scholarly journals Histone H1 Reduces the Frequency of Initiation inXenopus Egg Extract by Limiting the Assembly of Prereplication Complexes on Sperm Chromatin

1998 ◽  
Vol 9 (5) ◽  
pp. 1163-1176 ◽  
Author(s):  
Zhi Hong Lu ◽  
Donald B. Sittman ◽  
Piotr Romanowski ◽  
Gregory H. Leno
Keyword(s):  
Replication Origin ◽  
Histone H1 ◽  
Sperm Chromatin ◽  
Replication Forks ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Interesting Possibility ◽  
Replication Foci ◽  
Xenopus Egg ◽  
Sperm Nuclei

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopussperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.

scholarly journals Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times

2001 ◽  
Vol 152 (1) ◽  
pp. 15-26 ◽  
Author(s):  
J. Julian Blow ◽  
Peter J. Gillespie ◽  
Dennis Francis ◽  
Dean A. Jackson
Keyword(s):  
Dna Sequence ◽  
Replication Origins ◽  
Origin Firing ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Dna Replication Origins ◽  
Small Clusters

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [3H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

scholarly journals In vitro assembly of coiled bodies in Xenopus egg extract.

1994 ◽  
Vol 5 (6) ◽  
pp. 633-644 ◽  
Author(s):  
D W Bauer ◽  
C Murphy ◽  
Z Wu ◽  
C H Wu ◽  
J G Gall
Keyword(s):  
Rna Processing ◽  
Mrna Splicing ◽  
Immunofluorescent Staining ◽  
Rrna Processing ◽  
Xenopus Egg Extract ◽  
Coiled Bodies ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Sperm Nuclei

When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from < 1.0 to approximately 3.0 microns in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.

Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos

1997 ◽  
Vol 110 (17) ◽  
pp. 2053-2063 ◽  
Author(s):  
P. Bell ◽  
C. Mais ◽  
B. McStay ◽  
U. Scheer
Keyword(s):  
Rrna Genes ◽  
Sperm Chromatin ◽  
Xenopus Egg Extract ◽  
Xenopus Embryos ◽  
Architectural Element ◽  
Pol I ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Tata Box Binding Protein ◽  
Polymerase I

When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.

Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract

1997 ◽  
Vol 110 (21) ◽  
pp. 2745-2758 ◽  
Author(s):  
Z.H. Lu ◽  
D.B. Sittman ◽  
D.T. Brown ◽  
R. Munshi ◽  
G.H. Leno
Keyword(s):  
Dna Replication ◽  
Nuclear Lamina ◽  
Sperm Chromatin ◽  
Linker Histones ◽  
Nuclear Assembly ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Initiation Of Replication ◽  
Xenopus Egg ◽  
Assembly Analysis

We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.

Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts

1992 ◽  
Vol 101 (3) ◽  
pp. 509-515
Author(s):  
A.M. Sleeman ◽  
G.H. Leno ◽  
A.D. Mills ◽  
M.P. Fairman ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Salivary Glands ◽  
High Speed ◽  
De Novo ◽  
Replication Forks ◽  
Egg Extract ◽  
Replication Foci ◽  
Sperm Nuclei ◽  
Polytene Nucleus ◽  
Polytene Nuclei

We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin. Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.

Regulation of mammalian replication origin usage in Xenopus egg extract

1998 ◽  
Vol 111 (19) ◽  
pp. 2989-2998 ◽  
Author(s):  
D.S. Dimitrova ◽  
D.M. Gilbert
Keyword(s):  
Replication Origin ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Cell Nuclei ◽  
Entire Genome ◽  
Xenopus Egg Extract ◽  
Xenopus Embryos ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

Cell Colony Formation Induced by Xenopus Egg Extract as a Marker for Improvement of Cloned Blastocyst Formation in the Pig

2011 ◽  
Vol 13 (6) ◽  
pp. 521-526 ◽  
Author(s):  
Ying Liu ◽  
Olga Østrup ◽  
Juan Li ◽  
Gábor Vajta ◽  
Peter M. Kragh ◽  
...  
Keyword(s):  
Colony Formation ◽  
Blastocyst Formation ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Cell Colony

Aphidicolin-Sensitive DNA Polymerase Is Incorporated into the Chromatin during Nuclear Envelope Assembly in Xenopus Egg Extract

1995 ◽  
Vol 219 (1) ◽  
pp. 283-291
Author(s):  
Yoshihiro Takasuga ◽  
Machiko Murata ◽  
Jinpei Yamashita ◽  
Toshiow Andoh ◽  
Tatsuo Yagura
Keyword(s):  
Nuclear Envelope ◽  
Dna Polymerase ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg

scholarly journals Xenopus egg extract: A powerful tool to study genome maintenance mechanisms

2017 ◽  
Vol 428 (2) ◽  
pp. 300-309 ◽  
Author(s):  
Wouter S. Hoogenboom ◽  
Daisy Klein Douwel ◽  
Puck Knipscheer
Keyword(s):  
Genome Maintenance ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg
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