Degree Type

Dissertation

Date of Award

1995

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Robert W. Thornburg

Abstract

Plant uridine-5'-monophosphate (UMP) synthase has been studied at both the biochemical and molecular levels. Using a purification procedure of salt precipitation coupled with a series of chromatography techniques, UMP synthase was purified to more than 2,000-fold from crude extracts of Cucurbita pepo L. As with UMP synthases from other higher eukaryotes, the UMP synthase of squash has both orotate phosphoribosyl transferase and orotidine-5'-monophosphate (OMP) decarboxylase activities. The molecular mass of the native UMP synthase was about 110 kDa and SDS-PAGE showed a subunit molecular mass of 50 kDa. The other biochemical properties of this UMP synthase are very similar to those of previously studied UMP synthase from tomato and tobacco. Antiserum was prepared against the purified UMP synthase protein, and western blot analysis indicates that the UMP synthases from a wide variety of plants sources are immunologically similar;From N-nitroso-N-methylurea (NMU)-mutagenized tobacco cells, we have selected cell lines which are resistant to 5-fluoroorotic acid (FOA). We found at least 3 classes of FOA-resistant cell lines. UMP synthase-defective cells, in which, of the de novo biosynthetic enzymes, only the UMP synthase has low activity. A cell line of rpy1 has low activities in UMP synthase, low aspartate transcarbamoylase and dihydroorotase. A cell line of rpy2 has elevated levels of both UMP synthase and aspartate transcarbamoylase activities. Further characterization indicated that the rpy2 cell lines lost their sensitivity to thymine levels. Regardless of the level of cellular thymine, the UMP synthase gene was constitutively expressed at a high level;In Nicotiana plumbaginifolia callus, the expression of UMP synthase is metabolically regulated. In the presence of high levels of thymine, the expression of the UMP synthase gene was repressed. Whereas when this metabolite is limited, the gene is up-regulated. RNA analysis indicates that this regulation occurs at the transcription level. Further investigation of this regulation suggested that the metabolic regulation of the UMP synthase gene expression involves a regulatory protein. This protein may function to communicate the cellular level of thymine metabolite to the gene expression system.

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Djoko Santoso

Language

en

Proquest ID

AAI9540938

File Format

application/pdf

File Size

146 pages

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