The long-term objective of this proposal is to elucidate the mechanisms regulating the tissue-specific, developmentally coordinated expression of pyruvate kinase (PK) isozyme genes. The hypothesis of this proposal is that many of these mechanisms will function in a broader sense to insure the selective expression of only particular subsets of all cellular genes. Complementary DNA (cDNA) libraries will be made to Poly A RNA isolated from rabbit liver, skeletal muscle, kidney and reticulocytes. cDNA clones for each of the 4 major PK isozymes mRNA's will be isolated by colony hybridization. These clones will be characterized by restriction enzyme mapping, DNA sequencing and cross-hybridization experiments. The steady-state level of each mRNA will be determined in these tissues as a function of development and the time frame of isozyme switching established using molecular hybridization methods. The nuclear-cytoplasmic distribution of the mRNA's will be quantitated. Potential differential processing of isozyme mRNA will be investigated by Northern hybirdization alalysis. To further define the role of gene structure in the regulation of isozyme gene switching, primary hepatocyte cell cultures will be established. Specific changes in the DNA methylation patterns, gene rearrangements and/or gene amplification will be examined using the cloned cDNA's as hybridization probes. The role of various factors (hormonal, cell-cell, other environmental) which may act to maintain a pattern of expression will be investigated. Human red blood cell PK deficiency represents a naturally occurring genetic variant to the normal patern of PK gene expression. PK mRNA in the reticulocytes of patients with this disease will be studied to determine where the defect exists and how the defect changes the normal processing, stability and translation of mRNA and what effect his alteration has on protein turnover.
