Adenovirus E1a Gene Product Expressed at High Levels in
            <i>Escherichia coli</i>
            Is Functional

Ferguson, B; Jones, Nicholas; Richter, Joel D.; Rosenberg, Martin

doi:10.1126/science.6374895

Cited by 116 publications

(78 citation statements)

References 30 publications

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“…It is difficult to assess in any quantitative manner the effects of the urea treatment on myc function. However, the E. (15). That myc and ElA are both proline-rich proteins thought to share structural (36) and functional (39) similarities is consistent with their apparent resistance to transient denaturation.…”

Section: Methodsmentioning

confidence: 67%

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Expression and characterization of the human c-myc DNA-binding protein.

Watt¹,

Shatzman²,

Rosenberg³

1985

Mol. Cell. Biol.

189

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In an effort to study in detail the nature of the protein product of the human protoorcogene c-myc, we have expressed the gene at high levels in Escherichia coli. The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E. coli.Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoind,pcible expression system is very efficient. The product was relatively stable and accumulated to approximately 10% of total cellular protein. A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses. A high-titer polyclonal antiserum was raised against the pure protein and shown tfo immunoprecipitate the p11$am--Yc fusion protein of MC-29-infected quail cells. This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burlitt lymphoma cell line. We conclude that this 64-kilodalton protein is the human c-myc gene product since the E. coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000. Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for doublestranded DNA.

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Section: Methodsmentioning

confidence: 67%

“…This atypical characteristic of myc suggests that its native structure may also be quite unusual. The adenovirus ElA gene product (15) and the c-fos gene from mice (45), which are also proline-rich, nuclear localized proteins, exhibit similar anomolous behavior on SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Expression and characterization of the human c-myc DNA-binding protein.

Watt¹,

Shatzman²,

Rosenberg³

1985

Mol. Cell. Biol.

189

View full text Add to dashboard Cite

show abstract

“…Guanidine hydrochloride might 'melt' the insoluble precipitate, and some fraction of the protein might renature binding specificity during the graded removal of the denaturant. In light of this interpretation, we note that bacterial expression of the adenovirus El A gene (Ferguson et al 1984) and the Dwsophila engrailed gene (Desplan et al 1985) leads to the expression of insoluble proteins that require exposure to chaotropic agents for solubilization. Alternatively, appropriate folding or multimerization of a foreign protein may not occur in E. coli, and the denaturation/renaturation process may help to overcome this problem.…”

Section: Discussionmentioning

confidence: 99%

In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.

Vinson¹,

LaMarco²,

Johnson³

et al. 1988

Genes Dev.

536

312

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“…Our results do not implicate any particular protein. One possible candidate is the adenovirus Ela 289-amino acid protein, which is known to trans-activate adenovirus early promoters (2,6,12,24,26,39,43). Although the mechanism of Ela trans-activation is not known, it is unlikely that this protein binds directly to DNA (13).…”

Section: Discussionmentioning

confidence: 99%

Downstream sequences affect transcription initiation from the adenovirus major late promoter.

Mansour¹,

Grodzicker²,

Tjian³

1986

Mol. Cell. Biol.

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We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and + 190 by showing that insertion of SV40 sequences within the intron after the first leader segment at + 190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.In a permissive host, the human adenoviruses, which have a double-stranded DNA genome of 36,000 base pairs, undergo a regulated program of gene expression that is divided into early and late phases, demarcated by the initiation of viral DNA replication. During the late phase of infection, viral transcription initiates primarily from the major late promoter, giving rise to primary transcripts that are processed by differential splicing and polyadenylation into more than 30 mRNA species. Each of the late mRNAs carries at its 5' end a common 203-nucleotide untranslated sequence that is spliced to a protein-coding sequence. This 5' region is called the tripartite leader because it is coded by three noncontiguous viral DNA segments that are located between the major start site for late transcription and the late proteincoding regions (for a review, see reference 59). Concomitant with the induction of late transcription, host cell protein synthesis is repressed, and late viral mRNAs are preferentially translated (16,46). The function of the tripartite leader is not fully understood, but there are data which suggest that the leader is a signal that identifies late viral mRNAs for selective translation (4,30,57 indicated that a nearly complete tripartite leader is necessary for optimal levels of hybrid mRNA translation during the late phase of infection. However, it was not possible to use these recombinant viruses to assess systematically the contribution of cis control sequences to late promoter activity because the Ela enhancer is located upstream of the hybrid transcription unit and is likely to affect its activity. Moreover, the hybrid transcription units are missing the intron sequences between the leader segments, and thus pote...

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Adenovirus E1a Gene Product Expressed at High Levels in Escherichia coli Is Functional

Cited by 116 publications

References 30 publications

Expression and characterization of the human c-myc DNA-binding protein.

Expression and characterization of the human c-myc DNA-binding protein.

In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.

Downstream sequences affect transcription initiation from the adenovirus major late promoter.

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