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.

Architecture of the sporulation-specific Cdc14 promoter from the oomycete Phytophthora infestans.

Abstract: The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts Evidence was also obtained for a second region within the promoter that activates Cdc14 transcription during sporulation which does not involve those repeats The CTYAAC motif also affects the specificity of transcription initiation Wild-type Cdc14 is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific Replacing the Cdc14 initiation region with the Inr-like region of the constitutive Piexo1 gene had no apparent effect on the pattern of transcription Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters

Immediate upstream promoter regions required for neurospecific expression of SNAP-25

Abstract: The promoter structure and regulation of Snap, a gene encoding the presynaptic t-SNARE SNAP-25 implicated in synaptic vesicle docking and fusion, was studied. Transcription start-site analysis revealed two major start sites located 42 nucleotides apart. Nucleotide sequence of a promoter region 2073 nucleotides upstream of the first transcription site contains three AP-1, one CRE sequence, and three Sp1-like sites close to the TATA box. Further upstream of these sites two TG repeats were found. The ability of regions within the 5' upstream sequence to promote basal neural-specific expression in tissue culture cells was evaluated using a series of constructs containing both Snap gene start sites with progressively restricted 5' sequence linked to the chloramphenicol acetyl transferase (CAT) reporter gene. CAT expression was maximal in neuron-like undifferentiated ND7 and PC12 cells transfected with constructs containing Snap sequences up to 127 bp from the start site. In contrast, nonneuronal fibroblast cell lines did not express significant amounts of CAT, suggesting that this short 127-bp sequence is sufficient to drive neural specific expression of SNAP-25. Band shift analysis of oligonucleotides spanning from -127 to -41 bp of the Snap promoter revealed three distinct DNA-protein complexes generated by brain nuclear extracts and one by liver nuclear extracts, indicating that transcription factors that bind to this 86-bp sequence located just upstream of the TATA box are involved in regulation of basal neurospecific expression of this gene.

Dual tissue-specific expression of apo-AII is directed by an upstream enhancer

Abstract: Apolipoprotein-AII (apo-AII) is one of a family of evolutionarily related proteins which play a crucial role in lipid transport and metabolism. The serum levels of human apo-AII have been shown to be inversely correlated to the incidence of coronary heart disease and its expression to be limited to the liver and intestine. Here we demonstrate that this dual tissue-specificity involves DNA sequences located in a 259 bp region centred 782 bp upstream from the transcription initiation site. These sequences function in an orientation-independent manner and are absolutely required for transcription from the apo-AII promoter. The regulatory region contains sequences which are homologous to the apo-AI, beta-globin and immunoglobulin gene promoters and to the immunoglobulin heavy-chain enhancer.

Inverse PCR-mediated cloning of the promoter for the rat vasopressin V2 receptor gene.

Abstract: The adenylyl cyclase-coupled vasopressin V2 receptor has been cloned recently and shown, in rats, to be produced from the predominant form of two alternate spliced variants. To begin to unravel the transcriptional regulation of this receptor, we have isolated the 5′ flanking region of the rat vasopressin V2 receptor gene and characterized its promoter sequence. The method of inverse polymerase chain reaction (PCR), which allows the amplification of DNA fragments adjacent to a segment of known sequence, was used as an alternative approach to genomic DNA library screening. Using a probe encompassing part of the coding region, first we identified by Southern blot analysis, a single BstX I hybridizing fragment of 2.3 kilobases (kb). This size predicted a BstX I restriction site 1.5 kb upstream to the gene coding region. Cloning of this fragment was accomplished through circularization of BstX I restriction digests and inverse PCR-mediated amplification. Sequence analysis of the gene 5′ flanking domain enabled the design of oligonucleotide primers with the usual forward/reverse orientation, and additional clones were generated from native genomic DNA using a high fidelity thermoresistant DNA polymerase. Reverse transcription-PCR (RT-PCR) and primer extension analysis mapped the major transcription start site 422 nucleotides upstream to the translation initiation codon. The promoter region lacks a TATA box but contains a CAAT box and a consensus binding site for transcription factor Spl. Multiple potential binding sites for the transcription factor PEA3 are clustered in two DNA portions located 0.6 kb and 1 kb upstream to the coding region. In addition, sequences homologous to glucocorticoid response elements are present and might be responsible for the regulation by adrenal steroids of vasopressin-dependent adenylyl cyclase activity in the kidney.