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.

Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters.

Abstract: The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far. Here we report the establishment of the GAL4/UAS binary expression system in Tribolium castaneum. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the Tribolium hsp68 gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in Tribolium - 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on Tribolium, Drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs. With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in Tribolium. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in Tribolium and probably also other model systems.

Molecular structure and transcriptional function of the rat vascular AT1a angiotensin receptor gene.

Abstract: Rat vascular angiotensin receptors (AT1a receptors) are encoded by two mRNA transcripts sharing an identical receptor coding sequence but differing in their 5' and 3' untranslated sequences. We screened male Sprague-Dawley rat genomic libraries to clone the vascular AT1a receptor gene. Two sets of overlapping clones were isolated that encode over 90 kb of genomic sequence around the AT1a receptor gene. Four overlapping clones were identified from the 5' flanking portion of the gene. These contain the promoter region and two exons, 141 bp and 89 bp in size, respectively, encoding the alternatively spliced 5' untranslated mRNA sequence. Six additional clones overlap each other but do not overlap the set of clones from the 5' flanking region of the gene. These contain a single 1977-bp exon that encodes 900 bp of the 5' and 3' untranslated sequences in addition to a 1077-bp open reading frame identical to that found in vascular smooth muscle cell AT1a receptor cDNAs. Primer extension and RNase protection studies indicate that the transcription start site for this gene begins 9 bp upstream from the most 5' sequence found within the AT1a receptor cDNAs. Our mapping studies of the cloned gene, which so far includes an uncloned gap within the second intron, indicate that the transcription start site is no less than 67 kb upstream from the receptor coding exon. Promoter-reporter assays were performed by transfection of vascular smooth muscle cells with deletions of a 3.2-kb promoter region fused to a luciferase cDNA reporter plasmid. Relatively strong basal transcriptional activity is observed from the 5'-most 2 kb of the promoter and diminishes markedly with deletions within 1 kb of the early promoter region, suggesting strong promoter elements in the more upstream regions of the gene. Deletion of a 53-bp early promoter region containing the transcription start site and a putative TATA box completely abolishes the ability of upstream elements to drive transcription of the luciferase cDNA. These results indicate that we have isolated the AT1a receptor gene and its functional promoter.

Cell Cycle-Regulated Transcription of theCLB2Gene Is Dependent on Mcm1 and a Ternary Complex Factor

Abstract: Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutations which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in theCLB2UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control ofCLB2transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.

Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter.

Abstract: As a highly active plant viral promoter that is able to function in a wide variety of cell types, the cauliflower mosaic virus (CaMV) 35S promoter has the potential for harboring a plant enhancer element. We tested this possibility and demonstrated that a 338 base pair fragment isolated from the region upstream of the 35S TATA box can increase the expression of a low-activity heterologous promoter up to the level observed for the intact 35S promoter. This fragment is fully active in both orientations when placed 150 base pairs upstream of the transcription start site. However, the activity of this fragment is sensitive to location, demonstrating a reduction in activity and loss of orientation-independent function when the distance from the transcription start site is increased. By assaying fragments of different sizes, we have also characterized regions that are functional in directing the stimulation of the heterologous promoter.

Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter

Abstract: Summary: The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YIRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YIRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YIRim101p and to override its effects.