In Vitro Cleavage of eIF4GI but not eIF4GII by HIV-1 Protease and its Effects on Translation in the Rabbit Reticulocyte Lysate System

2002 ◽  
Vol 318 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Théophile Ohlmann ◽  
Déborah Prévôt ◽  
Didier Décimo ◽  
Florence Roux ◽  
Jérôme Garin ◽  
...  
Keyword(s):  
Rabbit Reticulocyte Lysate ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Hiv 1

scholarly journals Seed extracts inhibiting protein synthesis in vitro

1980 ◽  
Vol 186 (2) ◽  
pp. 439-441 ◽  
Author(s):  
A Gasperi-Campani ◽  
L Barbieri ◽  
P Morelli ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Rabbit Reticulocyte Lysate ◽  
Intact Cells ◽  
Euonymus Europaeus ◽  
Lower Extent ◽  
Reticulocyte Lysate ◽  
Dialysis Membranes ◽  
Inhibiting Protein ◽  
Seed Extracts

Of 33 seed extracts examined, 12 inhibited protein synthesis in a rabbit reticulocyte lysate. This activity seems to be due to a protein, since (i) it was recovered with the (NH4)2SO4 precipitate, (ii) it was retained by dialysis membranes, and (iii) in all cases but one was destroyed by boiling. Only the extracts from the seeds of Adenia digitata and, to a lower extent, of Euonymus europaeus inhibited protein synthesis in intact cells.

scholarly journals Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena

1987 ◽  
Vol 244 (2) ◽  
pp. 331-335 ◽  
Author(s):  
P H Andreasen ◽  
H Dreisig ◽  
K Kristiansen
Keyword(s):  
Codon Usage ◽  
Genetic Code ◽  
Subcellular Fraction ◽  
Tetrahymena Thermophila ◽  
Rabbit Reticulocyte Lysate ◽  
Reading Frame ◽  
Universal Genetic Code ◽  
Reticulocyte Lysate ◽  
Translational Termination

The codon usage of Tetrahymena thermophila and other ciliates deviates from the ‘universal genetic code’ in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

scholarly journals Heat shock response of Trichinella spiralis and T. pseudospiralis

Parasitology ◽  
1996 ◽  
Vol 112 (1) ◽  
pp. 89-95 ◽  
Author(s):  
R. C. Ko ◽  
L. Fan
Keyword(s):  
Heat Shock ◽  
Trichinella Spiralis ◽  
Transcriptional Level ◽  
Sds Page ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Shock Proteins ◽  
First Time ◽  
Laser Densitometry

SUMMARYHeat shock proteins (HSPs) were documented for the first time in both somatic extracts and excretory/secretory (ES) products of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis. Larvae recovered from muscles of infected mice were heat shocked at 37, 40, 43 and 45 °C in RPMI 1640 medium containing L-[35S]methionine. Somatic extracts and ES products of heat-shocked worms were then analysed by SDS-PAGE, autoradiography and laser densitometry. Prominent bands of HSPs were observed at 43 °C which is the optimal heat shock temperature. The major HSPs in somatic extracts of T. spiralis were 20, 47, 50, 70, 80 and 86 kDa. When the temperature was increased from 37 to 43 °C, the greatest increase in absorbance was observed in HSPs 70 and 86. In vitro translation of mRNA in a nuclease-treated rabbit reticulocyte lysate system showed an increase in the synthesis of the 80 kDa protein. This suggests that the production of HSP 80 is regulated at the transcriptional level. The major HSPs in the ES products were 11, 45, 53 and 64 kDa. In T. pseudospiralis, the major HSPs in the somatic extracts were 20, 26, 31, 50, 53, 70, 80 and 86 kDa, and in the ES products, 11, 35, 37, 41 and 64 kDa.

scholarly journals Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to diphtheria toxin

1996 ◽  
Vol 313 (2) ◽  
pp. 647-653 ◽  
Author(s):  
Olav KLINGENBERG ◽  
Sjur OLSNES
Keyword(s):  
Fusion Protein ◽  
Dihydrofolate Reductase ◽  
Diphtheria Toxin ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Toxin B ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Diphtheria Toxin A

A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.

The biogenesis of the cyanellae of Cyanophora paradoxa . III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

1989 ◽  
Vol 238 (1290) ◽  
pp. 89-102 ◽  
Keyword(s):  
Protein A ◽  
D1 Protein ◽  
Subcellular Fractionation ◽  
Coupling Factor ◽  
Cyanophora Paradoxa ◽  
In Vitro Synthesis ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Precursor Molecules

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cyto­plasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [ 35 S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the γ subunit of coupling factor CF1, and subunit II of PS I are syn­thesized in the cytoplasm as precursor molecules that are 5–8 kDa larger than their mature sizes. Antibodies directed against the psb A gene prod­uct (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.

Sign in / Sign up

Export Citation Format

Share Document