scholarly journals Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4484-4491 ◽  
Author(s):  
A Mayeda ◽  
Y Ohshima
Keyword(s):  
Consensus Sequence ◽  
Donor Site ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Beta Globin ◽  
Vitro Assay ◽  
Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

scholarly journals Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro

1988 ◽  
Vol 8 (10) ◽  
pp. 4484-4491
Author(s):  
A Mayeda ◽  
Y Ohshima
Keyword(s):  
Consensus Sequence ◽  
Donor Site ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Beta Globin ◽  
Vitro Assay ◽  
Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2051-2060
Author(s):  
B W Stillman ◽  
Y Gluzman
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Extracts ◽  
293 Cells ◽  
Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA

1987 ◽  
Vol 7 (8) ◽  
pp. 3018-3020
Author(s):  
Y Zhuang ◽  
H Leung ◽  
A M Weiner
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Splice Sites ◽  
Small Nuclear Ribonucleoprotein ◽  
Antigen 5

The use of alternative 5' splice sites in the simian virus 40 early-transcription unit controls the ratio of large T to small t antigen during viral infection. To study the regulation of these alternative 5' splice sites, we made two mutants which improve the match of the large-T-antigen 5' splice site to the 5' splice site consensus sequence. Whether these mutants were assayed in vitro or in vivo, we found that the efficiency of large-T splicing is increased by improving the match of the large-T-antigen 5' splice site to the consensus. We conclude that the match of a 5' splice site is an important determinant of 5' splice site utilization and that the simian virus 40 large-T-antigen 5' splice site is almost certainly recognized by the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein particle.

scholarly journals Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

1985 ◽  
Vol 5 (8) ◽  
pp. 2051-2060 ◽  
Author(s):  
B W Stillman ◽  
Y Gluzman
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Extracts ◽  
293 Cells ◽  
Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

scholarly journals The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA.

1987 ◽  
Vol 7 (8) ◽  
pp. 3018-3020 ◽  
Author(s):  
Y Zhuang ◽  
H Leung ◽  
A M Weiner
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Splice Sites ◽  
Small Nuclear Ribonucleoprotein ◽  
Antigen 5

The use of alternative 5' splice sites in the simian virus 40 early-transcription unit controls the ratio of large T to small t antigen during viral infection. To study the regulation of these alternative 5' splice sites, we made two mutants which improve the match of the large-T-antigen 5' splice site to the 5' splice site consensus sequence. Whether these mutants were assayed in vitro or in vivo, we found that the efficiency of large-T splicing is increased by improving the match of the large-T-antigen 5' splice site to the consensus. We conclude that the match of a 5' splice site is an important determinant of 5' splice site utilization and that the simian virus 40 large-T-antigen 5' splice site is almost certainly recognized by the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein particle.

scholarly journals Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1.

1996 ◽  
Vol 16 (3) ◽  
pp. 810-817 ◽  
Author(s):  
R T Kamakaka ◽  
M Bulger ◽  
P D Kaufman ◽  
B Stillman ◽  
J T Kadonaga
Keyword(s):  
Dna Replication ◽  
Direct Interaction ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Chromatin Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

To study the relationship between DNA replication and chromatin assembly, we have purified a factor termed Drosophila chromatin assembly factor 1 (dCAF-1) to approximately 50% homogeneity from a nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA replication-coupled chromatin assembly revealed that both dCAF-1 and human CAF-1 mediate chromatin assembly preferentially with previously yet newly replicated DNA relative to unreplicated DNA. Moreover, the preferential assembly of the postreplicative DNA was observed at 30 min after inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it was no longer apparent at 120 min after inhibition of replication. These findings suggest that the coupling between DNA replication and chromatin assembly may not necessarily involve a direct interaction between the replication and assembly factors at a replication fork.

scholarly journals Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

1989 ◽  
Vol 264 (27) ◽  
pp. 16160-16164
Author(s):  
I C Taylor ◽  
W Solomon ◽  
B M Weiner ◽  
E Paucha ◽  
M Bradley ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Human Heat Shock Protein ◽  
Stimulation Of

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro

1986 ◽  
Vol 6 (7) ◽  
pp. 2317-2323
Author(s):  
D Zarkower ◽  
P Stephenson ◽  
M Sheets ◽  
M Wickens
Keyword(s):  
Hela Cell ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Polyadenylation Site ◽  
Single Base ◽  
Cleavage And Polyadenylation ◽  
Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

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