Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

scholarly journals Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2343-2353 ◽  
Author(s):  
R H Singer ◽  
G L Langevin ◽  
J B Lawrence
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Colloidal Gold ◽  
Messenger Rna ◽  
Signal To Noise Ratio ◽  
Simultaneous Detection ◽  
Gold Particles ◽  
Rna Molecules ◽  
Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

scholarly journals Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

scholarly journals Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.

1989 ◽  
Vol 37 (1) ◽  
pp. 97-104 ◽  
Author(s):  
R A Wolber ◽  
T F Beals ◽  
H F Maassab
Keyword(s):  
In Situ Hybridization ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ultrastructural Localization ◽  
Dna Probe ◽  
Gold Particles ◽  
Virus Rna ◽  
Dnase Treatment ◽  
Simplex Virus

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.

Specificity of Lectins Labeled with Colloidal Gold to the Exopolymeric Matrix Carbohydrates of the Sulfate-Reducing Bacteria Biofilm Formed on Steel

2020 ◽  
Vol 82 (5) ◽  
pp. 11-20
Author(s):  
D.R. Abdulina ◽  
◽  
L.M. Purish ◽  
G.O. Iutynska ◽  
◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Colloidal Gold ◽  
Steel Surface ◽  
Lectin Binding ◽  
Sulfate Reducing Bacteria ◽  
Gold Particles ◽  
Sulfate Reducing ◽  
Transmission Electron ◽  
Reducing Bacteria

The studies of the carbohydrate composition of the sulfate-reducing bacteria (SRB) biofilms formed on the steel surface, which are a factor of microbial corrosion, are significant. Since exopolymers synthesized by bacteria could activate corrosive processes. The aim of the study was to investigate the specificity of commercial lectins, labeled with colloidal gold to carbohydrates in the biofilm exopolymeric matrix produced by the corrosive-relevant SRB strains from man-caused ecotopes. Methods. Microbiological methods (obtaining of the SRB biofilms during cultivation in liquid Postgate B media under microaerophilic conditions), biochemical methods (lectin-binding analysis of 10 commercial lectins, labeled with colloidal gold), transmission electron microscopy using JEM-1400 JEOL. Results. It was shown using transmission electron microscopy that the binding of lectins with carbohydrates in the biofilm of the studied SRB strains occurred directly in the exopolymerіс matrix, as well as on the surfaces of bacterial cells, as seen by the presence of colloidal gold particles. For detection of the neutral carbohydrates (D-glucose and D-mannose) in the biofilm of almost all studied bacterial strains PSA lectin was the most specific. This lectin binding in biofilms of Desulfotomaculum sp. К1/3 and Desulfovibrio sp. 10 strains was higher in 90.8% and 94.4%, respectively, then for ConA lectin. The presence of fucose in the SRB biofilms was detected using LABA lectin, that showed specificity to the biofilm EPS of all the studied strains. LBA lectin was the most specific to N-аcetyl-D-galactosamine for determination of amino sugars in the biofilm. The amount of this lectin binding in D. vulgaris DSM644 biofilm was 30.3, 10.1 and 9.3 times higher than SBA, SNA and PNA lectins, respectively. STA, LVA and WGA lectins were used to detect the N-acetyl-Dglucosamine and sialic acid in the biofilm. WGA lectin showed specificity to N-acetyl-D-glucosamine in the biofilm of all the studied SRB; maximum number of bounded colloidal gold particles (175 particles/μm2) was found in the Desulfotomaculum sp. TC3 biofilm. STA lectin was interacted most actively with N-acetyl-D-glucosamine in Desulfotomaculum sp. TC3 and Desulfomicrobium sp. TC4 biofilms. The number of bounded colloidal gold particles was in 9.2 and 7.4 times higher, respectively, than using LVA lectin. The lowest binding of colloidal gold particles was observed for LVA lectin. Conclusions. It was identified the individual specificity of the 10 commercial lectins to the carbohydrates of biofilm matrix on the steel surface, produced by SRB. It was estimated that lectins with identical carbohydrates specificity had variation in binding to the biofilm carbohydrates of different SRB strains. Establishing of the lectin range selected for each culture lead to the reduction of the scope of studies and labor time in the researching of the peculiarities of exopolymeric matrix composition of biofilms formed by corrosiverelevant SRB.

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