scholarly journals Differential stability of c-myc mRNAS in a cell-free system.

1988 ◽  
Vol 8 (7) ◽  
pp. 2860-2868 ◽  
Author(s):  
R Pei ◽  
K Calame
Keyword(s):  
Full Length ◽  
Free System ◽  
In Vitro System ◽  
Rapid Degradation ◽  
Cell Free System ◽  
Exon 1 ◽  
A Cell ◽  
The Stability

We have developed a simple cell-free system for studying the stability of different mRNAs in vitro. We demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin C alpha heavy chain or glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.

Differential stability of c-myc mRNAS in a cell-free system

1988 ◽  
Vol 8 (7) ◽  
pp. 2860-2868
Author(s):  
R Pei ◽  
K Calame
Keyword(s):  
Full Length ◽  
Free System ◽  
In Vitro System ◽  
Rapid Degradation ◽  
Cell Free System ◽  
Exon 1 ◽  
A Cell ◽  
The Stability

We have developed a simple cell-free system for studying the stability of different mRNAs in vitro. We demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin C alpha heavy chain or glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.

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

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.

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

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
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.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

scholarly journals Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378 ◽  
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

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

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.

scholarly journals In vitro synthesis of pp60v-src: myristylation in a cell-free system.

1988 ◽  
Vol 8 (10) ◽  
pp. 4295-4301 ◽  
Author(s):  
I Deichaite ◽  
L P Casson ◽  
H P Ling ◽  
M D Resh
Keyword(s):  
Amino Acids ◽  
Synthetic Peptide ◽  
Covalent Attachment ◽  
Free System ◽  
In Vitro Synthesis ◽  
Cell Free System ◽  
Reticulocyte Lysates ◽  
A Cell

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.

scholarly journals A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system.

1995 ◽  
Vol 130 (5) ◽  
pp. 1027-1039 ◽  
Author(s):  
T Misteli ◽  
G Warren
Keyword(s):  
Fragmentation Pathway ◽  
Golgi Membrane ◽  
Free System ◽  
Cell Free System ◽  
Independent Mechanism ◽  
A Cell ◽  
Golgi Fragmentation ◽  
Dependent Pathway

Golgi stacks were previously shown to be converted into tubular networks when incubated in mitotic cytosol depleted of the coatomer subunit of COP I coats (Misteli and Warren, 1994). Similar, though smaller, networks are now shown to be an early intermediate on the Golgi fragmentation pathway both in vitro and in vivo. Their appearance mirrors the disappearance of Golgi cisternae and at their peak they constitute 35% of total Golgi membrane. They are consumed by two pathways, the first involving the budding of COP I-coated vesicles described previously (Misteli and Warren, 1994). The second involves a COP I-independent mechanism that leads eventually to a vesicle fraction that is larger in size and more heterogeneous than that produced by the COP I-mechanism. We suggest that both pathways operate concurrently at the onset of mitotic fragmentation. The COP I-independent pathway converts cisternae into tubular networks that then fragment. The COP I-dependent pathway partially consumes first the cisternae at the beginning of the incubation and then the tubular networks that form from them.

scholarly journals Cell-free styrene biosynthesis at high titers

2020 ◽  
Author(s):  
William S. Grubbe ◽  
Blake J. Rasor ◽  
Antje Krüger ◽  
Michael C. Jewett ◽  
Ashty S. Karim
Keyword(s):  
Fossil Fuels ◽  
Product Formation ◽  
Synthesis Methods ◽  
Reaction Conditions ◽  
Free System ◽  
Cell Free System ◽  
Order Of Magnitude ◽  
A Cell

AbstractStyrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, an order of magnitude greater than cellular synthesis methods. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.HighlightsA cell-free system for styrene biosynthesis was established. This in vitro system achieved styrene titers an order of magnitude greater than the highest reported concentration in vivo.

scholarly journals Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

1984 ◽  
Vol 99 (1) ◽  
pp. 248-259 ◽  
Author(s):  
J E Rothman ◽  
L J Urbani ◽  
R Brands
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Chinese Hamster ◽  
In Vitro Assays ◽  
Wild Type ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.

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