Selective initiation of replication at origin sequences of the recombinant-DNA molecule of Physarum polycephalum using synchronous plasmodial extracts.
Item
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Title
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Selective initiation of replication at origin sequences of the recombinant-DNA molecule of Physarum polycephalum using synchronous plasmodial extracts.
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Identifier
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AAI9009725
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identifier
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9009725
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Creator
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Daniel, Dianne C.
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Contributor
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Adviser: Edward M. 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 | Biology, General
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Abstract
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A cell-free system using synchronous plasmodial extracts initiates DNA replication selectively on the 60 kb rDNA palindrome of Physarum polycephalum. Initiation occurs at two positions corresponding to in vivo origins of replication estimated by electron microscopy. The system also initiates selectively within a plasmid, pPHR21, containing one of these origins. In this plasmid bubbles expand bidirectionally and generate DpnI-resistant DNA. Whole cell extracts made at prophase or early S phase, times when the nucleolus is disorganized, are most active in pPHR21 replication. An early S-phase nuclear extract accumulates replication intermediates, which are visualized at a level three fold higher than with the whole cell extract. Using the nuclear extract, selective labeling of restriction fragments locates the initiation point in a 3.2 kb BstEII fragment. Centers of bubbles mapped by electron microscopy cluster at the upstream border of a series of 31 bp repeats 2.4 kb from the initiation point for ribosomal gene transcription. Two-dimensional gel electrophoretic fractionation and electron microscopic analysis of rDNA replication intermediates, both in vivo and in vitro, provide evidence for an unusual secondary structure in early replication bubbles. Deletion mutants have been constructed in the origin region to help identify sequence elements regulating the initiation event.
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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.
