpv-1 contains several ... | 1986 | 3013523 |
| immunization against bovine papillomavirus infection. | the two large open reading frames denoted l1 and l2 in the non-transforming region of the bovine papillomavirus type 1 (bpv-1) genome have been molecularly cloned to expression in escherichia coli. antisera against the e. coli-derived l1 and l2 protein reacted with bpv-1 in both enzyme-linked immunosorbent assays and immunoprecipitation reactions. neutralization of bpv-induced transformation of mouse c127 cells was demonstrated most consistently with antisera against the l1 protein. e. coli-deri ... | 1986 | 3013520 |
| papillomavirus infection in cattle: viral and chemical cofactors in naturally occurring and experimentally induced tumours. | six different types of bovine papillomavirus (bpv-1 to bpv-6) have been identified and classified into two subgroups: subgroup a, which induce fibropapillomas, and subgroup b, which induce true epithelial papillomas. bpv-4, a member of subgroup b, is the aetiological agent of papillomas of the upper alimentary canal, which can become a focus for transformation to squamous-cell carcinomas in animals feeding on bracken fern. strong circumstantial evidence suggests that the progression to malignanc ... | 1986 | 3013519 |