Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.

Böhnlein, Ernst; Gruß, Peter

doi:10.1128/mcb.6.5.1401

Cited by 52 publications

(40 citation statements)

References 50 publications

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“…The mixtures were then analyzed on low-ionic-strength polyacrylamide gels. DNA fragments bound to protein migrate more slowly through such gels than unbound DNA fragments (17,20), and in the presence of an agent to minimize nonspecific protein-DNA interactions, such as poly(dI) poly(dC), discrete mobility shifts arising from specific protein-DNA interactions can be observed (3,5,45,56,57).…”

Section: Resultsmentioning

confidence: 99%

“…However, evidence from both functional (36,50,52,54,69) and structural (6, 16) studies has been rapidly accumulating that the action of viral and cellular enhancers involves their interaction with specific protein factors. Recently, the interaction of such transacting factors in nuclear extracts with specific domains of the SV40 (70), polyomavirus (3,18,45), and insulin gene (40) enhancers has been directly demonstrated in vitro by DNase * Corresponding author. …”

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confidence: 99%

“…The glycerol concentration of the extract was lowered to less than 5% by addition of 10 3.3 mM final concentration, and the samples were treated with DNase I (Worthington, 2,227 U/mg) at final concentrations of between 2 and 6 ,ug/ml for 1 min at room temperature. Digestions were terminated by adding EDTA to a final concentration of 12 mM and placing the samples on ice.…”

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confidence: 99%

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At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

Sealey¹,

Chalkley²

1987

Mol. Cell. Biol.

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We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI-and EFH-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.Enhancers are a class of cis-acting regulatory DNA sequences that can activate transcription from both homologous and heterologous eucaryotic promoters in a relatively orientation-, position-, and distance-independent manner.Enhancers have been identified in many viral genomes (for reviews, see references 23, 43, 55) and near or within a variety of cellular genes (1, 13, 22, 24, 26, 41, 44, 60 64), where they often potentiate transcription in a cell typespecific, and sometimes inducible, fashion. Different enhancer elements bear no extensive sequence homologies, although several short, degenerate consensus sequences occur in certain subsets of enhancers (27, 38, 68, 71, and references therein). Despite the lack of sequence homology, enhancers do seem to share an organizational consensus. Extensive site-specific mutagenesis and deletion analyses have established that the simian virus 40 (SV40) and polyomavirus enhancers (28, 29, 71), as well as several other well-characterized enhancers (4,14,32,46,67), are mosaics of multiple different and often redundant sequence motifs. Each relevant sequence motif appears to be contained within a separable functional domain that possesses little enhancer activity unless combined with another domain. The functional domains of an enhancer, although nonhomologous, can compensate for each other in satisfying this multiplicity requirement (29,30,32,63,65,71).The mechanism(s) by which this modular arrangement of multiple short sequence motifs synergistically leads to the long-range activation of transcription from a promotor is not known for any enhancer. However, evidence from both functional (36,50,52,54,69) and structural (6, 16) studies has been rapidly accumulating that the action of viral and cellular enhancers involves their interaction with specific protein factors. Recently, the interaction of such transacting factors in nuclear extracts with specific domains of the SV40 (70), polyomavirus (3,18,45), and insulin gene (40) enhancers has been directly demonstrated in vitro by DNase * Corresponding author.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

Sealey¹,

Chalkley²

1987

Mol. Cell. Biol.

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“…Base substitution mutations in the P (B) element result in their ability to allow Py to replicate in the mouse embryonal carcinoma cell line F9 (see reference 1 and references therein), presumably owing to increased enhancer function. DNase I-hypersensitive sites are located in both a (A) and P (B) regions (7,27), and nuclear proteins capable of binding to sites within the enhancer have been described (6,20,41,43). It has been speculated that the binding of specific proteins to the enhancer region modifies the interaction of the core ori with the replication complex (16).…”

Section: Discussionmentioning

confidence: 99%

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Prives¹,

Murakami²,

Kern³

et al. 1987

Mol. Cell. Biol.

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Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinitypurified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (on) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core on led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core on region replicated in vitro, but to a lesser extent than wild-type Py DNA. Plasmids with deletions on the late-gene side of the core on, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.Replication of polyomavirus (Py) DNA proceeds bidirectionally from a fixed point within the noncoding region of the viral genome (for a review, see reference 16 and references therein). This replication origin (ori) region (Fig. 1) contains multiple T-antigen (T Ag)-binding sites within a G+C-rich 32-base-pair (bp) sequence of dyad symmetry adjacent to a region in which 13 of 14 residues form A * T pairs. It thus bears structural features similar to the replication ori of simian virus 40 (SV40). However, the relationship of the Py ori to other regulatory signals in the noncoding segment is somewhat different from that of SV40. The number and arrangement of large T Ag high-affinity binding sites on the early side of both viral ori regions differ. Also, the relative affinities of Py and SV40 T Ags for binding sites within their respective ori regions are apparently dissimilar (12,14,28,47). Moreover, while both viruses encode enhancer elements, the Py enhancer is almost directly adjacent to the late boundary of the ori region and consists of a series of discrete elements that bear homologies to other enhancer elements including those of SV40, adenovirus Ela, and mouse immunoglobulin genes (2,22,23,26,31,38,46,51,53). By contrast, the SV40 enhancer, a 72-bp repeated sequence (see reference 22 and references therein), is separated from the core ori by a series of G+C-rich sequences (the 21-bp repeats) that form a distinct transcriptional control element to which the cellular transcription factor, Spl, binds (...

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“…The essential origin sequences are shown (striped bars). DNAinding sites for putative enhancer-specific proteins (open boxes) include PEAl, 2, and 3 (Martin et al 1988); EFC (Fujimura 1986;Ostapuck et al 1986); PEBl (Bohnlein and Gruss 1986;Piette and Yaniv 1986); and F441 (Kovesdi et al 1987). The enhancer region of three PyV host-range mutants selected for growth in undifferentiated mouse embryonal carcinoma F9 cells (Fujimura et al 1981) contains a point mutation at nucleotide 5233 (solid bar, F441), a 31-bp tandem duplication of the mutated region (crosshatched box.…”

Section: Gene Expression In Individual Mouse Oocytes and Embryos Is Pmentioning

confidence: 99%

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

Martínez‐Salas

Linney²,

Hassell³

et al. 1989

Genes Dev.

111

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Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation FlOl, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1 : 67 : 21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.

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Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.

Cited by 52 publications

References 50 publications

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

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