scholarly journals Integrated Self-Inactivating Lentiviral Vectors Produce Full-Length Genomic Transcripts Competent for Encapsidation and Integration

2004 ◽  
Vol 78 (16) ◽  
pp. 8421-8436 ◽  
Author(s):  
Aaron C. Logan ◽  
Dennis L. Haas ◽  
Tal Kafri ◽  
Donald B. Kohn
Keyword(s):  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Full Length ◽  
Leader Region ◽  
Packaging Cell Line ◽  
Promoter Assay ◽  
Packaging Cell ◽  
Hiv 1

ABSTRACT To make human immunodeficiency virus type 1 (HIV-1)-based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this deletion are thought to have no LTR-directed transcription and are called self-inactivating (SIN) lentivectors. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5′ LTR, which is responsible for the production of full-length vector transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a SIN lentivector and then observed the production of viable vector particles containing full-length SIN lentivector genomes. We have also attempted to identify sequences in the SIN lentivector which are responsible for transcriptional activation at the 5′ LTR. Using different segments of the vector LTR and leader region in a promoter assay, we have determined that the residual promoter activity is contained entirely within the leader region and that, although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5′ end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators downstream binding factor 1 (DBF1) and SP1 reduces transcription from the SIN LTR by up to 80%. Knowledge of the potential for mobilization of HIV-1-derived SIN lentivectors will be important for the design of future gene therapy trials with such vectors.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals α1-Adrenergic stimulation of FGF-2 promoter in cardiac myocytes and in adult transgenic mouse hearts

1999 ◽  
Vol 276 (3) ◽  
pp. H826-H833 ◽  
Author(s):  
Karen A. Detillieux ◽  
Johanna T. A. Meij ◽  
Elissavet Kardami ◽  
Peter A. Cattini
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Intraperitoneal Administration ◽  
Initiation Site ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Cardiomyocytes ◽  
Stimulation Of

Fibroblast growth factor (FGF-2), a mitogenic, angiogenic, and cardioprotective agent, is reported to be released from the postnatal heart by a mechanism of transient remodeling of the sarcolemma during contraction. This release can be increased with adrenergic stimulation. RNA blotting was used to assess whether FGF-2 synthesis in neonatal rat cardiomyocytes might also be regulated by adrenergic stimulation. FGF-2 RNA levels were increased after treatment with norepinephrine for 6 h or with the α-adrenergic agonist phenylephrine for 48 h. To assess an effect on transcription, neonatal rat cardiomyocytes were transfected with a hybrid rat FGF-2 promoter/luciferase gene (−1058FGFp. luc) and treated with norepinephrine or phenylephrine for 6 or 48 h, respectively. FGF-2 promoter activity was increased two- to sevenfold in an α1-specific manner. Putative phenylephrine-responsive elements (PEREs) were identified at positions −780 and −761 relative to a major transcription initiation site. However, deletion analysis of −1058FGFp. luc showed that the phenylephrine response was independent of the putative PEREs, cell contraction, and Ca2+ influx. In transgenic mice expressing −1058FGFp. luc, a significant three- to sevenfold stimulation of FGF-2 promoter activity was detected in the hearts of two independent lines 6 h after intraperitoneal administration of phenylephrine (50 mg/kg). This increase was still apparent at 24 h but was not detected at 48 h posttreatment. Analysis of FGF-2 mRNA in normal mouse hearts revealed accumulation of the 6.1-kb transcript at 24 h. Control of local FGF-2 synthesis at the transcriptional level through adrenergic stimulation may be important in the response to injury as well as in the maintenance of a healthy myocardium.

scholarly journals Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region

2000 ◽  
Vol 74 (17) ◽  
pp. 8176-8182 ◽  
Author(s):  
Ramón García-Escudero ◽  
Eladio Viñuela
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
African Swine Fever Virus ◽  
Point Mutations ◽  
African Swine Fever ◽  
Initiation Site ◽  
Fever Virus ◽  
Transcriptional Initiation ◽  
Viral Promoters ◽  
Tata Sequence

ABSTRACT A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show thatcis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.

Characterization of a chicken retinoid X receptor-γ gene promoter and identification of sequences that direct expression in retinal cells

2000 ◽  
Vol 347 (2) ◽  
pp. 485-490
Author(s):  
Clara AMEIXA ◽  
Paul M. BRICKELL
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Neural Retina ◽  
Specific Protein ◽  
Retinoid X Receptor ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Enhancer Element ◽  
Extracellular Signals

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

2010 ◽  
Vol 84 (21) ◽  
pp. 11470-11478 ◽  
Author(s):  
Baoling Ying ◽  
Ann E. Tollefson ◽  
William S. M. Wold
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Mrna Level ◽  
A549 Cells ◽  
Initiation Site ◽  
Transcription Unit ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Ccaat Box

ABSTRACT We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp −158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.

scholarly journals Transcriptional Activation of the Integrated Chromatin-Associated Human Immunodeficiency Virus Type 1 Promoter

1998 ◽  
Vol 18 (5) ◽  
pp. 2535-2544 ◽  
Author(s):  
Aboubaker El Kharroubi ◽  
Graziella Piras ◽  
Ralf Zensen ◽  
Malcolm A. Martin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-κB sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-κB was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-κB failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-κB, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-κB. Taken together, these results suggest that Sp1 and NF-κB are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.

A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3083-3091 ◽  
Author(s):  
Masato Kaji ◽  
Osamu Matsushita ◽  
Eiji Tamai ◽  
Shigeru Miyata ◽  
Yuki Taniguchi ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Clostridium Pasteurianum ◽  
Dna Curvature ◽  
Combined Effects ◽  
Gene Characterization

This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the −35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions −69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.

scholarly journals Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839 ◽  
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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