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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.
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TL;DR: The template and factor requirements for in vitro transcription of the GC-rich promoter of the murine adenosine deaminase gene are explored and inclusion of 12 base pairs immediately adjacent to the proximal Sp1 site resulted in a 5-fold boost in transcriptional activity and corresponds to a second Sp1 binding site.
32 citations
TL;DR: None of these deletions had any effect on the expression of usg-1, a gene of unknown function that is transcribed in the same direction as the am gene, and which terminates about 3.5 kb upstream of the URSamβ element.
Abstract: We have constructed deletions in the 5′ non-coding sequences of the cloned Neurospora crassa am gene. Vectors with a truncated fragment of the am gene were used in transformation experiments to introduce the deletions into the chromosome by homologous recombination. Analysis of glutamate dehydrogenase (GDH) expression by enzyme assay and immunoblots, as well as Northern and dot blots of poly (A)+ RNA, in the deletion strains indicates that there are two upstream regulatory sequences that control the level of gene expression. The closer of these two elements (URSamα) is at approximately 1.4 kb upstream of the transcriptional start site. The second elements (URSamβ) is located between 2.1 and 3.2 kb upstream of the transcription start site. Deletion of either of these two elements reduces am expression to about 50% of the wild-type level. Deletion of both elements reduce am expression to from 5–16% of the wild-type level. Deletion of 1.1 kb of sequence just downstream of URSamα, which brings this element to within 300 bp of the transcription start site, had no effect on am expression. Likewise, deletion of 3.5 kb of sequence upstream of URSamβ had no effect on expression. None of these deletions had any effect on the expression of usg-1, a gene of unknown function that is transcribed in the same direction as the am gene, and which terminates about 3.5 kb upstream of the URSamβ element.
32 citations
TL;DR: The 3' regulatory boundary of the skeletal alpha-actinactin gene promoter was defined in this paper, which indicated that divergent transcription may occur in vivo but with rapid turnover of nuclear transcripts.
Abstract: The chicken skeletal alpha-actin gene promoter region (-202 to -12) provides myogenic transcriptional specificity. This promoter contains partial dyad symmetry about an axis at nucleotide -108 and in transfection experiments is capable of directing transcription in a bidirectional manner. At least three different transcription initiation start sites, oriented toward upstream sequences, were mapped 25 to 30 base pairs from TATA-like regions. The opposing transcriptional activity was potentiated upon the deletion of sequences proximal to the alpha-actin transcription start site. Thus, sequences which serve to position RNA polymerase for alpha-actin transcription may allow, in their absence, the selection of alternative and reverse-oriented start sites. Nuclear runoff transcription assays of embryonic muscle indicated that divergent transcription may occur in vivo but with rapid turnover of nuclear transcripts. Divergent transcriptional activity enabled us to define the 3' regulatory boundary of the skeletal alpha-actin promoter which retains a high level of myogenic transcriptional activity. The 3' regulatory border was detected when serial 3' deletions bisected the element (-91 CCAAA TATGG -82) which reduced transcriptional activity by 80%. Previously we showed that disruption of its upstream counterpart (-127 CCAAAGAAGG -136) resulted in about a 90% decrease in activity. These element pairs, which we describe as CCAAT box-associated repeats, are conserved in all sequenced vertebrate sarcomeric actin genes and may act in a cooperative manner to facilitate transcription in myogenic cells.
32 citations
TL;DR: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombination Saccharomyces cerevisiae, and some important characteristics of the secreted rhG- CSF are demonstrated.
Abstract: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1–10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 600-609, 1998.
32 citations
TL;DR: It is demonstrated that 7S K DNA represents a novel class III gene, the promoter elements of which are located outside the coding sequence, and a procaryotic vector DNA sequence was efficiently transcribed by RNA polymerase III under the control of the7S K RNA gene upstream sequence in vitro and in vivo.
Abstract: We have analyzed the transcription of a recently isolated human 7S K RNA gene in vitro and in vivo. In contrast to hitherto characterized class III genes (genes transcribed by RNA polymerase III), the coding sequence of this gene is not required for faithful and efficient transcription by RNA polymerase III. In fact, a procaryotic vector DNA sequence was efficiently transcribed by RNA polymerase III under the control of the 7S K RNA gene upstream sequence in vitro and in vivo. S1-nuclease protection analyses confirmed that the 7S K 5'flanking sequence was sufficient for accurate transcription initiation. These data demonstrate that 7S K DNA represents a novel class III gene, the promoter elements of which are located outside the coding sequence.
32 citations