Disassembly of the Drosophila nuclear lamina in a homologous cell-free system

1995 ◽  
Vol 108 (5) ◽  
pp. 2027-2035 ◽  
Author(s):  
N. Maus ◽  
N. Stuurman ◽  
P.A. Fisher
Keyword(s):  
Nuclear Lamina ◽  
Meiotic Metaphase ◽  
Two Dimensional ◽  
Cell Free Extract ◽  
Free System ◽  
Nuclear Lamin ◽  
A Cell

Stage 14 Drosophila oocytes are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins. Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. A previously unidentified soluble lamin isoform is easily seen after in vitro disassembly. This isoform is detectable but present only in very small quantities in vivo and is apparently derived specifically from one of the two interphase lamin isoforms. Cell-free nuclear lamina disassembly is ATP-dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. This system will allow enzymological characterization of cell-free lamina disassembly as well as molecular analysis of specific Drosophila mutants.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

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.

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.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

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.

scholarly journals Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

2003 ◽  
Vol 163 (4) ◽  
pp. 813-824 ◽  
Author(s):  
Kazuhito Tomizawa ◽  
Satoshi Sunada ◽  
Yun-Fei Lu ◽  
Yoshiya Oda ◽  
Masahiro Kinuta ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Critical Role ◽  
Ring Formation ◽  
Free System ◽  
Rich Domain ◽  
A Cell ◽  
Dynamin I ◽  
Deficient Mice

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

scholarly journals N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465 ◽  
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

scholarly journals A cell-free framework for biological systems engineering

2015 ◽  
Author(s):  
Henrike Niederholtmeyer ◽  
Zachary Sun ◽  
Yutaka Hori ◽  
Enoch Yeung ◽  
Amanda Verpoorte ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Biological Systems ◽  
Biological Engineering ◽  
Free System ◽  
Node Negative ◽  
A Cell ◽  
Free Environment ◽  
Synthetic Networks

While complex dynamic biological networks control gene expression and metabolism in all living organisms, engineering comparable synthetic networks remains challenging1,2. Conducting extensive, quantitative and rapid characterization during the design and implementation process of synthetic networks is currently severely limited due to cumbersome molecular cloning and the difficulties associated with measuring parts, components and systems in cellular hosts. Engineering gene networks in a cell-free environment promises to be an efficient and effective approach to rapidly develop novel biological systems and understand their operating regimes3-5. However, it remains questionable whether complex synthetic networks behave similarly in cells and a cell-free environment, which is critical for in vitro approaches to be of significance to biological engineering. Here we show that synthetic dynamic networks can be readily implemented, characterized, and engineered in a cell-free framework and consequently transferred to cellular hosts. We implemented and characterized the “repressilator”6, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring these novel three-node and five-node oscillators to Escherichia coli, resulting in robust and synchronized oscillations reflecting the in vitro observation. We demonstrate that comprehensive circuit engineering can be performed in a cell-free system and that the in vitro results have direct applicability in vivo. Cell-free synthetic biology thus has the potential to drastically speed up design-build-test cycles in biological engineering and enable the quantitative characterization of synthetic and natural networks.

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 STUDIES ON A BACTERICIDAL AGENT EXTRACTED FROM A SOIL BACILLUS

1939 ◽  
Vol 70 (3) ◽  
pp. 249-256 ◽  
Author(s):  
René J. Dubos ◽  
Carlo Cattaneo
Keyword(s):  
Present Report ◽  
Active Form ◽  
Bactericidal Effect ◽  
Original Material ◽  
Cell Free Extract ◽  
Gram Positive ◽  
A Cell ◽  
Bactericidal Agent

A cell-free extract of cultures of an unidentified soil bacillus, which exerts a bactericidal effect on Gram-positive microorganisms, has been described in previous reports; the first active preparations which were obtained were found to contain a protein precipitable at pH 4.5. It is shown in the present report that the bactericidal agent can be obtained in an active form free of protein. The new purified preparations retain all the activity of the original material, both in vitro and in vivo.

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