DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells

Linney, Elwood; Donerly, Sue

doi:10.1016/0092-8674(83)90102-2

Cited by 131 publications

(95 citation statements)

References 42 publications

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“…Polyomavirus mutants that productively infect EC cells have been isolated, and several of the mutations have been mapped to the enhancer region of the early promoter (16,42,53). Expression data provide evidence that the primary block for polyomavirus replication in EC cells is a defect in early viral expression, and the enhancer mutations relieve the defect by activating the early promoter (29). These results have been extended to MMuLV restriction in EC cells since enhancer function of the M-MuLV long terminal repeat (LTR) was also reduced in the stem cells (28).…”

mentioning

confidence: 75%

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells.

Loh¹,

Sievert²,

Scott³

1987

Mol. Cell. Biol.

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Embryonal carcinoma (EC) cells are nonpermissive for retrovirus replication. Restriction of retroviral expression in EC cells was studied by using DNA transfection techniques. To investigate the activity of the Moloney murine leukemia virus (M-MuLV) enhancer and promoter sequences, the M-MuLV long terminal repeat and the defined long terminal repeat deletions were linked to neo structural gene sequences that encode resistance to the neomycin analog G418. Transient expression data and drug resistance frequencies support the findings that the M-MuLV enhancer is not active in EC cells but that promoter sequences are functional. In addition, a proviral DNA fragment that encodes the leader RNA sequence of a M-MuLV recombinant retrovirus was found to restrict expression specifically in EC cells. Deletion analysis of the leader fragment localized the inhibitory sequences to a region that spans the M-MuLV tRNA primer binding site. It is not known whether restriction occurs at a transcriptional or posttranscriptional level, but steady-state RNA levels in transient expression assays were significantly reduced.

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mentioning

confidence: 75%

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells.

Loh¹,

Sievert²,

Scott³

1987

Mol. Cell. Biol.

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“…Varying lengths of poly(dG) tract were inserted into the SmaI-SacI sites in the expression vector by the G-C tailing method employing terminal transferase to generate {dG}, TKCAT; n is the number of dG residues (for a detailed protocol, see Kohwi-Shigematsu and Kohwi 1991). The PyTKCAT construct contains a 191-bp PvuII-4 fragment {PyF101) of the polyoma enhancer sequence placed 5' of the TK promoter and the CAT gene (Linney and Donerly 1983). This PvuII-4 fragment has a tandem duplication of 54 bp having a single-point mutation in each repeat (Fujimura et al 1981 ).…”

Section: Plasmid Dnamentioning

confidence: 99%

“…One of the two PstI sites {the 3' site) shown in Figure 1A is the internal site in the TK promoter located 22 bp upstream of the HinclI site. The pTKCAT contains the CAT gene and an AATAAA sequence for the polyadenylation signal (Linney and Donerly 1983). Varying lengths of poly(dG) tract were inserted into the SmaI-SacI sites in the expression vector by the G-C tailing method employing terminal transferase to generate {dG}, TKCAT; n is the number of dG residues (for a detailed protocol, see Kohwi-Shigematsu and Kohwi 1991).…”

Section: Plasmid Dnamentioning

confidence: 99%

Altered gene expression correlates with DNA structure.

Kohwi¹,

Kohwi‐Shigematsu²

1991

Genes & Development

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We examined the participation of triplex DNA structure in gene regulation using a poly(dG)-poly(dC) sequence as a model. We show that a poly(dG)-poly(dC) sequence, which can adopt an intramolecular dG.dG.dC triplex under superhelical strain, strongly augments gene expression when placed 5' to a promoter. The activity of this sequence exhibits a striking length dependency: dG tracts of 27-30 bp augment the expression of a reporter gene to a level comparable to that observed with the polyoma enhancer in mouse LTK-cells, whereas tracts of 35 bp and longer have virtually no effect. A supercoiled plasmid containing a dG tract of 30 bp competes in vivo for a trans-acting factor as revealed by reduction in the reporter gene transcription driven by the (dG)29/promoter of the test plasmid, while dGs of 35 bp and longer in the competition plasmid failed to compete. In purified supercoiled plasmid DNA at a superhelical density of -0.05, dG tracts of 32 bp and longer form a triplex, whereas those of 30 bp and shorter remain double-stranded under a PBS solution. These results suggest that a localized superhelical strain can exist, at least transiently, in mouse LTK-cells, and before being relaxed by topoisomerases this rapidly induces dG tracts of 35 bp and longer to adopt a triplex preventing the factor from binding. Thus, these data suggest that a poly(dG)-poly(dC) sequence can function as a negative regulator by adopting an intramolecular triple helix structure in vivo.

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“…The polyomavirus DNase I hypersensitive sites overlap sequence elements which exert a major effect on RNA transcription and DNA replication (5,6,13,15,16,18,25,26), and which are homologous to adenovirus, simian virus 40, and immunoglobulin G transcriptional enhancers (1,11,12,21,27 (20). Either these or related proteins might induce DNase hypersensitivity by binding to DNA in a fashion analogous to that observed upstream of the chicken adult 3-globin gene (8,9).…”

mentioning

confidence: 99%

“…That region of the viral genome from the origin of replication to the sites of initiation of transcription of late mRNAs is hypersensitive to various nucleases and frequently lacks nucleosome morphology (3,14). These sequences are required both for DNA replication and early mRNA transcription and can serve as a transcriptional enhancer for cis-linked cellular genes (5,6,13,15,16,25,26).We examined the effect of mutations in this region on DNase I hypersensitivity. Our data indicated that altered chromatin structure is determined at the site of DNase I cleavage by the sequence AAGCAPuPuAAG flanked by small inverted repeats.…”

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confidence: 99%

Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

Bryan¹,

Folk²

1986

Mol. Cell. Biol.

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DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I. The chromatin of the 5' flanking regions of many active or potentially active genes is unusually susceptible to nuclease attack (7). This 5' hypersensitivity probably reflects the accessibility of specific sequences in the DNA, but its physical nature and functional significance remain speculative (2,7,17). Polyomavirus is a useful system with which to study sequences conferring DNase I hypersensitivity to chromatin since its DNA can be manipulated and then reintroduced into mouse cells, in which it is packaged into minichromosomes. That region of the viral genome from the origin of replication to the sites of initiation of transcription of late mRNAs is hypersensitive to various nucleases and frequently lacks nucleosome morphology (3,14). These sequences are required both for DNA replication and early mRNA transcription and can serve as a transcriptional enhancer for cis-linked cellular genes (5,6,13,15,16,25,26).We examined the effect of mutations in this region on DNase I hypersensitivity. Our data indicated that altered chromatin structure is determined at the site of DNase I cleavage by the sequence AAGCAPuPuAAG flanked by small inverted repeats.Herbomel et al. (14) previously demonstrated that the enhancer region of the polyomavirus strain A2 contains two regions exhibiting DNase I hypersensitivity, but their analysis did not establish whether cleavage by DNase I was determined by sequences within or outside of the enhancer. To precisely map these DNase I-hypersensitivity sites, we used the technique of indirect end labeling (28) with the BamHI site as reference point (Fig. 1A).Mouse 3T6 cells were infected with polyomavirus strain A3 at 0.5 PFU per cell. After 24 h, the nuclei were isolated and digested with DNase I to produce approximately 1 cut per 10 kilobases. The DNA was purified, digested with BamHI, and fractionated by agarose gel electrophoresis, and then probed with a radiolabeled fragment extending from nucleotide 4659 to 5048. Of the polyomavirus minichromosomes cut with DNase I, most are cleaved in a region 470 to 570 base pairs (bp) from the BamHI site (Fig. 1C). DNase 1 digestion of purified DNA produces an entirely different * Corresponding author. t Present address: Genex Corp., Gaithersburg, MD 20877. pattern ( Fig. 2) and shows that specific cutting between the BamHI site and the site of origin of DNA replication is dependent on chromatin structure. Using densitometric scans, we established the two main locations of cutting to within 5 bp. The two hypersensitive sequences resemble each other in that cleavage occurs within a region containing the sequence AAGCAPuPu...

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DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells

Cited by 131 publications

References 42 publications

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells.

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells.

Altered gene expression correlates with DNA structure.

Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

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