Two pathways in the biosynthesis of cadystins (γEC)nG in the cell-free system of the fission yeast

Small metal-binding peptides, cadystins, with the general structure of (γ-Glu-Cys)n-Gly ((γEC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120 000 × g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is γ-Glu-Cys (γEC) dipeptidyl transfer from both glutathione (γECG) and cadystins to glutathione and cadystins. The other one is γEC polymerization from (γEC)n and glutathione to (γEC)n+i, followed by glycine addition with glutathione synthetase.Key words: cadystin, fission yeast, cell-free biosynthesis, dipeptidyl transferase.

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Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

Journal of Virology ◽

10.1128/jvi.02737-07 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5967-5980 ◽

Cited By ~ 78

Author(s):

Judit Pogany ◽

Peter D. Nagy

Keyword(s):

High Efficiency ◽

Tomato Bushy Stunt Virus ◽

Free System ◽

Cell Free System ◽

Virus Rna ◽

A Cell ◽

Viral Replicase ◽

Template Specificity ◽

Rna Complex

ABSTRACT To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ∼30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.

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Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

Molecular and Cellular Biology ◽

10.1128/mcb.1.7.635-651.1981 ◽

1981 ◽

Vol 1 (7) ◽

pp. 635-651

Author(s):

D C Lee ◽

R G Roeder

Keyword(s):

Adenovirus Type ◽

Free System ◽

Ribonucleic Acids ◽

Cell Free System ◽

Cell Extracts ◽

A Cell ◽

Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

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Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

Biochemical Journal ◽

10.1042/bj1370513 ◽

1974 ◽

Vol 137 (3) ◽

pp. 513-524 ◽

Cited By ~ 27

Author(s):

W. Ian P. Mainwaring ◽

Peter A. Wilce ◽

Allan E. Smith

Keyword(s):

Prostate Gland ◽

Sodium Dodecyl ◽

Ventral Prostate ◽

Experimental Conditions ◽

Free System ◽

Cell Free System ◽

Messenger Ribonucleic Acid ◽

Selective Incorporation

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6–15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5′-fluoro-orotic acid into this 6–15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6–15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6–15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.

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Light-Dependent Sucrose Synthesis in Cell-Free (Reconstituted) Lysates of Evacuolated Mesophyll Protoplasts

Zeitschrift für Naturforschung C ◽

10.1515/znc-1990-3-412 ◽

1990 ◽

Vol 45 (3-4) ◽

pp. 217-222 ◽

Cited By ~ 2

Author(s):

Manfred Steingraber ◽

Hanns Frohnmeyer ◽

Rüdiger Hampp

Keyword(s):

Percoll Gradient ◽

Functional Interaction ◽

Free System ◽

Mesophyll Protoplasts ◽

Cell Free System ◽

Fructose 2 ◽

Fructose 1,6 Bisphosphatase ◽

Reconstituted System ◽

Oil Filtration

Abstract Oat mesophyll protoplasts were evacuolated by centrifugation on a Percoll gradient and used as starting material for establishing a functional cell-free system. For this purpose evacuolated protoplasts were osmotically lysed. From the resulting homogenate a cytosolic fraction was obtained by silicon oil filtration. This fraction was used to dilute preparations of lysed evacuolated protoplasts in order to reduce the number of organelles per volume cytosol. The latter was necessary to increase light-dependent oxygen evolution of the cell-free system. The resulting kind of “reconstituted” system showed light-dependent sucrose formation (about 100 nmol (mg Chi)-1 · hr-1) with bicarbonate as the only substrate. As this property depends on a functional interaction of chloroplast and cytosolic reactions, this cell-free system appeared to perform essential steps of partitioning of CO2 between starch and sucrose. Addition of about 12 μm fructose 2 ,6 -bisphosphate, an inhibitor of fructose 1,6 -bisphosphatase and an activator of the PPi-dependent fructose 6 -phosphate phosphotransferase, caused sucrose degradation in the light. Thus, this cell-free system allows both the study of cytosolic enzyme activities under quasi in vivo conditions and the manipulation of cellular reaction sequences by plasma membrane-impermeable compounds.

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Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

Biochemical Journal ◽

10.1042/bj3470363 ◽

2000 ◽

Vol 347 (2) ◽

pp. 363-368 ◽

Cited By ~ 1

Author(s):

Vijayalakshmi NAGARAJ ◽

David NORRIS

Keyword(s):

Homologous Recombination ◽

Specific Activity ◽

Yeast Cells ◽

Active Components ◽

Free System ◽

Cell Free System ◽

Molecule Formation ◽

A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

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Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Molecular Biology of the Cell ◽

10.1091/mbc.8.4.621 ◽

1997 ◽

Vol 8 (4) ◽

pp. 621-636 ◽

Cited By ~ 53

Author(s):

J M Caron

Keyword(s):

Posttranslational Modifications ◽

Posttranslational Modification ◽

Hydrophobic Interactions ◽

Covalent Attachment ◽

Protein Structure Analysis ◽

Free System ◽

Intracellular Membranes ◽

Cell Free System ◽

Cytoplasmic Proteins

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

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Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

The Scientific World JOURNAL ◽

10.1155/2016/2597376 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Tomoyuki Iwasaki ◽

Naoe Kaneko ◽

Yuki Ito ◽

Hiroyuki Takeda ◽

Tatsuya Sawasaki ◽

...

Keyword(s):

Drug Discovery ◽

Early Onset ◽

Adaptor Protein ◽

Dependent Manner ◽

Blau Syndrome ◽

Free System ◽

Cell Free System ◽

A Cell ◽

Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

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Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

Molecular and Cellular Biology ◽

10.1128/mcb.5.6.1238 ◽

1985 ◽

Vol 5 (6) ◽

pp. 1238-1246 ◽

Cited By ~ 105

Author(s):

J J Li ◽

T J Kelly

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

T Antigen ◽

Dna Molecules ◽

Free System ◽

Cell Free System ◽

Cell Extracts ◽

Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

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