scholarly journals Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.

1993 ◽  
Vol 121 (4) ◽  
pp. 729-742 ◽  
Author(s):  
Z Zachar ◽  
J Kramer ◽  
I P Mims ◽  
P M Bingham
Keyword(s):  
Experimental System ◽  
Genetic System ◽  
Nuclear Surface ◽  
Rna Transport ◽  
Mrna Metabolism ◽  
Its Gene ◽  
Nuclear Rna ◽  
Intranuclear Transport ◽  
Polytene Nuclei ◽  
Nascent Transcripts

We report studies using an enhanced experimental system to investigate organization of nuclear pre-mRNA metabolism. It is based on the powerful genetic system and polytene nuclei of Drosophila. We observe (at steady state) movement of a specific pre-mRNA between its gene and the nuclear surface. This movement is isotropic, at rates consistent with diffusion and is restricted to a small nuclear subcompartment defined by exclusion from chromosome axes and the nucleolus. Bulk polyadenylated nuclear pre-mRNA precisely localizes in this same subcompartment indicating that most or all pre-mRNAs use the same route of intranuclear movement. In addition to association with nascent transcripts, snRNPs are coconcentrated with pre-mRNA in this subcompartment. In contrast to constitutive splices, at least one regulated splice occurs slowly and may undergo execution remotely from the site of pre-mRNA synthesis. Details of our results suggest that retention of incompletely spliced pre-mRNA is a function of the nuclear surface. We propose a simple model--based on channeled diffusion--for organization of intranuclear transport and metabolism of pre-mRNAs in polytene nuclei. We argue that this model can be generalized to all metazoan nuclei.

Changes in nuclear RNA transport incident to carcinogenesis

1977 ◽  
Vol 13 (2) ◽  
pp. 139-147 ◽  
Author(s):  
D.E. Schumm ◽  
M. Hanausek-Walaszek ◽  
A. Yannarell ◽  
T.E. Webb
Keyword(s):  
Rna Transport ◽  
Nuclear Rna

scholarly journals 864 EXPLORING THE MECHANISM OF NUCLEAR RNA TRANSPORT WITH MUTANT HUMAN tRNA GENES

1985 ◽  
Vol 19 (4) ◽  
pp. 254A-254A
Author(s):  
Janet A Tobian ◽  
Jose G Castano ◽  
Michael A Zasloff
Keyword(s):  
Trna Genes ◽  
Rna Transport ◽  
Nuclear Rna

scholarly journals An assessment of some methodological criticisms of studies of RNA efflux from isolated nuclei

1983 ◽  
Vol 214 (3) ◽  
pp. 915-921 ◽  
Author(s):  
P S Agutter
Keyword(s):  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
General Mechanism ◽  
Nuclear Surface ◽  
Efflux Rate ◽  
Isolated Nuclei ◽  
Cytoplasmic Rna ◽  
Nuclear Rna

RNA efflux from isolated nuclei can be studied either as a means of elucidating the general mechanism of nucleo-cytoplasmic RNA transport, or as part of an investigation of the processing and utilization of particular gene transcripts. The present paper describes an assessment of three methodological criticisms of RNA-efflux measurements that are made for the former reason: for such measurements, it is sufficient to show that the post-incubation supernatant RNA is similar overall to homologous cytoplasmic mRNA, rather than to nuclear RNA, that is nevertheless of intranuclear origin, and that alterations to the medium during experiments do not markedly perturb this general nuclear restriction. The results seem to justify the following conclusions. (1) Although degradation of the nuclear RNA occurs during incubation in vitro, this process does not account for the appearance of RNA in the postnuclear supernatant. The degradation can be largely prevented by the addition of serine-proteinase inhibitors without altering the RNA efflux rate. (2) Some adsorption of labelled cytoplasmic RNA to the nuclear surface occurs during both isolation and incubation of the nuclei, and some desorption occurs during incubation. However, these effects introduce errors of less than 10% into the measurements of efflux rates. (3) Exogenous acidic polymers, including polyribonucleotides, disrupt nuclei and increase the apparent RNA efflux rate by causing leakage of nuclear contents. However, this effect can largely be overcome by including the nuclear stabilizers spermidine, Ca2+ and Mn2+ in the medium. In terms of this assessment, it appears that RNA efflux from isolated nuclei in media containing nuclear stabilizers serves as a reasonable model for transport in vivo.

scholarly journals A cryoprotectant induces conformational change in glyceraldehyde-3-phosphate dehydrogenase

2018 ◽  
Vol 74 (5) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Ju Kim
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Dna Replication ◽  
Membrane Fusion ◽  
Three Dimensional ◽  
Glycolytic Enzyme ◽  
Rna Transport ◽  
Nuclear Rna ◽  
Cellular Apoptosis ◽  
Dimensional Crystal

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, catalyses the conversion of D-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. While mammalian and yeast GAPDHs are multifunctional proteins that have additional functions beyond those involved in glycolysis, including reactions related to nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis, Escherichia coli GAPDH (ecGAPDH) has only been reported to function in glycolysis. The S-loop of GAPDH is required for interaction with its cofactor and with other proteins. In this study, the three-dimensional crystal structure of GAPDH treated with trehalose is reported at 2.0 Å resolution. Trehalose was used as a cryoprotectant for the GAPDH crystals. The structure of trehalose-bound ecGAPDH was compared with the structures of both NAD+-free and NAD+-bound ecGAPDH. At the S-loop, the bound trehalose in the GAPDH structure induces a 2.4° rotation compared with the NAD+-free ecGAPDH structure and a 3.1° rotation compared with the NAD+-bound ecGAPDH structure.

scholarly journals High Frequencies of Preprophase Bands in Soybean Protoplast Cultures

1989 ◽  
Vol 92 (4) ◽  
pp. 575-580
Author(s):  
HONG WANG ◽  
ADRIAN J. CUTLER ◽  
LARRY C. FOWKE
Keyword(s):  
Cell Line ◽  
High Frequency ◽  
Developmental Stages ◽  
Plant Cells ◽  
Experimental System ◽  
Nuclear Surface ◽  
High Frequencies ◽  
Index Ratio ◽  
Soybean Cell ◽  
Preprophase Bands

Protoplasts derived from a non- regenerable soybean cell line provide an excellent experimental system for studying plant preprophase bands (PPBs). Cultured protoplasts developed PPBs in high frequencies, permitting a detailed analysis of PPB development. From observations of thousands of PPBs, six distinct developmental stages were identified. This classification should prove useful in recognizing developmental stages of PPBs and in comparing results among different tissues and species. Perinuclear fluorescence appeared when PPBs were well developed. It consisted of microtubule strands radiating from the nuclear surface at its early stages and more extensive arrays on the surface at later stages. All protoplast cultures from 0.5 day to 5 days had a PPB index (ratio between number of PPBs and number of phragmoplasts) greater than unity, suggesting that in this cell line a high frequency of PPBs is apparently not related to the potential for organized growth (e.g. embryogenesis). Different methods of quantifying the occurrence of PPBs were evaluated. The PPB value (ratio between % of cells with PPB and the mitotic index) was compared with the PPB index and results indicate that it is a useful new parameter for studies of the distribution of PPBs in plant cells.

scholarly journals Construction of an infectious clone of Zika virus stably expressing an EGFP marker in a eukaryotic expression system

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Jing Gao ◽  
Jiayi Chen ◽  
Weizhi Lu ◽  
Jintai Cai ◽  
Linjuan Shi ◽  
...  
Keyword(s):  
Zika Virus ◽  
Infectious Clone ◽  
Expression System ◽  
Plaque Assay ◽  
Experimental System ◽  
Genetic System ◽  
Vero Cells ◽  
Eukaryotic Expression ◽  
Reverse Genetic System ◽  
Cmv Promoter

Abstract Background Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP. Methods A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot. Results Rescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy. Conclusion Our results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.

scholarly journals Surveillance-ready transcription: nuclear RNA decay as a default fate

Open Biology ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 170270 ◽  
Author(s):  
Stefan Bresson ◽  
David Tollervey
Keyword(s):  
Quality Control ◽  
Rna Decay ◽  
Control Mechanisms ◽  
Protein Coding ◽  
Nuclear Rna ◽  
Rna Quality Control ◽  
Spurious Transcription ◽  
Rna Biogenesis ◽  
Bona Fide ◽  
Nascent Transcripts

Eukaryotic cells synthesize enormous quantities of RNA from diverse classes, most of which are subject to extensive processing. These processes are inherently error-prone, and cells have evolved robust quality control mechanisms to selectively remove aberrant transcripts. These surveillance pathways monitor all aspects of nuclear RNA biogenesis, and in addition remove nonfunctional transcripts arising from spurious transcription and a host of non-protein-coding RNAs (ncRNAs). Surprisingly, this is largely accomplished with only a handful of RNA decay enzymes. It has, therefore, been unclear how these factors efficiently distinguish between functional RNAs and huge numbers of diverse transcripts that must be degraded. Here we describe how bona fide transcripts are specifically protected, particularly by 5′ and 3′ modifications. Conversely, a plethora of factors associated with the nascent transcripts all act to recruit the RNA quality control, surveillance and degradation machinery. We conclude that initiating RNAPII is ‘surveillance ready’, with degradation being a default fate for all transcripts that lack specific protective features. We further postulate that this promiscuity is a key feature that allowed the proliferation of vast numbers of ncRNAs in eukaryotes, including humans.

Fine Structure of Cytochalasin Induced Polyploid Cells

1968 ◽  
Vol 26 ◽  
pp. 122-123
Author(s):  
Awtar Krishan ◽  
Nestor Bohonos
Keyword(s):  
Fine Structure ◽  
Cytochalasin B ◽  
Present Report ◽  
Cell Monolayer ◽  
Polyploid Cell ◽  
Specific Cell ◽  
Nuclear Surface ◽  
Unsuccessful Attempt ◽  
Daughter Cells ◽  
Polyploid Cells

Cytochalasin B, a mould metabolite from Helminthosporium dermatioideum has been shown to interfere with specific cell activities such as cytoplasmic cleavage and cell movement. Cells undergoing nuclear division in the presence of cytochalasin B are unable to complete the separation of the resulting daughter cells. In time-lapse studies, the daughter cells coalesce after an initial unsuccessful attempt at separation and form large multinucleate polyploid cells. The present report describes the fine structure of the large polyploid cells induced in Earle's L-cell monolayer cultures by exposure to cytochalasin B (lγ/ml) for 92 hours.In the present material we have seen as many as 7 nuclei in these polyploid cells. Treatment with cytochalasin B for longer periods of time (6 to 7 days, with one medium change on the 3rd day) did not increase the number of nuclei beyond the 7 nuclei stage. Figure 1 shows a large polyploid cell with four nuclei. These nuclei are indistinguishable in their fine structure from those of the cells from control cultures but often show unusually large numbers of cytoplasmic invaginations and extensions of the nuclear surface (Figure 2).

Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

1995 ◽  
Vol 53 ◽  
pp. 764-765
Author(s):  
W.F. Marshall ◽  
A.F. Dernburg ◽  
B. Harmon ◽  
J.W. Sedat
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Condensation ◽  
Three Dimensional ◽  
Physiological Role ◽  
Nuclear Surface ◽  
Intact Cells ◽  
Molecular Determinants ◽  
Surface Harmonic ◽  
Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

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