Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions.

Osborne, Timothy F.; Schell, Richard; Burch-Jaffe, E.; Berget, S J; Berk, Arnold

doi:10.1073/pnas.78.3.1381

Cited by 27 publications

(8 citation statements)

References 40 publications

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“…M13 RF DNA was converted to unit-length linear molecules by treatment with Asu I. After phenol extraction and ethanol precipitation, the linear DNA was treated with BAL31, phenol extracted, and blunt-end ligated with T4 ligase essentially as described (11). In some cases, a 12-base pair EcoRI linker (C-A-T-G-A-A-T-T-C-A-T-G) was joined to the linear DNA prior to closure as described (11).…”

Section: Methodsmentioning

confidence: 99%

Viable deletions of the M13 complementary strand origin

Kim

Hines

Ray

1981

Proc. Natl. Acad. Sci. U.S.A.

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The single-stranded DNA of bacteriophage M13 is converted to a duplex replicative form by a mechanism involving RNA-primed initiation at a single unique site on the viral DNA. The DNA sequence that specifies the RNA primer is contained largely within one of two adjacent hairpin structures protected from DNase degradation by RNA polymerase. We have used in vitro techniques to construct a series of M13 mutants having deletions in the region of the complementary strand origin. Deletions of the duplex replicative form DNA range in size from 54 to 201 base pairs. The largest deletions remove both of the RNA polymerase-protected hairpins and the entire sequence specifying the primer RNA. Mutants lacking one or both hairpins form faint plaques, give reduced phage yields, and show a lag in phage production of >30 min. The rate of conversion of the single-stranded viral DNA to the parental replicative form is reduced both in vivo and in vitro. These results indicate that both the RNA polymeraseprotected hairpins and the RNA primer-coding sequence are important, but not essential, for replication. Other sequences within the origin region, or possibly elsewhere in the genome, may play a role in complementary strand initiation in these mutant phages. The M13 viral strand is initiated by extension of the 3' terminus generated by site-specific nicldng of the viral strand of the replicative form DNA by the M13 gene II protein. This specific nicldng site is retained in all of the M13 deletion mutants. Deletion end points do not extend into a 13-nucleotide sequence preceding the viral strand nicking site. We propose that a sequence including these 13 nucleotides is required for gene II protein action at this site.On infection, the circular single-stranded DNA of bacteriophage M13 is converted into a double-stranded replicative form (RF) by synthesis of the complementary strand (1, 2). Initiation of the complementary strand occurs within a 508-base pair intergenic region between genes II and IV on the phage chromosome (3-7). A RNA primer is synthesized by RNA polymerase acting at a unique site in the intergenic region (8, 9). In vitro, two hairpins in the intergenic region ofthe viral strand are protected from DNase degradation by Escherichia coli RNA polymerase in the presence ofE. coli single-strand binding (ssb) protein (7). The sequence coding for the primer for complementary strand synthesis is contained largely within one ofthese protected hairpins. Although both the initiation site and the nucleotide sequence of the RNA primer are known, the sequence that determines the specificity and site of initiation has not been identified.To determine which sequences in the origin region are essential for replication and to examine the role of potential hairpin structures, we have used in vitro techniques to introduce deletions into the region of the complementary strand origin. Plaque-forming phage have been isolated in which the sequence coding for the RNA primer for the complementary strand and both of the hairpin structu...

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Section: Methodsmentioning

confidence: 99%

Viable deletions of the M13 complementary strand origin

Kim

Hines

Ray

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Ideally, ElA transactivation should be examined in the absence of these other consequences of infection. (33). Sequences between nucleotides 520 and 1837 of adenovirus type 2 (Ad2) (37) Single-stranded DNA of M13 containing adenovirus sequences was prepared as described previously (29 20 ,ug of RNA in 50 pl of 0.4 M NaCl-40 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.4)-lmM EDTA-80% (vol/vol) formamide for 16 h. S1 digestion of the DNA-RNA hybrid and electrophoretic analysis of the products were as described previously (32).…”

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confidence: 99%

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

Brunet¹,

Berk²

1988

Mol. Cell. Biol.

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The adenovirus ElA proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level ElA expression in the absence of viral infection would facilitate biochemical studies of ElA-mediated transactivation. Toward this end, we introduced the adenovirus type 2 ElA gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable ElA mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of ElA expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying ElA concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same ElA concentration and that transcription from each promoter increased as the ElA concentration increased. These results indicate that ElA-mediated transactivation is proportional to the concentration of ElA protein. ElA-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the ElA proteins was sufficient to increase the transcriptional activity of nuclear extracts.

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“…It is often useful to decrease the length ofa given DNA fragment in a progressive, controlled manner (1)(2)(3)(4)(5)(6)(7). Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments.…”

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confidence: 99%

“…One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3,6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9). The primary disadvantage of these approaches is that the nucleases act simultaneously at both ends of the fragment so that desirable sequences at one end are not preserved.…”

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confidence: 99%

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

Putney¹,

Benkovic²,

Schimmel³

1981

Proc. Natl. Acad. Sci. U.S.A.

180

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2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP [caS]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to. be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand ofa double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease mI is limited and is followed by SI nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this way thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and SI nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.It is often useful to decrease the length ofa given DNA fragment in a progressive, controlled manner (1-7). Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments. One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3, 6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9). The primary disadvantage of these approaches is that the nucleases act simultaneously at both ends of the fragment so that desirable sequences at one end are not preserved.The Sp diastereomer of the nucleoside a-thiotriphosphates (which have a sulfur substituted for an oxygen at the a-phosphate) act as substrates for Escherichia coli DNA polymerase I in DNA synthesis (10). These compounds, although incorporated at an efficiency similar to that of the natural substrates, effectively block the 3' exonuclease activity of DNA polymerase I at the phosphorothioate nucleotide linkage (unpublished results). In work reported here we found that placement of an athionucleotide at only one of the 3' ends of a DNA fragment effectively blocks that end from digestion with exonuclease III. Digestion proceeds progressively from the opposite end and, after S1 nuclease treatment, the fragment is shortened with the integrity of the nucleotide sequence at the "capped" end preserved. Alternatively, if exonuclease III digestion is allowed to proceed to completion, a full-length single strand is generated from the original double strand.This work also demonstrates that, when 2'-deoxyadenosine 5'-O-(l-thiotriphosphate) (dATP [aS]) is inserted into 3' recessed ends ofrestriction fragments, the phosphorothioate linkage allows subsequent ligation ofthe DNA, restriction ofligated junctions, and in vivo replication of plasmids containing phosphorothioate nucleotide linkages. Purific...

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Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions.

Cited by 27 publications

References 40 publications

Viable deletions of the M13 complementary strand origin

Viable deletions of the M13 complementary strand origin

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

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