Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells

1986 ◽  
Vol 6 (1) ◽  
pp. 201-208
Author(s):  
T Leff ◽  
P Chambon
Keyword(s):  
Hela Cells ◽  
Upstream Region ◽  
Gene Products ◽  
Promoter Regions ◽  
Chimeric Promoter ◽  
293 Cells ◽  
Activation Of Transcription ◽  
Upstream Promoter ◽  
Sequence Elements ◽  
Responsive Element

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.

scholarly journals Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells.

1986 ◽  
Vol 6 (1) ◽  
pp. 201-208 ◽  
Author(s):  
T Leff ◽  
P Chambon
Keyword(s):  
Hela Cells ◽  
Upstream Region ◽  
Gene Products ◽  
Promoter Regions ◽  
Chimeric Promoter ◽  
293 Cells ◽  
Activation Of Transcription ◽  
Upstream Promoter ◽  
Sequence Elements ◽  
Responsive Element

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.

scholarly journals Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

2008 ◽  
Vol 100 (05) ◽  
pp. 821-828 ◽  
Author(s):  
Gauthami Jalagadugula ◽  
Danny N. Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts ◽  
Upstream Promoter ◽  
Hel Cells ◽  
The Difference ◽  
Responsive Element ◽  
Increased Activity

SummaryGαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5’ upstream region of the Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 5’ upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt-1042/-1037 eliminated the difference. Gel-shift studies on Gαq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence –1042/-1037.Silencing of endogenous EGR-1 inhibited Gαq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence –1042/-1037 bp in Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

scholarly journals Identification of a Methylation Imprint Mark within the Mouse Gnas Locus

2000 ◽  
Vol 20 (16) ◽  
pp. 5808-5817 ◽  
Author(s):  
Jie Liu ◽  
Shuhua Yu ◽  
Deborah Litman ◽  
Weiping Chen ◽  
Lee S. Weinstein
Keyword(s):  
Expression Patterns ◽  
Differential Methylation ◽  
Upstream Region ◽  
Gene Products ◽  
Alternative Promoters ◽  
Promoter Regions ◽  
Α Subunit ◽  
Somatic Tissues ◽  
Specific Mrnas ◽  
Translational Start

ABSTRACT The imprinted mouse gene Gnas produces the G protein α-subunit GSα and several other gene products by using alternative promoters and first exons. GSα is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLαs are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The GSα promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions −3400 to −939 relative to the GSα translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and GSα mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3.5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLαs promoter regions (Nespand Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of GSα.

scholarly journals Sequence Elements Necessary for Transcriptional Activation of BAD1 in the Yeast Phase of Blastomyces dermatitidis

2004 ◽  
Vol 3 (3) ◽  
pp. 785-794 ◽  
Author(s):  
Peggy J. Rooney ◽  
Bruce S. Klein
Keyword(s):  
Transcriptional Activation ◽  
Histoplasma Capsulatum ◽  
Upstream Region ◽  
Blastomyces Dermatitidis ◽  
Specific Gene ◽  
Specific Expression ◽  
Sequence Elements ◽  
Putative Transcription Factor ◽  
Yeast Phase ◽  
Responsive Element

ABSTRACT Blastomyces dermatitidis is a dimorphic fungal pathogen that converts from mycelia or conidia to a host-adapted yeast morphotype upon infection. Conversion to the yeast form is accompanied by the production of the virulence factor BAD1. Yeast-phase-specific expression of BAD1 is transcriptionally regulated, and its promoter shares homology with that of the yeast-phase-specific gene YPS3 of Histoplasma capsulatum. Serial truncations of the BAD1 upstream region were fused to the lacZ reporter to define functional areas in the promoter. Examination of P BAD1 -lacZ fusions in B. dermatitidis indicated that BAD1 transcription is upregulated in the yeast phase. The 63-nucleotide box A region conserved in the YPS3 upstream region was shown to be an essential component of the minimal BAD1 promoter. A matched P YPS3 -lacZ construct indicated that this same region was needed for minimal YPS3 promoter activity in B. dermatitidis transformants. Reporter activity in H. capsulatum transformants similarly showed a requirement for box A in the minimal BAD1 promoter. Several putative transcription factor binding sites were identified within box A of BAD1. Replacement of two of these predicted sites within box A—a cAMP responsive element and a Myb binding site—sharply reduced transcriptional activity, indicating that these regions are critical in dictating the yeast-phase-specific expression of this crucial virulence determinant of B. dermatitidis.

ZFARED: A Database of the Antioxidant Response Elements in Zebrafish

2020 ◽  
Vol 15 (5) ◽  
pp. 415-419
Author(s):  
Azhwar Raghunath ◽  
Raju Nagarajan ◽  
Ekambaram Perumal
Keyword(s):  
Target Genes ◽  
Antioxidant Response ◽  
Zebrafish Genome ◽  
Promoter Regions ◽  
Protein Coding ◽  
Response Elements ◽  
Toxic Agents ◽  
Upstream Promoter ◽  
Its Gene ◽  
Antioxidant Response Elements

Background: Antioxidant Response Elements (ARE) play a key role in the expression of Nrf2 target genes by regulating the Keap1-Nrf2-ARE pathway, which offers protection against toxic agents and oxidative stress-induced diseases. Objective: To develop a database of putative AREs for all the genes in the zebrafish genome. This database will be helpful for researchers to investigate Nrf2 regulatory mechanisms in detail. Methods: To facilitate researchers functionally characterize zebrafish AREs, we have developed a database of AREs, Zebrafish Antioxidant Response Element Database (ZFARED), for all the protein-coding genes including antioxidant and mitochondrial genes in the zebrafish genome. The front end of the database was developed using HTML, JavaScript, and CSS and tested in different browsers. The back end of the database was developed using Perl scripts and Perl-CGI and Perl- DBI modules. Results: ZFARED is the first database on the AREs in zebrafish, which facilitates fast and efficient searching of AREs. AREs were identified using the in-house developed Perl algorithms and the database was developed using HTML, JavaScript, and Perl-CGI scripts. From this database, researchers can access the AREs based on chromosome number (1 to 25 and M for mitochondria), strand (positive or negative), ARE pattern and keywords. Users can also specify the size of the upstream/promoter regions (5 to 30 kb) from transcription start site to access the AREs located in those specific regions. Conclusion: ZFARED will be useful in the investigation of the Keap1-Nrf2-ARE pathway and its gene regulation. ZFARED is freely available at http://zfared.buc.edu.in/.

scholarly journals Rice protein-binding microarrays: a tool to detect cis-acting elements near promoter regions in rice

Planta ◽  
2021 ◽  
Vol 253 (2) ◽  
Author(s):  
Joung Sug Kim ◽  
SongHwa Chae ◽  
Kyong Mi Jun ◽  
Gang-Seob Lee ◽  
Jong-Seong Jeon ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Binding ◽  
Gene Networks ◽  
Upstream Region ◽  
Transcriptional Level ◽  
Cis Elements ◽  
Promoter Regions ◽  
Entire Genome ◽  
Binding Motifs ◽  
Dna Binding Motifs

Abstract Main conclusion The present study showed that a rice (Oryza sativa)-specific protein-binding microarray (RPBM) can be applied to analyze DNA-binding motifs with a TF where binding is evaluated in extended natural promoter regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements. Abstract Transcription factors (TFs) regulate gene expression at the transcriptional level by binding a specific DNA sequence. Thus, predicting the DNA-binding motifs of TFs is one of the most important areas in the functional analysis of TFs in the postgenomic era. Although many methods have been developed to address this challenge, many TFs still have unknown DNA-binding motifs. In this study, we designed RPBM with 40-bp probes and 20-bp of overlap, yielding 49 probes spanning the 1-kb upstream region before the translation start site of each gene in the entire genome. To confirm the efficiency of RPBM technology, we selected two previously studied TFs, OsWOX13 and OsSMF1, and an uncharacterized TF, OsWRKY34. We identified the ATTGATTG and CCACGTCA DNA-binding sequences of OsWOX13 and OsSMF1, respectively. In total, 635 and 932 putative feature genes were identified for OsWOX13 and OsSMF1, respectively. We discovered the CGTTGACTTT DNA-binding sequence and 195 putative feature genes of OsWRKY34. RPBM could be applicable in the analysis of DNA-binding motifs for TFs where binding is evaluated in the promoter and 5′ upstream CDS regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements.

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 775-787 ◽  
Author(s):  
Hannes Nahrstedt ◽  
Christine Schröder ◽  
Friedhelm Meinhardt
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Mitomycin C ◽  
Bacillus Megaterium ◽  
Functional Complementation ◽  
Homologous Gene ◽  
Gene Products ◽  
Null Mutant ◽  
Promoter Regions ◽  
Increased Sensitivity

Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

scholarly journals The Role of EjSVPs in Flower Initiation in Eriobotrya japonica

2019 ◽  
Vol 20 (23) ◽  
pp. 5933 ◽  
Author(s):  
Yuanyuan Jiang ◽  
Jiangrong Peng ◽  
Zhike Zhang ◽  
Shoukai Lin ◽  
Shunquan Lin ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Expression Patterns ◽  
Active Role ◽  
Eriobotrya Japonica ◽  
Flower Initiation ◽  
Mads Box ◽  
Promoter Regions ◽  
Expression Levels ◽  
Flowering Regulation ◽  
Responsive Element

Flowering plants have evolved different flowering habits to sustain long-term reproduction. Most woody trees experience dormancy and then bloom in the warm spring, but loquat blooms in the cold autumn and winter. To explore its mechanism of flowering regulation, we cloned two SHORT VEGETATIVE PHASE (SVP) homologous genes from ‘Jiefanzhong’ loquat (Eriobotrya japonica Lindl.), namely, EjSVP1 and EjSVP2. Sequence analysis revealed that the EjSVPs were typical MADS-box transcription factors and exhibited a close genetic relationship with other plant SVP/DORMANCY-ASSOCIATED MADS-BOX (DAM) proteins. The temporal and spatial expression patterns showed that EjSVP1 and EjSVP2 were mainly expressed in the shoot apical meristem (SAM) after the initiation of flowering; after reaching their highest level, they gradually decreased with the development of the flower until they could not be detected. EjSVP1 expression levels were relatively high in young tissues, and EjSVP2 expression levels were relatively high in young to mature transformed tissues. Interestingly, EjSVP2 showed relatively high expression levels in various flower tissues. We analyzed the EjSVP promoter regions and found that they did not contain the C-repeat/dehydration-responsive element. Finally, we overexpressed the EjSVPs in wild-type Arabidopsis thaliana Col-0 and found no significant changes in the number of rosette leaves of Arabidopsis thaliana; however, overexpression of EjSVP2 affected the formation of Arabidopsis thaliana flower organs. In conclusion, EjSVPs were found to play an active role in the development of loquat flowering. These findings may provide a reference for exploring the regulation mechanisms of loquat flowering and the dormancy mechanisms of other plants.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

1984 ◽  
Vol 4 (10) ◽  
pp. 2072-2081
Author(s):  
J D Tratschin ◽  
M H West ◽  
T Sandbank ◽  
B J Carter
Keyword(s):  
Hela Cells ◽  
High Efficiency ◽  
Constitutive Expression ◽  
Chloramphenicol Acetyltransferase ◽  
Human Cells ◽  
Foreign Gene ◽  
Similar Amount ◽  
Adeno Associated Virus ◽  
293 Cells ◽  
Bacterial Plasmid

We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

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