Nuclear Envelope Formation In Vitro: A Sea Urchin Egg Cell-Free System

2008 ◽  
pp. 207-223 ◽  
Author(s):  
Richard D. Byrne ◽  
Vanessa Zhendre ◽  
Banafshé Larijani ◽  
Dominic L. Poccia
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Egg Cell ◽  
Free System ◽  
Cell Free System ◽  
Sea Urchin Egg ◽  
Nuclear Envelope Formation

Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro

1996 ◽  
Vol 109 (6) ◽  
pp. 1275-1283 ◽  
Author(s):  
P. Collas ◽  
D. Poccia
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Critical Element ◽  
Marker Enzyme ◽  
Sea Urchin Egg ◽  
Nuclear Envelope Formation

We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2 beta binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2 alpha binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2 beta. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.

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.

A cell free system to study reassembly of the nuclear envelope at the end of mitosis

Cell ◽  
1986 ◽  
Vol 44 (4) ◽  
pp. 639-652 ◽  
Author(s):  
Brian Burke ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Free System ◽  
Cell Free System ◽  
A Cell

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

Biochimie ◽  
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

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

1974 ◽  
Vol 137 (3) ◽  
pp. 513-524 ◽  
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.

Activation of protein synthesis in a sea urchin cell-free system

1981 ◽  
Vol 84 (2) ◽  
pp. 432-439 ◽  
Author(s):  
M.M. Winkler ◽  
R.A. Steinhardt
Keyword(s):  
Protein Synthesis ◽  
Sea Urchin ◽  
Free System ◽  
Cell Free System
Sign in / Sign up

Export Citation Format

Share Document