Expression of the human CMP-NeuAc:GM3 α2,8-sialyltransferase (GD3 synthase) gene through the NF-κB activation in human melanoma SK-MEL-2 cells
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Chromatin immunoprecipitation
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This chapter deals with natural and synthetic promoters frequently used in yeast. It describes principles and strategies exploited to produce synthetic promoters and their cognate transcription factors. The chapter points out the essential structural and functional features of yeast promoters. It highlights some examples of both regulated and constitutive natural yeast promoters. The characterization of these sequences allowed for the identification of structural and functional features that are exploited to build synthetic promoters and heterologous transcription factors. Modification of the binding affinity between the transcription factor and its cognate transcription factor binding site (TFBS) results in promoter strength modulation. The chapter discusses two main groups of synthetic promoters. One includes modified versions of natural promoters, and the other contains hybrid promoters. The chapter also describes Saccharomyces cerevisiae promoters. The characterization of S. cerevisiae promoters began by using them to drive expression of reporter genes.
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In the post-genomic era, identifying and characterzing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Now, chromatin immunoprecipitation assay (ChIP) is ideally suited for investigating DNA-protein interactions in vivo. The conditions of chromatin immunoprecipitation assay was optimized and utilized to screen unknown target genes of transcription factor AP-2 alpha. The results yielded a more complete understanding of AP-2α-regulated signaling pathways and further elucidated the complex transcriptional networks.
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ChIA-PET
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In order to explore the regulation mechanism of fibroin heavy chain gene in silkworm (Bombyx mori),the recombinant AcMNPV was used as the transfer vector and green fluorescent protein (EGFP) gene was used as the reporter gene to investigate the expression profiles of the reporter gene in silkworm tissues of the 5th instar larvae driven by differently truncated promoters of fibroin heavy chain gene (FibH) (-2 127 /+23,-925 /+23 and-238 /+23). The results showed that there was ectopic expression of EGFP in fat body and hemocyte which was driven by three FibH promoters of various lengths. Green fluorescence could be observed directly on silkworm cuticle under fluorescence microscope. The three promoters of various lengths could initiate transcription of EGFP efficiently in posterior silk gland and produced green fluorescent light. Promoters of 0.9KH and 0.2KH could initiate transcription of EGFP in middle silk gland and yielded green fluorescent light. That of 2.1KH could initiate transcription of EGFP but no green fluerescent light was detected. All three promoters could also drive ectopic expression of EGFP in Sf9 cultured cells. The above results revealed that the promoter of fibroin heavy chain gene does not have strict tissue specificity.
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Summary Our limited understanding of plant promoters does not allow us to recognize any core promoter elements for the majority of plant promoters. To understand the promoter architecture of Arabidopsis, we used the combined approach of in silico detection of novel core promoter elements and large‐scale determination of transcription start sites (TSSs). To this end, we developed a novel methodology for TSS identification, using a combination of the cap‐trapper and massively parallel signature sequencing methods. This technique, CT–MPSS, allowed us to identify 158 237 Arabidopsis TSS tags corresponding to 38 311 TSS loci, which provides an opportunity for quantitative analysis of plant promoters. The expression characteristics of these promoters were analyzed with respect to core promoter elements detected by our in silico analyses, revealing that Arabidopsis promoters contain two main types of elements with exclusive characteristics, the TATA type and the GA type. The TATA‐type promoters tend to be associated with the Y Patch and the Inr motif, and cause high expression with sharp‐peak TSS clusters. By contrast, the GA type produces broad‐type TSS clusters. Unlike mammalian promoters, plant promoters are not associated with CpG islands. However, plant‐specific GA‐type promoters share some characteristics with mammalian CpG‐type promoters.
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Fine-tuning of the expression of genes is crucial for cell factory construction. Promoters are the most important tools to control gene expression. However, native promoters are often limited by their transcriptional ability. In this study, we sought to overcome the limitations of native promoters in Saccharomyces cerevisiae through the construction of hybrid promoter libraries for both constitutive promoters and promoters induced by diauxic shift. A series of hybrid constitutive promoters were constructed by combing the upstream activation sequences and changing the core promoter elements. The transcriptional capacity of the strongest promoter was 2-fold higher than that of the yeast native TEF1 promoter. Aside from the constitutive promoters, hybrid promoters that were induced in the post-diauxic phase were also constructed. These promoters had low transcriptional ability during growth on glucose and automatically activated upon growth with a diauxic shift. The strength of these promoters was also increased by replacing the core promoter with strong core promoters. Our study provides a series of constitutive and diauxic shift-induced promoters with a broad range of transcriptional capacity and will facilitate synthetic biology and metabolic engineering application.
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Bidirectional promoters lie between adjacent genes, which are transcribed from opposite strands of DNA. The functional mechanisms underlying the activation of bidirectional promoters are currently uncharacterised. To define the core promoter elements of bidirectional promoters in human, we mapped motifs for TATA, INR, BRE, DPE, INR, as well as CpG-islands. We found a consistently high correspondence between C+G content, CpG-island presence and an average expression level increasing the median level for all genes in bidirectional promoters. These CpG-rich promoters showed discrete initiation patterns rather than broad regions of transcription initiation, as are typically seen for CpG-island promoters. CpG-islands encompass both TSSs within bidirectional promoters, providing an explanation for the symmetrical co-expression patterns of many of these genes. In contrast, TATA motifs appear to be asymmetrically positioned at one TSS or the other. Our findings demonstrate that bidirectional promoters utilize a variety of core promoter elements to initiate transcription. CpG-islands dominate the regulatory landscape of this group of promoters.
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Chloramphenicol acetyltransferase
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SUMMARY The leap from simple unicellularity to complex multicellularity remains one of life's major enigmas. The origins of metazoan developmental gene regulatory mechanisms are sought by analyzing gene regulation in extant eumetazoans, sponges, and unicellular organisms. The main hypothesis of this manuscript is that, developmental enhancers evolved from unicellular inducible promoters that diversified the expression of regulatory genes during metazoan evolution. Promoters and enhancers are functionally similar; both can regulate the transcription of distal promoters and both direct local transcription. Additionally, enhancers have experimentally characterized structural features that reveal their origin from inducible promoters. The distal co‐operative regulation among promoters identified in unicellular opisthokonts possibly represents the precursor of distal regulation of promoters by enhancers. During metazoan evolution, constitutive‐type promoters of regulatory genes would have acquired novel receptivity to distal regulatory inputs from promoters of inducible genes that eventually specialized as enhancers. The novel regulatory interactions would have caused constitutively expressed genes controlling differential gene expression in unicellular organisms to become themselves differentially expressed. The consequence of the novel regulatory interactions was that regulatory pathways of unicellular organisms became interlaced and ultimately evolved into the intricate developmental gene regulatory networks (GRNs) of extant metazoans.
Multicellular organism
Gene regulatory network
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