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.

273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS

2009 ◽  
Vol 21 (1) ◽  
pp. 234 ◽  
Author(s):  
C.-Y. Chiang ◽  
P.-C. Tang
Keyword(s):  
Somatic Cells ◽  
Egfp Expression ◽  
3T3 Cells ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.

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.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig

2014 ◽  
Vol 26 (7) ◽  
pp. 1017 ◽  
Author(s):  
Ying Liu ◽  
Olga Østrup ◽  
Rong Li ◽  
Juan Li ◽  
Gábor Vajta ◽  
...  
Keyword(s):  
Donor Cell ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Long Term Effect ◽  
Donor Cells ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7–22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

scholarly journals The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control.

1997 ◽  
Vol 17 (8) ◽  
pp. 4312-4321 ◽  
Author(s):  
J R Wu ◽  
D M Gilbert
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Decision Point ◽  
Cell Nuclei ◽  
Phase Selection ◽  
Restriction Point ◽  
Xenopus Egg ◽  
Point Control

At a distinct point during G1 phase (the origin decision point [ODP]), Chinese hamster ovary (CHO) cell nuclei experience a transition (origin choice) that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. We have investigated the relationship between the ODP and progression of CHO cells through G1 phase. Selection of the DHFR origin at the ODP was rapidly inhibited by treatment of early G1-phase cells with the protein kinase inhibitor 2-aminopurine (2-AP). Inhibition of the ODP required administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point). Cells deprived of either serum or isoleucine from metaphase throughout early G1 phase acquired the capacity to replicate in Xenopus egg extract (replication licensing) and subsequently passed through the ODP on the same schedule as cells cultured in complete growth medium. After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual loss of replication licensing. However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until the point at which all nuclei had lost the capacity to initiate replication. These results provide evidence that the ODP requires a mitogen-independent protein kinase that is activated after replication licensing and prior to R-point control.

scholarly journals Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

1995 ◽  
Vol 15 (6) ◽  
pp. 2942-2954 ◽  
Author(s):  
D M Gilbert ◽  
H Miyazawa ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
Nuclear Membrane ◽  
S Phase ◽  
G1 Phase ◽  
Site Specific ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

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.

Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract

1996 ◽  
Vol 109 (9) ◽  
pp. 2275-2286 ◽  
Author(s):  
C. Zhang ◽  
H. Jenkins ◽  
M.W. Goldberg ◽  
T.D. Allen ◽  
C.J. Hutchison
Keyword(s):  
Nuclear Matrix ◽  
Extraction Procedure ◽  
Nuclear Lamina ◽  
The Body ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Whole Mounts

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from “lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei

2000 ◽  
Vol 113 (5) ◽  
pp. 887-898 ◽  
Author(s):  
C.J. Li ◽  
J.A. Bogan ◽  
D.A. Natale ◽  
M.L. DePamphilis
Keyword(s):  
Prolonged Exposure ◽  
Selective Activation ◽  
Initiation Factors ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Dna Substrate

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

scholarly journals XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

2009 ◽  
Vol 20 (11) ◽  
pp. 2684-2696 ◽  
Author(s):  
Iva Kronja ◽  
Anamarija Kruljac-Letunic ◽  
Maïwen Caudron-Herger ◽  
Peter Bieling ◽  
Eric Karsenti
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Microtubule Growth ◽  
And Function ◽  
Xenopus Egg Extracts ◽  
Reduced Density

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ΔEB1 or ΔXMAP215 spindles leads to faulty kinetochore–microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215–EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215–EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.

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.

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