In vitro nuclear reconstitution could be induced in a plant cell-free system

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

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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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Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

Biochimie ◽

10.1016/j.biochi.2012.09.031 ◽

2013 ◽

Vol 95 (2) ◽

pp. 320-328

Author(s):

Seiki Nakao ◽

Sufang Zhang ◽

Markku Vaara ◽

Juhani E. Syväoja ◽

Marietta Y. Lee ◽

...

Keyword(s):

Dna Replication ◽

Mammalian Cell ◽

Gap Filling ◽

Free System ◽

Cell Free System ◽

Replication Assay ◽

System A

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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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Enhancement by Activated Protein C of tPA-induced Lysis in a Cell-free System

10.1055/s-0038-1644379 ◽

1987 ◽

Author(s):

J C Fredenburgh ◽

D Collen ◽

M E Nesheim

Keyword(s):

Protein C ◽

Activated Protein C ◽

Protein S ◽

Plasminogen Activation ◽

Free System ◽

Cell Free System ◽

Normal Plasma ◽

Lysis Time ◽

A Cell

The profibrinolytic activity of human activated protein C (APC) was studied in a cell-free system using human plasma. Normal and Ba+* citrate adsorbed human plasmas were dialyzed against 150mM NaCl, 20mM Hepes, pH 7.4 and diluted to an A280 of 16. Reactions were initiated by the addition of aliquots of plasma to cuvettes containing human melanoma tPA and human thrombin at final concentrations of 1 and 30nM, respectively. The effects of Ca+* and varying concentrations of APC on clotlysis times were examined by monitoring turbidity at 600nM while maintaining the temperature at 37°C. The lysis time, defined as the midpoint of turbidity change, was 128 min for normal plasma containing 10 mM Ca+* and showed progressive and saturable shortening to about 90 min at > 50nM APC. In the absence of Ca+*, lysis time was 55 min for normal plasma and did not shorten in response to APC. With Ba+* citrate adsorbed plasma, the lysis time was 82 min in the presence of 10mM Ca+*, and shortened to 42 min without Ca+*. APC had no effect on lysis time in Ba+* adsorbed plasma either with or without Ca+*. Both bovine and human APC were equally potent. Electrophoresis in DodSO4 and autoradiography of plasma samples containing 125I-labelled plasminogen indicated enhanced rates of plasminogen activation in the presence of APC. These data indicate that APC decreases lysis time in vitro at the level of plasminogen activation. This effect is dependent on Ca+* and may involve additional vitamin K-dependent protein ( s).

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In vitroTranslation of Natural mRNAs in a Cell-free System Containing Components from Interferon-treated Chicken Fibroblasts and Factor Preparations from Mouse Ascites Cells or Rabbit Reticulocytes

Zeitschrift für Naturforschung C ◽

10.1515/znc-1975-5-616 ◽

1975 ◽

Vol 30 (5-6) ◽

pp. 398-405 ◽

Cited By ~ 3

Author(s):

G. Hiller ◽

G. Viehhauser ◽

I. Winkler ◽

D. Pohl ◽

C. Jungwirth ◽

...

Keyword(s):

Chick Embryo ◽

In Vitro Translation ◽

Interferon Treatment ◽

Mixed Cell ◽

Free System ◽

Free Protein ◽

Cell Free System ◽

Mouse Ascites ◽

Rabbit Reticulocytes

Abstract Interferon Mechanism, in vitro Translation, Viral and Cellular mRNA, Chick Embryo Fibroblasts, Vaccinia Infection The effect of interferon has been studied in a mixed cell-free protein synthesizing system. Hemo globin (Hb) and Encephalomyocarditis virus (EMC) -RNA can be efficiently translated in vitro in a system containing S-30 lysates or run-off ribosomes from primary chick embryo fibroblasts (CEF) and a postmicrosomal supernatant from mouse ascites cells or a ribosomal-wash preparation from rabbit reticulocytes. Ribosomes prepared from CEF pretreated with high doses of homologous inter feron (500 units/ml) were able to translate Hb-RNA in the presence of heterologous factors with the same efficiency as ribosomes prepared from control cells. Translation of EMC-RNA was slightly reduced if ribosomes from interferon-treated cells were used in the mixed cell-free system, confirming previous reports. No inhibitory effect caused by interferon treatment of CEF cells could be detected on in vitro translation of natural mRNAs if the cells had, in addition to interferon treatment, been infected with vaccinia virus. Possible reasons for the different observations made with our cell-free protein synthesizing system from CEF and with cell-free systems prepared from mouse cells are discussed.

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Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

Biochemical Journal ◽

10.1042/bj3100461 ◽

1995 ◽

Vol 310 (2) ◽

pp. 461-467 ◽

Cited By ~ 1

Author(s):

C A Feghali ◽

T M Wright

Keyword(s):

Transcription Factor ◽

Tyrosine Phosphatase ◽

Cell Fractionation ◽

Dependent Manner ◽

Sodium Orthovanadate ◽

Ifn Gamma ◽

Free System ◽

Cell Free System ◽

A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

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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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