Selective extraction of polyoma DNA from infected mouse cell cultures

Hirt, Bernhard

doi:10.1016/0022-2836(67)90307-5

Cited by 5,207 publications

(2,453 citation statements)

References 6 publications

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“…Cell extracts were analyzed using 10% SDS-PAGE (Laemmli, 1970)+ [ 35 S]-labeled proteins were detected from dried gels by autoradiography+ Alternatively proteins were detected by immunoblotting using Immobilon P membranes (Millipore) with mouse anti-myc Mab 9E10, 1:2,000, or rabbit anti-bglucuronidase (1:5,000, 5 Primer3 Prime, Inc+), or anti-actin (1:1,000, Sigma) primary antibodies followed by peroxidase-labeled sheep anti-mouse IgG or donkey anti-rabbit IgG secondary antibodies (1:3,000, Amersham) as appropriate and chemiluminescence reagents (Pierce)+ Plasmid DNA recovery from selected cells Plasmid DNA was recovered from selected COS-7 cells using the procedure described by Hirt (1967) and transformed into competent E. coli (DH5a) cells+ Plasmids were isolated from amplified individual colonies or from pools of colonies as described above using a Promega Wizard miniprep kit or the BIO-101 (Anachem) maxiprep kit+…”

Section: Sds-page and Western Blotsmentioning

confidence: 99%

A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Robertson

Seamons

Belsham

1999

RNA

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Section: Sds-page and Western Blotsmentioning

confidence: 99%

A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Robertson

Seamons

Belsham

1999

RNA

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“…Strongly stained cells were isolated by¯uorescence activated cell sorting using a FACStar (Becton Dickinson, Mountain View, CA, USA). Plasmid DNA of the stained cells was isolated by lysis according to the method described by Hirt (1967). Genomic DNA and proteins were separated by centrifugation.…”

Section: Antibodiesmentioning

confidence: 99%

Metastasis-association of the rat ortholog of the human epithelial glycoprotein antigen EGP314

Würfel

Rösel

Seiter³

et al. 1999

Oncogene

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Screening for surface molecules expressed by metastasizing rat tumors had revealed evidence for metastasisassociation of a molecule also expressed on epithelial cells. The similarity to the expression pro®le of the panepithelial glycoprotein EGP314 prompted us to isolate and sequence the gene and to explore functional features of the molecule in transfected tumor lines. The molecule D5.7A, named according to the antibody, D5.7, used for selection, indeed, is the ortholog of EGP314 with 92% and 80% identity to the murine and the human molecules. Like EGP314, D5.7A has a particular cleavage site, a small cleavage product being resolved under reducing conditions from the membrane anchored part of the molecule. Transfection of a low metastasizing ®brosarcoma, pheochromoblastoma and adenocarcinoma revealed that expression of D5.7A facilitates tumor progression. Depending on the origin of the tumor, D5.7A transfectants either metastasized via the lymphatic system (pheochromoblastoma, adenocarcinoma) or hematogeneously (®brosarcoma). Particularly after proteolytic cleavage, D5.7A facilitated cell ± cell adhesion and provided a proliferative signal upon crosslinking. Thus, the rat ortholog of EGP314 is involved in metastasis formation. Importantly, its functional activities apparently rely on proteolytic cleavage. These ®ndings provide a ®rst evidence on how a panepithelial marker can be involved in tumor progression.

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“…Virus was allowed to adsorb at 37°C for I h, and 100 ml of culture medium containing 2 % fetal calf serum (FCS) were then added to each roller bottle. The infection was allowed to proceed for 70 h. Cells were harvested and viral and cellular DNA were separated by precipitation of the cellular DNA according to the procedure described by Hirt [21]. The supernatant fluid was extracted three times with phenol , precipitated with ethanol , dialysed against buffer (10 mM Tris-HCI, pH 7.2; I mM EDTA; 0.2 M NaCI) and run twice in CsCI gradients to minimize RNA contamination .…”

Section: Isolation Of Dnamentioning

confidence: 99%

Mitochondrial DNA arranged into chromatin-like structures after injection into amphibian oocyte nuclei

Zentgraf

Trendelenburg

Spring

et al. 1979

Experimental Cell Research

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SUMMARYPurified mitochondrial DNA (mitDNA) from ovaries ofXenopus lae vis was injected into the nuclei (germinal vesicles) of large viteUogenic oocytes of the same organism and examined by electron microscopy ofthe spread nuclear contents. Normally located nuclei of untreated oocytes as weil as peripherally translocated nuclei of centrifuged oocytes were used. In addition, oocyte nuclei isolated and incubated under liquid paraffin oil were injected with DNA . The integrity oftranscriptional structures of endogenous chromosomal (Iampbrush chromosomes) and extrachromosomal (nucleoli) genes of the injected nuclei was demonstrated . Microinjected mitDN A was identified as circles of chromatin exhibiting polynucleosome-like organization and a me an contour length of 2.6 J.Lm, corresponding to a compaction ratio of the mitDN A of about 2 : I. This DNA packing ratio is similar to that observed after preparation of various kinds of native chromatin in low salt buffers . The chromatin circles formed from injected mitDNA only very rarely exhibited lateral fibrils suggestive of transcriptional activity. These results suggest that purified mitDNA can be transformed to normally structured chromatin when exposed to oocyte nuclear contents but is rarely , if at all , transcribed in this form and in this environment. Circular double-stranded DN A molecules injected into oocyte nuclei of Xenopus laevis are relatively stable and can be assembled into chromatin-like configurations. This has been demonstrated for SV40 DNA [1][2][3][4][5], plasmid DNA [6] and extrachromosomal ribosomal DNA (rDNA) from an insect [7]. Moreover, transcription of genes contained in such injected DNA molecules has been demonstrated [8] , e.g., for SV40 DNA [9][10][11][12][13] , and for various plasmids such as those containing genes coding for histones of Drosophila [9,11] or sea urchin [14][15][16], genes coding for 5S rRNA ofXenopus [17], genes coding for tRN As of a nematode [18] or Xenopus laevis [15,19], and a plasmid containing a major portion of the pre-rRNA gene of Xenopus [6]. Transcription of DNA injected into Xenopus oocyte nuclei has also been demonstrated for the naturally occurring circular pre-rRNA genes from ovaries of the water beetle, Dytiscus marginalis [7]. In the present study we show that another eukaryotic kind of circular DNA, which in nature is not associated with histones and nuclear transcriptional complexes , namely the mitochondrial genome, can be organized into Exp Cell Res 122 (/979 )

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Selective extraction of polyoma DNA from infected mouse cell cultures

Cited by 5,207 publications

References 6 publications

A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Metastasis-association of the rat ortholog of the human epithelial glycoprotein antigen EGP314

Mitochondrial DNA arranged into chromatin-like structures after injection into amphibian oocyte nuclei

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