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.

The measurement of the production of during erythroid differentiation of the Friend erythroleukemia cell

1987 ◽  
Vol 65 (3) ◽  
pp. 188-194 ◽  
Author(s):  
E. Schmedt ◽  
L. Kleiman
Keyword(s):  
Rna Synthesis ◽  
Rna Polymerase Iii ◽  
Erythroid Differentiation ◽  
Gene Probe ◽  
5S Rna ◽  
Cytoplasmic Rna ◽  
Nuclear Rna ◽  
Nuclear Synthesis

The production of [Formula: see text] during Friend cell erythroid differentiation has been studied. In vitro measurements of total nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of [Formula: see text] was measured by hybridization of labelled nuclear RNA to a [Formula: see text] gene probe. During erythroid differentiation the percentage of nuclear RNA represented by [Formula: see text] remains constant (0.065%), so that the absolute synthesis of [Formula: see text] fluctuates during differentiation, in parallel with the fluctuations in the synthesis of total nuclear RNA. The relative synthesis of [Formula: see text]in vivo was studied by labelling cells with 35Pi, isolating the resulting radioactive tRNA – 5S RNA population, and hybridizing this population to a [Formula: see text] gene probe. The ratio of [Formula: see text] in newly synthesized cytoplasmic RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of [Formula: see text] during differentiation probably also occur for the nuclear synthesis of most tRNA and 5S RNA species. Attempts were made to measure the relative steady-state concentration of [Formula: see text] using both aminoacylation and in vitro end labelling of tRNA followed by hybridization to a [Formula: see text] gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating tRNA concentrations in the cell.

Testicular biosynthesis and epididymal endoproteolytic processing of rat sperm surface antigen 2B1

1996 ◽  
Vol 109 (10) ◽  
pp. 2561-2570
Author(s):  
R. Jones ◽  
A. Ma ◽  
S.T. Hou ◽  
R. Shalgi ◽  
L. Hall
Keyword(s):  
Plasma Membrane ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Close Correlation ◽  
Epididymal Maturation ◽  
Domain Organisation ◽  
Sperm Antigens ◽  
Pass Through

Binding of mammalian spermatozoa to the zona pellucida of homologous eggs is mediated by specific molecules on their surface membranes. In the present investigation we describe the biogenesis, epididymal processing and cellular distribution of a plasma membrane antigen (2B1) on rat spermatozoa that has a potential role in mediating zona binding. 2B1 is expressed postmeiotically in the testis as a precursor glycoprotein (approximately 60 kDa) that first appears on the plasma membrane of stage 6 to 8 round spermatids. Northern and western blot analyses show that there is a close correlation between the timing of transcription and expression of the glycoprotein on the cell surface. During spermatid elongation 2B1 is excluded from the head domain and is sequestered onto the sperm tail. As spermatozoa pass through the caput epididymidis 2B1 is endoproteolytically cleaved at a specific arginine residue (Arg 312) to produce a heterodimeric glycoprotein (approximately 40 kDa and approximately 19 kDa) containing intramolecular disulphide bridges. Endoproteolysis at Arg 312 also takes place during culture of washed testicular or caput spermatozoa in vitro and can be prevented by serine proteinase inhibitors or enhanced by trypsinisation. However, neither processing in vivo or in vitro has any effect on the domain organisation of 2B1 antigen i.e. it remains localised to the tail. These results support the hypothesis that sperm antigens that are important for fertilization are synthesized as precursor molecules in the testis and are then “activated' during epididymal maturation and capacitation, thereby ensuring that they only become fully functional at the site of fertilization.

Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

1987 ◽  
Vol 58 (02) ◽  
pp. 753-757 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Ramos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Bleeding Diathesis ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

In vivo and in vitro effect of proteinase inhibitors on gut proteinases

2005 ◽  
Vol 1722 (2) ◽  
pp. 156-167 ◽  
Author(s):  
V TAMHANE ◽  
N CHOUGULE ◽  
A GIRI ◽  
A DIXIT ◽  
M SAINANI ◽  
...  
Keyword(s):  
Proteinase Inhibitors ◽  
Vitro Effect

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

scholarly journals Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

2004 ◽  
Vol 78 (14) ◽  
pp. 7619-7633 ◽  
Author(s):  
Guohua Zhang ◽  
Jiuchun Zhang ◽  
Anne E. Simon
Keyword(s):  
Solution Structure ◽  
Rna Virus ◽  
Structural Features ◽  
General Mechanism ◽  
Minus Strand ◽  
Turnip Crinkle Virus ◽  
Structural Elements ◽  
Transcription In Vitro

ABSTRACT Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3′-terminal bases. Deletion of satC 3′-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3′ end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3′-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3′ terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3′ end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3′-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.

In-vivo phytochrome control of in vitro transcription rates in isolated nuclei from oat seedlings

Planta ◽  
1984 ◽  
Vol 161 (5) ◽  
pp. 444-450 ◽  
Author(s):  
Egon M�singer ◽  
Eberhard Sch�fer
Keyword(s):  
In Vitro Transcription ◽  
Isolated Nuclei

scholarly journals Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

2004 ◽  
Vol 78 (16) ◽  
pp. 8732-8745 ◽  
Author(s):  
Amy L. Odegard ◽  
Kartik Chandran ◽  
Xing Zhang ◽  
John S. L. Parker ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Structural Changes ◽  
General Mechanism ◽  
Membrane Penetration ◽  
Outer Capsid Protein ◽  
Core Proteins ◽  
Peptide Release ◽  
Outer Capsid

ABSTRACT Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

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