Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

Stabel, Silvia; Doerfler, Walter; Friis, Robert R.

doi:10.1128/jvi.36.1.22-40.1980

Cited by 114 publications

(58 citation statements)

References 39 publications

(54 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…From aliquots of highly purified intraor extracellular Adl2, the viral DNA was extracted and cleaved with restriction endonuclease EcoRI or BamHI, and the fragments were separated by electrophoresis on an agarose gel. After blotting and hybridization to 32P-labeled Adl2 DNA, the amounts of intra-and extracellular Adl2 DNA were compared by autoradiography and spectrophotometric scanning of the autoradiograms (15). The relative amounts of Adl2 DNA extracted from intraor extracellular virions were taken as direct measures of the mass of Adl2 particles.…”

Section: Resultsmentioning

confidence: 99%

“…DNA preparations were cleaved with the restriction endonuclease EcoRI, BamHI, HindIII, Mspl, or PstI. Fragments were separated by electrophoresis on 0.5 to 1.5% agarose gels, blotted to nitrocellulose (BA85) filters (14), and hybridized to 32P-labeled Adl2 DNA as described before (15). Adl2 DNA was labeled by nick translation (13), and DNA-DNA hybridization experiments were carried out as outlined elsewhere (19).…”

Section: Inoculation Of Hek Hela or Kb Cells With Extracellularmentioning

confidence: 99%

“…Adl2 DNA was labeled by nick translation (13), and DNA-DNA hybridization experiments were carried out as outlined elsewhere (19). Adl2-specific fragments were eventually visualized by autoradiography and quantified by scanning spectrophotometry as described earlier (15). Cleavage patterns of DNA preparations extracted from intracellular and extracellular virions were compared.…”

Section: Inoculation Of Hek Hela or Kb Cells With Extracellularmentioning

confidence: 99%

“…Amounts of DNA which corresponded to equivalent portions of intracellular or extracellular Adl2 particles were cut with EcoRI (a) or BamHI (b), the fragments were separated by electrophoresis on a 0.5% agarose gel and transferred to nitrocellulose filters (14), and Ad12 DNA was visualized by hybridization to 32P-labeled Adl2 DNA followed by autoradiography. The viral DNA bands, in particular those corresponding to EcoRI fragment B, C, or D or BamHI fragment A, B, or E, were quantitated by spectrophotometric scanning (15). by hybridization to 32P-labeled Adl2 DNA.…”

Section: D-[6-3h]glucosamineor [35s]methionine-labeled Extraormentioning

confidence: 99%

“…by hybridization to 32P-labeled Adl2 DNA. Amounts of viral DNA present in each DNA preparation were compared by scanning the intensity of viral DNA bands in autoradiograms (15).…”

Section: D-[6-3h]glucosamineor [35s]methionine-labeled Extraormentioning

confidence: 99%

See 4 more Smart Citations

Increased infectivity of extracellular adenovirus type 12

Brüggemann¹,

Klenk²,

Doerfler³

1985

J Virol

View full text Add to dashboard Cite

Human adenovirus type 12 was propagated on human embryonic kidney cells, and the specific infectivities of intra-and extracellular virus particles were compared between 48 and 104 h after infection. Released virions exhibited a specific infectivity of up to 10 times higher than that of intracellular particles. The increased infectivity was apparently not due to enhanced rates of adsorption or penetration of extracellular virus. There may be a delay in the onset of viral DNA replication in intracellular virus-infected cells. Differences in the composition of intra-and extracellular virions were not recognized. Differences might also be sought in late expression or assembly of progeny virions or both. The data indicated that the virions released from the infected cells differed from those retained in the nucleus with respect to their specific infectivities. Active mechanisms of virus release have not yet been investigated.In the course of a systematic investigation on the infectivity of human adenovirus type 12 (Adl2), we discovered that extracellular virions liberated from human embryonic kidney (HEK) cells exhibited considerably higher specific infectivity than intracellular particles. In the past, most experiments conventionally carried out with adenoviruses were performed with intracellular virions extracted from infected cells. The extracellular virus comprising only a fraction of the total virus yield was often discarded.The high specific infectivity of extracellular virions raised interesting questions with respect to late steps of maturation and about the release of adenovirions which were assembled in the nucleus of the cell. Extracellular virions were not previously investigated for differences in structure, composition, or infectivity in comparison to intracellular virions. It was conceivable that adenovirions were further modified after the assembly stage in the nucleus and that one of these modifying events might be required for the efficient release of the virions. The mechanism of release of infectious adenovirions from the cell nucleus was not yet studied in detail. In principle there are two alternatives: (i) virions could be liberated passively, as it were, as a consequence of the destruction of cells in late stages of infection, or (ii) there might be an active process of virion release. If extracellular virions differed significantly from intracellular particles, it was conceivable that the mechanisms of infection and uncoating, perhaps also the late events in viral gene expression or assembly and release, exhibited striking differences depending on whether intracellular or extracellular virions were used for inoculation.In this communication we will demonstrate that the total extracellular infectivity of Adl2 represents about 25 to 30% of the infectivity of the intracellular virions, but only 2.5 to 3% of the mass of intracellular virions. The specific infectivity of extracellular adenovirions of type 2 (Ad2) or type 12, as determined by focus-forming assay (12), was approximately 10 times high...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Inoculation Of Hek Hela or Kb Cells With Extracellularmentioning

confidence: 99%

Section: Inoculation Of Hek Hela or Kb Cells With Extracellularmentioning

confidence: 99%

Section: D-[6-3h]glucosamineor [35s]methionine-labeled Extraormentioning

confidence: 99%

“…by hybridization to 32P-labeled Adl2 DNA. Amounts of viral DNA present in each DNA preparation were compared by scanning the intensity of viral DNA bands in autoradiograms (15).…”

Section: D-[6-3h]glucosamineor [35s]methionine-labeled Extraormentioning

confidence: 99%

See 3 more Smart Citations

Increased infectivity of extracellular adenovirus type 12

Brüggemann¹,

Klenk²,

Doerfler³

1985

J Virol

View full text Add to dashboard Cite

show abstract

Conclusions derived from a survey of junction sites

Doerfler

1999

Foreign DNA in Mammalian Systems

View full text Add to dashboard Cite

Patch homologies and the integration of adenovirus DNA in mammalian cells.

Gahlmann¹,

Leisten²,

Vardimon³

et al. 1982

The EMBO Journal

Self Cite

View full text Add to dashboard Cite

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2‐3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5, corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120‐130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca ‐ to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.

show abstract

Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

Cited by 114 publications

References 39 publications

Increased infectivity of extracellular adenovirus type 12

Increased infectivity of extracellular adenovirus type 12

Conclusions derived from a survey of junction sites

Patch homologies and the integration of adenovirus DNA in mammalian cells.

Contact Info

Product

Resources

About