scholarly journals Cell-specific expression of the human gastrin gene: evidence for a control element located downstream of the TATA box.

1987 ◽  
Vol 7 (12) ◽  
pp. 4329-4336 ◽  
Author(s):  
L E Theill ◽  
O Wiborg ◽  
J Vuust

Fragments of 5'-flanking and noncoding exon I sequences of the human gastrin gene were analyzed in transient expression assays after transfection of a variety of cell lines with the pSVCAT vector system. In the presence of the simian virus 40 (SV40) enhancer, the gastrin gene fragment from nucleotides -250 to +57, relative to the cap site, was as efficient a promoter as the SV40 early promoter itself. In the absence of the SV40 enhancer, gastrin gene 5'-flanking sequences had no promoter activity except in the murine neuroblastoma cell line N18TG2. In this cell line, the fragment from -1300 to +57 stimulated transcription as actively as the SV40 early promoter with its enhancer. This cell-specific gastrin gene promoter activity was in accordance with the finding that gastrin is synthesized in certain neuronal cells. Promoter activity declined with decreasing distance from the 5' end to the cap site and disappeared after removal of the gastrin gene TATA box. In vector constructions containing short vector-linker sequences homologous to a functionally important region of the SV40 enhancer, the gastrin gene fragment from -17 to +57 showed considerable promoter activity, exclusively in N18TG2. It is concluded that the truncated gastrin gene promoter plus the first exon contains a cell-specific element that may act in collaboration with upstream elements to facilitate the accumulation of transcripts.

1987 ◽  
Vol 7 (12) ◽  
pp. 4329-4336
Author(s):  
L E Theill ◽  
O Wiborg ◽  
J Vuust

Fragments of 5'-flanking and noncoding exon I sequences of the human gastrin gene were analyzed in transient expression assays after transfection of a variety of cell lines with the pSVCAT vector system. In the presence of the simian virus 40 (SV40) enhancer, the gastrin gene fragment from nucleotides -250 to +57, relative to the cap site, was as efficient a promoter as the SV40 early promoter itself. In the absence of the SV40 enhancer, gastrin gene 5'-flanking sequences had no promoter activity except in the murine neuroblastoma cell line N18TG2. In this cell line, the fragment from -1300 to +57 stimulated transcription as actively as the SV40 early promoter with its enhancer. This cell-specific gastrin gene promoter activity was in accordance with the finding that gastrin is synthesized in certain neuronal cells. Promoter activity declined with decreasing distance from the 5' end to the cap site and disappeared after removal of the gastrin gene TATA box. In vector constructions containing short vector-linker sequences homologous to a functionally important region of the SV40 enhancer, the gastrin gene fragment from -17 to +57 showed considerable promoter activity, exclusively in N18TG2. It is concluded that the truncated gastrin gene promoter plus the first exon contains a cell-specific element that may act in collaboration with upstream elements to facilitate the accumulation of transcripts.


Author(s):  
Katarzyna Billing-Marczak ◽  
Leonora Buzanska ◽  
Lois Winsky ◽  
Marcin Nowotny ◽  
Tomasz Rudka ◽  
...  

2007 ◽  
Vol 32 (1) ◽  
pp. 60-66 ◽  
Author(s):  
Masahiro Ikeda ◽  
Masahiro Kakuyama ◽  
Takehiro Shoda ◽  
Kazuhiko Fukuda

1998 ◽  
Vol 72 (1) ◽  
pp. 609-616 ◽  
Author(s):  
Ralph Gareus ◽  
Andreas Gigler ◽  
Andrea Hemauer ◽  
Marianne Leruez-Ville ◽  
Frédéric Morinet ◽  
...  

ABSTRACT Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-κB/c-Rel, and GC-box binding cellular factors.


2004 ◽  
Vol 33 (1) ◽  
pp. 51-58 ◽  
Author(s):  
G Canettieri ◽  
A Franchi ◽  
R Sibilla ◽  
E Guzman ◽  
M Centanni

The regulation of expression of type II deiodinase (D2) is a critical mechanism to maintain appropriate intracellular concentrations of tri-iodothyronine in selected tissues. One of the major regulators of D2 concentrations is cAMP, which potently increases human type II deiodinase (hD2) gene transcription in some tissues via a conserved cAMP response element (CRE) located in the promoter region. In addition, the regulatory region of the hD2 gene contains several TATA box/transcription start site (TSS) units, suggesting the presence of different transcripts that might be characterised by different biological properties. However, it is still unclear whether one ore more TATA box/TSS units are needed in response to cAMP or to other signals able to modulate hD2 transcription. In this study we have analysed the ability of cAMP to regulate hD2 in JEG3 cells, a human choriocarcinoma cell line highly responsive to cAMP. Transient transfection assays of different hD2 gene promoter constructs revealed that cAMP induces transcription starting from the most 5' TSS, located about 80 nucleotides from the CRE. RT-PCR studies have revealed that cAMP activates the expression of a long-lived transcript in JEG3 cells. Site-directed mutagenesis and deletion analysis of promoter constructs have shown that a single CRE/TATA box/TSS unit is needed to confer responsiveness to cAMP. By using chromatin immunoprecipitation studies, we have also demonstrated that the response to cAMP involves the binding of transcription factor CRE binding protein (CREB) to the CRE located in the hD2 promoter. In summary, in JEG3 cells cAMP induces transcription of a long-lived hD2 RNA via CREB and a single CRE/TATA box/TSS unit. This study provides new insights to the regulation of expression of hD2 in placenta.


2021 ◽  
Vol 12 ◽  
Author(s):  
Li Xu ◽  
Zhiying Li ◽  
Sheng Wang

Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.


2010 ◽  
Vol 84 (13) ◽  
pp. 6308-6317 ◽  
Author(s):  
Aspen Workman ◽  
Clinton Jones

ABSTRACT Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen of cattle. Like other members of the subfamily Alphaherpesvirinae, BoHV-1 establishes latency in sensory neurons and has the potential to reactivate from latency. Dexamethasone (DEX) treatment of latently infected calves or rabbits consistently leads to reactivation from latency. The BoHV-1 transcript encoding the infected cell protein 0 (bICP0) is consistently detected during reactivation from latency, in part because the bICP0 early promoter is activated by DEX. During DEX-induced reactivation from latency, cyclin expression is stimulated in infected sensory neurons. Cyclin-dependent kinase activity phosphorylates Rb (retinoblastoma tumor suppressor gene product) family proteins and consequently releases the E2F family of transcription factors, suggesting that E2F family members stimulate productive infection and/or reactivation from latency. In this study, we provide evidence that repression of E2F1 by a specific small interfering RNA (siRNA) reduced productive infection approximately 5-fold. E2F1 or E2F2 stimulated bICP0 early promoter activity at least 100-fold in transient transfection assays. Two E2F-responsive regions (ERR) were identified within the early promoter, with one adjacent to the TATA box (ERR1) and one approximately 600 bp upstream from the TATA box (ERR2). Mobility shift assays suggested that E2F interacts with ERR1 and ERR2. E2F1 protein levels were increased at late times after infection, which correlated with enhanced binding to a consensus E2F binding site, ERR1, or ERR2. Collectively, these studies suggest that E2F1 stimulates productive infection and bICP0 early promoter activity, in part because E2F family members interact with ERR1 and ERR2.


2007 ◽  
Vol 558 (1-3) ◽  
pp. 1-6 ◽  
Author(s):  
Masahiro Ikeda ◽  
Masahiro Kakuyama ◽  
Takehiro Shoda ◽  
Yasumasa Iwasaki ◽  
Kazuhiko Fukuda

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