scholarly journals Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

1991 ◽  
Vol 11 (1) ◽  
pp. 299-308 ◽  
Author(s):  
N J Marini ◽  
R M Benbow
Keyword(s):  
Xenopus Laevis ◽  
Plasmid Dna ◽  
Supramolecular Assembly ◽  
Micrococcal Nuclease ◽  
Dna Molecules ◽  
End Joining ◽  
Stage Of Development ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Xenopus Laevis Embryos

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency

1991 ◽  
Vol 11 (1) ◽  
pp. 299-308
Author(s):  
N J Marini ◽  
R M Benbow
Keyword(s):  
Xenopus Laevis ◽  
Plasmid Dna ◽  
Supramolecular Assembly ◽  
Micrococcal Nuclease ◽  
Dna Molecules ◽  
End Joining ◽  
Stage Of Development ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Xenopus Laevis Embryos

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

scholarly journals Postfertilization deadenylation of mRNAs in Xenopus laevis embryos is sufficient to cause their degradation at the blastula stage.

1997 ◽  
Vol 17 (1) ◽  
pp. 209-218 ◽  
Author(s):  
Y Audic ◽  
F Omilli ◽  
H B Osborne
Keyword(s):  
Xenopus Laevis ◽  
Untranslated Region ◽  
Sequence Information ◽  
Blastula Stage ◽  
Cytoplasmic Polyadenylation ◽  
Cis Acting ◽  
Stage Of Development ◽  
Chimeric Rnas ◽  
Xenopus Laevis Embryos

Although the maternal Xenopus laevis Eg mRNAs are deadenylated after fertilization, they are not immediately degraded and they persist in the embryos as poly(A)- transcripts. The degradation of these RNAs is not detected until the blastula stage of development (6 to 7 h postfertilization). To understand the basis for this delay between deadenylation and degradation, it is necessary to identify the cis-acting element(s) required to trigger degradation in blastula stage embryos. To this end, several chimeric RNAs containing different portions of the 3' untranslated region of Eg2 mRNA were injected into two-cell X. laevis embryos. We observed that only the RNAs that contained the cis-acting elements that confer rapid deadenylation were subsequently degraded at the blastula stage. This suggested that deadenylation may be sufficient to trigger degradation. By injecting chimeric RNAs devoid of Eg sequence information, we further showed that only deadenylated RNAs were degraded in X. laevis embryos. Last, introduction of a functional cytoplasmic polyadenylation element into a poly(A)- RNA, thereby causing its polyadenylation after injection into embryos, protected the RNA from degradation. Hence, in X. laevis embryos, the postfertilization deadenylation of maternal Eg mRNAs is sufficient to cause the degradation of an mRNA, which, however, only becomes apparent at the blastula stage. Possible causes for this delay between deadenylation and degradation are discussed in the light of these results.

scholarly journals Nonhomologous End-Joining Ligation Transfers DNA Regulatory Elements between Cointroduced Plasmids

2004 ◽  
Vol 24 (19) ◽  
pp. 8323-8331 ◽  
Author(s):  
Toshio Ishikawa ◽  
Eun Jig Lee ◽  
J. Larry Jameson
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Molecules ◽  
End Joining ◽  
Exogenous Dna ◽  
Strand Breaks

ABSTRACT Cointroduction of plasmids into mammalian cells is commonly used to investigate transcription factor regulation of reporter genes or to normalize transfection efficiency. We report here that cotransfected DNA molecules commonly transfer enhancer elements from one plasmid to another. Using separate Renilla or Firefly luciferase reporters, we found that an estrogen response element (ERE) originally linked to one of the reporters stimulated expression of the non-ERE-containing reporter. Similar enhancer transfer was seen with the cytomegalovirus enhancer. This enhancer transfer effect was not seen when cells were transfected separately with the reporters and the extracts were then combined before luciferase assays. The degree of enhancer transfer increased with transfected plasmid concentration and was greater when linearized rather than circular plasmid DNA was used. We hypothesized that double-strand breaks and heteroligation of cointroduced DNA molecules mediated the transfer of regulatory elements from one molecule to another. PCR of transfected plasmid DNA confirmed nonhomologous end-joining (NHEJ) ligation of DNA fragments originally present in separate plasmids. The NHEJ reaction was enhanced by UV light treatment to introduce double-strand breaks, and it was greater after liposome-mediated transfection than after calcium-phosphate-mediated transfection. NHEJ also occurred after adenoviral transfer of DNA into cells. We conclude that NHEJ mediates the transfer of regulatory DNA elements among cointroduced DNA molecules. These findings indicate the need for caution when interpreting results of transfection experiments containing more than one plasmid and suggest a mechanism whereby viruses or other exogenous DNA might recombine to activate unrelated genes.

scholarly journals Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

1985 ◽  
Vol 5 (6) ◽  
pp. 1220-1228 ◽  
Author(s):  
J R Patton ◽  
D A Ross ◽  
C B Chae
Keyword(s):  
Protein Complexes ◽  
Mapping Method ◽  
Micrococcal Nuclease ◽  
Beta Globin ◽  
Nuclear Rna ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Rna Mapping ◽  
Rna Protein Complexes ◽  
Endogenous Nuclease

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

scholarly journals Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei

1985 ◽  
Vol 5 (6) ◽  
pp. 1220-1228
Author(s):  
J R Patton ◽  
D A Ross ◽  
C B Chae
Keyword(s):  
Protein Complexes ◽  
Mapping Method ◽  
Micrococcal Nuclease ◽  
Beta Globin ◽  
Nuclear Rna ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Rna Mapping ◽  
Rna Protein Complexes ◽  
Endogenous Nuclease

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

scholarly journals Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid

1975 ◽  
Vol 151 (3) ◽  
pp. 497-503 ◽  
Author(s):  
J F Krall ◽  
S H Socher ◽  
N T Van ◽  
B W O'Malley
Keyword(s):  
Dna Sequences ◽  
Thermal Denaturation ◽  
Tissue Specificity ◽  
Deoxyribonucleic Acid ◽  
Micrococcal Nuclease ◽  
Chromosomal Protein ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Limited Degree ◽  
Chromatin Proteins

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.

scholarly journals Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles

2019 ◽  
Vol 14 (2) ◽  
pp. 196-213
Author(s):  
Patrizia Bonfanti ◽  
Anita Colombo ◽  
Melissa Saibene ◽  
Luisa Fiandra ◽  
Ilaria Armenia ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Iron Nanoparticle ◽  
Xenopus Laevis Embryos ◽  
Ingested Nanoparticles
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