Sequence-specific interaction of histones with the simian virus 40 enhancer region in vitro.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of PEX, an endopeptidase of uncertain function. This defect is shared by Hyp mice, the murine homologue of the human disease, in which a 3′ Pex deletion has been documented. In the present study, we report that immortalized osteoblasts derived from the simian virus 40 (SV40) transgenic Hyp mouse (TMOb- Hyp) have an impaired capacity to mineralize extracellular matrix in vitro. Compared with immortalized osteoblasts from the SV40 transgenic normal mouse (TMOb-Nl), osteoblast cultures from the SV40 Hyp mouse exhibit diminished 45Ca accumulation into extracellular matrix (37 ± 6 vs. 1,484 ± 68 counts ⋅ min−1 ⋅ μg protein−1) and reduced formation of mineralization nodules. Moreover, in coculture experiments, we found evidence that osteoblasts from the SV40 Hyp mouse produce a diffusible factor that blocks mineralization of extracellular matrix in normal osteoblasts. Our findings indicate that abnormal PEX in osteoblasts is associated with the accumulation of a factor(s) that inhibits mineralization of extracellular matrix in vitro.

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

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.642 ◽

1985 ◽

Vol 5 (4) ◽

pp. 642-648 ◽

Cited By ~ 26

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2317-2323.1986 ◽

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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Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1476-1482.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1476-1482

Author(s):

H Ariga

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Rna Polymerase Iii ◽

Simian Virus 40 ◽

Cell Extract ◽

Simian Virus ◽

T Antigen ◽

Sv40 T Antigen ◽

Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

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Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.75-83.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 75-83

Author(s):

Y Berko-Flint ◽

S Karby ◽

D Hassin ◽

S Lavi

Keyword(s):

De Novo ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Dna Molecules ◽

Viral Origin ◽

Cell Extracts ◽

Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

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Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5088-5098.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5088-5098

Author(s):

J Yang ◽

D B DeFranco

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Tumor Antigen ◽

Nuclear Import ◽

Simian Virus 40 ◽

Simian Virus ◽

Large Tumor ◽

Large Tumor Antigen ◽

Cell Cytosol

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.

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A novel protein factor is required for use of distal alternative 5' splice sites in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5945-5953.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5945-5953

Author(s):

J E Harper ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Deae Cellulose ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

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