Clonal selection of autonomously replicating sequences from human HL60 cells in vitro.
Item
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Title
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Clonal selection of autonomously replicating sequences from human HL60 cells in vitro.
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Identifier
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AAI9009745
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identifier
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9009745
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Creator
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Jenab, Shirzad.
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Contributor
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Adviser: Edward Johnson
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Date
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1989
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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Each eukaryotic chromosome consists of linear double stranded DNA molecules that are bound to many nuclear proteins to form complex aggregates that then can be fit into distinct chromosomes. Although the lengths may vary in different eukaryotes, a controlled and efficient replication of the genome occurs once in every cell division and is essential to the survival of the animals. To replicate all of their genomes during the 10 hour synthesis phase of the cell cycle, the cells are thought to unwind 10{dollar}\sp4{dollar} initiation sites and form bubbles where DNA synthesis starts. In spite of this number of replication origins, the mechanisms and sequences involved in initiation of eukaryotic replication are not known, perhaps due to the large size of the genome, the complexity of DNA with nuclear proteins and the unavailability of appropriate assays.;This is a project to replicate in vitro the SV40 origin-containing plasmids using cellular extracts prepared from permissive (monkey COS7, CV1) and semi-permissive (human HL60) cell lines. I have analyzed the modes and intermediates of replication by two dimensional gel electrophoresis and by electron microscopy. Furthermore, I have used the replication efficient HL60 extracts to select and characterize cloned human (HL60) DNA fragments which can initiate replication as was tested by the above system. These replicating clones have then been used for in vivo transfection studies. Availability of these plasmids will enhance studies of human DNA replicons and the factors controlling over the initiation processes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
