scholarly journals Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.

1988 ◽  
Vol 106 (1) ◽  
pp. 1-12 ◽  
Author(s):  
M A Sheehan ◽  
A D Mills ◽  
A M Sleeman ◽  
R A Laskey ◽  
J J Blow
Keyword(s):  
Dna Replication ◽  
Soluble Fraction ◽  
Replication Initiation ◽  
Sperm Chromatin ◽  
Nuclear Pore ◽  
Free System ◽  
Cell Free System ◽  
Nuclear Assembly ◽  
Single Membrane ◽  
A Cell

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.

Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

1990 ◽  
Vol 97 (1) ◽  
pp. 177-184
Author(s):  
L.S. Cox ◽  
G.H. Leno
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
High Speed ◽  
De Novo ◽  
Soluble Fraction ◽  
Sperm Chromatin ◽  
Nuclear Assembly ◽  
Egg Extract ◽  
Egg Extracts ◽  
Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

scholarly journals The transformation of the cytoplasmic oestradiol–receptor complex into the nuclear complex in a uterine cell-free system

1972 ◽  
Vol 128 (3) ◽  
pp. 611-616 ◽  
Author(s):  
Michael Gschwendt ◽  
Terrell H. Hamilton
Keyword(s):  
High Speed ◽  
Soluble Fraction ◽  
Supernatant Fraction ◽  
Receptor Complex ◽  
Free System ◽  
Cell Free System ◽  
Nuclear Complex ◽  
Nuclear Particle ◽  
A Cell ◽  
A Subunit

Experiments performed with a cell-free system in tris–EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol–receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2°C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37°C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37°C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.

scholarly journals DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function

1992 ◽  
Vol 101 (1) ◽  
pp. 43-53 ◽  
Author(s):  
L.S. Cox
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Cell Nucleus ◽  
Wheat Germ ◽  
Nuclear Dna ◽  
Nuclear Transport ◽  
Sperm Chromatin ◽  
Cell Free Extract ◽  
Nuclear Assembly ◽  
A Cell

The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of N-acetylglucosamine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.

scholarly journals The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants

1998 ◽  
Vol 111 (20) ◽  
pp. 3017-3026 ◽  
Author(s):  
M. Hughes ◽  
C. Zhang ◽  
J.M. Avis ◽  
C.J. Hutchison ◽  
P.R. Clarke
Keyword(s):  
Dna Replication ◽  
Nuclear Import ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Wild Type ◽  
Ran Gtpase ◽  
Free System ◽  
Nuclear Assembly

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

scholarly journals Effect of detergents on sterol synthesis in a cell-free system of yeast

1982 ◽  
Vol 23 (6) ◽  
pp. 803-810
Author(s):  
S Hata ◽  
T Nishino ◽  
N Ariga ◽  
H Katsuki
Keyword(s):  
Sterol Synthesis ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of Endosomal Proteolysis in a Cell-free System

1989 ◽  
Vol 264 (10) ◽  
pp. 5392-5399
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
P D Stahl
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell
Sign in / Sign up

Export Citation Format

Share Document