Base specific fractionation of double stranded DNA: affinity chromatography on a novel type of adsorbant

Bûnemann, Hans; Müller, Werner

doi:10.1093/nar/5.3.1059

Cited by 63 publications

(8 citation statements)

References 18 publications

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“…Hybridization of the tk probe to HindIIIdigested DNA from this line detected a single 12-kbp fragment (data not shown). To enrich for this 12-kbp fragment, DNA from line 5.5 was digested with HindlIl and fractionated by chromatography on a malachite green-containing gel (4). Samples from the column fractions were subjected to gel electrophoresis and hybridization with a tk probe to identify fractions containing the 12-kbp tk fragment.…”

Section: Resultsmentioning

confidence: 99%

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

Steele¹,

Bakken²,

Reeder³

1984

Mol. Cell. Biol.

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We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of sitespecific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

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Section: Resultsmentioning

confidence: 99%

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

Steele¹,

Bakken²,

Reeder³

1984

Mol. Cell. Biol.

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“…Xenopus DNA was cut with restriction endonuclease HindIII and fractionated on a malachite green gel (18). The fractions were then electrophoresed on an agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Transcription of repetitive sequences on Xenopus lampbrush chromosomes.

Jamrich¹,

Warrior²,

Steele³

et al. 1983

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

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We reinvestigated the lampbrush chromosomes of Xenopus laevis and found them well suited for the study of transcription by in situ hybridization to nascent RNA transcripts. Using this technique, we analyzed the transcription of three repetitive sequences that do not show any sequence homology and that differ in their degree of interspersion. We found that they are located on different parts of the chromosomes: two are clustered, one is interspersed. All three of these sequences are transcribed at the lampbrush chromosome stage and transcripts from both strands of each sequence can be detected. The amount of transcription is apparently not proportional to the number of copies of the repetitive sequence at a given chromosomal locus, suggesting that other sequences are involved in the regulation of their transcription. The large size of the newt chromosomes is correlated with a very large genome, which, however, is a distinct disadvantage for experiments involving cloning and characterization of specific DNA sequences. For this reason we felt that it would be useful to search for an experimental system with a considerably smaller genome but with cytologically usable lampbrush chromosomes. The obvious choice was Xenopus laevis. The Xenopus genome is under investigation in several laboratories and the lampbrush chromosomes have already been described (9, 10), although they have been generally considered poorly suited for detailed cytological studies. We reinvestigated these chromosomes and found them quite adequate for the study of transcription and protein distribution by in situ hybridization and indirect immunofluorescence, respectively. In this report, we demonstrate that some of the long transcription units on Xenopus lampbrush chromosomes contain repetitive sequences. Transcripts of repetitive sequences have been reported in a variety of tissues, including the lampbrush chromosomes of newt oocytes (11)(12)(13)(14)(15). Studies of amphibian lampbrush MATERIALS AND METHODSIn Situ Hybridization to Mitotic Chromosomes. Mitotic chromosome squashes were prepared from gut cells of adult Xenopus and hybridized in situ according to Pardue and Gall (16).In Situ Hybridization to Lampbrush Chromosomes. 3H-Labeled complementary RNA (cRNA) was prepared from singlestranded probes and was hybridized in situ to nascent RNA chains on the chromosomes according to Diaz et al. (12) and Pukkila (17).Preparation of Cloned DNA and Radioactive Probes. Xenopus DNA was cut with restriction endonuclease HindIII and fractionated on a malachite green gel (18). The fractions were then electrophoresed on an agarose gel. The 750-base-pair band that is visible in the first fractions of the gradient was eluted and cloned in plasmid pBR322 by standard procedures. The identity of the cloned DNA was confirmed by hybridization to genomic DNA in a Southern blot. The HindII1 fragment was then recloned in a phage M 13 vector. In the course of this work we learned that the same repetitive DNA fragment was independently cloned by Lam and Carr...

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“…ified NaDodSO4 lysis procedure (32) followed by acridine yellow-agarose column chromatography (33 (Fig. 2).…”

mentioning

confidence: 99%

Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows.

Hauswirth¹,

Laipis²

1982

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

333

164

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Base specific fractionation of double stranded DNA: affinity chromatography on a novel type of adsorbant

Cited by 63 publications

References 18 publications

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

Transcription of repetitive sequences on Xenopus lampbrush chromosomes.

Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows.

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