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.

Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter.

Abstract: The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5′ promoter and a 1.2-kb 3′ terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin ( Act ) or globulin ( Glb ) gene promoters and the reporter gene construct Glb-β-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uid A gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 ( Gt1 ) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 μg⋅mg −1 total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.

A novel promoter in the mouse rDNA spacer is active in vivo and in vitro.

Abstract: We have identified a novel RNA polymerase I (pol I) transcription initiation site within the 'non-transcribed' spacer of mouse rDNA. This spacer promoter is located about 2 kb upstream of the 45S pre-rRNA promoter and directs specific transcription initiations both in a cell-free system using truncated templates and in vivo after transfection into mouse cells. The spacer promoter contains an 11 out of 16 bases match to the core element of the major ribosomal gene promoter and is oriented in the same direction. It exerts a significantly lower transcriptional activity as compared to the 45S pre-rRNA promoter. The elongation of transcripts initiated at the spacer promoter is stopped at a termination signal located 170 bp upstream of the pre-rRNA start site. Since it has been previously shown that, in addition to its terminator function, the same sequence motif acts as an upstream element of the adjacent gene promoter, the function of the spacer promoter may be to capture free pol I molecules and drive them to the gene promoter in order to achieve the high level of transcription characteristic of eukaryotic rRNA genes.

Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region.

Abstract: The ie-1 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a transregulatory protein (IE1) which accelerates the expression of early and late virus genes. Transcription of ie-1 occurs immediately upon infection from a conserved CAGT motif and continues into the late phases. To examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed mutagenesis. The ie-1 upstream activating region, extending from nucleotide -546 to the TATA element at -34, stimulated ie-1 basal promoter activity more than 1,000-fold when transfected into uninfected Spodoptera frugiperda SF21 cells. However, when introduced into the genome of AcMNPV recombinants, the ie-1 upstream activating region had only a minimal twofold effect early in infection. Instead, maximum steady-state levels of early ie-1 RNAs required sequences within the 5' noncoding leader region extending from +11 to +24 relative to the RNA start site (+1). The +11 to +24 noncoding region did not influence the stability of ie-1 transcripts. When assayed by in vitro transcription, deletion of the +11 to +24 region reduced the levels of ie-1 runoff RNAs. Thus, this downstream activating sequence controlled the rate of early ie-1 transcription. A larger overlapping region from +11 to +36 affected steady-state levels of ie-1 RNAs late (24 h) in infection. Deletion of sequences that included the conserved CAGT start site abolished early ie-1 transcription. Thus, ie-1 is the first example of an early baculovirus gene in which essential cis-acting regulatory elements reside within the 5' noncoding region and include sequences comprising the RNA start site.

The Saccharomyces cerevisiae MET3 gene: Nucleotide sequence and relationship of the 5′ non-coding region to that of MET25

Abstract: In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is coregulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5′ and 3′ flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250–280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.

Heterogeneously initiated transcription from the pre-B- and B-cell-specific mb-1 promoter: analysis of the requirement for upstream factor-binding sites and initiation site sequences.

Abstract: The mb-1 gene, encoding a membrane immunoglobulin-associated protein, is developmentally regulated and expressed specifically in pre-B and mature B lymphocytes. Analysis of the TATA-less mb-1 promoter indicated that it directs initiation of transcription from multiple sites. Promoter sequences between -68 and +70 conferred the correct pattern of cell type-specific transcription upon a heterologous gene. Two nuclear factor-binding sites that are important for promoter function were identified between -59 and -38. Both sites interacted with ubiquitous nuclear factors in vitro. One of these factors was identified as Sp1. Multimerized copies of both factor-binding sites augmented expression from a heterologous minimal promoter in both lymphoid and nonlymphoid cells, suggesting that additional mb-1 promoter sequences are involved in determining the correct cell type specificity. Analysis of the heterogeneity of transcription initiation indicated that a mutation which increased the distance between upstream sequences and the region of initiation resulted in the utilization of a novel set of initiation sites. Moreover, an insertion of a TATA element into the mb-1 promoter at -30 biased initiation of transcription to +1 but did not abolish the use of the other sites. Mutation of an initiator sequence homology encompassing one of the major initiation sites had only a minor effect on its utilization. From these data, we conclude that upstream factor-binding sites in the TATA-less mb-1 promoter define a region in which initiation of transcription occurs at multiple sites.