Abstract：
BACKGROUND:Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative. RESULTS:Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency. CONCLUSIONS:Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.
journal_name
BMC Mol Bioljournal_title
BMC molecular biologyauthors
Voss C,Schmitt B,Werner-Simon S,Lutz C,Simon W,Anderl Jdoi
10.1186/1471-2199-15-7subject
Has Abstractpub_date
2014-04-03 00:00:00pages
7issn
1471-2199pii
1471-2199-15-7journal_volume
15pub_type
杂志文章abstract：BACKGROUND:Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. RESULTS:By expressing the human 14-3-3epsilon, as...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-8-27
更新日期：2007-04-12 00:00:00
abstract：BACKGROUND:Improving the treatment of renal cell carcinoma (RCC) will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7), also known as B-FABP and BLBP, which is expressed normally ...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-12-31
更新日期：2011-07-19 00:00:00
abstract：BACKGROUND:Retinal ganglion cells (RGCs) are preferentially lost in glaucoma or optic neuritis. In the present study, we investigated the protective effect of mircoRNA 100 (miR-100) against oxidative stress induced apoptosis in RGC-5 cells. RESULTS:Rat RGC-5 cells were cultured in plates and H2O2 was added to induce o...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/s12867-014-0025-1
更新日期：2014-11-18 00:00:00
abstract：BACKGROUND:Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate alpha-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), an...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-11-25
更新日期：2010-03-31 00:00:00
abstract：BACKGROUND:Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-7-42
更新日期：2006-11-13 00:00:00
abstract：BACKGROUND:The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in re...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-6-2
更新日期：2005-01-19 00:00:00
abstract：BACKGROUND:Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis. R...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-10-97
更新日期：2009-10-25 00:00:00
abstract：BACKGROUND:Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skelet...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-50
更新日期：2008-05-19 00:00:00
abstract：BACKGROUND:GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-10
更新日期：2008-01-23 00:00:00
abstract：BACKGROUND:Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which check...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-8-119
更新日期：2007-12-21 00:00:00
abstract：BACKGROUND:Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping ...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-102
更新日期：2008-11-12 00:00:00
abstract：BACKGROUND:SRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones. RESULTS:We identified four intervals of high homology upstr...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-85
更新日期：2008-10-14 00:00:00
abstract：BACKGROUND:Cyclic AMP-dependent protein kinase (PKA) plays a central role in regulation of energy metabolism. Upon stimulation of testicular Sertoli cells by follicle stimulating hormone (FSH), glycolysis is activated to increase the production of nutrients for the germ cells, and a new regulatory subunit of cAMP-depen...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-3-10
更新日期：2002-06-21 00:00:00
abstract：BACKGROUND:CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPdelta is highly expressed in G0 growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPdelta have been associated with impaired contact...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-83
更新日期：2008-10-01 00:00:00
abstract：BACKGROUND:The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency o...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-8-100
更新日期：2007-10-31 00:00:00
abstract：BACKGROUND:The human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two reg...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-10-32
更新日期：2009-04-17 00:00:00
abstract：BACKGROUND:Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/s12867-016-0075-7
更新日期：2016-11-04 00:00:00
abstract：BACKGROUND:Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acc...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-8-96
更新日期：2007-10-25 00:00:00
abstract：BACKGROUND:Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease. RESULTS:Insertion of a TEV protease cleavage site into Cdc23 allows i...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-6-13
更新日期：2005-06-07 00:00:00
abstract：BACKGROUND:Conserved two-component system ColRS of Pseudomonas genus has been implicated in several unrelated phenotypes. For instance, deficiency of P. putida ColRS system results in lowered phenol tolerance, hindrance of transposition of Tn4652 and lysis of a subpopulation of glucose-grown bacteria. In order to disco...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-10-46
更新日期：2009-05-16 00:00:00
abstract：BACKGROUND:Members of the Sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. Among these members, Sox30 had been considered to exist only in mammals since its discovery, and its e...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-11-98
更新日期：2010-12-11 00:00:00
abstract：BACKGROUND:The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect repli...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/s12867-015-0053-5
更新日期：2016-01-05 00:00:00
abstract：BACKGROUND:Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of ba...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-13-20
更新日期：2012-06-25 00:00:00
abstract：BACKGROUND:Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-12-32
更新日期：2011-08-01 00:00:00
abstract：BACKGROUND:When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS:Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, di...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-6-10
更新日期：2005-04-26 00:00:00
abstract：BACKGROUND:The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important t...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-2-12
更新日期：2001-01-01 00:00:00
abstract：BACKGROUND:The L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in axon guidance. In many human epithelial carcinomas L1CAM is overexpressed and thereby augments cell motility, invasion and metastasis formation. L1CAM positive carcinomas are associated with bad prognosi...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-11-64
更新日期：2010-08-27 00:00:00
abstract：BACKGROUND:The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amp...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-96
更新日期：2008-10-30 00:00:00
abstract：BACKGROUND:In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we use...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-8-112
更新日期：2007-12-19 00:00:00
abstract：BACKGROUND:Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expr...
journal_title：BMC molecular biology
pub_type： 杂志文章
doi：10.1186/1471-2199-9-43
更新日期：2008-04-29 00:00:00
