Integration of Adenovirus DNA into the Cellular Genome

Doerfler, Walter; Burger, Harold; Ortı́n, Juan; Fanning, Ellen; Brown, Dennis T.; Mestphal, M.; Winterhoff, Ute; Weiser, Barbara; Schick, J.

doi:10.1101/sqb.1974.039.01.063

Cited by 25 publications

(24 citation statements)

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“…Restriction endonuclease EcoRI (21) was isolated according to the procedure of R. N. Yoshimori (Ph.D. thesis, Univ. of California, San Francisco, 1971), as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

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Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells

Ortı́n¹,

Scheidtmann²,

Greenberg³

et al. 1976

J Virol

105

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“…Restriction endonuclease EcoRI (21) was isolated according to the procedure of R. N. Yoshimori (Ph.D. thesis, Univ. of California, San Francisco, 1971), as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…It is conceivable that viral DNA persisting in an integrated state in the cellular genome is subject to cellular transcriptional controls. This possibility exists also in productively infected cells, as evidence has been adduced for an integrated state of Ad2 DNA in productively infected cells as well (5,10,41).…”

mentioning

confidence: 95%

Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells

Ortı́n¹,

Scheidtmann²,

Greenberg³

et al. 1976

J Virol

105

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“…The 40-100S DNA from Ad2-infected KB cells was also analyzed by reassociation kinetics (2,18,21). So far, the analysis was limited to the 16-18 hr period.…”

Section: Quantitation Of Intracellularmentioning

confidence: 99%

Intracellular forms of adenovirus DNA: integrated form of adenovirus DNA appears early in productive infection.

Schick¹,

Baczko²,

Fanning³

et al. 1976

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

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In KB cells productively infected with adenovirus type 2, alkali-stable >100S and 40-1OOS viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) In previous publications (1, 2) an alkali-stable, high-molecular-weight (50-90 S) form of viral DNA was described in productively infected cells, and evidence was presented that this DNA represented viral sequences integrated into cellular DNA. Integrated viral sequences in productively infected cells were also described in other virus systems (3). With adenovirus, integrated viral genomes were previously found in abortively infected cells (4-6), and it was shown that in adenovirus-transformed cells specific fragments of the viral genome persisted (7), probably in an integrated form (8).In for 30-60 min. The extract was then made 0.5 M in NaOH with 6 M NaOH and was finally layered on top of an alkaline sucrose density gradient. In other experiments the Ad2-infected or mock-infected KB cells were first incubated at 370 with 250 ,ug of Pronase or proteinase K per ml for 1 or 18 hr at neutral pH, and incubation was subsequently continued in 0.5 M NaOH at 40 for 18 hr (in the case of 1 hr incubation with Pronase) or the lysate was layered on an alkaline sucrose gradient immediately after adding alkali (in the case of 18 hr incubation with Pronase).Resedimentation (14), and the peak of the cellular DNA was isolated on a neutral sucrose gradient (Fig. 7a). Samples of 0.5-1.0 ml of cellular DNA (1-2 jig) were dialyzed into 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA. Subsequently, 2-5 Mg of 14C-labeled Ad2 marker DNA was added, and the DNA was cleaved with the EcoRI restriction endonuclease under conditions outlined elsewhere (2, 12). The digestion products were analyzed by electrophoresis on 13 cm long, cylindrical, 1.5% polyacrylamide-0.8% agarose gels at 40 V for 10-12 hr (12); the gels were cut into 1 mm slices; and the DNA was eluted from each slice by ultrasonic treatment (microtip) for 3 min in 1.0 ml of 0.45 M NaCl, 0.045 M sodium citrate, pH 7.6 (3 X SSC) (16). The 3H-and "4C-labeled DNA from each slice was hybridized to unlabeled Ad2 DNA fixed to nitrocellu-

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“…In one out of the seven integrations analyzed, a substitution was observed 9 nucleotides downstream of the integration event. The presence of microhomologies between the viral terminus and chromosomal DNA has been observed in recombination studies of both viral vectors (28,36,43) and wild-type adenovirus (5,6). Two out of seven integrations had occurred within genes, and in three of the remaining five integrations, the vector DNA had recombined into a locus between 11 and 34 kb upstream or downstream of a gene.…”

Section: Downloaded Frommentioning

confidence: 98%