Specific Fragmentation of DNA of Adenovirus Serotypes 3, 5, 7, and 12, and Adeno-Simian Virus 40 Hybrid Virus Ad2+ND1 by Restriction Endonuclease R· EcoRI

The products of complete digestion of duplex DNA of each of seven human adenoviruses with restriction endonuclease R. EcoRI ranged from two fragments for adenovirus 7 DNA (Ad7) to six fragments for Ad12 and Ad2 DNA. Viral serotypes from the same subgroups appeared to have related cleavage sites; Ad3 DNA and Ad7 (cl E46-LL) DNA were each cleaved into three fragments, and Ad7 (cl 19) DNA lacked one of the cleavage sites present in Ad3 and Ad7 (cl E46-LL) DNA. One of the cleavage sites in Ad2 DNA was deleted in the DNA' of adeno-SV40 hybrid virus Ad2(+)ND1, and three of the cleavage sites in Ad2 DNA were missing in Ad5 DNA. Thus, Ad2(+)ND1 DNA was cleaved into five and Ad5 DNA into three fragments. Each fragment represented a unique segment of viral DNA since each fragment was obtained in equimolar amounts and since the sum of the molecular weights of the fragments equaled the molecular weight of the homologous intact adenovirus DNA.     

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease.

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.     

DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites. The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfI sites were not restricted. No rearrangements of the plasmids carrying SfiI sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA.     

Overabundance of rare-cutting restriction endonuclease sites in the human genome.

A human chromosome 3-specific cosmid library was constructed from a somatic cell hybrid containing human chromosome 3 as its only human component. This library was screened to identify 230 human recombinants which contained an average insert size of 37 kilobases. DNA prepared from 54 of these cosmids, representing 2000 kilobases of human DNA, was then tested for restriction endonuclease sites for EcoRI, HindIII, KpnI, XhoI, and DraI, as well as those of the rare-cutting restriction endonucleases NotI, SfiI, NruI, MluI, SacII, and BssHII. Sites for the latter enzymes were much more abundant than would be expected from theoretical calculations, reflecting non-random clustering of these sites. This has important implications for the use of these enzymes in the construction of physical maps of chromosomes. Some individual cosmids contained large numbers of rare sites, offering an alternative means of physically mapping chromosomes based upon identifying clusters of rare restriction sites. These clusters appear to be spaced an average of 1000 kb apart.     

Cloning, physical mapping and cross-hybridization of the canine adenovirus types 1 and 2 genomes

Cloned HindIII fragments of the type 1 and type 2 canine adenovirus (canAd) genomes were mapped with respect to the BamHI, EcoRI, HincII, PaeR7 and PstI restriction endonuclease cleavage sites. Considering the large differences found between the canAd-1 and canAd-2 DNA molecules, in terms of number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the canAd-1 and canAd-2 physical maps. Both viruses are genetically related to the extent of 57%. Our results confirm the existence of two distinct Ad species in the dog.     

Physical organization of subgroup B human adenovirus genomes.

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.     

Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rate of 1–2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.     

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites

Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.     

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.     

Specific properties of two enteric adenovirus 41 clones mapped within early region 1A.

Enteric adenovirus type 40 (Ad40) and Ad41 form the sixth subgenus of human adenoviruses. They are associated with infantile diarrhea but cannot be isolated in conventional cell cultures. The genome of the fastidious enteric Ad41 has been cloned, and the cleavage sites of the genome produced by restriction endonucleases BamHI, EcoRI, HpaI, NruI, PvuI, and SalI have been mapped. To develop useful hybridization methods for direct detection of adenoviruses, a restriction fragment library containing Ad41 DNA, with plasmid pBR322 as vector, has been constructed. Clones have been isolated which contain 8 of 10 possible BamHI fragments of Ad41, inserted into the BamHI cleavage site of the vector. Two of these clones are particularly useful for the detection of adenoviruses. One clone detects members of all human adenovirus subgenera, and the second clone is specific for enteric adenoviruses, in particular Ad41. A conspicuous absence of detectable homology was noted at 1.5 to 3.3 map units of the Ad41 genome in hybridizations against other serotypes of adenoviruses, including the closely related enteric Ad40. This sequence corresponds to the 5' portion of early region Ia.     

Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.     

Restriction maps of human adenovirus types 2, 5 and 3 for BclI, ClaI, pvul and SphI endonucleases

The sites of cleavage by BclI, ClaI, PvuI and SphI in the DNA from adenovirus (Ad) serotypes 2, 5 and 3 have been located. Certain site coordinates were in accord with nucleotide sequences already published. A small difference in size between the PvuI-E fragments from Ad2 and Ad5 confirmed the existence of a deletion in the N-terminal moiety of Ad5 hexon gene, as previously implied by interserotypic recombinants (Boursnell and Mautner, Virology 112 (1981) 198-209) and more recently by amino acid sequencing (Von Bahr-Lindstr?m et al., Virology 118 (1982) 352-362).     

Physical mapping of human adenovirus type 7 DNA using a simple and sensitive method of terminal labeling

The tyrosine-containing peptide covalently attached to each 5'-terminus of adenovirus type 7 (Ad 7; Greider) DNA was labeled with 125I. The 5'-labeled DNA was subjected to digestion with several restriction endonucleases and the size of the labeled terminal fragments was determined. Partial hydrolysis by these endonucleases generated a series of labeled fragments which were fused to the terminal fragments and could, therefore, be detected by autoradiography. From the sizes of the partial products the location of the cleavage sites of the enzymes on Ad7 DNA could be determined. The subgenomic DNA extracted from incomplete particles by protease treatment could also be labeled with 125I, since it was found to contain the tyrosine-containing peptide covalently attached to the preferentially packaged left end of the genome.     

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.     

Restriction endonuclease mapping of adenovirus 35, a type isolated from immunocompromised hosts.

The DNA of adenovirus 35 (Ad35), a type recently associated with infections in immunocompromised hosts, was mapped by the use of BamHI, SmaI, PstI, EcoRI, and HpaI restriction endonucleases. In addition to standard mapping procedures, we used the in vitro adenovirus DNA replication system with origins at both physical ends of the linear molecule to determine the terminal fragments. Deletions of single restriction endonuclease sites in a group of closely related adenovirus isolates from patients with acquired immunodeficiency syndrome helped determine the location of some DNA fragments on the genome.     

Homologous recombination of adenovirus DNA in mammalian cells: enhanced recombination following UV-irradiation of the virus.

We have used adenovirus as a molecular probe to examine the recombination of viral DNA following infection of mammalian cells. The technique gives a quantitative measure of homologous recombination between adenovirus type 2 (Ad2) and Ad5PyMTR3. Ad5PyMTR3 is an insertion mutant of Ad5 containing polyoma virus (Py) DNA inserted into a deleted E1 region of the Ad5 genome. Cells were coinfected with Ad2 and Ad5PyMTR3 and at an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. Although Ad2 and Ad5 have more than 99% DNA homology, they differ sufficiently in their restriction endonuclease patterns, such that recombinant viral DNA molecules containing the Py insert could be detected and quantified by Southern blotting and hybridization to a radioactive Py DNA probe. Using this method we are able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. Recombination was detected in Chinese hamster ovary cells, monkey kidney cells, human HeLa cells, normal human fibroblasts and SV40 transformed human fibroblasts. In experiments using HeLa cells, the frequency of recombination between the Py insert on Ad5PyMTR3 and a number of unique restriction enzyme sites on Ad2 increased with the distance from the Py insert to the restriction site. Also in HeLa cells, recombination increased with increasing amounts of viral DNA synthesis and with increasing UV dose to the virus. UV-irradiation of both coinfecting viruses with 1500 J/m2 resulted in a more than 100-fold reduction in the amount of viral DNA synthesized and about a 3-fold increase in the frequency of recombination.