... the Vicinity of an. Intracisternal A Particle Genome in Syrian Hamster Tumor Cells ...... complex in baby hamster kidney cells infected with adenovirus type 12.
JOURNAL OF VIROLOGY, Sept. 1987, p. 2719-2726 0022-538XI871092719-08$02.00/0 Copyright C) 1987, American Society for Microbiology
Vol. 61, No. 9
Insertion of Adenovirus Type 12 DNA in the Vicinity of an Intracisternal A Particle Genome in Syrian Hamster Tumor Cells URSULA LICHTENBERG, CHRISTIANE ZOCK, AND WALTER DOERFLER*
Institute of Genetics, University of Cologne, 5000 Cologne, Federal Republic of Germany Received 17 February 1987/Accepted 19 May 1987
In the adenovirus type 12 (Ad12)-induced hamster tumor T1111(2) about 10 Ad12 genome equivalents were integrated at different sites. One of the integrated copies proved unstable and was lost from the cellular genome or rearranged upon passage of the cell line, H1111(2), established from this tumor. This unstable site of junction between the left terminus of Adl2 DNA and hamster DNA and the preinsertion site from BHK21 hamster cells was cloned, sequenced, and analyzed. The junction site showed several peculiarities. At the left terminus of Adl2 DNA, the first 64 nucleotides were deleted. At a distance of 127 nucleotides to the left from this junction site, an internal dispersed fragment of Adl2 DNA comprising nucleotides 1290 to 1361 of the authentic Adl2 DNA sequence was inserted into cellular DNA in an inverted orientation relative to the complete Adl2 genome that was located in its vicinity. The 127-nucleotide sequence between the intact Adl2 genome and the separate 72-base-pair (bp) Adl2 DNA fragment was cellular, but it was not identical to the preinsertion sequence at this location. The sequences flanking the termini of the dispersed 72-bp Adl2 DNA fragment were characterized by direct repeats of 9 or 10 nucleotides. To the left of Adl2 nucleotide 1361 in the separate 72-bp fragment, about 620 cellular nucleotides followed which were identical at the occupied and at the preinsertion sites. It was conceivable that the separate 72-bp Adl2 DNA fragment and the cellular sequence of 127 bp to its right had been transposed en bloc from another unknown location. Abutting the 620 nucleotides of cellular DNA to the left of this block, the 3'-terminal sequence of an endogenous, intracisternal A particle (IAP) genome of hamster cells was detected. The possible significance of the proximity of an IAP sequence to an inserted Adl2 genome with respect to the transformation event, to the instability at this site, or to the transcriptional activity of this region is not known. The 620 bp of cellular DNA between the 72-bp Adl2 DNA fragment and the end of the long terminal repeat of the hamster IAP sequence was apparently of a unique type. Transcriptional activity was not found in the approximate region between nucleotides -620 (to the left) and +350 (to the right) relative to the site of Adl2 DNA insertion, but was found outside these boundaries.
In previous work, nine different junction sequences between integrated adenovirus type 12 (Adl2) DNA or adenovirus type 2 (Ad2) DNA and cellular DNAs were analyzed in detail (7, 8, 11, 12, 15, 16, 42, 44, 45, 48). Comparison of sequence data for these sites did not reveal a common nucleotide sequence at which insertion had occurred preferentially. At several sites, patch homologies between viral and cellular sequences or peculiar secondary structulres were observed (8, 16, 44, 48). In a few instances (48; this report), the cellular nucleotide sequence analysis was extended far enough to detect host DNA sequences of functional significance. At a distance of 490 nucleotides from the inserted Adl2 genome in the Adl2-induced hamster tumor cell line CLAC1 (48), the nucleotide sequence of a 4.5S cellular RNA (19, 20) was detected (45). We have discovered that the preinsertional cellular DNA sequences at or close to the sites of adenoviral DNA insertion were transcriptionally active (17, 45; this report). This finding prompted the hypothesis that transcriptional activity at cellular sites constituted one of the preconditions for the insertion of foreign DNA (17, 45). In the present study, one of the junction sites between the left terminus of Adl2 DNA and hamster cell DNA isolated from the Adl2-induced tumor T1111(2) (27, 29) and the cellular preinsertion site were cloned, sequenced, and analyzed. *
MATERIALS AND METHODS
Origin of the T1111(2) tumor and of cell line H1111(2). The origin of the Ad12-induced hamster tumor T1111(2) and some of its characteristics were described elsewhere (27, 29). The cell line established from this tumor, H1111(2) (27), and BHK21(B3) hamster cells (51) were propagated in Dulbecco modified Eagle medium (3) supplemented with 10% fetal calf serum. Propagation of Adl2 on human KB cells and isolation of Adl2 DNA. Techniques for propagation and isolation were described earlier (9, 13). Genomic cloning and subcloning of DNA fragments. T1111(2) DNA was cleaved with EcoRI, and one of the off-size DNA fragmnents (arrow in Fig. 1) was selected by velocity sedimentation on a sucrose density gradient (8). The selected DNA fragments were ligated with the terminal fragments (K arms) of XgtWES AKB DNA (56). The ligation product was packaged in vitro (21). The K plaques were screened for the presence of sequences from the left Adl2 DNA terminus by the plaque transfer technique (4) and by hybridization to the 32P-labeled cloned EcoRI C fragment of Adl2 DNA (gift of A. van der Eb, Leiden). The preinsertion site was cloned in a similar way. The clone containing the preinsertion site was selected by using a 32P-labeled subfragment (pl, Fig. 2b) from the junction clone. Subfragments of the junction or preinsertion fragment were cloned into the
Corresponding author. 2719
2720
LICHTENBERG ET AL.
J. VIROL.
C41-
kbp
A 11.8
B a7-C 5A
D 4.0
EcoRI|
C
=
-
E
241----
F
0.61----
I'D I
B
I
E F,
A
FIG. 1. Patterns of integration of Adl2 DNA in the DNA of the Adl2-induced hamster tumor T1111(2) and in the hamster tumor line H1111(2) which was derived from this tumor. The origin of the tumor and the tumor cell line as well as the Adl2 integration patterns were published earlier (27). The scheme presents the distribution of
Adl2 DNA sequetlces as seen in an autoradiogram of EcoRl-cut DNAs which were electrophoresed, blotted, and hybridized to 32P-labeled Adl2 DNA. The DNA from the tumor cell line H1111(2) was analyzed at various passages (3 and 13) in culture. Adl2 DNA was mixed with BHK21 DNA as carrier and cleaved with EcoRI. The fragments generated were coelectrophoresed as size markers. Several of the off-size bands, including that of 7.25 kbp in tumor DNA, proved unstable in the tumor cell line. The 7.25-kbp band (arrow) was derived from the left end of Adl2 DNA, as it hybridized to the left-terminal EcoRI C fragment of Adl2 DNA (27; data not shown). This fragment was cloned and further investigated in this study. The scheme also presents the EcoRI map of Adl2 DNA and the fragment sizes.
polylinker site of M13mp8 or M13mp9 DNA (35) and in some experiments into pBR322 DNA. Determinations of nucleotide sequences. The method of
Sanger et al. (41) was used, with ox-35S-labeled deoxynucleotide triphosphates (dNTPs). In some of the experiments, DNA fragments were 3'-end labeled by using [ot32P]dNTPs and Klenow polymerase (23) or 5'-end labeled by using [y-32P]ATP and polynucleotide kinase (39). The nucleotide sequence was then determined by the method of Maxam and Gilbert (33, 34) with 0.2-mm-thick polyacrylamide-urea gels (18, 34). Isolation and analyses of cellular RNAs. Cytoplasmic RNAs from BHK21 cells or from the Adl2-transformed hamster cell lines T637 and HA12/7 (49, 54) were isolated and analyzed by standard methods (1, 2, 43, 46). Computer analyses. Nucleotide sequences were analyzed by the computer programs of the University of Wisconsin Genetics Computer Group (WordSearch, BestFit, and Map). All analyses were performed on a VAX750/11 computer
(Digital Equipment Corp.). RESULTS
Cloning and restriction mapping of the site of junction between Adl2 DNA and hamster DNA in the hamster tumor
T1111(2) and of the preinsertion site from BHK21 cells. The integration patterns of Adl2 DNA in the Adl2-induced hamster tumor T1111(2) and in passages 3 and 13 of the cell line H1111(2) established from this tumor (27) are shown schematically in Fig. 1. Two off-size bands (7.25 and 5.2 kilobase pairs [kbp]) corresponded to the left terminai Adl2 DNA linked to cellular DNA and proved unstable in cell line H1111(2) (Fig. 1). Since the loss or rearrangements of integrated Adl2 genomes from the cellular genome did not occur infrequently at early passages after tumor induction (27), the anatomy of one of the unstable sites was investigated. The 7.25-kbp fragment from T1111(2) DNA (arrow in Fig. 1) was cloned into XgtWES KB DNA (clone X17) and subcloned into pBR322 DNA (clone p17). The results of analyses on these clones by Southern blot hybridization are shown in Fig. 2a. The excised 895-bp fragment from subclone pl (Fig. 2b) served as the hybridization probe. This fragment hybridized to the junction site (arrow in Fig. 2a) in DNA from the tumor T1111(2) and also to the unoccupied site in the same tumor DNA or in BHK21 DNA, an EcoRI fragment of 3.1 kbp (arrow in Fig. 2a). This preinsertion fragment was also cloned from BHK21 DNA by using the excised and gel-purified insert from clone pl as the hybridization probe and )gtWES XAB DNA as the vector (clone X16). The fragment was also subcloned into pBR322 DNA (clone pl6) (Fig. 2a). The restriction maps of the clones described above were determined (Fig. 2b). The maps also included the nucleotide sequencing strategies for these segments (horizontal arrows in Fig. 2b). The data presented in Fig. 2a and b demonstrated that the cellular DNA sequences at the occupied and unoccupied sites were colinear, except for the cellular sequence between the intact Adl2 DNA molecule and the inverted, additional fragment of Adl2 DNA (see below). Colinearity of integrated Adl2 DNA with virion DNA and conservation of cellular DNA sequences at the junction site. The results of previous analyses (Fig. 1) demonstrated that the entire integrated Adl2 DNA molecule was colinear with virion DNA (27), except for the terminal fragments, which were linked to cellular DNA. Colinearity between the inserted and virion Adl2 DNA was assessed, at least for the left-terminal EcoRI C fragment of Adl2 DNA. Adl2 DNA or the junction clone (X17) DNA was cut with EcoRI and then with HhalI or MspI. DNA fragments were separated by electrophoresis on a 1.5% agarose gel, blotted, and hybridized to the 32P-labeled EcoRI C fragment of Adl2 DNA. The results (not shown) demonstrated that all fragments comigrated except for the Adl2 junction fragments (Fig. 2b). Thus, the integrated viral DNA was colinear with virion Adl2 DNA. Similar analyses (data not shown) ascertained that the cellular DNA sequences in the junction and preinsertion clones were colinear except at the site of integration of the inverted additional Adl2 DNA fragment (Fig. 2b). Determination of the nucleotide sequences at the site of Adl2 DNA integration and at the corresponding unoccupied site. At the junction site of Adl2 DNA with cellular DNA in tumor T1111(2) DNA, a total nucleotide sequence of 899 bp, and in the corresponding preinsertion sequence in hamster BHK21 DNA, a sequence of 1,311 bp were determined (Fig. 3a). In addition, a segment of 198 bp (Fig. 2b, p16 subclone) in the intracisternal A particle (lAP) sequence, to the left of the long terminal repeat (LTR), was determined (bp 7237 to 7435; see Fig. 2 in reference 38). This sequence is not shown here. The nucleotide sequences in Fig. 3a were arranged so that the bottom row represented the sequence in the tumor
VOL. 61, 1987
