Upstream activating sequence

About: Upstream activating sequence is a research topic. Over the lifetime, 1633 publications have been published within this topic receiving 100112 citations.

Promoter architecture and response to a positive regulator of archaeal transcription

Abstract: The archaeal transcription apparatus is chimeric: its core components (RNA polymerase and basal factors) closely resemble those of eukaryotic RNA polymerase II, but the putative archaeal transcriptional regulators are overwhelmingly of bacterial type. Particular interest attaches to how these bacterial-type effectors, especially activators, regulate a eukaryote-like transcription system. The hyperthermophilic archaeon Methanocaldococcus jannaschii encodes a potent transcriptional activator, Ptr2, related to the Lrp/AsnC family of bacterial regulators. Ptr2 activates rubredoxin 2 (rb2) transcription through a bipartite upstream activating site (UAS), and conveys its stimulatory effects on its cognate transcription machinery through direct recruitment of the TATA binding protein (TBP). A functional dissection of the highly constrained architecture of the rb2 promoter shows that a 'one-site' minimal UAS suffices for activation by Ptr2, and specifies the required placement of this site. The presence of such a simplified UAS upstream of the natural rubrerythrin (rbr) promoter also suffices for positive regulation by Ptr2 in vitro, and TBP recruitment remains the primary means of transcriptional activation at this promoter.

Hypoxia Inducible Double Plasmid System for Myocardial Ischemia Gene Therapy

Abstract: Coronary artery disease frequently involves repeated bouts of myocardial ischemia. To automatically up-regulate the cardioprotective transgenes under hypoxic ischemia, a "vigilant vector" gene therapy system was developed and tested in a rat embryonic myocardial cell line (H9c2). In the vigilant vector, a hypoxia response element-incorporated promoter was used as a switch to turn on the gene expression in response to hypoxic signal. Furthermore, a novel double plasmid system was designed to elevate the potency of the vigilant vector. Instead of putting the promoter and the reporter gene in the same plasmid (single plasmid system), we separated them into two plasmids: the transactivator plasmid and reporter plasmid (double plasmid system). The hypoxia response element (HRE)-incorporated promoter increased the expression of a chimeric transcription factor consisting of the yeast GAL4 DNA binding domain and the human nuclear (transcription) factor-kappaB (NF-kappaB) p65 activation domain. The powerful chimeric regulator binds specifically to the upstream activating sequence for GAL4 in the reporter plasmid and activates the transcription of the transgene. Our experiments showed that the HRE-mediated expression could quickly increase 2.08 +/- 0.75-fold within 6 hours of hypoxia and further augmented 7.12 +/- 1.52-fold when the hypoxia condition was prolonged to 24 hours. The hypoxia-inducible double plasmid system dramatically amplified the transgene expression under both hypoxia and normoxia by 412.79 +/- 185.27-fold and 205.35 +/- 65.44-fold, respectively, relative to the single plasmid system. From these results, we concluded that this hypoxia inducible double plasmid system could be used therapeutically to switch on genes that have proven beneficial effects in myocardial ischemia.

Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element.

Abstract: The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a −12/−24 σN-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5’ end to the RNA was identified at position 641, 12 bp downstream from the −12/−24 promoter. Footprinting experiments showed that the G residues at positions −26 and −9 are hypermethylated, and that the region from −10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the σN-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

Identification of the DNA sequences controlling the expression of the MAT alpha locus of yeast.

Abstract: We have excised a 28-base-pair DNA fragment from the MAT alpha intergenic region and tested its ability to direct diploid-specific transcriptional repression. This fragment (1643-1671, 5'-GCTTCCCAATGTAGAAAAGTACA-TCATA-3') lies within a region required for the normal diploid-specific repression of the MAT alpha transcripts. First, the fragment was inserted into a 53-base-pair MAT alpha deletion that expresses alpha 1 and alpha 2 constitutively. Insertion of the fragment restores proper diploid regulation to the MAT alpha transcripts: alpha 1 mRNA is strongly repressed and alpha 2 mRNA is reduced by a factor of approximately equal to 10 from its haploid level. The fragment works equally well in either orientation, and two copies of the fragment do not lead to stronger repression than a single copy. We also inserted the fragment at three sites upstream of the CYC1-lacZ fusion gene. Insertions placing the regulatory fragment between the CYC1 upstream activator sequence (UAS) and the coding region make beta-galactosidase efficiently in alpha haploids but produce 1/40th the enzyme in a/alpha diploids. This diploid-specific repression requires functional MATa-1 gene product. Insertion of the MAT fragment on the opposite side of the UAS (37 base pairs upstream of the UAS) also caused diploid repression of the fusion gene, but only by a factor of 7. When the regulatory fragment is inserted at a large distance on the far side of the UAS (375 base pairs), it has little if any effect on beta-galactosidase expression. We postulate that this sequence is the operator recognized by the diploid-specific repressor.