This proposal aims to study aspects of human triosephosphate isomerase (TPI) gene expression. TPI functions in glycolysis and gluconeogenesis and is required for cell growth and maintenance. The effects of disease- associated and in vitro-generated frameshift and nonsense mutations on human TPI RNA metabolism have been assessed by RNA blotting, primer extension, S1 nuclease transcript mapping and, in some instances, the polymerase chain reaction. Results indicate that any mutation that causes translation to terminate prematurely 50 or more nucleotides upstream of the final intron results in a decreased level of TPI MRNA. This decreased level is about five-fold below normal, is characteristic of both nuclear and cytoplasmic cell fractions, is dependent upon the presence of introns, and is not due to an increased rate of cytoplasmic MRNA decay. Comparable techniques will be used to analyze: 1) the effect of nonsense codons in other genes in order to determine the generality of the association of the association of the premature termination of translation with aberrant nuclear RNA metabolism and 2) the process by which nonsense codons in the TPI gene mediate the reduction of TPI MRNA. Results could reveal a link between MRNA translation in the cytoplasm and nuclear RNA processing/export or a mechanism by which nonsense codons are recognized in the nucleus, either of which would be an important finding. Other studies will continue characterizing the TPI gene promoter. These studies will aim to 1) isolate and characterize the transcriptional activator that binds immediately upstream of the transcription start site, 2) characterize the cis-acting element(s) responsible for serum- induced transcription and 3) analyze the sequences that negatively regulate transcription. Finally, three disease-associated TPI gene lesions that are presumed to be uncharacterized mutations will be studied in order to understand better how changes in TPI gene structure affect TPI gene expression.
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