Studies of base modification in deoxyribonucleic acid by mass spectrometry.

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Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Crain, Pamela Forbes.
Contributor Scott, Marshall F.; Cheng, Chin Shu Dr.
Title Studies of base modification in deoxyribonucleic acid by mass spectrometry.
Date 1983-06
Description Mass spectrometry has been applied to two types of problems relating to the occurrence of modified bases in deoxyribonucleic acid (DNA), in which small sample amounts have dictated the requirement for a highly sensitive analytical method. One problem concerns accurate measurement of levels of occurrence of known modified bases, and the other illustrates determination of the structure of an uncharacterized modified deoxyribonucleoside. A direct chemical assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography - mass spectrometry with selected ion monitoring. 5-Methylcytosine is determined relative to thymine using 5-({('2)H(,3)}methyl)cytosine and {methyl-('2)H(,3)}thymine. The method yields quantitative measurements down to 0.002 mol %, or approximately 1 5-methylcytosine in 50,000 bases, based on measurement of 500 nanograms of DNA. The method has been applied to analysis of DNAs of very low or uncharacterized 5-methylcytosine content. DNAs of Drosophila melanogaster, Aedes albopictus, Sulfolobus solfataricus, Methanococcus voltae, and two strains of yeast, were found not to contain 5-methylcytosine to an extent of greater than 1 in 50,000 bases. Halobacterium volcanii DNA was found to contain 0.24% 5-methylcytosine relative to thymine content. The origin of 5-methylcytosine in bacteriophage (PHI)X-174 DNA was also investigated. The new assay was used to analyze both single-stranded (virion) and double-stranded RF I forms of (PHI)X-174 DNA isolated from phage propagated in E. coli C lacking the DNA cytosine methylase (dcm('-)), and in wild-type E. coli C (dcm('+)). Both forms of phage DNA from E. coli C (dcm('-)) were found to contain 5-methylcytosine. High resolution mass spectrometry was used to establish the structure of a new modified deoxyribonucleoside from bacteriophage Mu DNA. The modification elaborated by the Mu mom gene converts deoxyadenosine to N-(9-(beta)-D-2'-deoxyribofuranosyl-purin-6-yl)glycinamide. This is the seventh known form of base modification in DNA, and the first characterization of a hypermodified purine.
Type Text
Publisher University of Utah
Subject DNA; Ion Recordings
Subject MESH Cytosine; Spectrum Analysis, Mass
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Studies of base modification in deoxyribonucleic acid by mass spectrometry." Spencer S. Eccles Health Sciences Library. Print version of "Studies of base modification in deoxyribonucleic acid by mass spectrometry." available at J. Willard Marriott Library Special Collection. QP 6.5 1983 C73.
Rights Management © Pamela Forbes Crain.
Format Medium application/pdf
Identifier us-etd2,55
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Funding/Fellowship NIH Grant GM 26982.
ARK ark:/87278/s6nk3vmt
Setname ir_etd
Date Created 2012-04-23
Date Modified 2021-05-06
ID 193263
Reference URL https://collections.lib.utah.edu/ark:/87278/s6nk3vmt
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