Identification of a Ty1 regulatory sequence responsive to STE7 and STE12

Ty1 activation of gene expression observed in haploid cell types of Saccharomyces cerevisiae requires the STE7 and STE12 gene products. An activator sequence within Ty1 that is responsive to these two regulators has been defined. Complex formation between a factor in whole-cell extracts and the DNA regulatory element showed the same dependence on the STE7 and STE12 gene products as did reporter gene expression. Base pair substitutions within the binding site abolished the ability to form the factor-DNA complex and to activate gene expression. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for gene activation. Because the predicted protein for the STE7 gene product is homologous to protein kinases, we suggest that protein phosphorylation may directly or indirectly regulate formation of this DNA-protein complex.

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Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

Biochemistry Research International ◽

10.1155/2018/6406372 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Gwang Sik Kim ◽

Young Chul Lee

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Transcriptional Activation ◽

Reporter Gene Expression ◽

Temperature Sensitive ◽

Internal Deletion ◽

Cell Extracts ◽

Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

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Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

Functional Plant Biology ◽

10.1071/pp9960075 ◽

1996 ◽

Vol 23 (1) ◽

pp. 75 ◽

Cited By ~ 8

Author(s):

SR Mudge ◽

WR Lewis-Henderson ◽

RG Birch

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Ccd Camera ◽

Reporter Gene Expression ◽

In Vitro Assays ◽

E Coli ◽

Cell Extracts ◽

Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

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The Virion Host Shutoff Protein of Herpes Simplex Virus Inhibits Reporter Gene Expression in the Absence of Other Viral Gene Products

Virology ◽

10.1006/viro.1995.1431 ◽

1995 ◽

Vol 211 (2) ◽

pp. 491-506 ◽

Cited By ~ 33

Author(s):

Annette Schmidt Pak ◽

David N. Everly ◽

Kimberli Knight ◽

G.Sullivan Read

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Viral Gene ◽

Gene Products ◽

Virion Host Shutoff ◽

Virion Host Shutoff Protein ◽

Simplex Virus

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Multiple factors regulating the expression of human thromboxane synthase gene

Biochemical Journal ◽

10.1042/bj3190783 ◽

1996 ◽

Vol 319 (3) ◽

pp. 783-791 ◽

Cited By ~ 11

Author(s):

Kuan-Der LEE ◽

Seung Joon BAEK ◽

Rong-Fong SHEN

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Promoter Activity ◽

Reporter Gene Expression ◽

Regulatory Sequence ◽

Dependent Manner ◽

Cis Elements ◽

Thromboxane Synthase ◽

Multiple Factors ◽

Repressive Effect

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, ∼75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (∼55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

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Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression

Gene ◽

10.1016/j.gene.2012.01.071 ◽

2012 ◽

Vol 498 (2) ◽

pp. 231-236 ◽

Cited By ~ 3

Author(s):

Siew Ching Ngai ◽

Rozita Rosli ◽

Norshariza Nordin ◽

Abhi Veerakumarasivam ◽

Syahril Abdullah

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Regulatory Element ◽

Reporter Gene Expression ◽

Lentivirus Vector ◽

Low Level ◽

Polypurine Tract ◽

Central Polypurine Tract ◽

Posttranscriptional Regulatory Element

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Two Novel Adenovirus Vector Systems Permitting Regulated Protein Expression in Gene Transfer Experiments

Journal of Virology ◽

10.1128/jvi.72.10.8358-8361.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8358-8361 ◽

Cited By ~ 48

Author(s):

Magnus Molin ◽

Maria C. Shoshan ◽

Karin Öhman-Forslund ◽

Stig Linder ◽

Göran Akusjärvi

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Reporter Gene ◽

Adenovirus Vector ◽

Cell Types ◽

Reporter Gene Expression ◽

Reporter Protein ◽

Ru 486 ◽

Vector Systems ◽

Wide Range

ABSTRACT Two new adenovirus vector systems based on the tetracycline-regulated Tet-ON- (Gossen, M., et al., Science 268:1766–1769, 1995) and the RU 486-regulated progesterone antagonist (Wang, Y., et al., Proc. Natl. Acad. Sci. USA 91:8180–8184, 1994)-induced gene expression systems are described. We show that both systems permit a tight control of chloramphenicol acetyltransferase reporter gene expression in a variety of cell types, with induction levels of approximately 1,800-fold (Tet-ON system) and 600-fold (RU 486-regulated system), respectively. A significant advantage of our vector systems is that reporter protein expression can be adjusted over a wide range by varying the amount of inducer. The Tet-ON system is also shown to permit an efficient control of reporter gene expression in mice.

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