Ad12 particles enter the cytoplasm.

Ad12 DNA can be found in the nuclei of hamster cells (13,22).

Ad12 DNA associates with the chromosomes (23).

Ad12 DNA can integrate into the host chromosome (13,24–26).

Some of the early Ad12 functions are transcribed, although inefficiently (22,27).

By DNA array analyses, a limited number of changes in cellular transcription patterns can be documented upon the infection of BHK21 cells with Ad12 (Dorn, A. and Doerfler, W. unpublished results).

Ad12 DNA replication cannot be detected (3,22,28).

Ad12 VA and L1 RNAs are not transcribed (29).

The region downstream of the major late promoter (MLP) of Ad12 DNA carries a 33-bp-long mitigator element,* which inactivates the MLP in hamster cells (30).

Late Ad12 genes are not detectably transcribed (28,31).

Per definitionem, new Ad12 virions are not produced in the abortive system (3).

There is no cytopathic effect in Ad12-infected hamster cells (the infected cells continue to replicate [3]).

As one of the biological consequences of the abortive infection of hamster cells, Ad12 induces undifferentiated tumors in newborn hamsters. (Ad2 fails to do so. The abortive mode of infection enables the Ad12 genome to become permanently fixed in the host genome and to survive for many cell generations. Productive infections would kill all infected cells.)

The genomic DNA is then alkali-denatured in 0.3 M NaOH for 15 min at 37°C and for 3 min at 95°C.

Subsequently, treat the DNA with sodium bisulfite. Mix the denatured DNA solution (66 μL) with 1.2 mL of the bisulfite solution (Subheading 2., item 3), overlay with mineral oil, and incubate at 55°C for 16 h in a water bath in the dark.

From the above reaction mixture (step 3), purify the DNA using glassmilk.

For desulfonation, add 3 μL of 10 M NaOH and incubate for 10 min at 37°C.

The solution is thereupon neutralized, the DNA ethanol-precipitated, dried, and redissolved in 20 μL H2O.

Amplify selected segments in the promoters by PCR with appropriate oligodeoxyribonucleotide (oligo) primers.

Clone reaction products into the pGEMT vector (Promega, Madison, WI) after purification by using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) and transfect into E. coli XL1BlueMRF' (Stratagene, La Jolla, CA) applying standard procedures.

Isolate a number of clones and determine the nucleotide sequences with an Applied Biosystems 377 DNA sequencer.The bisulfite reaction (49,50) converts all unmethylated C residues into U residues, which are copied during PCR amplification into T residues, whereas the 5-methyldeoxycytidine (5-mC) residues are refractory to the bisulfite conversion reaction. Thus, a C residue in the eventually determined nucleotide sequence proves the presence of a 5-mC residue in this position in the original genomic nucleotide sequence. All bona fide C residues score as Ts.

Amplification conditions are as follows: After the initial denaturation step at 94°C for 5 min, 35 cycles of denaturation at 94°C for 15 s, annealing for 15 s at temperatures between 51°C and 57°C, and an extension reaction at 72°C for 30 s follows. A final extension step at 72°C for 5 min terminates the reaction.

The RT reaction is carried out at 48°C for 45 min, followed by PCR with 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 1 min and elongation at 68°C for 2 min. Subsequently, a final elongation step at 68°C for 7 min is applied.

All reverse transcription (RT)-PCR reaction products are analyzed by electrophoresis on 2% agarose gels and are stained with ethidium bromide.

Products in the size range predicted from the published DNA sequence are purified from the gel, subcloned into the pGEM-T vector, and the DNA is sequenced.

At 18 h after transfection, infect the cells with 100 plaque-forming units of Ad12 per cell.

At 28 h p.i., suspend about 10⁷ cells in phosphate-buffered saline containing 1% Igepal CA-630, 0.1% sodium dodecyl sulfate (SDS), and 4 mM Pefabloc SC protease inhibitor.

The cells are disrupted by ultrasonic treatment for 2 min in a branson B-12 sonifier. After removal of cell debris, treat cell extracts for 1 h at 4°C with 1.0 μg of normal rabbit immunoglobulin G and 20 μL of protein A—agarose conjugate.

Subsequently the beads are pelleted, and the lysate is incubated at 4°C overnight with 15 μL of Ad12-pTP rabbit antiserum and 30 μL of protein A—agarose.

Immunoprecipitates are collected by centrifugation for 5 min at 1000g and are washed for times with phosphate-buffered saline.

For the release of Ad12 DNA from this complex, suspend the beads in 50 μL of 10 m M Tris-HCl (pH 8.0), 2 mM ethylene diamine tetraacetic acid, 10 mM NaCl, 1% SDS, and treat for 3 h at 55°C with 1μg of Proteinase K per mL.

This protocol is described in ref.34. The DNA is then analyzed by standard restriction and blotting procedures.

Prepare single-stranded (ss) M13-Ad12 DNAs (we used 28 different clones) using the ss-M13-DNA purification kit.

Denature the ssDNAs in 0.4 M NaOH, 5 mM ethylene diamine tetraacetic acid, fix on positively charged nylon membranes, and hybridize to ³²P-labeled cDNA. (We used amounts of 20 or 100 ng of Ad12-M13 DNA for HeLa-Ad12 or BHK21-Ad12 DNA array analysis, respectively.)

For reverse transcription and ³²P-labeling of cDNA, total RNA from mock- or Ad12-infected cells is isolated by using a Macherey/Nagel kit. In this reaction, 5 μg of RNA, 2 µg of oligo (dT) 12–18 primers, and 1 mM of each of the dNTPs are denatured at 65°C for 5 min, chilled on ice, then mixed with 5 μCi of (γ-³²P)- dCTP (3000 Ci/mmol), 10 mM dithiothreitol, superscript II reaction buffer, and 300 U of superscript II reverse transcriptase and are incubated at 42°C for 1.5 h.

Subsequently, the ³²P-labeled probes are purified by gel filtration on Sephadex G-50 columns and are hybridized to DNA arrays on membranes at 42°C for 20 h in DIG hybridization buffer.

Wash membranes three times for 20 min in 0.5X standard sodium citrate, 1% SDS at 65°C and expose for 14 h (HeLa-Ad12) or 48 h (BHK21-Ad12) to X-ray films. For many years, research in the author's laboratory was supported by the Deutsche Forschungsgemeinschaft Bonn, Germany (through SFBs 74 and 274 and grant DO 165/17), by the Wilhelm Sander Foundation, Munich, by Amaxa GmbH, Cologne, and recently by the Institute for Clinical and Molecular Virology, University Erlangen—Nürnberg.