Arrangement of sequences in the inverted terminal repetition of adenovirus 18 DNA.

Abstract

In contrast to the single-stranded circular molecules produced with denatured DNA from other adenoviruses, there was associated with nearly all circular molecules of adenovirus type 18 a visible, duplex projection.These projections had a mean contour length of 0.31 ± 0.12 ,sm, equivalent to approximately 3% of genome length. Individual (3)(4)(5). At present the DNA of only one other group of viruses, the adenovirus-associated viruses (AAV), has been found to contain a similar type of repetition (6). Because of these repetitious sequences, the ends of single-stranded DNA molecules can self-anneal to form circles closed by a duplex hydrogenbonded segment. Although the arrangement of sequences within the repetition should be apparent from the morphology of the closure region, in the case of adenovirus DNA this region apparently has been too short to visualize by electron microscopy. We have recently found, however, that preparations of denatured human adenovirus type 18 (Ad 18) DNA can yield up to 80% single-stranded circular molecules which contain a visible projection or "panhandle." In the present report we show that these projections are duplex structures and that they are produced by annealing of the inverted terminal repetition with corresponding complementary sequences at the opposite end of the strand. They thus represent the hydrogen-bonded segments closing circular molecules and consequently provide direct, visual evidence for the arrangement of sequences within the inverted terminal repetition of an adenovirus DNA. MATERIALS AND METHODSViruses and Viral DNA. Human adenovirus serotypes 18 and 31 were prototype strains and were obtained from Flow Laboratories (Rockville, Md.) and H. Shimojo, respectively. Both viruses had been plaque-purified and were free of AAV contamination. Stock pools were prepared by passage in primary human embryonic kidney cells (HEM Research, Inc., Rockville, Md.). Viruses were produced in suspension cultures of KB cells and purified as described (7). The extraction of intact adenovirus DNA from bands of CsCl-purified virus was carried out as before (8)

Genome structure of incomplete particles of adenovirus

“…In general, this inverted repetition is too short for the double-stranded region to be seen in the electron microscope. However, Garon et al (9) have reported single-stranded circles that contain visible projections in preparations of Adl8 DNA. When Ad2 or Ad3 DNA molecules extracted from incomplete particles are denatured and allowed to renature for a short time at a low concentration (<1 ,ug/ml), conditions favoring intramolecular reannealing, a significant proportion of the molecules appear as singlestrand circles with long double-stranded projections (panhandles).…”

Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis.

“…The function of the repetitious sequences is not definitely known, but their location and the possibility that they contain a terminal palindrome suggest a role in DNA synthesis (5,7). Furthermore, this repetition may relate to adenovirus dependence, since the adenovirus genome is the only other viral DNA known to possess a similar type of terminal redundancy (8)(9)(10).…”

“…[3H]Thymidine chases were performed by pelleting pulse-labeled, infected cells at 600 X g and resuspending cells in warmed growth medium supplemented with thymidine, 100 ,4g/ml, and 2'-deoxycytidine, 10 ,ug/ml. These conditions permit continued DNA synthesis without further incorporation of [3H]thymidine (13).…”

Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII

“…When complete and incomplete BAV3 DNA were denatured and then renatured, many single-stranded circular DNAs were seen by the electron microscope (16). However, in those figures, no double-stranded "panhandle" structures were detected; this is not the case in human adenovirus type 18 (6), which has the long inverted terminal repetitions (about 3% of the genome) in its DNA. These facts show that both complete and incomplete BAV3 DNA contained inverted terminal repetitions and that the length of repetition is rather short.…”