scholarly journals Characterization of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene promoter and identification of a repressor element

1998 ◽  
Vol 336 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Judy GROVER ◽  
Peter J. ROUGHLEY
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Transcription Start Site ◽  
Gene Promoter ◽  
Start Site ◽  
Leucine Rich Repeat ◽  
Transcription Start ◽  
Repeat Protein ◽  
Repressor Element ◽  
Leucine Rich Repeat Protein

The 5´-flanking region of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene has been characterized for both promoter and repressor activity by using a variety of reporter gene constructs and transient transfection into chondrocytes or fibroblasts. The human PRELP gene lacks a TATA box, and in its absence a Sp1-binding site residing 29 bp upstream of the transcription start site is essential for initiating gene expression. In contrast, an Ets-binding site residing 497 bp upstream of the transcription start site can lead to the repression of gene expression. The analysis of nuclear proteins by gel retardation studies with the repressor element identified a common protein, presumably an Ets family member, present in neonatal chondrocytes and skin fibroblasts that do not express the PRELP gene. The factor was not detected in nuclear protein preparations from adult chondrocytes in which the PRELP gene is expressed.

scholarly journals Location of promoter elements necessary and sufficient to direct testis-specific expression of the Hst70/Hsp70.2 gene

2004 ◽  
Vol 379 (3) ◽  
pp. 739-747 ◽  
Author(s):  
Dorota ŚCIEGLIŃSKA ◽  
Natallia VYDRA ◽  
Zdzisław KRAWCZYK ◽  
Wiesława WIDŁAK
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Gene Promoter ◽  
Specific Gene ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Specific Expression ◽  
High Level ◽  
Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 are expressed specifically in pachytene primary spermatocytes and spermatids. Here we demonstrate that a 165 bp fragment of the Hst70 gene promoter, containing the T1 transcription start site region, entire exon 1 and 42 bp 5´ region of the intron, is sufficient to drive testis-specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice with the same developmentally regulated pattern as the endogenous Hsp70.2 gene. We show further that high-level tissue-specific gene expression requires additional sequences localized upstream of the T2 transcription start site. Electrophoretic mobility-shift assay analysis revealed that only testes of juvenile rats, when Hst70 gene expression is repressed, contain proteins that specifically bind to the Oct (octamer) sequence localized directly downstream of the T1 site.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

scholarly journals Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

2005 ◽  
Vol 187 (9) ◽  
pp. 3158-3170 ◽  
Author(s):  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Transcription Start Site ◽  
Regulatory Protein ◽  
Dnase I ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Dnase I Footprinting

ABSTRACT The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the σ54-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions −54 to +137 with respect to the start site, and from −63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel σ54-dependent promoter architecture, with the activator FlrC binding downstream of the σ54-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.

scholarly journals Alpha-thalassemia resulting from deletion of regulatory sequences far upstream of the alpha-globin structural genes

Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1589-1595
Author(s):  
L Romao ◽  
L Osorio-Almeida ◽  
DR Higgs ◽  
J Lavinha ◽  
SA Liebhaber
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Globin Gene ◽  
Regulatory Sequences ◽  
Start Site ◽  
Structural Genes ◽  
Transcription Start ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Globin Gene Expression

We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5′ to the transcription start site of the alpha 2-globin gene. This alpha- thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression.

scholarly journals A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Isaac Shamie ◽  
Sascha H Duttke ◽  
Karen J la Cour Karottki ◽  
Claudia Z Han ◽  
Anders H Hansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Cho Cells ◽  
Genome Engineering ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Silent Genes

Abstract Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal bop Gene Promoter

1999 ◽  
Vol 181 (8) ◽  
pp. 2513-2518 ◽  
Author(s):  
Nitin S. Baliga ◽  
Shiladitya DasSarma
Keyword(s):  
Transcription Start Site ◽  
Direct Evidence ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Tata Box ◽  
Saturation Mutagenesis ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Transcriptional Terminator

ABSTRACT Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeonHalobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum− bop) Halobacterium mutant for purple (Pum+ bop +) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

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