PHYSICAL MAPPING OF A VIRAL GENOME BY SITE SPECIFIC ENHANCEMENT AND STUDIES OF THE EFFECT OF CIRCULAR STRUCTURE AND PERMUTATION ON THE RATE OF DNA RENATURATION.
Item
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Title
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PHYSICAL MAPPING OF A VIRAL GENOME BY SITE SPECIFIC ENHANCEMENT AND STUDIES OF THE EFFECT OF CIRCULAR STRUCTURE AND PERMUTATION ON THE RATE OF DNA RENATURATION.
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Identifier
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AAI8023714
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identifier
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8023714
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Creator
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KINBERG-CALHOUN, JUDY.
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Contributor
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James G. Wetmur
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Date
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1980
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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, Microbiology
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Abstract
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One of the studies reported here is aimed at producing fine structure physical genomic maps and takes advantage of the relative instability of mismatched base pairs in DNA. A separate set of studies examines the effect of excluded volume on the rate at which DNA strands renature. DNA molecules of varying shape or permutation are used to explore this phenomenon.;For feasibility studies of site specific enhancement mapping, heteroduplex DNA molecules were formed using T7 deletion mutant C63 and the wild type bacteriophage. These heteroduplexes contain a deletion loop of approximately 600 bases whose location is known. First, the heteroduplexes were incubated in 0.03 M chloroacetaldehyde in a solvent consisting of 2.4 M tetraethylammonium chloride (Et(,4)NCl), at pH 4.7. Chloroacetaldehyde reacts with and covalently modifies deoxycytosine and deoxyadenosine residues in DNA. These reactions occur much more readily with single stranded DNA than double stranded DNA. Chloroacetaldehyde will also react at or near defects in the DNA structure such as the ends, single strand breaks, and mismatch sites in DNA heteroduplexes. In 2.4 M Et(,4)NCl, DNA melting is independent of base composition. At pH 4.7 in this solvent, chloroacetaldehyde reacts with cytosine and adenosine residues at the same rate. Next, the modified substrates are incubated with endonuclease S1. This enzyme specifically cleaves single stranded regions of DNA. In a heteroduplex molecule of T7 containing a deletion loop of 600 bases, reaction with chloroacetaldehyde results in denaturation from the site of the deletion loop. Endonuclease S1 is better able to recognize and cleave the DNA strand opposite the deletion loop when the heteroduplex has been pretreated with chloroacetaldehyde than in the absence of such pretreatment.;Site specific enhancement mapping was attempted by forming heteroduplex DNA molecules using the DNA from an amber mutant of bacteriophage T7 and from its spontaneously occurring revertant. The conditions of chloroacetaldehyde or S1 incubation were varied in an attempt to find conditions in which heteroduplexes containing a single base mismatch could be cleaved by endonuclease S1. We were unable to find conditions which allowed detection of any such cleavage.;The possibility of an effect of excluded volume of DNA renaturation reates was tested by examining the rate at which circularly permuted linear molecules renature with one another and by comparing the rates at which a linear DNA molecule renatures with its linear complement and its circular complement. The results of trials in which molecules ranging from 9 to 42% out-of-phase were allowed to renature before being examined in the electron microscope are consistent with there being little or no detectable effect of circular permutation of DNA renaturation rates. When a single stranded linear DNA molecule was presented with two topologically distinct but otherwise identical complements, renaturation was approximately three times faster with the linear complement than with the circular one. This result is consistent with an effect of excluded volume on the rate of DNA renaturation.;Finally, an excluded volume theory has been derived which takes into account the probability of random overlap of segments of two DNA chains which are interacting to form a nucleation site. The experimental results agree qualitatively and semi-quantitatively with the excluded volume theory.
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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.
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Program
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Biomedical Sciences
