DNA sequences affecting specific initiation of transcription in vitro from the EIII promoter of adenovirus 2.

Lee, Dong Hoon; Roeder, Robert G.; Wold, William S.M.

doi:10.1073/pnas.79.1.41

Cited by 39 publications

(11 citation statements)

References 22 publications

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“…This is in contrast to the rabbit P-globin (7), simian virus 40 early region (20), and adenovirus late system (13), where the efficiency of transcription seems to be unaffected or only slightly reduced by deletion of sequences downstream to the TATA box. However, results similar to ours have been obtained with the silk fibroin gene (27) and adenovirus early region III (17). These results suggest the existence of two classes of TATAdependent promotion in the in vitro transcription systems.…”

Section: Discussionsupporting

confidence: 78%

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

Mitsialis¹,

Manley²,

Guntaka³

1983

Mol. Cell. Biol.

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The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.Upon infection of sensitive cells by retroviruses, the genomic RNA is converted into a duplex DNA which is eventually integrated into the host chromosomal DNA (2). In the process of this conversion of the RNA, two long terminal repeats (LTRs) of 350 base pairs (bp), with nucleotide sequences derived from the 5' as well as the 3' ends of the RNA genome, are generated at the termini of the DNA molecule (12,26

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Section: Discussionsupporting

confidence: 78%

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

Mitsialis¹,

Manley²,

Guntaka³

1983

Mol. Cell. Biol.

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“…Presently available systems supporting the PolII-dependent initiation of transcription in vitro include partially fractionated extracts that require the addition of exogenous polymerase (45) and whole cell crude extracts containing endogenous polymerase activity (29). These systems are capable of accurate initiation of transcription on cloned DNA preparations and are able to recognize at least some of the primary DNA sequence signals that are important for correct initiation of transcription in vivo (6,12,16,23,31). They are seriously deficient, however, in the efficiency with which a given promoter is utilized (typically several orders of magnitude lower in terms of RNA transcripts produced per template copy) and in the ability to reproduce the differential levels of transcription observed in vivo from regulated promoters.…”

Section: Construction and Transfection Of Mmtv Ltr-mentioning

confidence: 99%

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

Ostrowski¹,

Richard-Foy²,

Wolford³

et al. 1983

Mol. Cell. Biol.

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The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative Si mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.The regulation of mouse mammary tumor virus (MMTV) expression by glucocorticoids has attracted considerable interest as a model for hormone action (13, 37). The early availability of homogeneous cDNA probes from purified virion preparations led to the demonstration that an increase in newly synthesized virus-specific RNA can be detected very quickly after the addition of hormone to MMTV-producing tumor cells in culture (40, 50), suggesting that RNA levels increase as a direct result of a stimulation in the rate of transcription. The subsequent finding that hormone regulation of MMTV expression is maintained in nonmurine cells chronically infected with MMTV (38) indicated that the virus either encodes the sequences necessary for hormone responsiveness or preferentially integrates in regions of cellular DNA containing such regulatory signals.More recently, the long terminal repeat (LTR) of the provirus has been fused to coding sequences that provide a selective function in mammalian cells, and the resulting chimeras have been introduced into cultured cells by calcium phosphate-mediated transfection. These investigations implied that LTR sequences alone are sufficient to establish hormone regulation of the selectable gene product (17,24). Chimeric molecules are assimilated in these transfection exper...

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“…One of the elements closest to the coding sequences of mammalian genes is an evolutionarily conserved sequence, C/T,CATT,A/G, which straddles DNA sequences complementary to the 5' terminus of the mRNA (the cap site) (8). Although this sequence is dispensible for gene expression in vivo, in vitro experiments have revealed that deletion of sequences surrounding this element reduces the efficiency of transcription by a factor of 10-to 20-fold (38,52). Another sequence also required for transcription in vitro (11,46,61,89) but dispensible for expression in vivo (4) is the GoldbergHogness box or TATA box located between positions -20 and -30 (M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978).…”

mentioning

confidence: 99%

Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

et al. 1984

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To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' unidirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5-to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

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DNA sequences affecting specific initiation of transcription in vitro from the EIII promoter of adenovirus 2.

Cited by 39 publications

References 22 publications

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

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