Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

Download Full-text

B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1815 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1815-1822 ◽

Cited By ~ 8

Author(s):

J M Gimble ◽

D Levens ◽

E E Max

Keyword(s):

Protein Binding ◽

B Cell ◽

Base Pair ◽

Consensus Sequence ◽

Dimethyl Sulfate ◽

Simian Virus 40 ◽

Human Immunoglobulin ◽

Core Element ◽

Immunoglobulin Kappa

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4484 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4484-4491 ◽

Cited By ~ 17

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.

Download Full-text

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.541 ◽

1989 ◽

Vol 9 (2) ◽

pp. 541-550 ◽

Cited By ~ 46

Author(s):

S E Porter ◽

J J Champoux

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Unknown Origin ◽

Simian Virus ◽

Asymmetric Distribution ◽

Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

Download Full-text

An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5312 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5312-5320 ◽

Cited By ~ 20

Author(s):

Z W Qian ◽

J Wilusz

Keyword(s):

Binding Site ◽

Rna Binding ◽

Specific Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

Polyadenylation Signal ◽

Band Shift

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

Download Full-text

A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6397 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6397-6407 ◽

Cited By ~ 51

Author(s):

J Wilusz ◽

T Shenk

Keyword(s):

Spatial Relationship ◽

Simian Virus 40 ◽

Simian Virus ◽

Polyadenylation Signal ◽

Cross Linking ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Hnrnp C ◽

C Proteins ◽

Nuclear Ribonucleoprotein

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.

Download Full-text

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1170-1183.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1170-1183

Author(s):

M Yamaguchi ◽

E A Hendrickson ◽

M L DePamphilis

Keyword(s):

Dna Polymerase ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Simian Virus ◽

Sequence Specificity ◽

Dna Polymerase Alpha ◽

Dna Primase ◽

Template Sequence

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.

Download Full-text

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.541-550.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 541-550

Author(s):

S E Porter ◽

J J Champoux

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Unknown Origin ◽

Simian Virus ◽

Asymmetric Distribution ◽

Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.3018-3020.1987 ◽

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.

Download Full-text

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

Journal of Virology ◽

10.1128/jvi.75.14.6392-6401.2001 ◽

2001 ◽

Vol 75 (14) ◽

pp. 6392-6401

Author(s):

John D. Purviance ◽

Andrea E. Prack ◽

Brett Barbaro ◽

Peter A. Bullock

Keyword(s):

Dna Synthesis ◽

Critical Role ◽

Simian Virus 40 ◽

Simian Virus ◽

Nucleoside Triphosphate ◽

Replication System ◽

The Core ◽

In Vitro Replication ◽

Primase Complex

ABSTRACT The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) α-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol α-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.

Download Full-text