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A Stenlund

Publications and source records attributed to A Stenlund.

36 records · Page 2Linked to original sources
PubMed1991

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Animals↗
PubMed1991

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression.

Animals↗
PubMed1990

The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor.

The E2 open reading frame (ORF) of the bovine papillomavirus (BPV-1) encodes a family of site-specific DNA-binding proteins. The full-length protein is a transcriptional activator, whereas the polypeptides that contain only the carboxy-terminal domain are repressors. Here we show that the trans-activator can work as a repressor of transcription for one of the BPV-1 promoters by binding to a DNA sequence required for basal activity of the promoter. This operator site is defined as a 12-bp sequence that lies immediately downstream of the cap site. The operator DNA contains sequences that are defined genetically and biochemically as being important for basal level promoter activity. Furthermore, this site has been shown to be protected in a DNase footprint assay using fractionated HeLa cell extracts. The repression does not simply result from E2 blocking RNA polymerase initiation or elongation, because a strong E2-binding site placed at the operator has no repressive effect on transcription when the basal target sequence is placed independently upstream of the promoter. Thus, this is an interesting parallel to a theme well known in prokaryotes, where some site-specific DNA-binding proteins can work as either activators or repressors. In this system, as well as in the prokaryotic systems, the precise position of the binding site relative to other cis signals at the promoter determines the nature of the effects.

Base Sequence↗
PubMed1989

Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome.

The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction. Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms. Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes. In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA. Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1. Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome. In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein. Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold. The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site. The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role.

Animals↗
PubMed1989

Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs.

Genetic and biochemical evidence has established that the E2 open reading frame (ORF) of bovine papillomavirus type 1 encodes at least two different site-specific DNA-binding proteins, one which activates and the other which represses expression from a viral promoter (P. F. Lambert, B. A. Spalholz, and P. M. Howley, Cell 50:69-78, 1987). We have obtained data which show that a second form of the repressor gene is expressed in transformed cells harboring stable viral plasmids. The structural details of this gene have been discerned by cDNA cloning, by RNase protection, and by primer extension analysis of in vivo RNA. Moreover, data from in vitro transcription experiments support the notion that this form of the E2 repressor is expressed from a novel viral promoter and that a small exon from another ORF is linked to an active repressor domain in E2. Thus, two different forms of the repressor are expressed from different promoters and might be independently regulated either in the cell cycle or in different tissue types. We show by functional in vivo assays utilizing a cDNA vector encoding this gene that the trans-acting factor has in vivo activities similar to those of the known repressor. Our screen of a cDNA library for cDNA clones representing bovine papillomavirus transcripts has also revealed a number of other novel structures defining new donor and acceptor RNA-processing sites. Notably, clones which conceptually can be translated to yield an E7 protein, the viral M gene, and the entire E2 ORF have been characterized. Finally, truncated versions of putative E8 cDNAs were also obtained.

Bovine papillomavirus 1↗
PubMed1987

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 transcription. Both in vitro and in vivo assays have been used to define this promoter and show that a sequence located just 3' to the transcriptional start site is required for activity. This DNA sequence element, which has been defined through deletions, coincides with a binding site for a cellular factor and is also required for a functional origin of replication. Possible models for how a transcription factor may play a role in the regulation of DNA replication are discussed.

Acetyltransferases↗
PubMed1987

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.

Base Sequence↗
PubMed1986

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 amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.

Bovine papillomavirus 1↗
PubMed1986

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 promoter regions. A major cap site is located at coordinate 1 and another putative cap site at coordinate 31. A third candidate cap site maps around coordinate 39.

Animals↗
PubMed1985

Messenger RNAs from the transforming region of bovine papilloma virus type I.

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.

Animals↗
PubMed1984

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.

Amino Acid Sequence↗
PubMed1983

Sequences of bovine papillomavirus type 1 DNA--functional and evolutionary implications.

A swedish isolate of bovine papillomavirus type 1 (BPV1) was cloned and a 4807 base pairs long sequence was determined, covering the entire late region of the BPV1 genome and strategic parts of the early region. The sequence reveals two large uninterrupted reading frames, designated L1 and L2, which encode tentative polypeptides with molecular weights of 55.5 and 50.0 K. The promoter region for early transcription and the common 3'-end of early transcripts were identified in the sequence by performing S1 nuclease analysis of RNA isolated from a BPV1 transformed cell line. A detailed comparison between the established sequence and sequences from human papillomavirus type 1a (HPV1a) (1) and another BPV1 isolate (2) was also carried out. Only five differences were found when the sequences of the two BPV1 isolates were compared, two of which are located in reading frame L2. The results revealed furthermore that the genomes of BPV1 and HPV1a appear to be organized in a very similar fashion and the homology between reading frames L1 in BPV1 and HPV1 were particularly striking. A comparison of the established BPV1 sequence with sequences from SV40 and polyomavirus revealed no significant homology.

Animals↗
PubMed1983

Secretion of the hepatitis B virus surface antigen from mouse cells using an extra-chromosomal eucaryotic vector.

Recombinant DNA molecules which contained a subgenomic fragment of the hepatitis B virus (HBV) genome, the pML2 vector and the bovine papillomavirus type 1 (BPV) genome were constructed. The HBV fragment includes the entire transcription unit for the hepatitis B surface antigen (HBsAg). After propagation in Escherichia coli, the recombinant plasmids were cleaved with endonucleases SalI and PvuI to eliminate most of the bacterial sequences before transfection of mouse C127 cells. Foci were observed 10--14 days after transfection. Cells from selected foci were cloned and the supernatants were assayed for the presence of HBsAg. Most of the clones tested were found to secrete HBsAg particles into the growth medium. These particles appear to be similar to the 22 nm particles present in the serum of HBV chronic carriers. SDS-polyacrylamide gel electrophoresis revealed that the particles contain two polypeptides, probably representing the glycosylated and unglycosylated forms of the HBsAg major polypeptide. An analysis of DNA from the transformed clones revealed that they contain multiple extra-chromosomal copies of the recombinant, which, however, had suffered rearrangement.

Animals↗
PubMed1983

European elk papillomavirus: characterization of the genome, induction of tumors in animals, and transformation in vitro.

The European elk papillomavirus (EEPV) genome was cloned in the BamHI cleavage site of the pBR322 vector. The cloned genome was used for construction of a physical map, employing restriction endonucleases BamHI, BglII, HindIII, PvuII, SacI, and XhoI. The sequence homology between the EEPV and bovine papillomavirus type 1 genomes was elucidated by performing hybridizations in different concentrations of formamide. Sequence homology could only be revealed under less stringent conditions, i.e., Tm - 43 degrees C. Nucleotide sequence information was also collected from the regions which lie adjacent to the three HindIII sites that are present in the EEPV genome. The results made it possible to align the EEPV and bovine papillomavirus type 1 genomes. Transformation by EEPV was demonstrated with the C127 mouse cell line, and fibrosarcomas were induced in young hamsters after subcutaneous injection. The transformed cells and the tumors contain multiple, nonintegrated copies of the EEPV genome. Virus particles could not be detected either in tumors or in transformed cells.

Animals↗
PubMed1982

A common sequence in the inverted terminal repetitions of human and avian adenoviruses.

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.

Adenoviridae↗
PubMed1982

Sequence homology between avian and human adenoviruses.

Studies of hybridization between fowl adenovirus type 1 (chicken embryo lethal orphan virus) DNA and human adenovirus type 2 DNA revealed two short but distinct regions which cross-hybridized under stringent conditions. One of the homologous regions was located between map positions 18.1 and 19.3 and did not correspond to any gene recognized so far. The second region mapped in the hexon gene between position 57 and 58.

Adenoviridae↗
PubMed1980

Construction of restriction enzyme fragment libraries containing DNA from human adenovirus types 2 and 5.

Restriction-fragment libraries containing adenovirus type 2 (Ad2) DNA have been constructed, using the pBR322 plasmid (Bolivar et al., 1977) as a vector. Clones have been isolated which contain all the HindIII fragments of Ad2 DNA except the terminal G- and K-fragments inserted into the HindIII cleavage site of the vector. All the 13 SmaI-fragments of Ad2 DNA were separately inserted into the PstI site of the pBR322 vector after addition of homopolymeric poly(dG) tails to the fragments and poly(dC) tails to the linearized plasmid. Two large fragments of adenovirus type 5 (AD5) DNA, located between map positions 17.0 and 59.5 and between map positions 59.5 and 97.3, respectively, were cloned using bacteriophage lambda as a vector. All clones, which are described in the present report, are available upon request.

Adenoviruses, Human↗