Construction of artificial promoters sensitively responsive to sonication in vitro
Akihiko WatanabeSatoshi KakutaniRyohei OgawaSung‐il LeeToru YoshidaAkihiro MoriiGo KagiyaLoreto B. FerilHideki FuseTakashi Kondo
11
Citation
24
Reference
10
Related Paper
Citation Trend
Keywords:
Sonication
CAAT box
Chalcone synthase(CHS),coded by the chs gene super-family,is a key enzyme in flavonoid biosynthesis.Two independent promoters was isolated for chsA,named PchsA-L(550 bp) and PchsA-S(354 bp)(GenBank accession number EF199747 and EF199748 respectively),from the genomic DNA of Petunia hybrida.PchsA-L differs with PchsA-S mainly in that PchsA-L has a 182 bp fragment from 88～269 bp,and the sequence 103～201 bp has the characteristics of a typical intron.Both promoter sequences contain conserved sequences of TATA box,CCAAT box,cap site(CCATAA),and the flower-specific promoter sequences TACPyAT box,anther box(TAGAAGTGACAGAAAT),G-box(CACGTG),box1 element(ATGTCACGTGCCATC) and box2 element(TGTGTTGAAGGTTTGCTA).The petunia plant used for promoter cloning was a diploid with 14 chromosomes.Southern blotting showed that both promoters had multiple copies in the genome.The two promoters segregated in the offspring but the segregation did not meet the ratio of 1∶2∶1.qRT-PCR analysis showed no significant difference in chsA gene expression in non-UV-treated and UV-treated floral organ of plants with one or both promoters.The expression of chsA in the UV-treated seedling leaves was increased compared with UV-treated floral organ,and PchsA-L-driven chsA expression in the UV-treated seedling leaves was very significantly increased than that driven by PchsA-S while there was no chsA gene expression in non-UV-treated seedling leaves.The results show the presence of two independent promoters PchsA-L and PchsA-S for chsA in the petunia genome;the 182 bp intron-like sequence in PchsA-L promoter could significantly increased chsA gene expression in UV-treated seedling leaves.
Petunia
TATA box
CAAT box
Cloning (programming)
Cite
Citations (0)
We analyzed an extended core promoter regions covering [-70,+60] segment relative to the transcription start site of human promoters contained in the Eukaryotic Promoter Database. The analysis was made by using the Match program ver. 1.9 with an optimized setting and the TRANSFAC Professional database ver. 7.2. This analysis revealed that the most common transcription factor binding site in the examined collection of core promoters appears to be initiator (characterized by GEN_INI), which is expected. The other less obvious sites found were Spz1, E2F-1, ZF5, and C/EBP. The 'cap' site was also in this most common group. Over-representation of these sites relative to the non-promoter background data ranged from 0.3167 to 32.1645. These sites were characterized by being present in more than 60% of promoter sequences. Interestingly, the TATA-box has been found in only 11.63% of all examined promoters. The study is complemented by separate analyses of promoter groups having different GC content. These additional analyses revealed that the most common promoter elements found also include AP-2, CdxA, Pax-2, SRY, STAT1 and STAT5A. It was also observed that a number of promoter elements show strong preference either for the GC-rich or the GC-poor core promoters.
TATA box
CAAT box
Transcription
Cite
Citations (64)
To improve the protection efficiency of the recombinant Marek' s disease viruses (MDV) in chickens with or without maternal antibodies,the work of selecting the optimal promoters for the construction of recombinant MDV was carried out. Combined with the efficient genetic manipulation, the composed promoters was constructed by use of the MDV gB core promoter with the regulatory elements from the early immediately promoter and enhancer of hCMV, the promoter and enhancer of SV40 or the partial enhancer of hCMV. And these composed promoters were ligased to the luciferase to construct the eukaryotic expressing vectors and named PhCMV-gB, Psv-gB and Pen-gB, respectively. In vitro, these vectors and internal standard plasmid (pSV-beta-LacZ) were transiently co-transfected into secondary CEF by FuGene 6 Transfection Reagent. Furthermore, cells were harvested 48 hours after transfection. Then the luciferase activity was detected by a luciferase assay kit, at the same time, the beta-galactosidase enzyme activity was detected by a beta-galactosidase enzyme assay kit, and the luciferase activity was corrected by the beta-galactosidase enzyme activity to get the relative luciferase activity. The relative luciferase activity was used as the transcriptional activity. By comparison of the relative luciferase activity of every promoter, it was found that these composed promoters could more effectively drive the reporter gene expression than the full legth of gB promoter did. Among them, PhCMV-gB robustly drove the reporter gene expression. On the other hand, PSV-gB and Pen-gB appeared to have the same strength; But compared with the commercial strong promoters, the transcriptional activity of the composed promoter were less than as or the same as that of the strong promoters. Therefore, at a sense, it can be proposed that these composed promoters have not only the characteristic of MDV gB promoter, but also that of the commercial strong promoters. These provide the choices for further developing the new-type recombinant MDV vaccine.
Cite
Citations (0)
We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.
TATA box
CAAT box
Transcription
Cite
Citations (114)
In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.
CAAT box
TATA box
FOXO1
DNA binding site
Transcription
Cite
Citations (11)
[Objective] The ras promoters were cloned from genomic DNA of Auricuralia auricular so as to provide promoters for breeding better species by genetic technology.[Method] PCR was used to clone promoters with the genomic DNA of A.auricular strain YBS-3 as template,and then the sequences of four ras promoters were obtained.The analysis of promoter sequences was made by three promoter analysis software:Promoter prediction,Place and TFSEARCH ver.1.3.[Result] Four fragments contained core elements of promoter including TATA-box and CAAT-box,and many other important cis-elements such as GATA BOX,GCGC BOX,CCAAT BOX1,etc.At the same time,there was at least one transcriptional start site in these sequences.And several transcription factor binding sites were detected in these sequences.[Conclusion] Four promoter fragments were all having promoter function theoretically.
TATA box
CAAT box
Cloning (programming)
Transcription
DNA binding site
genomic DNA
Cite
Citations (0)
Promoter is an important component of genes that determines the transcription of mRNA to achieve the orderly development and differentiation of organisms. In present study,we identified 1 341 promoters in silkworm( Bombyx mori) genomic and full-length cDNA sequence databases. Sequence analysis showed that GC content and AT content had one and two peak values in the promoter region from- 500 to 200 bp,respectively. Elements of TATA-box,Initiator( Inr),CAAT-box and GC-box were present in 256,453,200 and 334 promoters,respectively. The first three elements were dominantly located in the region from- 500 to 200 bp,and the last element was mostly located at the upstream far from- 1 600 to- 300 bp. Silkworm-based genomic microarray data showed that 23 promoters are for testis-biased gene expression. Cis-element analysis indicated that these promoters for testis-biased expression had 6 predominant motifs,among which 3 motifs had significantly higher occurrence than the promoters for non-tissue specific expression,suggesting that these 3 motifs play an important role in the regulation of testis-biased gene transcription.
TATA box
CAAT box
Transcription
Cite
Citations (0)
The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.
TATA box
DNA binding site
CAAT box
TATA-binding protein
Transcription
General transcription factor
Cite
Citations (33)
CAAT box
Cite
Citations (12)
CAAT box
TATA box
Upstream activating sequence
Cite
Citations (68)