Origin and spatial distribution of maternal messenger RNA during oogenesis of an insect, Oncopeltus fasciatus

1979 ◽  
Vol 39 (1) ◽  
pp. 63-76
Author(s):  
D.G. Capco ◽  
W.R. Jeffery
Keyword(s):  
Spatial Distribution ◽  
Messenger Rna ◽  
Germinal Vesicle ◽  
Oncopeltus Fasciatus ◽  
Follicle Cells ◽  
Rna Molecules ◽  
Maternal Mrna ◽  
Chorion Formation ◽  
Oocyte Cytoplasm ◽  
Rna Accumulation

In order to investigate the origin and spatial distribution of maternal mRNA during oogenesis, in situ hybridization with [3H]-poly(U) was utilized for the detection of poly(A)-containing RNA [poly(A)+RNA] in histological sections of Oncopeltus fasciatus ovaries. In the germarium poly(A)+RNA was found to accumulate in the trophocyte cytoplasm concomitant with the maturation of these cells. Poly(A)+RNA was also detected in the trophic cores and nutritive tubes suggesting that these channels participate in the transport of trophocyte-derived mRNA to the oocytes. Although large amounts of poly(A)+RNA were also detected in the cytoplasm of the follicle cells, particularly during late vitellogenesis when pseudopod-like processes projected into the ooplasm, no evidence was obtained for the transport of poly(A)+RNA from these processes to the oocytes. The content of poly(A)+RNA in the oocyte cytoplasm continually increased during oogenesis. In stage 2–4 oocytes poly(A)+RNA accumulation occurred in the apparent absence of transcriptional activity in the germinal vesicle nuclei suggesting that most maternal mRNA molecules synthesized during early oogenesis are of trophocyte origin. Poly(A)+RNA also continued to accumulate after chorion formation, when the nutritive tubes are longer active in RNA transport. This implies that other sources of maternal mRNA may exist during late oogenesis. The distribution of poly(A)+RNA molecules in the oocyte cytoplasm appeared to be uniform throughout oogenesis with one exception. During late vitellogenesis poly(A)+RNA activity was significantly enhanced in the anterior and posterior periplasmic cytoplasms relative to the lateral periplasm and the endoplasm. After chorion formation these variations disappeared. The results suggest that maternal mRNA molecules arise from at least 2 sources during oogenesis. During late vitellogenesis these molecules appear to be subject to differential localization in the polar perimeters of the oocyte cytoplasm.

Maternal messenger RNA distribution in silkmoth eggs. I. Clone Ec4B is associated with the cortical cytoskeleton

Development ◽  
1990 ◽  
Vol 108 (3) ◽  
pp. 497-505
Author(s):  
W.H. Kastern ◽  
C.A. Watson ◽  
S.J. Berry
Keyword(s):  
Messenger Rna ◽  
Amino Acid Sequences ◽  
Follicle Cells ◽  
Messenger Rnas ◽  
Maternal Mrna ◽  
Hybridization Data ◽  
Interesting Possibility ◽  
Genome Analyses ◽  
Cortical Cytoskeleton

We have constructed a cDNA library from mature egg RNA of the silkmoth, Hyalophora cecropia. Differential screening of the library using cDNA made against mRNAs from the yolky cytoplasm (soluble fraction) and the cortical cytoplasm (cytoskeletal-associated or cortical fraction) resulted in several clones that hybridized to a higher degree to the cDNA from the cytoskeletal-associated fraction. We selected and analyzed the clone giving the strongest signal (designated Ec4b) for its distribution in situ and found that it bound to mRNAs in the nurse cell cytoplasm, in the cortex and in the follicle cells of oocytes. Hybridization of the insert from Ec4b to both detergent-soluble and -insoluble (cortical) RNA on dot blots further supported the observation that the mRNA corresponding to Ec4b was enriched in this cytoskeletal fraction. The mRNA for Ec4b was approximately 500 bases long and the gene seems to be a member of a large multigene family in the H. cecropia genome. Analyses of the nucleotide and amino acid sequences reveal similarity to lepidopteran chorion genes and a lesser but convincing similarity to vertebrate cytokeratins. The filter and in situ hybridization data point to the association of specific messenger RNAs with the cortical cytoskeleton of silkmoth oocytes. Aspects of the structure of the protein encoded by this mRNA suggest that it is a structural component necessary for formation of the cellular blastoderm of the embryo. The association of this maternal mRNA with the cortical cytoskeleton presents the interesting possibility that mRNA bound to the cytoskeleton may be capable of participating in the synthesis of new cytoskeleton or related structures during blastoderm formation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Fluctuating Diffusivity of RNA-Protein Particles: Analogy with Thermodynamics

Entropy ◽  
2021 ◽  
Vol 23 (3) ◽  
pp. 333
Author(s):  
Yuichi Itto
Keyword(s):  
Internal Energy ◽  
Messenger Rna ◽  
Living Cells ◽  
Statistical Fluctuation ◽  
Rna Molecules ◽  
Average Value ◽  
Thermodynamic Entropy ◽  
Laws Of Thermodynamics ◽  
Protein Particles ◽  
Quantity Of Heat

A formal analogy of fluctuating diffusivity to thermodynamics is discussed for messenger RNA molecules fluorescently fused to a protein in living cells. Regarding the average value of the fluctuating diffusivity of such RNA-protein particles as the analog of the internal energy, the analogs of the quantity of heat and work are identified. The Clausius-like inequality is shown to hold for the entropy associated with diffusivity fluctuations, which plays a role analogous to the thermodynamic entropy, and the analog of the quantity of heat. The change of the statistical fluctuation distribution is also examined from a geometric perspective. The present discussions may contribute to a deeper understanding of the fluctuating diffusivity in view of the laws of thermodynamics.

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.

Light-Induced Gene Expression Using Messenger RNA Molecules

2010 ◽  
Vol 20 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Sigurd Bøe ◽  
Stein Sæbøe-Larssen ◽  
Eivind Hovig
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Rna Molecules

scholarly journals The Impact of Dietary Compounds in Functional Foods on MicroRNAs Expression

2021 ◽  
Author(s):  
Wittaya Chaiwangyen
Keyword(s):  
Messenger Rna ◽  
Positive Impact ◽  
Biologically Active ◽  
Transcriptional Gene Silencing ◽  
Cellular Mirnas ◽  
Rna Molecules ◽  
Post Transcriptional Gene Silencing ◽  
Molecular Machinery ◽  
Different Origins ◽  
The Impact

MicroRNAs (miRNAs) are a class of non-coding endogenous RNA molecules that are involved in post-transcriptional gene silencing via binding to their target messenger RNA, leading to mRNA degradation or translational repression. MicroRNAs can be modulated by several factors including hormones, transcription factors, and dietary compounds. These biologically active compounds have positive impact on the progression of human pathology including non-communicable diseases, which indicating that administration of diet may have potential as therapeutic agents in modulating the risk of chronic diseases. Interestingly, evidence emerging in recent years suggests that dietary miRNAs can be absorbed in human circulation, modulated human gene expression and biological functions. The exploitation of the miRNA functioning within different origins, cellular miRNAs and dietary miRNAs will help us to understand the molecular machinery as well as the regulatory mechanisms involved in fundamentally important biological processes. Therefore, this knowledge may be applied of natural bioactive compounds in preventive or therapeutic approaches.

"Cap" sequences is poly(A)-rich RNA from dry wheat embryos

1981 ◽  
Vol 59 (10) ◽  
pp. 868-870 ◽  
Author(s):  
Byron G. Lane
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Messenger Rna ◽  
High Performance ◽  
Higher Plants ◽  
Rna Molecules ◽  
Wheat Embryos ◽  
The Subject ◽  
Widespread Interest

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5′-terminai "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5′)ppp(5′)N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C), and uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5′)ppp(5′)A, with roughly equivalent amounts of m7G(5′)ppp(5′)G and m7G(5′)ppp(5′)C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; these differences have not been previously noted in the literature and are the subject of brief comment in this paper.

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation

1988 ◽  
Vol 90 (4) ◽  
pp. 543-553 ◽  
Author(s):  
J. Gautier ◽  
J.K. Pal ◽  
M.F. Grossi de Sa ◽  
J.C. Beetschen ◽  
K. Scherrer
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Immunoblot Analysis ◽  
Ambystoma Mexicanum ◽  
Meiotic Maturation ◽  
Crescent Formation ◽  
Oocyte Growth ◽  
Oocyte Cytoplasm

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.

Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 769-779
Author(s):  
R.C. Smith ◽  
W.M. Bement ◽  
M.A. Dersch ◽  
E. Dworkin-Rastl ◽  
M.B. Dworkin ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Xenopus Oocytes ◽  
Cell Metabolism ◽  
Germinal Vesicle ◽  
Annulate Lamellae ◽  
Maternal Mrna ◽  
Nonspecific Effects ◽  
Abnormal Accumulation ◽  
Cellular Mrnas

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv). Ultrastructural analysis detected further abnormalities including blebbing of the plasma membrane, anomalous cytoskeletal structures, hyperorganised annulate lamellae, hyperinvagination of the gv, and formation of irregular nucleoli within the gv. Analysis of newly synthesised proteins by [35S]methionine radiolabelling of oocytes demonstrated that ODN injection can trigger a general decrease in both label uptake and protein synthesis. Qualitative effects on protein synthesis could also be observed, particularly a decrease in synthesis of high molecular weight proteins. The severity of ODN-induced effects is dose-dependent and highly variable from ODN to ODN. The previously reported delay in progesterone-induced maturation observed in oocytes depleted of the maternal mRNA D7 by ODN-directed degradation (Smith R. C., Dworkin M. B. and Dworkin-Rastl E. (1988) Genes and Devpt. 2, 1296–1306) is most likely a result of nonspecific ODN effects in the oocyte. Oocytes injected with effective antisense D7 ODNs that do not display detectable side effects matured with normal kinetics.

Polycation Coated Polymeric Particles as Vehicles of RNA Delivery Into Immune Cells

2010 ◽  
Author(s):  
Ying Zheng ◽  
Wilson S. Meng
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Messenger Rna ◽  
Flow Cytometric Analysis ◽  
Confocal Imaging ◽  
Dependent Manner ◽  
Carrier System ◽  
Foxp3 Mrna ◽  
Rna Molecules ◽  
Polymeric Particles

The purpose of this work is to develop a carrier system for delivering RNA molecules aimed to downregulate specific functions in T cells. In many forms of cancer, T cells that express the protein Forkhead Box P3 (Foxp3) are associated with cancer progression. These cells can be identified by CD4 and CD25, molecules express on the cell surface. Studies have shown that downregulation of Foxp3 can increase the ability of other immune cells to destroy tumors. A class of RNA molecules, commonly referred to as “siRNA”, bind to and degrade specific messenger RNA (mRNA) in a sequence-dependent manner such that expression of the encoded protein is terminated. Because mRNA molecules are located inside cells, a carrier system is required to facilitate the uptake of siRNA, which does not passively diffuse through the plasma membrane. To this end, nanosized polymeric particles coated with the polycation, ornithinex10-histidinex6 (or O10H6) were used to adsorb siRNA that bind to the mRNA encoding Foxp3. The RNA-loaded particles are spherical and uniform in size (normally distributed, polydispersity index = 0.072). Loading of RNA to the particles was confirmed using gel electrophoresis. RNA complexed with the particles are protected from serum destabilization: 83.1% of RNA were recovered compared to 36.1% in RNA that were not associated with the particles. Association with the particles increased the uptake of the RNA in mouse T cells from 3.2±0.2% (free RNA) to 20.1±3.9%. Specifically, uptake of the RNA in T cells that express CD4 increased from 2.7±0.2% to 27.1±1.3% when particles were employed. These differences are statistically significant in three experiments conducted (p < 0.01). Internalization of the RNA into T cells was confirmed using confocal imaging. Flow cytometric analysis showed that the particle-complexed RNA reduced the percentage of T cells that express both CD4 and CD25 in mice carrying tumors from 24.0% when free RNA molecules were used to 13.5%. In these cells, the level of Foxp3 mRNA was reduced by 30%. In conclusion, the particles facilitate the uptake of siRNA molecules into a population of T cells that is known to promote cancer growth.

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