Ultrastructural analysis of early pre-messenger RNA processing events using the Miller chromatin spreading method

1992 ◽  
Vol 50 (1) ◽  
pp. 554-555
Author(s):  
Ann Beyer ◽  
Yvonne Osheim
Keyword(s):  
Rna Processing ◽  
Messenger Rna ◽  
Structural Features ◽  
Ultrastructural Analysis ◽  
Electron Microscopic ◽  
Mrna Transcripts ◽  
Carbon Coated ◽  
Nascent Transcripts ◽  
Active Genes

We use the Miller chromatin spreading technique for electron microscopic visualization of transcriptionally active genes. In this method, cells are hypotonically disrupted and cellular contents are diluted into water at pH 8-9 and fixed with formaldehyde. The dispersed cellular contents are centrifuged onto a carbon-coated EM grid; the majority of the material that is deposited on the grid consists of entangled masses of dispersed chromatin, some regions of which are transcriptionally active (Fig 1). Our interests lie in ultrastructural analysis of co-transcriptional RNA processing events on pre-mRNA transcripts, which we analyze by mapping structural features on successive nascent transcripts on a given gene. The two processing events that we have been able to study by this approach are the removal of introns by splicing and generation of the 3’ end of the transcript.

Evaluation of sectioning techniques for Electron Microscopic analyses of low-density polyethylene foams

1986 ◽  
Vol 44 ◽  
pp. 880-881
Author(s):  
P. L. McCarthy ◽  
C. W. Price
Keyword(s):  
Cell Walls ◽  
Surface Preparation ◽  
Structural Features ◽  
Low Density Polyethylene ◽  
Low Density ◽  
Electron Microscopic ◽  
Freeze Fracturing ◽  
Planar Surfaces ◽  
Carbon Coated ◽  
Sem Analyses

Cell sizes of low-density polyethylene foams in the range of 0.5 to 5μm are most conveniently measured by SEM analyses. Unfortunately, the cell walls are relatively weak and fragile membranes that either collapse or are severely distorted by conventional surface preparation and sectioning techniques. Sectioning damage can be circumvented to some extent by freeze fracturing. However, fractures tend to propagate through the weakest structural features, they can be associated with severe deformation, even at liquid nitrogen temperatures, and they frequently do not yield planar surfaces for reliable statistical measurements. Therefore, alternate sectioning techniques were evaluated. The most promising techniques are vibrotome sectioning and ultramicrotomy. These techniques are compared with freeze fracturing using SEM examinations of carbon-coated specimens prepared from the same batch of foam.

scholarly journals DNA Damage–Induced BARD1 Phosphorylation Is Critical for the Inhibition of Messenger RNA Processing by BRCA1/BARD1 Complex

2006 ◽  
Vol 66 (9) ◽  
pp. 4561-4565 ◽  
Author(s):  
Ho-Shik Kim ◽  
Hongjie Li ◽  
Murat Cevher ◽  
Alissa Parmelee ◽  
Danae Fonseca ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Processing ◽  
Messenger Rna

scholarly journals Pre-Messenger RNA Processing Factors in the Drosophila Genome

2000 ◽  
Vol 150 (2) ◽  
pp. F37-F44 ◽  
Author(s):  
Stephen M. Mount ◽  
Helen K. Salz
Keyword(s):  
Rna Processing ◽  
Messenger Rna ◽  
Drosophila Genome ◽  
Processing Factors

Distribution of the myosin I-like ninaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes

1992 ◽  
Vol 101 (1) ◽  
pp. 247-254 ◽  
Author(s):  
J.L. Hicks ◽  
D.S. Williams
Keyword(s):  
Compound Eye ◽  
Photoreceptor Cells ◽  
Amino Acid Sequences ◽  
Ultrastructural Analysis ◽  
Multivesicular Bodies ◽  
Filamentous Actin ◽  
Electron Microscopic ◽  
Myosin I ◽  
Specific Proteins ◽  
Mutant Phenotypes

The Drosophila ninaC gene encodes for two head-specific proteins of 132 kDa and 174 kDa. Their predicted amino acid sequences indicate that they may have myosin I and kinase properties. We have: (1) determined the cellular and subcellular distributions of the ninaC proteins in the Drosophila retina by electron microscopic immunocytochemistry with an antibody specific for epitopes shared by both proteins; (2) characterized the ultrastructure of the mutant phenotype. The proteins were detected only in the photoreceptor cells, but were detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they were found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdomeres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. Our ultrastructural analysis showed that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) was absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, was 3-fold greater in comparison with wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

Features of the architectonics of the microstructure of the primary remex of Owls (Strigiformes) due to the specifics of the flight

2021 ◽  
Vol 66 (2) ◽  
pp. 232-246
Author(s):  
E. O. Fadeeva
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Cross Section ◽  
Dorsal Surface ◽  
Structural Features ◽  
Electron Microscopic Investigation ◽  
Original Research ◽  
Electron Microscopic ◽  
Microstructural Characteristics ◽  
Scanning Electron

Conducted electron microscopic investigation of the primary remex fine structure of thirteen species of Owls (Strigiformes), using a scanning electron microscope (SEM). It is shown that Owls (Strigiformes) have a number of specific primary remex microstructural characteristics. First of all, these are the features of the structure of the pennaceous barb: a cross section configuration, a pith architectonics on the cross section and longitudinal sections, a cuticular structur of the barb. A number of the unique features in the microstructure of the vanules of the pennaceous barb have been found for the first time (at the scanning electron microscope level, at a large SEM magnification). First of all, these are the structural features of the distal barbules and the structure of the apical portion of the barb with the elongated proximal barbules and the distal barbules tightly contiguous to the ramus and closed with each other. Mentioned characteristics make for the thick velvet-like dorsal surface of the vane and the presence of a complex of peculiar “bunches” (fringes) forming the cleft edge (a fringed edge) of the inner vane – exceptionally specific adaptive characteristics in Strigiformes. Рresentenced original research results suggest that Owls (Strigiformes) have a number specific microstructural characteristics of the primary remex and also a number of the unique features in the microstructure of the primary remex which reflecting the ecological and morphological adaptations conditioned by the flight specificity.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals A dual reporter approach to quantify defects in messenger RNA processing

2009 ◽  
Vol 395 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Ayan Banerjee ◽  
Mimi C. Sammarco ◽  
Scott Ditch ◽  
Ed Grabczyk
Keyword(s):  
Rna Processing ◽  
Messenger Rna ◽  
Dual Reporter

Messenger RNA Processing in Eukaryotes

2013 ◽  
pp. 59-64
Author(s):  
K. Potter ◽  
N. Cremona ◽  
J.A. Wise
Keyword(s):  
Rna Processing ◽  
Messenger Rna

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

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