Date of Award

9-26-2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

David O. Lambeth

Abstract

Nucleoside diphosphate kinase (NDP kinase) is an enzyme which transfers the gamma phosphoryl group of a nucleoside triphosphate donor to a nucleoside diphosphate acceptor. NDP kinases have been implicated in a wide range of biological phenomena including tumor metastasis, differentiation, gene regulation, signal transduction, and mitochondrial energetics. Prior experimentation documented in the early biochemical literature has given strong evidence for the presence of a mitochondrial NDP kinase. In particular, pigeon liver mitochondria possess an unusually high level of NDP kinase activity confined to the matrix space. Despite extensive work on vertebrate NDP kinases in the last decade, no genes had been cloned as of 1996 that clearly coded for a mitochondrial NDP kinase. Mitochondrial matrix NDP kinase had been previously purified from pigeon liver in this laboratory. In addition, most of the cDNA sequence for a cytosolic isoenzyme counterpart had been also determined in this lab prior to this work. The purpose of this work was to determine the sequence for this mitochondrial matrix NDP kinase, and to study its genetic relationship to its cytosolic isoenzyme counterpart. The laboratory efforts in these studies have made use of PCR-based strategies to clone cDNA sequences. Long-distance PCR systems were used to amplify genomic genes. Computer-based local alignment tools were used with publicly available sequence databases to identify candidate human and mouse genes. Automated sequencing was used for gene sequencing. Tissue distribution studies were performed using quantitative RT-PCR. This research has resulted in a general understanding of the genetic relationship between the pigeon mitochondrial matrix and cytosolic NDP kinases. The deduced protein sequences share only 53% identity. The mitochondrial matrix protein is expressed primarily in liver and kidney, while the cytosolic protein is expressed in all tissues studied. The two genes share a common ancestral gene and have divergently evolved to carry out distinct functions. The matrix enzyme is likely involved with GTP/GDP metabolism, while the cytosolic enzyme carries out more diverse roles. Orthologous sequences were obtained for chicken, but no highly homologous human counterpart to the matrix enzyme was detected. In addition to this work, computer-based gene searching has resulted in the identification of three additional novel human NDP kinases with only modest homology to the currently known human NDP kinases.

Download

Share

COinS