Enzymatic methods for sequence analysis applied to DNA restriction and methylation sites.
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Biomedical subjects
Publications and source records attributed to T J Kelly.
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The simian virus 40 (SV40) DNA segments present in a series of adenovirus-SV40 hybrids have been mapped with respect to the sites of cleavage of SV40 DNA by restriction endonucleases. Two approaches have been used. First, nucleic acid hybridizations were performed between equimolar quantities of the denatured DNAs of SV40 and each hybrid virus and the radiolabeled transcripts of 11 DNA fragments obtained by cleavage of SV40 DNA by restriction endonuclease from Hemophilus influenzae. Secondly, selected fragments of SV40 DNA produced by the H. influenzae or H. parainfluenzae restriction endonucleases were used to form heteroduplex DNA molecules with adenovirus and adenovirus-SV40 hybrid DNA, which were then analyzed by electron microscopy. The two sets of data were consistent and have permitted alignment of the map of the SV40 segments of the hybrid viruses with the H. influenzae and H. parainfluenzae cleavage maps of SV40. Since cells infected with some of the hybrid viruses contain one or more SV40-specific antigens, the genetic determinants of these antigens could be localized on the cleavage map.
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A series of viable recombinants between adenovirus 2 (Ad2) and simian virus 40 (SV40) (nondefective Ad2-SV40 hybrids) have been isolated. The members of this series (designated Ad2(+)ND(1) through Ad2(+)ND(5)) differ from one another in the early SV40-specific antigens and the SV40-specific RNA species which they induce in infected cells. They also contain different amounts of SV40 DNA as shown by RNA-DNA hybridization techniques. We have examined the structure of the DNA molecules from these hybrids, using electron microscope heteroduplex mapping techniques. Each hybrid was found to contain a single segment of SV40 DNA of characteristic size covalently inserted at a unique location in the adenovirus 2 DNA molecule. The SV40 segments of the various hybrids formed an overlapping series with a common end point. When the results of the electron microscopic study were combined with data on antigen induction, it was found that a self-consistent map could be constructed which related specific regions of the SV40 genome to the induction of specific antigens. The order of these early SV40 antigen inducing regions in the SV40 DNA segments contained in the nondefective hybrids is: U antigen, tumor specific transplantation antigen, and T antigen with the U antigen region being nearest the common end point.
The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2(+)ND(4), is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R(1) restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2(+)ND(4), called Ad2(+)ND(4del), was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R(1) endonuclease cleavage point.
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.
Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.
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Radiologic technologists are called upon to provide patient care as well as produce radiographs. In accordance with quality assurance programs in health care institutions, we are expected to know and use accepted standard methods of care. This article provides the reader with background on the causes and treatments of vasovagal syncope. Sections include signs and symptoms, anatomy and physiology and common methods of treatment.
CT following myelography is superior to myelography alone in the diagnosis of spine disease. When using water soluble contrast, post myelogram orders include keeping the patient's head elevated. Examination by computed tomography necessitates the patient's head be lowered. This study demonstrates that post myelographic CT does not produce a significant increase in headache.
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