The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.     

Nucleotide sequence at the site of junction between adenovirus type 12 DNA and repetitive hamster cell DNA in transformed cell line CLAC1.

The hamster cell line CLAC1 originated from a tumor induced by injecting human adenovirus type 12 (Ad12) into newborn hamsters. Each cell contained about 12 copies of viral DNA colinearly integrated at two or three different sites. We have cloned and sequenced a DNA fragment comprising the site of junction between the left terminus of Ad12 DNA and cellular DNA. The first 174 nucleotides of Ad12 DNA were deleted at the site of junction. Within 40 nucleotides, there were one tri-, two tetra-, one penta-, and one heptanucleotide which were identical in the 174 deleted viral nucleotides and the cellular sequence replacing them. In addition, there were patch-type homologies ranging from octa- to decanucleotides between viral and cellular sequences. There is no evidence for a model assuming adenovirus DNA to integrate at identical cellular sites. The cellular DNA sequence corresponding to the junction fragment was cloned also from BHK21 (B3) hamster cells and sequenced. Up to the site of linkage with viral DNA, this middle repetitive cellular DNA sequence was almost identical with the equivalent sequence from CLAC1 hamster cells. Taken together with the results of previously published analyses (11, 12), the data suggest a model of viral (foreign) DNA integration by multiple short sequence homologies. Multiple sets of short patch homologies might be recognized as patterns in independent integration events. The model also accounts for the loss of terminal viral DNA sequences.     

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

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Species dependence of the major late promoter in adenovirus type 12 DNA.

In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the expression of most of the late viral functions. We have inserted the major late promoter (MLP) of Ad2 or of Ad12 DNA in front of the chloramphenicol acetyl transferase gene in the pSVO-CAT construct. Upon transfection into uninfected human and hamster cells, the pAd12MLP-CAT construct shows no significant activity; the pAd2MLP-CAT construct exhibits low activity. In Ad12-infected human cells, both constructs are active. These findings support the notion that other viral factors are required for MLP activity of Ad2 or Ad12 DNA in permissive human cells. In Ad2-infected hamster cells, both the pAd2MLP-CAT and the pAd12MLP-CAT constructs are active. Apparently, the Ad12 MLP can be activated by Ad2 functions, as already demonstrated for the entire Ad12 genome in double-infected cells or in Ad2- or Ad5-transformed cells superinfected with Ad12. In Ad12-infected hamster cells, however, the MLP of Ad12 DNA is inactive but that of Ad2 DNA shows activity. Thus the MLP of Ad12 DNA somehow differentiates between cellular auxiliary functions of different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.     

Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression?

An inverse correlation has been described between the levels of DNA methylation in specific segments of adenovirus DNA integrated into the genomes of transformed and tumor cells and the extent to which these segments are expressed as messenger RNA. In the adenovirus type 2 (Ad2)-transformed hamster cell lines HE2 and HE3, the virus-specific DNA binding protein (DBP) is not expressed, and the DNA in the DBP gene is completely methylated in all 5'-CCGG-3' sites. At least part of the late promoter/leader sequence of the DBP gene is present in cell lines HE2 and HE3. In line HE1, on the other hand, the DBP is expressed, and the DNA in the DBP gene is unmethylated at the 5'-CCGG-3' (HpaII) sites. The late promotor/leader sequence of the DBP gene is expressed in cytoplasmic RNA isolated from line HE1. The effect of DNA methylation has also been tested in vitro in a microinjection system using Xenopus laevis oocytes. Unmethylated DNA fragments of Ad2 (E2a region) have been found to serve as active templates. When the same fragments are methylated at the 5'-CCGG-3' sites by the HpaII DNA-methyltransferase, viral RNA synthesis is inhibited upon microinjection into oocyte nuclei. These results provide direct evidence for the notion that DNA methylated at highly specific sites is somehow involved in the regulation of gene expression.     

Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.     

Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns.

We investigated whether, upon the integration of multiple copies of adenovirus type 12 (Ad12) DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change with consecutive passages of cells in culture. By the injection of purified Ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, were established. These cell lines carried different copy numbers of Ad12 DNA per cell in an integrated form and differed in morphology. Cell line HT5 had been passed twice through hamsters as tumor cells and was subsequently passaged in culture. Patterns of Ad12 DNA integration were determined by restriction cleavage of the nuclear DNA with BamHI, EcoRI, HindIII, MspI, or PstI followed by Southern blot hybridization using 32P-labeled Ad12 DNA or its cloned terminal DNA fragments as hybridization probes. In this way, the off-size fragments, which represented the sites of linkage between Ad12 and cellular DNAs, were determined. At early passage levels in culture, the integration sites of Ad12 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the five different tumor cell lines. Upon repeated passage, however, the off-size fragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions, Ad12-transformed tumor cell lines that carry the foreign (Ad12) genome in selective, probably very similar sites of the cellular genome evolve.     

Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells.

The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.     

Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with (3)H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The (3)H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral (3)H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.     

Integration of viral DNA into the genome of the adenovirus type 2-transformed hamster cell line HE5 without loss or alteration of cellular nucleotides

Hamster cell line HE5 has been established from primary LSH hamster embryo cells by transformation with adenovirus type 2 (Ad2) (1). Each cell contains two to three copies of integrated Ad2 DNA (2, 3). We cloned and sequenced the sites of junction between viral and cellular DNAs. The terminal 10 and 8 nucleotides of Ad2 DNA were deleted at the left and right sites of junction, respectively. The integrated viral DNA had an internal deletion between map units 35 and 82 on the Ad2 genome. At the internal site of deletion, the remaining viral sequences were linked via a GT dinucleotide of unknown origin. From HE5 DNA, the unoccupied sequence corresponding to the site of insertion was also cloned and sequenced. Part of this sequence was shown to be expressed as cytoplasmic RNA in HE5 and primary LSH hamster embryo cells. The viral DNA had been inserted into cellular DNA without deletions, rearrangements or duplications of cellular nucleotides at the site of insertion. Thus, insertion of Ad2 DNA appeared to have been effected by a mechanism different from that of bacteriophage lambda in Escherichia coli and from that of retroviral genomes in vertebrates. It was conceivable that the terminal viral protein (4) was somehow involved in integration either on a linear or a circularized viral DNA molecule.     

The adenovirus type 12 - mouse cell system: permissivity and analysis of integration patterns of viral DNA in tumor cells.

The integration patterns of persisting adenovirus type 12 (Ad12) DNA were analyzed in two Ad12-induced tumors of Balb/c and CBA/J mice and in one tumor cell line derived from an Ad12-induced retinoblastoma of C3H origin. In all three tumors the Ad12 genome was integrated colinearly and various copy numbers of viral DNA were found. Analysis of the Ad12 integration patterns revealed relatively simple offsize band patterns regardless of Ad12 copy numbers. The degree of methylation at the 5''-CCGG-3'' sites in the inserted Ad12 genome was determined using the isoschizomeric restriction endonuclease pair HpaII and MspI. Methylation was rather incomplete in the primary tumor tissues but almost complete in the retinoblastoma line carried in culture for many passages. The levels of expression of the viral genome in the Balb/c tumor and in the retinoblastoma line were determined by in vitro translation of RNA isolated from these cells and selected with appropriate restriction endonuclease fragments of Ad12 DNA. In both instances the 59 K, 19 K, and 17 K proteins of the E1b region were expressed. Proteins of the E1a region appeared very faint in the size class between 22 K and 42 K. The permissivity of Ad12 and the replication of Ad12 DNA in mouse cells were investigated by blotting restricted DNA from cells soon after, and a long time after, infection and by hybridization with 32P-labeled Ad12 DNA. Neither primary mouse kidney cells nor the established L929 mouse cell line supported viral DNA replication. These results raise the question to what extent host cell factors determine Ad12 DNA replication in mammalian cells.     

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

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.     

Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.

The insertion stability and DNA methylation patterns of integrated adenovirus type 12 (Ad12) genomes were investigated in Ad12-induced tumors and in tumor cell lines established from them as a function of time of passage under culture conditions. Upon subcultivation of cells from some of the tumors, the viral genomes were eliminated, apparently in a stepwise process with segments of the left termini of Ad12 DNAs persisting the longest. Morphological variants of these tumor cells lost all viral DNA and yet retained the oncogenic phenotype. All 13 independently isolated clones from one revertant line were devoid of Ad12 DNA. It could not be ruled out that very short sequence elements of viral DNA, such as promoters or enhancing sequences, could have persisted in these variants. The extent of viral DNA methylation was minimal in Ad12-induced tumors, although the viral genome was not extensively expressed, if at all. Upon passage in culture, the levels of viral DNA methylation increased. It was interesting that establishment of the final methylation pattern of integrated Ad12 DNAs required many cell generations after the fixation of foreign DNA in the host genome. The shift in methylation was nonrandom. The late parts of the inserted viral genomes became methylated more extensively than did the early gene segments.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.