Purification of RNA-free plasmid DNA using alkaline extraction followed by ultrogel A2 column chromatography

Micard, D.; Sobrier, Marie-Laure; Couderc, J.L.; Dastugue, B.

doi:10.1016/0003-2697(85)90636-0

Cited by 36 publications

(14 citation statements)

References 12 publications

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“…"D" fragments generated by a Hind III digest of D. melanogaster mitochondrial DNA (39). Plasmid DNAs were purified as described in (40) and 32P-labeled by nick-translation (41) to a specific activity of 10 dpm/ug. Hybridizations were performed for 36 h. at 400C in 50X (v/v) formamide, 1 x Denhart's solution, 0.6 M NaCl, 60 mM Sodium Citrate and 50 ug.…”

Section: Methodsmentioning

confidence: 99%

Illegitimate recombination in the histone multigenic family generates circular DNAs in Drosophila embryos

Pont

Degroote

Picard

1988

Nucleic Acids Research

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From extrachromosomal covalently closed circular DNA molecules purified from Drosophila melanogaster embryos, we have isolated 24 clones homologous to the histone tandemly repeated gene family. Some of the clones harbor one of the two main types of genomic repeated units of 4.8 and 5.0 kb. and probably result from homologous recombination. The remaining clones have a size ranging from 0.2 to 2.5 kb. and most of them carry a single fragment of the repeated unit. Nucleotide sequences of the junction region of six of these clones indicate they are generated by illegitimate recombination between short (8-15 bp.) imperfect direct repeats. The data suggest that most of the histone homologous circular DNA molecules are deleted histone units.

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Section: Methodsmentioning

confidence: 99%

Illegitimate recombination in the histone multigenic family generates circular DNAs in Drosophila embryos

Pont

Degroote

Picard

1988

Nucleic Acids Research

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“…Recombinants ss (single-straned) t1 3 DNA were prepared according to MESSING (27). Recombinant duplex DNAs were purified either by the BIRBOIM and DOLY method followed by a CsCl gradient or by the MICARD et al method (28,29). Restriction fragments of cloned mtDNA sequencs were gel-purified.…”

Section: Methodsmentioning

confidence: 99%

RNA mapping onDrosophilamitochondrial DNA: precursors and template strands

et al. 1986

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Drosophila melanogaster mitochondrial DNA (mtDNA) is closely related to the mammalian and amphibian mtDNA except for gene organization. In Drosophila, genes are distributed in clusters alternatively coded on each strand. Besides the eleven major foreseeable transcripts previously described (MERTEN and PARDUE, 1981, J. Mol. Biol., 153, 1-21), we have characterized two poly A+ transcripts, one major and one minor which could correspond respectively to the ND3 and ND6 reading frames, and 27 poly A+ minor transcripts (0.2 to greater than 3.2 kb) which are distributed along the mtDNA except in the rRNAs, ND 1 and A+ T rich regions. The mapping and length of 25 of these transcripts strongly suggest a precursor role. They would be processed at the level of tRNA or tRNA-like sequences. Most of them are transcribed from the template strand of each gene cluster and their distribution is in agreement with the hypothesis of several transcription origins and terminations located near the extremities of each gene cluster. Quantitatively our results show a large variation in each presumptive mature transcript compared to the other, even in a given gene cluster, suggesting a specific degradation of some of the mature transcripts.

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“…Monomer fragments and tandemly repeated DNA templates containing the Lytechinus variegatus 5S rRNA gene sequence were derived from plasmid p5S207-12 or p5S172-12 (13). Plasmids were purified from Escherichia coli HB101 by the alkaline lysis method (14) followed by CsCl gradient banding. Oligonucleosome templates consisting of 12 repeats of a 207-base-pair (bp) (207-12 template) or a 172-bp (172-12 template) DNA sequence were prepared by Hha I digestion of plasmid p5S207-12 or p5S172-12, followed by exclusion chromatography on Ultrogel A2 (15).…”

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confidence: 99%

DNA and protein determinants of nucleosome positioning on sea urchin 5S rRNA gene sequences in vitro.

Dong

Hansen

Holde

1990

Proc. Natl. Acad. Sci. U.S.A.

174

156

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DNA and protein determinants of nucleosome positioning have been examined after in vitro reconstitutions of native or modified histone octamers onto tandem repeats of 207-and 172-base-pair DNA sequences containing the Lytechinus variegatus SS rRNA gene and onto monomeric sequences derived from these by digestion with various restriction endonucleases. In all cases, a major nucleosome position as well as a number of minor positions have been observed, which indicates that the generation of multiple positions is an inherent property of the SS rRNA gene sequence. Interestingly, all positions observed differ by multiples of 10 base pairs. Data obtained under different reconstitution conditions demonstrate that the observed distributions of nucleosomes on these DNA templates are equilibrium distributions. This study has also examined the positioning of histone octamers from which histone "tails" had been removed by tryptic digestion. Results indicate that the histone tails are not determinants of nucleosome positioning. Although our results suggest that the mechanical properties of the SS rDNA are the fundamental factors determining nucleosome positioning, they are insufficient to direct all nucleosomes into a single location.It is well established that nucleosomes can be positioned over specific DNA sequences in vivo and in vitro (1). Although it has been demonstrated that the flanking boundaries established by neighboring site-specific proteins can influence nucleosome positioning in vivo (2), reconstitution of nucleosomes onto short DNA templates in vitro indicates that nucleosome positioning can also occur when such boundaries do not exist (3, 4). In many cases, results strongly suggest that the fundamental determinants of nucleosome positioning reside in DNA sequence-dependent mechanical properties such as bending and flexibility (5)(6)(7)(8)(9)).An example of sequence-dependent nucleosome positioning that has generated considerable interest is nucleosome formation on sea urchin 5S rRNA gene sequences. Simpson and Stafford (3) (14) followed by CsCl gradient banding. Oligonucleosome templates consisting of 12 repeats of a 207-base-pair (bp) (207-12 template) or a 172-bp (172-12 template) DNA sequence were prepared by Hha I digestion of plasmid p5S207-12 or p5S172-12, followed by exclusion chromatography on Ultrogel A2 (15). EcoRi and Msp I monomer templates were prepared by restriction endonuclease digestion of the tandemly repeated 207-12 oligonucleosome template, followed by purification through 2% low-melting agarose gels (16). Rsa I and Xmn I monomer templates were prepared by restriction endonuclease digestion of the intact plasmid p5S207-12 and subsequent purification through Ultrogel A2 (15).Preparation of Native and Trypsinized Histone Octamers. Native nucleosome core particles were purified from chicken erythrocytes as described (17). Trypsinized core particles were prepared from native core particles by digestion with immobilized trypsin, followed by purification through sucrose gradients (17). ...

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Purification of RNA-free plasmid DNA using alkaline extraction followed by ultrogel A2 column chromatography

Cited by 36 publications

References 12 publications

Illegitimate recombination in the histone multigenic family generates circular DNAs in Drosophila embryos

Illegitimate recombination in the histone multigenic family generates circular DNAs in Drosophila embryos

RNA mapping onDrosophilamitochondrial DNA: precursors and template strands

DNA and protein determinants of nucleosome positioning on sea urchin 5S rRNA gene sequences in vitro.

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