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J A Rose

Publications and source records attributed to J A Rose.

At least 55 records · Page 3Linked to original sources
PubMed1973

Self-complementarity of terminal sequences within plus or minus strands of adenovirus-associated virus DNA.

At least 70% of plus or minus strands of adenovirus-associated virus DNA contain self-complementary sequences at or near their termini. Self-annealing of these sequences generates circular molecules that are closed by duplex, hydrogen-bonded segments. The self-annealed segments are sensitive to exonuclease III and have a thermal stability comparable to that of double-stranded DNA molecules. Length measurements of double-stranded adenovirus-associated virus DNA molecules show a bimodal distribution, with the larger component being 10% shorter than SV40 DNA. The presence of self-complementary terminal sequences in single-stranded molecules of viral DNA has been observed previously only with DNA from adenoviruses. It is thus especially notable that adenovirus-associated virus replication is unconditionally dependent on a helper adenovirus. A possible role for terminal self-complementary sequences in viral DNA replication is suggested.

Adenoviridae↗
PubMed1972

A unique form of terminal redundancy in adenovirus DNA molecules.

A unique form of terminal redundancy has been observed in DNA molecules extracted from several human adenovirus serotypes. Electron microscopic studies reveal that single-stranded circular molecules are formed when native DNA is denatured and then annealed. Temperatures approaching the T(m) of native DNA are required to convert circles to linear molecules, indicating a high degree of self-complementarity between terminal base sequences of DNA strands. Single-stranded circles are not generated if a limited number of nucleotides (2-4%) are removed from the 3' ends of native DNA by digestion with Escherichia coli exonuclease III before denaturation and annealing. The lenght of the redundant segment appears to differ among major serotypic groups, and a possible association between increased length of the redundant segment and increased oncogenic capability of virus serotype is suggested. Evidence for the configuration of the duplex closure region of circular molecules is also presented.

Adenoviridae↗
PubMed1972

Adenovirus-associated virus multiplication. VII. Helper requirement for viral deoxyribonucleic acid and ribonucleic acid synthesis.

The adsorption of adenovirus-associated virus (AAV) type 2 by KB cells and the subsequent penetration of the AAV genome to the cell nucleus was measured with and without helper adenovirus type 2 (Ad2). It was found that the helper virus did not enhance either process. On the other hand, a synthesis of AAV deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) was not detected by nucleic acid hybridization after KB cells were infected with AAV type 2 alone, whereas both AAV DNA and RNA synthesis were readily detected when cells were additionally infected with Ad2 or herpes simplex virus type 1, a partial helper of AAV replication. AAV RNA synthesis was initially observed 10 to 11 hr after simultaneous infection with Ad2, but the interval between AAV infection and AAV transcription could be reduced to 4 to 5 hr when cells were first infected with Ad2 for 10 hr. It was estimated that AAV DNA synthesis accounted for 3% of the total DNA in cells after a simultaneous infection with Ad2. These findings, together with the previous observation that adenovirus provides a helper function(s) after AAV uncoating, suggest that AAV are defective only subsequent to the uncoating process, and that helper viruses may provide a factor(s) needed for initiating synthesis of AAV DNA, RNA, or both.

Adenoviridae↗
PubMed1972

Adenovirus-associated virus multiplication. 8. Analysis of in vivo transcription induced by complete or partial helper viruses.

We have analyzed ribonucleic acid (RNA) synthesized in KB cells co-infected with adenovirus-associated virus (AAV) type 2 and either adenovirus type 2 (Ad2) or herpes simplex virus type 1 (HSV-1). With either type of helper virus, synthesis of AAV RNA was readily detected by deoxyribonucleic acid (DNA)-RNA hybridization. As is the case for AAV RNA synthesized with helper Ad2, the AAV RNA synthesized with HSV-1 as helper annealed only to the thymidine-rich (minus) AAV DNA strand. In addition, AAV RNA synthesized with either type of helper (i) contained similar nucleotide sequences as determined by hybridization inhibition tests and (ii) had a mean molecular weight of approximately 7.5 x 10(5) based on sedimentation in dimethylsulfoxide-sucrose gradients. These experiments suggest that the restricted helper function of HSV-1 is not due to abnormal transcription of the AAV genome. Since the mean molecular weight of AAV RNA is equivalent to 40 to 50% of the AAV genome, as few as two or three AAV RNA species may be transcribed in vivo. In contrast to adenovirus RNA, cleavage of AAV RNA after transcription was not observed.

Adenoviridae↗
PubMed1972

Adenovirus-associated virus multiplication. IX. Extent of transcription of the viral genome in vivo.

Nucleic acid hybridization procedures were used to measure the extent of transcription of adenovirus-associated virus (AAV) deoxyribonucleic acid (DNA) in KB cells in the presence of either adenovirus or herpes simplex virus as the helper. Annealing of AAV ribonucleic acid to AAV DNA was monitored by a hybridization inhibition assay on nitrocellulose filters or by hydroxyapatite chromatography. These experiments confirmed the previous observation that, in the presence of either type of helper virus, only one strand of AAV DNA (the thymidine-rich or "minus" strand) is transcribed in vivo. However, it was found that only 70 to 80% of this strand appears to be transcribed in vivo. Furthermore, studies with minus strands employing hydroxyapatite chromatography and nuclease S(1), which specifically degrades single-stranded DNA, indicated that up to 20% of the minus strand is self-complementary. It seems likely that these self-complementary sequences account for the bulk of that portion of the minus strand (20 to 30%) which is not transcribed in vivo.

Adenoviridae↗
PubMed1971

Simian virus 40 integration site in an adenovirus 7-simian virus 40 hybrid DNA molecule.

The E46(+) strain of Adenovirus 7 is a mixed-virus population containing defective Adenoviurs 7-SV40 hybrid particles and helper, nonhybrid Adenovirus 7 particles. We have applied electron microscopic mapping techniques to obtain a physical map of the genome of the hybrid particles present in E46(+)PL1, a substrain of E46(+) derived from a single two-hit plaque. DNA molecules extracted from purified E46(+)Pl1 virions were found to be linear duplexes, with a mean lenght of 10.9 mum. When these molecules were denatured and renatured, a unique heteroduplex was formed that presumably derived one of its strands from an Adenovirus 7-SV40 hybrid molecule and the other from a nonhybrid Adenovirus 7 molecule. This heteroduplex was double-stranded, except for a short region near one end where the two strands were not paired. On the basis of measurements of the lengths of the single-and double-stranded regions in the heteroduplex, the structure of the Adenovirus 7-SV40 hybrid genome can be reconstructed as follows: The hybrid genome contains 16% less Adenovirus 7 DNA than the nonhybrid Adenovirus 7 genome. This deletion consists of the segment of DNA that maps between 0.05 and 0.21 molecular lenghts in the nonhybrid Adenovirus 7 DNA molecule. The deleted DNA has been partially replaced by an amount of heterologous DNA equivalent to 75% of the complete SV40 genome. A model for the generation of the hybrid genome is presented.

Adenoviridae↗
PubMed1971

Propagation and purification of high-titer human cytomegalovirus.

High-titered yields of human cytomegalovirus (CMV), strain AD 169, were produced in WI-38 cells in large roller bottles. Maximum plaque titers were observed by the 4th day after infection at which time infectivity in the medium was 200 times greater than that associated with the cells. Virus released into the medium was recovered by sedimentation in a sucrose gradient in a continuous-flow centrifuge rotor. Maximal viral infectivity was found at a sucrose concentration of 42%, equivalent to a density of 1.18 g/cm(3). Deoxyribonucleic acid extracted from these preparations was about 80% viral and 20% cellular as judged by equilibrium centrifugation in cesium chloride density gradients.

Cell Line↗
PubMed1971

Structural proteins of adenovirus-associated viruses.

The structural proteins of adenovirus-associated virus (AAV) types 1, 2, and 3 were analyzed by acrylamide gel electrophoresis. In each case, one major protein (C) and two minor proteins (A and B) were identified. Component C had an estimated molecular weight of 62,000 daltons, and the molecular weights of components A and B were found to be 87,000 and 73,000 daltons, respectively. Coelectrophoresis of adenovirus and AAV proteins revealed an overlap only between the adenovirus fiber-penton component and the AAV C polypeptide. Among AAV serotypes, homologous components were electrophoretically identical, except that the C component of AAV-2 was of slightly lower molecular weight than the C components of AAV-1 and AAV-3. The relative incorporation of (14)C-arginine and (14)C-mixed amino acids into the three polypeptides of AAV-2 was similar, indicating an absence of an arginine-rich component. In addition, AAV-2 was found to have a substantially lower arginine content than helper adenoviruses.

Adenoviridae↗
PubMed1971

Adenovirus-associated virus multiplication. VI. Base compostion of the deoxyribonucleic acid strand species and strand-specific in vivo transcription.

The two complementary strand species of 5-bromodeoxyuridine-substituted, adenovirus-associated virus type 2 (AAV-2) deoxyribonucleic acid were preparatively separated in CsCl density gradients and further purified by sedimentation through 5 to 20% sucrose. The base composition of each strand species was determined, and it was found that the species banding at a greater density in CsCl (heavy strands) had an expected higher thymidine content (26.5%) than that 21.7%) of the less dense species (light strands). Furthermore, the base composition of in vivo-synthesized, AAV-specific ribonucleic acid was similar to that of light-strand deoxyribonucleic acid, and this ribonucleic acid apparently hybridized only with heavy strands. These observations indicate that the heavy-strand species alone serves as the transcriptional template in vivo. This study represents the first instance in which the base composition and specificity of in vivo transcription have been determined for each of the complementary strands of an animal virus deoxyribonucleic acid.

Adenoviridae↗
PubMed1970

Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands.

Single-stranded adenovirus-associated virus type 2 deoxyribonucleic acid (AAV-2 DNA) has been isolated from the virion after enzymatic pretreatment of the particles by heating at 53 C for 1 hr in 0.015 m NaCl plus 0.0015 m sodium citrate in the presence of 1% sodium dodecyl sulfate. Double-stranded AAV-2 DNA present as a marker is not denatured by this treatment. AAV-2 single-stranded DNA is composed of two complementary species which can be separated in neutral CsCl when 5-bromodeoxyuridine has been substituted for thymidine in the DNA. The present report is the first documented instance of the separation of complementary strands of an animal virus DNA.

Adenoviridae↗
PubMed1969

Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA.

Extracted adenovirus-associated virus DNA is known to be double-stranded, and, therefore, it has been assumed that these virus particles contain a double-stranded genome. Recent findings, however, have suggested that the DNA in virus particles is equivalent to only half the molecular weight of extracted molecules. A density analysis of DNA extracted from a mixture of virus particles containing either bromodeoxyuridine-substituted or unsubstituted DNA shows that virions contain single-stranded DNA which, when released, forms duplex structures. A similar circumstance is as yet unknown among other viruses.

Adenoviridae↗