scholarly journals Programmable cell-free transcriptional switches for antibodies detection

2021 ◽  
Author(s):  
Aitor Patiño Diaz ◽  
Sara Bracaglia ◽  
Simona Ranallo ◽  
Tania Patiño ◽  
Alessandro Porchetta ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Cost Effective ◽  
In Vitro Transcription ◽  
Rna Aptamer ◽  
Transcription System ◽  
Specificity And Sensitivity ◽  
Sensing Platform ◽  
A Cell ◽  
Antibody Levels

We report here the development of a cell-free in-vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA aptamer. The system couples the unique programmability and responsiveness of DNA-based systems with the specificity and sensitivity offered by in-vitro genetic circuitries and commercially available transcription kits. We demonstrate that cell-free transcriptional switches can efficiently measure antibody levels directly in blood serum. Thanks to the programmable nature of the sensing platform the method can be adapted to different antibodies: we demonstrate here the sensitive, rapid and cost-effective detection of three different antibodies and the possible use of this approach for the simultaneous detection of two antibodies in the same solution.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter

1991 ◽  
Vol 11 (1) ◽  
pp. 117-125
Author(s):  
M Falzon ◽  
E L Kuff
Keyword(s):  
Promoter Activity ◽  
In Vitro Transcription ◽  
Site Directed Mutagenesis ◽  
Long Terminal Repeats ◽  
Transcription System ◽  
A Cell ◽  
Induced Changes ◽  
Intracisternal A Particle

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

scholarly journals Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

1991 ◽  
Vol 11 (1) ◽  
pp. 117-125 ◽  
Author(s):  
M Falzon ◽  
E L Kuff
Keyword(s):  
Promoter Activity ◽  
In Vitro Transcription ◽  
Site Directed Mutagenesis ◽  
Long Terminal Repeats ◽  
Transcription System ◽  
A Cell ◽  
Induced Changes ◽  
Intracisternal A Particle

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases

2013 ◽  
Vol 433 (2) ◽  
pp. 92-94
Author(s):  
Qiang Wang ◽  
Leiyun Weng ◽  
Hongbing Jiang ◽  
Shijian Zhang ◽  
Tetsuya Toyoda
Keyword(s):  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Viral Rna ◽  
Transcription System

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene

1991 ◽  
Vol 11 (5) ◽  
pp. 2832-2841
Author(s):  
N Mechti ◽  
M Piechaczyk ◽  
J M Blanchard ◽  
P Jeanteur ◽  
B Lebleu
Keyword(s):  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
First Intron ◽  
Nuclear Extracts ◽  
Intron 1 ◽  
A Cell ◽  
Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

Accurate transcription of a plant mitochondrial gene in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039
Author(s):  
P J Hanic-Joyce ◽  
M W Gray
Keyword(s):  
Mitochondrial Gene ◽  
Plant Mitochondria ◽  
In Vitro Transcription ◽  
Dna Templates ◽  
In Vitro System ◽  
Transcription System ◽  
Translation Initiation Codon ◽  
Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals Regulation of protamine gene expression in an in vitro homologous system.

1996 ◽  
Vol 43 (2) ◽  
pp. 369-377 ◽  
Author(s):  
J M Jankowski ◽  
P D Cannon ◽  
F Van der Hoorn ◽  
L D Wasilewska ◽  
N C Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Transcription System ◽  
Proposed Model ◽  
Binding Factor ◽  
Gc Box ◽  
Responsive Element

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

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