scholarly journals Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

1986 ◽  
Vol 6 (12) ◽  
pp. 4697-4708 ◽  
Author(s):  
R H Costa ◽  
E Lai ◽  
J E Darnell
Keyword(s):  
Hela Cells ◽  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Proximal Promoter ◽  
Start Site ◽  
Beta Globin ◽  
Human Hepatoma Cells ◽  
Enhancer Element ◽  
A Cell

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific

1986 ◽  
Vol 6 (12) ◽  
pp. 4697-4708
Author(s):  
R H Costa ◽  
E Lai ◽  
J E Darnell
Keyword(s):  
Hela Cells ◽  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Proximal Promoter ◽  
Start Site ◽  
Beta Globin ◽  
Human Hepatoma Cells ◽  
Enhancer Element ◽  
A Cell

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Control of adenovirus late promoter expression in two human cell lines

1985 ◽  
Vol 5 (9) ◽  
pp. 2433-2442
Author(s):  
E D Lewis ◽  
J L Manley
Keyword(s):  
Hela Cells ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Enhancer Element ◽  
Cis Acting ◽  
Early Region ◽  
293 Cells

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.

scholarly journals Control of adenovirus late promoter expression in two human cell lines.

1985 ◽  
Vol 5 (9) ◽  
pp. 2433-2442 ◽  
Author(s):  
E D Lewis ◽  
J L Manley
Keyword(s):  
Hela Cells ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Enhancer Element ◽  
Cis Acting ◽  
Early Region ◽  
293 Cells

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.

scholarly journals Role for DNA replication in beta-globin gene activation.

1988 ◽  
Vol 8 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
T Enver ◽  
A C Brewer ◽  
R K Patient
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Covalent Modification ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Template Copy

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

Comparison of intron-dependent and intron-independent gene expression

1988 ◽  
Vol 8 (10) ◽  
pp. 4395-4405
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Fold Increase ◽  
Simian Virus ◽  
Transcription Unit ◽  
Vector System ◽  
Beta Globin ◽  
Animal Cells ◽  
Transcriptional Enhancer ◽  
Kinase Gene

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

A novel myoblast enhancer element mediates MyoD transcription

1992 ◽  
Vol 12 (11) ◽  
pp. 4994-5003
Author(s):  
S J Tapscott ◽  
A B Lassar ◽  
H Weintraub
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Leukemia Virus ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Enhancer Element ◽  
Distal Regulatory Region ◽  
Avian Muscle ◽  
Moloney Leukemia Virus

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function

1983 ◽  
Vol 3 (7) ◽  
pp. 1295-1309
Author(s):  
R W Scott ◽  
S M Tilghman
Keyword(s):  
Transient Expression ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Alpha Fetoprotein ◽  
Tata Box ◽  
Deletion Mutants ◽  
Promoter Function ◽  
Flanking Region

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

A negative regulatory element with properties similar to those of enhancers is contained within an Alu sequence

1989 ◽  
Vol 9 (2) ◽  
pp. 355-364
Author(s):  
J D Saffer ◽  
S J Thurston
Keyword(s):  
Nuclear Protein ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Bidirectional Promoter ◽  
Simian Virus ◽  
Beta Globin ◽  
Base Pairs ◽  
Negative Regulatory Element ◽  
Alu Sequence ◽  
Green Monkey

A negative regulatory element has been found within a member of the African green monkey Alu family of interspersed repeated sequences. This "reducer" element decreased transcription in both directions from a cellular simian virus 40-like bidirectional promoter independently of both orientation and position. The reducer was not promoter specific since it also decreased expression from the simian virus 40 early and human beta-globin promoters. In addition, the reducer decreased transcription from a polymerase III promoter. The reducer was contained in 38 base pairs of an Alu family member and interacted specifically with a monkey cell nuclear protein.

scholarly journals Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4484-4491 ◽  
Author(s):  
A Mayeda ◽  
Y Ohshima
Keyword(s):  
Consensus Sequence ◽  
Donor Site ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Beta Globin ◽  
Vitro Assay ◽  
Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

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