An encapsidated, subgenomic messenger RNA encodes the coat protein of carnation mottle virus

1984 ◽  
Vol 4 (11) ◽  
pp. 949-956 ◽  
Author(s):  
Sally-Ann Harbison ◽  
T. Michael A. Wilson ◽  
Jeffrey W. Davies
Keyword(s):  
Coat Protein ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mottle Virus ◽  
Free System ◽  
Translation Strategy ◽  
Internal Initiation ◽  
Carnation Mottle Virus

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of Mr 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with α-chymotrypsin and SDS/polyacrylamide-gel electrophoresis.

Changes in the messenger RNA population during sporulation of Eimeria maxima

Parasitology ◽  
1991 ◽  
Vol 102 (1) ◽  
pp. 1-8 ◽  
Author(s):  
J. Ellis ◽  
T. Thurlby
Keyword(s):  
Gene Transcription ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Polyacrylamide Gel ◽  
Eimeria Maxima

SUMMARYMessenger RNA has been extracted from oocysts of Eimeria maxima. Using the techniques of in vitro translation and SDS–polyacrylamide gel electrophoresis, we have been able to show that major changes in gene transcription occur during sporulation. Following an overall reduction in the abundance of many mRNAs, several genes identified as the result of an increase in the abundance of their transcripts, are highly expressed during the latter stages of sporulation. A study of two genes whose transcription is down-regulated has provided evidence which shows that both single copy and repetitive sequences are regulated during sporulation of the oocyst.

Synthesis of Albumin with Exogenous Mouse-Liver Messenger RNA in a Homologous Cell-Free System

1974 ◽  
Vol 52 (5) ◽  
pp. 429-432 ◽  
Author(s):  
A. J. Faber ◽  
S. H. Miall ◽  
T. Tamaoki
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Free System ◽  
Cell Free System ◽  
Membrane Bound

RNA extracted from total and membrane-bound polysomes of mouse liver was capable of directing protein synthesis in a homologous cell-free system in the presence of a 0.5 M KCl ribosomal wash fraction. Analysis of the products by polyacrylamide gel electrophoresis and immunoprecipitation showed that newly formed albumin could account for up to 8% of the total protein synthesized.

Narcissus mosaic virus: a potexvirus with an encapsidated subgenomic messenger RNA for coat protein

1983 ◽  
Vol 3 (9) ◽  
pp. 837-846 ◽  
Author(s):  
Margaret N. Short ◽  
Jeffrey W. Davies
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Small Rna ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subgenomic Rna ◽  
Virus Rna ◽  
Narcissus Mosaic Virus

Narcissus-mosaic-virus RNA is translated into a coat-protein-sized product in wheat-germ cell-free extracts. This protein was shown to be very similar to authentic coat protein by partial proteolysis in SDS/polyacrylamide-gel electrophoresis, and by serology. Fractionation of the RNA revealed a small RNA molecule of approx. 840 nucleotides, which alone coded for the coat protein. This subgenomic RNA was found to be encapsidated in a short virus particle.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

Organophosphorus insecticide resistance in a new strain of Culex quinquefasciatus (Diptera: Culicidae) from Tanga, Tanzania

1993 ◽  
Vol 83 (1) ◽  
pp. 67-74 ◽  
Author(s):  
A. Khayrandish ◽  
R.J. Wood
Keyword(s):  
Insecticide Resistance ◽  
Culex Quinquefasciatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Laboratory Culture ◽  
Fourth Instar ◽  
Resistance Allele ◽  
Susceptible Control ◽  
The Mean

AbstractFourth instar larvae of a new strain of Culex quinquefasciatus Say from Tanzania (TANGA) were tested for insecticide resistance. Initially, the resistance ratio (RR) to chlorpyrifos was 41.8, to temephos 30.8, to propoxur 3.7. After 2–3 years of laboratory culture, resistance to chlorpyrifos and propoxur had declined (chlorpyrifos 5.7, 3.8; propoxur 1.9, permethrin 1.9). Significant synergism was found between s, s, s-tributyl trithiophosphate (DEF) and chlorphyrifos, reducing the RR from 8.0 to 2.5. Synergism between piperonyl butoxide and permethrin was less than in a susceptible control strain. Twelve esterase isozymes of different relative mobilities (Rm) on polyacrylamide gel electrophoresis were identified, ten of which remained when the strain was reinvestigated two years (approximately 32 generations) later. Null activity for all but one of these bands was observed in some larvae. Four esterase bands (Rm 0.25, 0.27, 0.31, 0.34, designated A2, A3, B2, B3) showed polymorphism in activity, with very intense bands in some larvae. The mean frequency of bands with activity greater than standard, declined as organophosphorus (OP) resistance declined, but resistance was unconnected with the frequency of nulls at these positions. In mass larval assays of in vitro sensitivity of acetylcholinesterase (AChE) to propoxur, the I50 exceeded 10x10−4M, compared with 0.1x10−4M in a reverted resistant strain (RANGOON). Single larvel assays revealed heterogeneity, which was interpreted on the basis of an AChE resistance allele (AceR) with a frequency of 0.23.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins inPichia pastorisand Encapsulation of Fluorescent Myoglobin

2011 ◽  
Vol 1317 ◽  
Author(s):  
Yuanzheng Wu ◽  
Hetong Yang ◽  
Hyun-Jae Shin
Keyword(s):  
Coat Protein ◽  
Large Scale ◽  
Materials Science ◽  
Cesium Chloride ◽  
Size Exclusion ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

Abstract:Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemblein vitrointo virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein inPichia pastorisand production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference ofP. pastorisand cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed intoP. pastorisGS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids byP. pastorisfermentation now available for utilization in pharmacology or nanotechnology fields.

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