scholarly journals Characterization of the promoter of human adipocyte hormone-sensitive lipase

1997 ◽  
Vol 328 (2) ◽  
pp. 453-461 ◽  
Author(s):  
Jacques GROBER ◽  
Henrik LAURELL ◽  
Régis BLAISE ◽  
Béatrice FABRY ◽  
Stéphane SCHAAK ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Rnase H ◽  
Hormone Sensitive Lipase ◽  
Start Codon ◽  
Start Site ◽  
Rnase Protection ◽  
Human Adipocyte ◽  
Hormone Sensitive

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5ʹ untranslated region (5ʹ-UTR). A single 5ʹ-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5ʹ-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5ʹ-UTR is mutually exclusive. The short and long 5ʹ-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5ʹ-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

scholarly journals Regulation of the furA and catC Operon, Encoding a Ferric Uptake Regulator Homologue and Catalase-Peroxidase, Respectively, in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (13) ◽  
pp. 3767-3774 ◽  
Author(s):  
Ji-Sook Hahn ◽  
So-Young Oh ◽  
Jung-Hye Roe
Keyword(s):  
Streptomyces Coelicolor ◽  
Transcriptional Start Site ◽  
Protein Product ◽  
Negative Regulator ◽  
Start Codon ◽  
Multicopy Plasmid ◽  
Ferric Uptake Regulator ◽  
Start Site ◽  
Reading Frame ◽  
Catalase Peroxidase

ABSTRACT We isolated the catC gene, encoding catalase-peroxidase in Streptomyces coelicolor, using sequence homology with the katG gene from Escherichia coli. Upstream of the catC gene, an open reading frame (furA) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the furA gene was cotranscribed with the catC gene. The transcriptional start site of the furA-catC mRNA was mapped to the translation start codon ATG of the furA gene. The putative promoter contains consensus −10 and −35 elements similar to those recognized by ςHrdB, the major sigma factor of S. coelicolor. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the furA gene was introduced into S. lividans on a multicopy plasmid, the increased production of catC transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the furA-catC operon. FurA protein bound to its own promoter region between −59 and −39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni2+, Mn2+, Zn2+, or Fe2+. Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.

scholarly journals A Common Hormone-Sensitive Lipase i6 Gene Polymorphism Is Associated With Decreased Human Adipocyte Lipolytic Function

Diabetes ◽  
2001 ◽  
Vol 50 (10) ◽  
pp. 2410-2413 ◽  
Author(s):  
J. Hoffstedt ◽  
P. Arner ◽  
M. Schalling ◽  
N. L. Pedersen ◽  
S. Sengul ◽  
...  
Keyword(s):  
Gene Polymorphism ◽  
Hormone Sensitive Lipase ◽  
Human Adipocyte ◽  
Hormone Sensitive

scholarly journals Regulation of Expression of the adhE Gene, Encoding Ethanol Oxidoreductase in Escherichia coli: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS

1999 ◽  
Vol 181 (24) ◽  
pp. 7571-7579 ◽  
Author(s):  
Jorge Membrillo-Hernández ◽  
E. C. C. Lin
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Start Site ◽  
Start Codon ◽  
Start Site ◽  
Rnase Iii ◽  
Regulation Of Expression ◽  
Experimental Conditions ◽  
Stem Loop ◽  
Translational Start ◽  
Transcriptional Start Sites

ABSTRACT The adhE gene of Escherichia coli, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of adhEis dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the lacZ gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activatesadhE transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.

scholarly journals Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 668-676 ◽  
Author(s):  
M Villa-Garcia ◽  
L Li ◽  
G Riely ◽  
PF Bray
Keyword(s):  
Transcription Start Site ◽  
Phorbol Esters ◽  
K562 Cells ◽  
Start Codon ◽  
Dependent Manner ◽  
Start Site ◽  
Transcription Start ◽  
Specific Expression ◽  
Rnase Protection ◽  
Isolation And Characterization

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5′ to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3′ intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5′ to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a “promoter-less” control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5′ beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

scholarly journals Identification and transcriptional regulation of the mir-218-1 alternative promoter

2020 ◽  
Author(s):  
Brenna A. Rheinheimer ◽  
Lukas Vrba ◽  
Bernard W Futscher ◽  
Ronald L Heimark
Keyword(s):  
Transcriptional Regulation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Alternative Promoter ◽  
Ductal Adenocarcinoma ◽  
Start Site ◽  
Public Datasets ◽  
Intron 4

AbstractBackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 expression and that mir-218-1 has a novel transcriptional start site separate from the SLIT2 promoter. This novel transcriptional start site showed transcriptional activity and was regulated by NF-kB.Conclusionsmir-218-1 is transcribed from an independent and novel transcriptional start site located within intron 4 of the SLIT2 gene in pancreatic ductal adenocarcinoma. Additionally, mir-218-1 expression is regulated by Nf-kB at this alternative transcriptional start site in pancreatic cancer.

Transcriptional regulation of human abraxas brother protein 1 expression by yin yang 1

2020 ◽  
Author(s):  
Pan Song ◽  
Jian Hong ◽  
Yuan Wang ◽  
Xuelian Yao ◽  
Yiqun Zhan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Codon ◽  
Mobility Shift ◽  
Cis Acting ◽  
Yin Yang 1 ◽  
Activating Transcription Factor 4 ◽  
Yin Yang

Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5′ rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located −13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region −89 to −59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.

scholarly journals The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

2002 ◽  
Vol 184 (6) ◽  
pp. 1801-1805 ◽  
Author(s):  
Brigitta Kurenbach ◽  
Dagmar Grothe ◽  
María Eugenia Farías ◽  
Ulrich Szewzyk ◽  
Elisabeth Grohmann
Keyword(s):  
Amino Acids ◽  
Transcriptional Start Site ◽  
Conjugative Plasmid ◽  
Start Codon ◽  
Start Site ◽  
Gene Encoding ◽  
Cleavage Activity ◽  
Reverse Transcription Pcr ◽  
Single Operon

ABSTRACT The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriTpIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg2+ or Mn2+ and is highest at temperatures of between 42 and 45°C.

scholarly journals Transcriptional activation of vesicular monoamine transporter 2 in the pre-B cell line Ea3.123

1999 ◽  
Vol 337 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Fiona WATSON ◽  
Damian G. DEAVALL ◽  
Janet A. MACRO ◽  
Rachel KIERNAN ◽  
Rod DIMALINE
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Vesicular Monoamine Transporter ◽  
Start Site ◽  
Monoamine Transporter ◽  
Vesicular Monoamine Transporter 2

Uptake and storage of monoamines in secretory granules is accomplished by vesicular monoamine transporters, and it is likely that vesicular monoamine transporter 2 (VMAT2) is important for histamine transport in vivo. In the present study we have used the pre-B-cell line Ea3.123 to investigate the mechanisms involved in the transcriptional activation of the VMAT2 gene. In Ea3.123 cells, VMAT2 mRNA abundance was increased following mobilization of intracellular calcium, and this increased mRNA expression was paralleled by changes in l-histidine decarboxylase mRNA, suggesting that VMAT2 may be responsible for sequestration of histamine into secretory vesicles in this cell line. We cloned the 5´-flanking region of the VMAT2 gene and determined its transcriptional start site by primer extension of rat VMAT2 mRNA. There was no TATA or TATA-like sequence upstream of this region; instead there were GC-rich elements, Ca2+/cAMP-response-element- and SP1-binding motifs. Approx. 900 bp upstream of the transcriptional start site was a purine–pyrimidine repeat sequence that may form a Z-DNA structure. A series of 5´-deletional VMAT2-promoter segments cloned upstream of a luciferase reporter were capable of driving transcription and indicated the presence of multiple regulatory elements, while stimulation with ionomycin or PMA resulted in an increased level of the transcriptional activity of the 5´-promoter segments studied.

Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32

1989 ◽  
Vol 9 (5) ◽  
pp. 2067-2074
Author(s):  
M L Atchison ◽  
O Meyuhas ◽  
R P Perry
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequences ◽  
Binding Sites ◽  
Protein Gene ◽  
Transcriptional Start Site ◽  
Ribosomal Protein Gene ◽  
Regulatory Elements ◽  
Sequence Motif ◽  
Start Site ◽  
Nuclear Factors

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

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