scholarly journals Correction: Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap® Analog by In Vitro Transcription

2021 ◽  
Vol 1 (12) ◽  
Author(s):  
Jordana M. Henderson ◽  
Andrew Ujita ◽  
Elizabeth Hill ◽  
Sally Yousif‐Rosales ◽  
Cory Smith ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
In Vitro Transcription

scholarly journals Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap ® Analog by In Vitro Transcription

2021 ◽  
Vol 1 (2) ◽  
Author(s):  
Jordana M. Henderson ◽  
Andrew Ujita ◽  
Elizabeth Hill ◽  
Sally Yousif‐Rosales ◽  
Cory Smith ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
In Vitro Transcription

scholarly journals Prolactin upstream factor I mediates cell-specific transcription.

1988 ◽  
Vol 8 (12) ◽  
pp. 5432-5438 ◽  
Author(s):  
Z D Cao ◽  
E A Barron ◽  
Z D Sharp
Keyword(s):  
Gene Transcription ◽  
Rna Synthesis ◽  
Transcriptional Activity ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
In Vitro Mutagenesis ◽  
Pituitary Cells ◽  
Factor I ◽  
Prolactin Gene

DNA sequence-specific chromatography was used to purify prolactin upstream factor I (PUF-I) approximately 10,000- to 20,000-fold from rat GH3 cells. The purified transcription factor reconstituted enhanced pituitary-specific prolactin RNA synthesis in nonpituitary in vitro transcription assays. In vitro mutagenesis demonstrated that the capacity to stimulate prolactin gene transcription was directly correlated with PUF-I binding to an A+T-rich region located from -63 to -36 in the prolactin 5'-flanking DNA. We propose that PUF-I is a critical modulator of transcriptional activity in pituitary cells and has a central role in the stimulation of prolactin gene transcription in the mammalian pituitary lactotroph.

scholarly journals EFFECT OF ACTINOMYCIN D ON RNA SYNTHESIS AND ANTIBODY FORMATION IN THE ANAMNESTIC RESPONSE IN VITRO

1964 ◽  
Vol 119 (6) ◽  
pp. 881-893 ◽  
Author(s):  
J. Donald Smiley ◽  
John G. Heard ◽  
Morris Ziff
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Lymphoid Cells ◽  
Limiting Factor ◽  
High Concentration ◽  
Antibody Synthesis ◽  
Anamnestic Response ◽  
Low Concentrations

Antibody synthesis in anamnestic lymphoid cells, measured by incorporation of leucine-C14 into specific antibody, was inhibited at moderate concentrations of actinomycin D. This was accompanied by marked inhibition of synthesis of RNA as measured by incorporation of H3-cytidine monophosphate. However, at low concentrations of actinomycin D, antibody synthesis was unaffected or even increased while RNA synthesis continued to be inhibited. The results obtained suggest that messenger RNA for antibody synthesis, either because it is relatively stable or present in excess, does not become a limiting factor until its synthesis is maximally inhibited. Puromycin, an inhibitor of amino acid coupling, abolished antibody synthesis in low concentration. 6-Mercaptopurine had no effect on the synthesis of antibody or RNA even at high concentration. The data obtained support the view that antibody synthesis follows pathways similar to those utilized for the formation of other types of proteins.

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

scholarly journals Construction of a synthetic messenger RNA encoding a membrane protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1464-1469 ◽  
Author(s):  
J L Rubenstein ◽  
T G Chappell
Keyword(s):  
G Protein ◽  
Messenger Rna ◽  
In Vitro Transcription ◽  
Membrane Insertion ◽  
Wheat Germ Extract ◽  
E Coli ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Synthetic Mrna

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

scholarly journals Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6488-6503 ◽  
Author(s):  
Yong-Heng Huang ◽  
Nelly Said ◽  
Bernhard Loll ◽  
Markus C Wahl
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Rna Folding ◽  
In Vitro Transcription ◽  
Structural Basis ◽  
Macromolecular Crystallography ◽  
Rna Transcription ◽  
Signal Element

AbstractRibosomal RNA synthesis in Escherichia coli involves a transcription complex, in which RNA polymerase is modified by a signal element on the transcript, Nus factors A, B, E and G, ribosomal protein S4 and inositol mono-phosphatase SuhB. This complex is resistant to ρ-dependent termination and facilitates ribosomal RNA folding, maturation and subunit assembly. The functional contributions of SuhB and their structural bases are presently unclear. We show that SuhB directly binds the RNA signal element and the C-terminal AR2 domain of NusA, and we delineate the atomic basis of the latter interaction by macromolecular crystallography. SuhB recruitment to a ribosomal RNA transcription complex depends on the RNA signal element but not on the NusA AR2 domain. SuhB in turn is required for stable integration of the NusB/E dimer into the complex. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination, that SuhB also can reduce intrinsic termination, and that SuhB-AR2 contacts contribute to these effects. Together, our results reveal functions of SuhB during ribosomal RNA synthesis and delineate some of the underlying molecular interactions.

In vitro transcription of RNA in nuclei, nucleoli and chromatin from physarum polycephalum

1977 ◽  
Vol 26 (1) ◽  
pp. 267-279
Author(s):  
K.E. Davies ◽  
I.O. Walker
Keyword(s):  
Rna Polymerase ◽  
Physarum Polycephalum ◽  
Rna Synthesis ◽  
In Vitro Transcription ◽  
Polymerase Activity ◽  
Controlled Conditions ◽  
Rna Polymerase Activity ◽  
Alpha Amanitin

Methods for isolating nuclei, nucleoli and chromatin from Physarum polycephalum which retain high levels of endogenous RNA polymerase activity are described. Under carefully controlled conditions with respect to mono- and divalent cation concentrations RNA synthesis in nuclei displayed linear kinetics for at least 30 min and the RNA products had a similar size distribution to nuclear RNA synthesis observed in vivo. Chromatin showed 60% of the nuclear transcriptional activity but no conditions were found where faithful transcription of the template occurred. Isolated nucleoli were 5-fold more active than nuclei and the endogenous RNA polymerase activity was insensitive to alpha-amanitin. Under carefully controlled conditions, the nucleoli appeared to support the accurate transcription, re-initiation and processing of rRNA chains in vitro.

scholarly journals Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

Methods ◽  
2010 ◽  
Vol 52 (4) ◽  
pp. 322-331 ◽  
Author(s):  
Bradley S. Carter ◽  
Jonathan S. Fletcher ◽  
Robert C. Thompson
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
In Vitro Transcription ◽  
Rna Expression ◽  
Rna Probes

scholarly journals In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells

1994 ◽  
Vol 297 (2) ◽  
pp. 297-301 ◽  
Author(s):  
J R Patton
Keyword(s):  
Cell Growth ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Significant Inhibition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Specific Step ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

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