Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity.

The DNA of human papillomavirus (HPV) obtained from a pool of plantar warts is cleaved by bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) restriction endonucleases at one and four specific sites, respectively. These sites were localized on the previously established cleavage map of HPV DNA, using the Hind, HindIII, HpaI, and EcoRI endonuclease restriction sites as reference. The four HpaII sites were mapped, clockwise, at 1.4, 41.1, 44.3, and 52.8% of the genome length from the unique BamI cleavage site taken as point zero. The HpaII site mapped at 1.4% of the genome length was absent in 40 to 50% of the molecules, thus showing a genetic heterogeneity of HPV DNA.     

Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis.

Five human papillomavirus (HPV) DNAs from lesions of an epidermodysplasia verruciformis patient were cloned in lambda L 47: DNA of HPV 5, which predominated in the carcinoma; DNA of a variant type of HPV 8, which was not detected in the carcinoma DNA by Southern blot hybridization but only by cloning; and DNAs of three papillomaviruses that were isolated from warts. Southern blot and liquid phase DNA-DNA hybridization under stringent conditions showed that the three viruses from warts were new types, which we named HPVs 19, 20, and 25. These viruses cross-hybridized between 3 and 29% among themselves and with HPVs 5 and 8. After physical mapping with several restriction enzymes, the colinear genomes were aligned with HPV 8 DNA to define early and late regions. HPVs 8, 19, and 25 shared homology in different parts of their genomes.     

Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis.

The DNAs of the human papillomaviruses (HPVs) associated with the benign lesions of two patients suffering from epidermodysplasia verruciformis (patients JD and JK) were analyzed by using 12 restriction endonucleases. None of the restriction endonucleases were one-cut enzymes for the HPV DNA obtained from patient JD, referred to as the prototypical HPV-5, whereas five of them were one-cut enzymes for the DNA of the major virus found in patient JK, referred to as HPV-9. The molecular cloning of the two fragments resulting from the cleavage of HPV-5 DNA by endonuclease HindIII and of the single fragment obtained after treatment of HPV-9 DNA with endonuclease BamHI was performed in Escherichia coli after the fragments were inserted in plasmid pBR322. A cleavage map of the two cloned genomes was constructed. Little sequence homology (4 to 5%) was detected between HPV-5 and HPV-9 DNAs by DNA-DNA hybridization experiments in liquid phase at saturation; this homology was reproducibly higher than that (2 to 3%) detected under the same conditions between these DNAs and HPV-1a DNA. In addition, blot hybridization experiments performed under stringent conditions showed no or little sequence homology between the DNAs of HPV-5 and HPV-9 and those of HPV prototypes of types 1, 2, 3, 4, and 7 associated with skin warts. These results confirm that HPV-5 and HPV-9 are two distinct HPV types.     

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.     

Analysis of the genome of type 7 simian adenovirus using restrictases.

The effect of the restricting endonucleases R.EcoRI, R.BamI and R.SalI on the genome of type 7 simian adenovirus (SA-7) has been studied. Since the DNA has only one site of R.EcoRI recognition the enzyme cleaves SA-7 DNA into two fragments with the molecular weights 12.0 and 10.0 . 10(6). The restrictase R.BamI cleaves the SA-7 DNA at six sites producing 7 fragments with the molecular weights 6.6, 5.9, 3.8, 2.7, 1.3, 0.7 and 0.6 . 10(6). R.SalI cleavage yields 6 fragments with the molecular weights 8.1, 5.5, 4.3, 2.45, 1.2 and 0.6 . 10(6). The R.BamI and R.SalI fragments are arranged in the orders E-A-D-F-C-G-B and A-B-D-F-E-C, respectively. The only R.EcoRI recognition site is localized in the C fragment produced by R.BamI and in the B fragment produced by R.SalI.     

Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively. These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12. The HPV-3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322. The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA. The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps. Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA. Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA. Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-1a, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-11a, respectively). One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.     

Restriction enzyme cleavage of ultraviolet-damaged DNA

SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

Anogenital warts contain several distinct species of human papillomavirus.

Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2. In 12 tissue samples lacking sequence homology to either HPV-1 or HPV-2 under stringent conditions, HPV-related sequences were detected when the hybridization was performed under less stringent conditions, indicating that an HPV distinct from both HPV-1 and HPV-2 is also associated with these lesions. This anogenital HPV also appeared to be distinct from the other characterized types of HPV. These data indicate that at least three HPVs are associated with anogenital wart disease.     

Location in bacteriophage lamdba DNA of cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H.

The sites in Escherichia coli bacteriophage lambda DNA cleaved by the site-specific endonuclease isolated from Bacillus amyloliquefaciens H (BamI) are found to be at 0.114, 0.466, 0.580, 0.713, and 0.861 lambda units. The sites were located by analysis of the products of digestion of lambda DNA and lambda-ara transducing phage DNA, and verified by double digestion with BamI and EcoRI.     

Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.     

Molecular cloning of viral DNA from human genital warts.

The DNA of human papilloma virus type 6 (HPV 6) has been cloned in Escherichia coli K-12 by using pBR322 as vector. The DNA was cloned at the BamHI and EcoRI cleavage sites. This DNA was mapped by employing further restriction endonucleases and by terminal labeling. No major differences were noted as compared to HPV 6 DNA originating directly from a genital wart. The existence of at least two DNA subtypes (HPV 6a and 6b) became apparent.     

Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA.

The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map.     

Effects of 2''-deoxy-2''-azidocytidine on polyoma virus DNA replication: evidence for rolling circle-type mechanism.

Rolling circle-type molecules were found in polyoma virus-infected cells after inhibition of DNA synthesis with 2'-deoxy-2'-azidocytidine. The circular DNA molecules were always relaxed and of polyoma length. Most of the attached tails were less than two times the length of the polyoma genome, but tails with a length of up to 4.75 times the genome were also found. After cleavage of the total pool of replicating molecules with either endo R.EcoRI or endo R.BamI, Y-shaped molecules with replicated portions of various lengths were generated from rolling circle-type molecules. Moreover, after cleavage, Y-shaped molecules with three unequal arms were found, which could be explained as derived from the tail in rolling circle-type molecules starting from the normal origin, i.e., 29% from the endo R.EcoRI cleavage site. Rolling circle-type molecules were also found during a normal, noninhibited infection cycle. In such cells, a relatively higher frequency of rolling circle-type molecules was observed late during infection. Compared with control cultures, cultures inhibited with 2'-deoxy-2'-azidocytidine showed a greater amount of rolling circle-type molecules relative to normal replicative intermediates. 2'-Deoxy-2'-azidocytidine has previously been shown to inhibit the initiation of new rounds of replication; thus, the result obtained here indicates that a rolling circle-type mechanism is independent of the reinitiation of DNA synthesis.     

Cleavage of type 2 adenovirus DNA by HaeIII endonuclease. I. Catalog of HaeIII fragments

Tye 2 adenovirus DNA was divided into 14 fragments by sequential use of BamI, HsuI, SmaI, anc EcoRI endonuclease. Each fragment was purified by gel electrophoresis and subsequently cleaved with HaeIII endonuclease. From the number of fragments produced, we could calculate the number of HaeIII cleavage sites: there are a total of 187 sites. HaeIII sites were not randomly distributed along the adenovirus chromosome. Most sites were clustered in the G + C-rich left half of the chromosome. The sum of the molecular weights of the HaeIII fragments is 22.4 . 10(6), within 2 % of the molecular weight of adenovirus DNA (22.9 . 10(6).     

Does the specific recognition of DNA by the restriction endonuclease EcoRI involve a linear diffusion step? Investigation of the processivity of the EcoRI endonuclease.

The time course of the EcoRI endonuclease catalysed cleavage of three substrates, two plasmid DNAs and one oligonucleotide, each with two EcoRI sites, was measured. The two plasmid DNAs with the EcoRI sites 318 and 96 base pairs apart are cut in a distributive fashion, while the oligonucleotide with the EcoRI sites 8 base pairs apart is cut in a partially processive manner. It is concluded that a linear diffusion of the EcoRI endonuclease on its substrate across long stretches of DNA is not likely to be operative during the recognition process. Microscopic dissociation-reassociation processes, however, increase the probability of the enzyme to attack further sites located in the immediate vicinity of a given site.     

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.     

Sequences spanning the EcoRI substrate site.

Substrate recognition by the EcoRI restriction endonuclease was investigated by analysis of the nucleotide sequences at the sites of enzymatic cleavage in various DNA molecules. 5'-end labeling and homochromatographic fingerprinting led to the determination of a 17-base-pair sequence spanning the EcoRI site of simian virus 40 DNA and a 15-base-pair sequence overlapping the EcoRI site of Col El plasmid DNA. Three other DNAs were similarly tested, although extended sequences were not determined in these cases. The EcoRI site was shown to be symmetric double-stranded equivalent of -N-G-A-A-T-T-C-N-.