Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit

1983 ◽  
Vol 3 (6) ◽  
pp. 1108-1122
Author(s):  
M Lusky ◽  
L Berg ◽  
H Weiher ◽  
M Botchan
Keyword(s):  
Thymidine Kinase ◽  
Dna Sequences ◽  
Base Pair ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Stable Transformation ◽  
High Molecular Weight Dna ◽  
Papilloma Virus ◽  
Recombinant Plasmids ◽  
Bovine Papilloma Virus

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

scholarly journals Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

1983 ◽  
Vol 3 (6) ◽  
pp. 1108-1122 ◽  
Author(s):  
M Lusky ◽  
L Berg ◽  
H Weiher ◽  
M Botchan
Keyword(s):  
Thymidine Kinase ◽  
Dna Sequences ◽  
Base Pair ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Stable Transformation ◽  
High Molecular Weight Dna ◽  
Papilloma Virus ◽  
Recombinant Plasmids ◽  
Bovine Papilloma Virus

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

scholarly journals Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro.

1984 ◽  
Vol 4 (12) ◽  
pp. 2911-2920 ◽  
Author(s):  
H Mishoe ◽  
J N Brady ◽  
M Radonovich ◽  
N P Salzman
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Base Pair ◽  
Transcriptional Control ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Control Element ◽  
Late Promoter ◽  
Transcription Control

We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

scholarly journals Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.

1982 ◽  
Vol 2 (8) ◽  
pp. 949-965 ◽  
Author(s):  
S E Conrad ◽  
M R Botchan
Keyword(s):  
Thymidine Kinase ◽  
Dna Sequences ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Fragments ◽  
Viral Origin ◽  
Isolation And Characterization ◽  
Human Dna ◽  
Sv40 Dna

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

scholarly journals Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.

1986 ◽  
Vol 6 (4) ◽  
pp. 1320-1325 ◽  
Author(s):  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Base Pair ◽  
Recombination Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Extrachromosomal Dna ◽  
Cos Cells ◽  
Antigen Gene

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.

Electron Microscopy of stained and unstained bovine papilloma virus: Comparison with polyoma virus

1989 ◽  
Vol 47 ◽  
pp. 820-821
Author(s):  
T.S. Baker ◽  
N. H. Olson ◽  
W. W. Newcomb ◽  
J. C. Brown ◽  
C. Olson
Keyword(s):  
Electron Microscopy ◽  
High Speed ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Supernatant Fraction ◽  
Epithelial Tissue ◽  
Papilloma Virus ◽  
Moderate Resolution ◽  
Microscopy Techniques ◽  
Bovine Papilloma Virus

Bovine papilloma virus type 1 (BPV-1) is a member of the papillomavirus genus, one of two in the Papovaviridae family. Papovaviruses are characterized by similarities in their capsid structure, genetics, biochemical composition and role in the formation of benign and cancerous tumors. The discoveries that the capsid structures of polyoma and simian virus 40 (SV40) (members of the genus polyomavirus) consist of 72 pentameric capsomeres raises the fundamental question of whether or not the capsids of the larger and more complex papilloma viruses have a similar, unexpected arrangement of the capsid subunits. The recent development of cryo-electron microscopy techniques, which facilitate direct visualization of the “native” morphology of biological specimens at moderate resolution (1-4 nm), provides an opportunity to critically examine the structure of BPV-1.The BPV-1 used in this study was originally isolated from a calf in 1965, passaged again in 1986, and stored in 50% glycerin and phosphate buffered saline. Virus was extracted and purified following the protocol of Cowsert et al. (1987): epithelial tissue rich in BPV-1 was mixed with an equal volume of buffer (1M NaCl, 20mM Tris, pH 7.5) and disrupted in a Waring blender. After high speed clarification, the supernatant fraction was mixed with an equal volume of Freon 113 and centrifuged at low speed. Virus from the aqueous phase was pelleted, resuspended in CsCl (ρ=l.33 g/cm3), density banded, pelleted and finally resuspended in 30 mM KCl and 6 mM Tris (pH 7.5) to a protein concentration of ˜3 mg/ml.

Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells

1988 ◽  
Vol 8 (12) ◽  
pp. 5280-5291
Author(s):  
H B Lieberman ◽  
P F Lin ◽  
D B Yeh ◽  
F H Ruddle
Keyword(s):  
Enzyme Activity ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Fold Increase ◽  
Simian Virus ◽  
Mrna Levels ◽  
Dot Blot ◽  
Kinase Gene ◽  
Cloned Gene

We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.

Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro

1984 ◽  
Vol 4 (12) ◽  
pp. 2911-2920
Author(s):  
H Mishoe ◽  
J N Brady ◽  
M Radonovich ◽  
N P Salzman
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Base Pair ◽  
Transcriptional Control ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Control Element ◽  
Late Promoter ◽  
Transcription Control

We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region

1982 ◽  
Vol 2 (8) ◽  
pp. 949-965
Author(s):  
S E Conrad ◽  
M R Botchan
Keyword(s):  
Thymidine Kinase ◽  
Dna Sequences ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Fragments ◽  
Viral Origin ◽  
Isolation And Characterization ◽  
Human Dna ◽  
Sv40 Dna

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

Correlation of gene expression and transformation frequency with the presence of an enhancing sequence in the transforming DNA

1984 ◽  
Vol 4 (2) ◽  
pp. 368-370
Author(s):  
P E Berg ◽  
W F Anderson
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transformation Frequency ◽  
Hamster Strain ◽  
Sarcoma Virus

The transformation frequency of cultured mammalian cells is increased 10- to 100-fold when certain DNA sequences are present in the transforming DNA. We wanted to determine whether enhancers, which stimulate gene expression, can cause this phenomenon. Three plasmids, each containing a galactokinase K (galK) gene, were used to transform galK- Chinese hamster cells. One plasmid has no enhancer, another has the simian virus 40 (72-base-pair repeat) enhancer, and the third has the Harvey sarcoma virus (73-base-pair repeat) enhancer. The presence of either enhancer significantly increased the appearance of GalK+ colonies. Galactokinase transient assays in this Chinese hamster strain in the presence of the same plasmids demonstrated an increase in GalK enzyme levels when either enhancer was present. These data indicate that there is a strong correlation between galK expression and transformation frequency that is dependent on the presence of an enhancer in the transforming DNA.

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