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)

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.

scholarly journals Isolation and Characterization of a Novel Member of the Relaxin/Insulin Family from the Testis of the Frog Rana esculenta*

Endocrinology ◽  
2001 ◽  
Vol 142 (7) ◽  
pp. 3231-3238 ◽  
Author(s):  
Gianluca de Rienzo ◽  
Francesco Aniello ◽  
Margherita Branno ◽  
Sergio Minucci
Keyword(s):  
In Situ Hybridization ◽  
Amino Acid ◽  
Messenger Rna ◽  
Rana Esculenta ◽  
Amino Acid Sequences ◽  
Characteristic Functional ◽  
Isolation And Characterization ◽  
Single Transcript ◽  
A Domains

Abstract A complementary DNA (cDNA) encoding a frog relaxin/insulin member family (fRLX) from testis cDNA library was isolated and characterized. The fRLX cDNA predicted a 155-amino acid protein with a low homology to mammalian RLF and relaxin. Northern blot analysis revealed a single transcript expressed in the interstitial compartment, RT-PCR, evidenced that fRLX is expressed at low levels in the oviduct and ovary too. The predicted mature fRLX protein, composed of the signal peptide, B, C, and A domains, has conserved amino acid sequences in the characteristic functional domains. A different expression of the transcript was found during the frog reproductive cycle, with a peak in Spring. After administration of ethane dimethane sulfonate, by in situ hybridization, fRLX messenger RNA disappeared from the interstitial compartment and reappeared again at the time of generating of a new population of Leydig cells (LC), strongly indicating that LC are the interstitial cell type expressing fRLX. Preliminary results obtained by in situ hybridization, performed on testis of hypophysectomized frogs evidenced a pituitary control of fRLX expression. This study is the first cloning of a relaxin/insulin family member in a nonmammalian vertebrate. In addition, because fRLX expression changes during the annual cycle suggesting its involvement in spermatogenesis, fRLX may be considered a new marker for the study of spermatogenesis in the Rana esculenta.

scholarly journals IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

1974 ◽  
Vol 63 (2) ◽  
pp. 402-413 ◽  
Author(s):  
P. R. Harrison ◽  
D. Conkie ◽  
N. Affara ◽  
J. Paul
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Fetal Liver ◽  
Liver Cells ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Fetal Liver Cells

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.

scholarly journals Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.

1980 ◽  
Vol 87 (2) ◽  
pp. 480-487 ◽  
Author(s):  
J F Catterall ◽  
J P Stein ◽  
P Kristo ◽  
A R Means ◽  
B W O'Malley
Keyword(s):  
Dna Sequences ◽  
Messenger Rna ◽  
Amino Acid Sequences ◽  
Structural Domains ◽  
Complementary Dna ◽  
Homologous Sequences ◽  
Functional Regions ◽  
Bacterial Plasmids ◽  
Dna Mixture

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.

Defining protective responses to pathogens: cytokine profiles in leprosy lesions

Science ◽  
1991 ◽  
Vol 254 (5029) ◽  
pp. 277-279 ◽  
Author(s):  
M Yamamura ◽  
K Uyemura ◽  
RJ Deans ◽  
K Weinberg ◽  
TH Rea ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Interleukin 2 ◽  
Interleukin 10 ◽  
Interleukin 4 ◽  
Cell Mediated Immunity ◽  
Messenger Rnas ◽  
Interleukin 5 ◽  
Immunological Mechanisms

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

scholarly journals Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization.

1982 ◽  
Vol 95 (1) ◽  
pp. 1-7 ◽  
Author(s):  
W R Jeffery
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Significant Proportion ◽  
Insoluble Fraction ◽  
Radioactive Isotopes ◽  
Follicle Cells ◽  
Insoluble Residue ◽  
Framework Analysis ◽  
Triton X

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent-extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

DEGRADATION OF MESSENGER RNA IN MAMMALIAN CELLS

1972 ◽  
Vol 71 (2_Suppla) ◽  
pp. S369-S380 ◽  
Author(s):  
Francis T. Kenney ◽  
Kai-Lin Lee ◽  
Charles D. Stiles
Keyword(s):  
Messenger Rna ◽  
Mammalian Cells ◽  
Messenger Rnas ◽  
Tyrosine Transaminase

ABSTRACT Analyses of the response of hydrocortisone-induced tyrosine transaminase in cultured H-35 cells to inhibitors of translation (cycloheximide, puromycin) suggest: (1) that bound ribosomes stabilize messenger RNA in vivo; (2) that messenger is degraded at a rate determined by the rate of translation. Since specific messenger RNAs of mammalian cells are degraded at quite different rates, there may be extensive heterogeneity either in the rate at which ribosomes traverse different messengers or in the number of ribosomes which translate specific messenger RNAs.

scholarly journals Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Muchen Pan ◽  
Ana L. Alvarez-Cabrera ◽  
Joon S. Kang ◽  
Lihua Wang ◽  
Chunhai Fan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Molecular Interactions ◽  
Messenger Rna ◽  
Rna Binding ◽  
Capsid Proteins ◽  
Rna Transcription ◽  
Infectious Particle ◽  
Virion Assembly ◽  
Rna Capping

AbstractMammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

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.

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