Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses

1985 ◽  
Vol 5 (10) ◽  
pp. 2842-2846
Author(s):  
M A Lopata ◽  
B Sollner-Webb ◽  
D W Cleveland
Keyword(s):  
Distal Region ◽  
Analytical Tool ◽  
Dna Probe ◽  
Dna Transfection ◽  
Potential Complication ◽  
Animal Cells ◽  
Rna Molecules ◽  
S1 Mapping ◽  
Powerful Analytical Tool

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

scholarly journals Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.

1985 ◽  
Vol 5 (10) ◽  
pp. 2842-2846 ◽  
Author(s):  
M A Lopata ◽  
B Sollner-Webb ◽  
D W Cleveland
Keyword(s):  
Distal Region ◽  
Analytical Tool ◽  
Dna Probe ◽  
Dna Transfection ◽  
Potential Complication ◽  
Animal Cells ◽  
Rna Molecules ◽  
S1 Mapping ◽  
Powerful Analytical Tool

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

S1 mapping of purified nascent transcripts of simian virus 40

1984 ◽  
Vol 4 (4) ◽  
pp. 625-633
Author(s):  
D E Lycan ◽  
K J Danna
Keyword(s):  
Affinity Chromatography ◽  
Salt Concentration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Rna Molecules ◽  
S1 Mapping ◽  
Nascent Rna ◽  
Lower Temperature ◽  
Nascent Transcripts

We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.

scholarly journals Determination of nanoparticle localisation within subcellular organelles in vitro using Raman spectroscopy

2015 ◽  
Vol 7 (23) ◽  
pp. 10000-10017 ◽  
Author(s):  
Esen Efeoglu ◽  
Mark Keating ◽  
Jennifer McIntyre ◽  
Alan Casey ◽  
Hugh J. Byrne
Keyword(s):  
Raman Spectroscopy ◽  
Multivariate Analysis ◽  
Local Environment ◽  
Analytical Tool ◽  
Subcellular Organelles ◽  
Analysis Techniques ◽  
Powerful Analytical Tool

Raman spectroscopy with the aid of Multivariate Analysis techniques is a powerful analytical tool to determine the localisation of nanoparticles and their local environment within subcellular organelles.

scholarly journals S1 mapping of purified nascent transcripts of simian virus 40.

1984 ◽  
Vol 4 (4) ◽  
pp. 625-633 ◽  
Author(s):  
D E Lycan ◽  
K J Danna
Keyword(s):  
Affinity Chromatography ◽  
Salt Concentration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Rna Molecules ◽  
S1 Mapping ◽  
Nascent Rna ◽  
Lower Temperature ◽  
Nascent Transcripts

We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

scholarly journals Effect of synthetic bone replacement material of different size on shear stress resistance within impacted native and thermodisinfected cancellous bone: an in vitro femoral impaction bone grafting model

2021 ◽  
Author(s):  
C. Fölsch ◽  
P. Sahm ◽  
C. A. Fonseca Ulloa ◽  
G. A. Krombach ◽  
M. Kampschulte ◽  
...  
Keyword(s):  
Cancellous Bone ◽  
Bone Grafting ◽  
Shear Force ◽  
Distal Region ◽  
Impaction Bone Grafting ◽  
Proximal Point ◽  
Local Antibiotics ◽  
Joint Replacements ◽  
Native Bone

AbstractAntibiotic carrier particles of variable size might influence mechanic properties within impacted thermodisinfected and native cancellous bone different. Herafill®G containing calciumsulfate and calciumcarbonate provides high local concentrations of gentamicin being important for revision surgery in infected joint replacements. Native and thermodisinfected cancellous bone derived from 6 to 7 months old piglets was used for in vitro impaction bone grafting and supplemented each with Herafill®G granules of two different sizes. Micromovement of implants related to shear force was measured in 29 specimens distributed in 6 groups. Thermodisinfected cancellous bone revealed a significant higher shear force resistance than native bone with a mean difference of 423.8 mdeg/Nm (p < 0.001) ranging within 95% confidence interval from 181.5 to 666.0 mdeg/Nm. Adding small granules to thermodisinfected bone did not reduce shear force resistance significantly since adding large granules to native bone improved it by 344.0 mdeg/Nm (p < 0.003). Shear force resistance was found higher at the distal region of the implant compared to a proximal point of measurement throughout all specimens. Less impaction impulses were necessary for thermodisinfected bone. Thermodisinfected cancellous bone might achieve a higher degree of impaction compared with native bone resulting in increased resistance against shear force since impaction was found increased distally. Supplementation of thermodisinfected bone with small granules of Herafill®G might be considered for application of local antibiotics. Large granules appeared more beneficial for supplementation of native bone. Heterogeneity of bone graft and technical aspects of the impaction procedure have to be considered regarding the reproducibility of femoral impaction bone grafting.

scholarly journals Fluorogenic Aptasensors with Small Molecules

Chemosensors ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 54
Author(s):  
Eun-Song Lee ◽  
Jeong Min Lee ◽  
Hea-Jin Kim ◽  
Young-Pil Kim
Keyword(s):  
Small Molecules ◽  
In Vitro Selection ◽  
Molecular Beacon ◽  
Future Directions ◽  
Rna Molecules ◽  
Amplification Process ◽  
Target Molecules ◽  
And Performance ◽  
Fluorescence Turn On

Aptamers are single-stranded DNA or RNA molecules that can be identified through an iterative in vitro selection–amplification process. Among them, fluorogenic aptamers in response to small molecules have been of great interest in biosensing and bioimaging due to their rapid fluorescence turn-on signals with high target specificity and low background noise. In this review, we report recent advances in fluorogenic aptasensors and their applications to in vitro diagnosis and cellular imaging. These aptasensors modulated by small molecules have been implemented in different modalities that include duplex or molecular beacon-type aptasensors, aptazymes, and fluorogen-activating aptamer reporters. We highlight the working principles, target molecules, modifications, and performance characteristics of fluorogenic aptasensors, and discuss their potential roles in the field of biosensor and bioimaging with future directions and challenges.

scholarly journals HPLC-ESI-QTOF-MS as a Powerful Analytical Tool for Characterising Phenolic Compounds in Olive-leaf Extracts

2012 ◽  
Vol 24 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Rosa Quirantes-Piné ◽  
Jesús Lozano-Sánchez ◽  
Miguel Herrero ◽  
Elena Ibáñez ◽  
Antonio Segura-Carretero ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Analytical Tool ◽  
Olive Leaf ◽  
Leaf Extracts ◽  
Powerful Analytical Tool

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

1989 ◽  
Vol 9 (10) ◽  
pp. 4479-4487
Author(s):  
M Cotten ◽  
G Schaffner ◽  
M L Birnstiel
Keyword(s):  
Antisense Rna ◽  
Nuclear Extract ◽  
Mrna Processing ◽  
Rna Molecules ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Processing ◽  
Antisense Dna ◽  
Nuclear Ribonucleoprotein ◽  
Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

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