Date of Award

1980

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Thomas O. Sitz

Committee Member

Kenneth Somers

Committee Member

Frank E. Scully, Jr.

Committee Member

James H. Yuan

Call Number for Print

Special Collections LD4331.C45R9

Abstract

5.8S rRNA is a low molecular weight ribosomal RNA which is found hydrogen bonded to 28S rRNA in the eucaryotic cell. There are two nucleotides which have 2'-0-methylations; a guanine residue at position 77 is fully methylated and a uridine residue at position 14 which is partially methylated. This partial 2'-0-methylation of the uridine residue has been found to vary with the tissue source, with the highest level in normal tissue and the lowest level in neoplastic tissue. In order to study the significance of this site-specific methylation, a reliable and convenient method of analysis was needed.

Early studies on the level of methylation of the uridine residue at position 14 in 5.8S rRNA were done using a pancreatic RNase digest followed by fractionation using the two-dimensional Sanger map technique. This method produces reliable results but was expensive and time consuming. Four alternate methods of analysis were developed, each of which satisfy different requirements in the study of 2'-0-methylation in the 5.8S rRNA molecule. Each of these techniques were found to resolve the dinucleotides and inorganic phosphate resulting from a combined T2 RNase and alkaline phosphatase digest of 5.8S rRNA. Three analyses were accomplished using high voltage paper electrophoresis: one method resolved the dinucleotides on Whatman 3MM paper in pH 3.5 buffer, the second used DEAE paper in pH 3.5 buffer, and the third method resolved the dinucleotides on DEAE paper in seven percent formic acid buffer. The fourth method of analysis was two-dimensional thin layer chromatography. One dimensional electrophoresis on Whatman 3MM paper was found to be convenient, reliable, and allowed many sample fragments to be resolved simultaneously.

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DOI

10.25777/34yz-2f91

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