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.

scholarly journals CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut

2006 ◽  
Vol 189 (5) ◽  
pp. 1856-1865 ◽  
Author(s):  
Sami S. A. Ashgar ◽  
Neil J. Oldfield ◽  
Karl G. Wooldridge ◽  
Michael A. Jones ◽  
Greg J. Irving ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Protein A ◽  
Phase Variation ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Insertion Mutant ◽  
Genes Encoding ◽  
Monospecific Antisera ◽  
Recombinant Polypeptides

ABSTRACT Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.

Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting

1992 ◽  
Vol 12 (9) ◽  
pp. 4242-4248 ◽  
Author(s):  
H Ilves ◽  
O Kahre ◽  
M Speek
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Genomic Structure ◽  
Protein A ◽  
Open Reading Frames ◽  
Initiation Of Translation ◽  
Reticulocyte Lysate ◽  
Internal Initiation ◽  
Overlapping Open Reading Frames

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

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.

scholarly journals In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

1983 ◽  
Vol 96 (3) ◽  
pp. 633-638 ◽  
Author(s):  
D L Paul ◽  
D A Goodenough
Keyword(s):  
Plasma Membranes ◽  
Proteolytic Processing ◽  
Membrane Insertion ◽  
Membrane Association ◽  
Lens Fiber ◽  
In Vitro Synthesis ◽  
Reticulocyte Lysate ◽  
Immune Precipitation ◽  
Dog Pancreas

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

scholarly journals Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

1992 ◽  
Vol 12 (9) ◽  
pp. 4242-4248 ◽  
Author(s):  
H Ilves ◽  
O Kahre ◽  
M Speek
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Genomic Structure ◽  
Protein A ◽  
Open Reading Frames ◽  
Initiation Of Translation ◽  
Reticulocyte Lysate ◽  
Internal Initiation ◽  
Overlapping Open Reading Frames

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

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

Cyanophora paradoxa: Fatty Acids and Fatty Acid Synthesis in vitro

1986 ◽  
Vol 41 (1-2) ◽  
pp. 169-171 ◽  
Author(s):  
Hans Kleinig ◽  
Peter Beyer ◽  
Carmen Schubert ◽  
Bodo Liedvogel ◽  
Friedhelm Lütke-Brinkhaus
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Oxygen Evolution ◽  
Fatty Acid Synthesis ◽  
Membrane Lipids ◽  
Fatty Acid Pattern ◽  
Acid Synthesis ◽  
Cyanophora Paradoxa ◽  
In Vitro Synthesis

Abstract The fatty acid pattern of Cyanophora paradoxa membrane lipids is highly unusual with 16:0, 20:3 and 20:4 as the main acids. The 20:4 acid is preferentially distributed among the cyanelle lipids. In isolated cyanelles a relatively low in vitro synthesis of only saturated and monounsatu­rated fatty acids from [l-14C]acetate was observed which corresponds to the relatively low photo­synthetic oxygen evolution.

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