Cleavage maps of human adenovirus type 7 DNA by restriction endonuclease HindIII and EcoRI

Adenovirus type 7 DNA (Grider strain) was cleaved into ten and two specific fragments by the restriction endonuclease HindIII and EcoRI, respectively. These specific fragments were mapped on the adenovirus type 7 genome by analyzing partial digestion products and by the double-digestion experiment.     

Orthopoxvirus DNA: strain differentiation by electrophoresis of restriction endonuclease fragmented virion DNA.

Procedures were developed for purifying intact intracytoplasmic poxvirus particles from infected cells and for isolating DNA from virions by equilibrium centrifugation in sodium diatrizoate density gradients. The buoyant density of twelve closely related orthopoxviruses purified in these gradients was determined to be 1.25 g/ml, and that of the isolated virion DNAs was 1.1 g/ml. Virion DNA from each of the 12 selected prototype and wild-type viruses was cleaved with three separate site-specific restriction endonucleases, Hin d III, Sal I, and Bam HI, and the fragments (molecular weights 0.5 × 10⁶ to 20 × 10⁶) were separated by agarose gel electrophoresis. Characteristic DNA fragment migration patterns observed in the gels permitted classification of the viruses. By comparing profiles of Hin d III cleaved DNAs we were able to group the viruses into 4 species: cowpox, vaccinia, monkeypox (2 isolates), and variola (8 isolates). Viruses from variola major infection could be differentiated from viruses from variola minor infection. Isolates within species (strains) were also differentiated, mainly by comparing the gel electrophoresis profiles of Sal I digested DNA from the viruses.     

Adeno-associated virus DNA structure Restriction endonuclease maps and arrangement of terminal sequences

Linear duplex molecules of adeno-associated virus type 2 (AAV2)-DNA were cleaved with the restriction endonucleases R.HhaI, R.HaeIII, R.HpaII, and R.AluI. The resulting specific fragments were analyzed by electrophoresis in polyacrylamide-gel slabs and their physical order on the AAV genome map was determined. This work provides a map of approximately 75 additional cleavage sites on AAV-DNA. The inverted terminal repetitious sequence was estimated to be 80 to 120 nucleotides long. The results obtained, considered together with previous data, are inconsistent with the model of two permutations of AAV-DNA strands having different startpoints. Rather, it appears that the “permutation” in AAV-DNA results from an unusual secondary structure located within the inverted repetition, between 20 and 100 nucleotides from the terminus.     

A comparison of the DNA of the "R" and "T" strains of mosquito iridescent virus.

The buoyant density (1.7135 g/cm³), percentage GC (53.9%), and melting temperature (T_m = 76.4°) of the DNA of “regular” mosquito iridescent virus (RMIV) and of “turquoise” mosquito iridescent virus (TMIV) are similar although the molecular weights of the two DNAs are different; 243.3 × 10⁶ and 286.7 × 10⁶, respectively. Analyses show that RMIV contains two identical duplex DNA molecules which are about 15% smaller than the single duplex DNA molecule of TMIV. Reassociation studies show that RMIV and TMIV contain about 17 and 30% repetitious DNA, respectively. Homology studies show the two DNAs to be 100% homologous in their DNA nucleotide sequences. We conclude that the DNA of RMIV and TMIV contain identical sequences and that the portion of the TMIV-DNA molecule accounting for the higher molecular weight is composed of repeated sequences common to both strains.     

Establishment and characterization of rat cell lines transformed by restriction endonuclease fragments of adenovirus 12 DNA

Rat cells (3Y1) were transformed by the HindIII-G fragment of adenovirus 12 DNA and cloned in solft agar cultures, and transformed cell lines (GY1, GY2, and GY3 cells) were established. Part of the HindIII-G fragment of adenovirus 12 DNA was the only viral DNA sequence present in GY1 cells. The characteristics of CY1 (a rat cell line transformed by the EcoRI-C fragment of adenovirus 12 DNA), GY1, and WY3 cells (a rat cell line transformed by the whole viral DNA of adenovirus 12) were examined. The growth of each transformed cell line was unlimited as compared to that of untransformed 3Y1 cells. Complement fixation and immunofluorescence suggested that CY1 and GY1 cells contained T antigen, while WY3 cells contained both T antigen and DNA-binding protein. CY1, GY1, and WY3 cells induced tumors in rats after transplantation. In the serum of rats bearing CY1 or GY1 cell tumors only antibody to T antigen was detectable, while antibodies to both T antigen and virus-induced DNA-binding protein were present in the serum of rats bearing WY3 cell tumors. These results show that the transforming gene is located on the left end (7.2%) of adenovirus 12 DNA and suggest that T antigen is among the gene products coded for by this region of the genome.     

DNA packaging in the lambdoid phages: the role of lambda genes Nu1 and A

Of all the morphogenic genes of the coliphage λ, only Nu1 and A are both necessary and sufficient for terminase activity in vitro. Previously we have reported that extracts of Nu1^?- and Aam^?-infected cells will not complement each other to promote packaging in an in vitro assay (A. Becker, H. Murialdo, and M. Gold, Virology, 78, 277–290, 1977). We report here that such complementation is possible under certain conditions; furthermore, complementation appears to proceed in a noncatalytic fashion. The product of gene Nu1 (gpNu1) therefore does not appear to be involved in the synthesis of the product of gene A (gpA). We discuss the hypothesis that λ terminase is a heterodimer consisting of gpNu1 and gpA subunits, and show that neither of these subunits alone is able to promote either the DNA-cutting or prohead-filling events of packaging.     

A comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus

Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group. Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and. bovine RS viruses have similar polypeptide profiles. However, PVM does not resemble the other two viruses closely. The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide. Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain. The mobilities of the other viral polypeptides showed little variation. The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin.     

Hydroxylamine mutagenesis of HSV DNA and DNA fragments: introduction of mutations into selected regions of the viral genome.

Procedures for the induction and isolation of temperature-sensitive (ts) mutants by in vitro mutagenesis of purified HSV DNA and DNA fragments have been developed. In order to establish conditions for the mutagenesis of viral DNA fragments with hydroxylamine (HA), virion-associated and intact DNA were mutagenized first. Under the conditions of mutagenesis employed, HA was shown to be a more powerful mutagen of purified DNA than of virion-associated DNA. Mutagenesis of intact DNA resulted in the identification of three new complementation groups. A mixture of EcoRI fragments A and B was next mutagenized. Mutations in DNA fragments were recovered by cotransfection with intact, wild-type DNA. Six ts mutants were isolated from the progeny of the mixed infection. Five of these mutants were analyzed further. One mutant, ts508, failed to complement tsB2 which had previously been shown to lie in fragment B. Like tsB2 and other members of this group, ts508 is DNA^?. The remaining four HA-induced mutants constitute three new DNA⁺ complementation groups. Marker rescue experiments indicate that mutants in two of these groups lie in fragment A.     

T4 DNA injection: II. Protection of entering DNA from host exonuclease V

Gene 2 amber mutants of bacteriophage T4 produce intact particles in nonpermissive (su^?) cells. The particles are noninfective on Escherichia coli B since the DNA of 2.Su^? particles is functionally excluded and rapidly degraded to acid-soluble fragments early after infection. Cells which were previously infected with immunity minus (imm^?) phage or cells lacking exonuclease V, neither degrade nor exclude 2.Su^? DNA. Exo V^? mutants of E. coli are fully permissive for gene 2 defective particles. Therefore gene 2 apparently protects the parental T4 genome against degradation by exonuclease V during and after penetration in E. coli. We postulate an additional role for gene 2 protein as a connector between DNA termini and the apical vertices of the head which aids in morphogenesis and possibly in ejection of DNA from the virion.     

Cloning of DNA fragments of the right end of phage mu and location of the HindIII, SalI, PstI, and BamHI restriction sites on the genetic map of mu

The construction of three new plasmids containing the right, variable end of phage Mu DNA is described. They complete a collection of pKN plasmids derived by cloning specific restriction fragments of Mu DNA into plasmid cloning vehicles. The collection contains different length Mu DNA segments which encompass the entire phage genome. The Mu-specific genetic information of each cloned fragment was determined by marker rescue analysis of plasmid-containing strains with Mu amber mutant phages characterized by O'Day et al.(K. O'Day, D. W. Schultz, W. Ericsen, L. Rawluk, and M. M. Howe, 1979, Virology93, 320–328). This analysis demonstrated the location of the HindIII, SalI, PstI, and BamHI restriction sites on the genetic map of Mu and located the new essential genes V, W, and Y on specific restriction fragments of the physical map of Mu DNA. This plasmid bank has thus allowed a further refinement in the correlation of the genetic and physical maps of Mu DNA and should provide a useful source of specific DNA segments for further genetic and biochemical analysis of Mu functions.     

Analysis by restriction enzyme cleavage of human varicella-zoster virus DNAs.

The DNAs from three varicella virus isolates and two herpes zoster virus isolates were digested with either Eco R_I or Hin D III site-specific endonucleases. The restriction patterns of the DNAs obtained from the five isolates were then compared following separation of the specific fragments by electrophoresis through 0.5% agarose gels. The number and mobilities of all DNA bands were found to be indistinguishable for all five isolates. Further analysis of Eco R_I-digested varicella-zoster virus DNA revealed the presence of both molar and submolar DNA fragments as found previously for other human herpesvirus DNAs.     

Replication of adenovirus type 5 DNA in KB cells: Localization and fate of parental DNA during replication

The localization and fate of parental adenovirus type 5 (Ad5) DNA in KB cells at the time viral DNA replication occurs has been investigated by electron-microscope autoradiography and biochemical methods. At 18 hr after infection the majority of the parental Ad5 DNA molecules is present in the nucleus. About 60% of these molecules are involved in viral DNA replication by this time, but about 40% have not yet replicated at all. Electron-microscope autoradiography of cells infected with ³H-labeled Ad5 reveals that replicating parental Ad5 DNA molecules are randomly distributed throughout the nucleus, while nonreplicating viral DNA has a preference to locate in the periphery of the nucleus. Based on the experiments reported in this communication, the intranuclear fate of parental Ad5 DNA is discussed.     

Mouse adenovirus: growth of plaque-purified FL virus in cell lines and characterization of viral DNA

The FL strain of mouse adenovirus (AdFL) was plaque-purified and grown in mouse 3T6 cells. Purified virions resemble human adenoviruses by electron microscopy, and viral DNA shares physical properties with human adenovirus DNA. AdFL-DNA is a linear duplex with a molecular weight of 20 × 10⁶, shows evidence of protein covalently linked at each end, and forms single-strand circles after denaturation, indicating inverted repeat sequences at or near the ends of the molecule. However, less than 10% nucleotide sequence homology was found between AdFL-DNA and the DNAs of Ad2, 7, or 12. Also, the C + G content of AdFL-DNA (44%) is lower than that of human adenovirus DNAs. While there was reactivity between extracts of AdFL-infected cells and antiserum against human adenovirus, sera against the T antigens of human adenoviruses of groups A, B, and C did not react with AdFL-infected cell lysates. Six restriction endonucleases were used to cleave AdFL-DNA and to construct cleavage maps of the DNA. These maps differed from those of Ad2 or Ad5.     

The isolation of restriction fragments containing the primary and secondary (galT) bacterial att sites of phage lambda.

Transducing derivatives of bacteriophage λ have been used to isolate small DNA restriction fragments which contain the primary bacterial att site (BOB′) for phage insertion and the secondary bacterial att site (ΔOΔ′) at galT. Four hybrid att sites, BOP′, POB, ΔOP′, and POΔ′, have also been isolated in small restriction fragments. Acrylamide-agarose or agarose gel electrophoresis profiles of two analogous isogenic sets of transducing phage were compared with profiles of wild-type λ to identify the att-containing fragments. Each set consisted of phages transducing bacterial DNA originating to the right (e.g., POB′ or POΔ′) or left (e.g., BOP′ or ΔOP′) of the prophage and a recombinant phage, transducing all of the bacterial DNA of both the rightward and leftward tranaducing lines (e.g., a regenerated BOB′ or ΔOΔ′). The att-containing fragment of each phage was identified by virtue of the fact that it was the only fragment which was unique to the gel profile of that phage. For the mutants transducing the primary bacterial att site, the restriction enzymes HindII + III and MboII were used sequentially to obtain fragments containing BOB′, POB′, and BOP′ of approximately 560, 580, and 340 base pairs in length, respectively. For the mutants transducing the secondary att site at galT, restriction enzymes HindIII, BamHI, EcoRI, and Hinfl were used to identify fragments containing ΔOΔ′, POΔ′, and ΔOP′ of approximately 755, 475, and 580 base pairs in length, respectively. These fragments can be isolated in sufficient quantity for biochemical studies on att site interactions and in sufficient purity for sequence analysis.     

Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.     

DNA packaging in the lambdoid phages: identification of the products of phi 80 genes 1 and 2

We have identified the products of genes 1 and 2 of the lambdoid coliphage φ80. These genes code for the subunits of the terminase enzyme that promotes DNA packaging during phage particle assembly. These data serve by analogy to identify the subunits of λ terminase as the products of Nu1 and A, respectively. Identification of the gene 2 product was aided by the isolation of a mutant which causes the overproduction of that polypeptide. Mutants which affect the mobility of the product of gene 1 on 12.5% SDS-polyacrylamide gels were isolated as pseudorevertants of two amber mutations in the gene. Certain of these gene 1 pseudorevertants affect the mobility of the product of gene 2. The hypothesis that these pseudorevertants arise by recombination with a cryptic prophage carried by the host is proposed. The possibility that genes 1 and 2 overlap out of phase for a portion of their coding sequences is discussed. Surprisingly, wild-type φ80 produces approximately 280-fold more of the gene 1 than gene 2 product.     

Replication of herpesvirus DNA. II. Sedimentation characteristics of newly synthesized DNA.

The sedimentation behavior of viral DNA synthesized during short labeling periods at various times after infection was investigated. These experiments indicated that viral DNA synthesis may be divided into the following two phases: (1) During the early phase, newly synthesized DNA is associated with structures which sediment with S values up to approximately twice that of mature viral DNA; (2) during the later phase, newly synthesized DNA is associated with structures that sediment much more rapidly. Both at early and later times after infection, approximately 20% of the newly synthesized DNA sediments in sucrose gradients more slowly than does mature viral DNA. Furthermore, after isopycnic centrifugation in CsCl, most of the newly synthesized viral DNA sediments in sucrose gradients more slowly than does mature viral DNA. The smaller than unit-size, newly synthesized viral DNA molecules are breakage products resulting from the fragility of newly synthesized DNA. These molecules fragment, not only because of their fragility in the regions of the replicative forks but also because of the presence of fragile sites at other positions along the newly synthesized DNA molecule. Experiments dealing with the transfer of parental DNA to progeny virions show that most parental viral DNA strands that are transferred to progeny virions retain their integrity. Breakage and reunion of parental viral DNA strands with progeny DNA is a relatively rare event.