Regulatory aspects of the human gene for the glycolytic enzyme triosephosphate isomerase (TPI) will be examined by analyzing the effects of diseaseassociated and in vitrogenerated mutations on gene expression. Hereditary TPI deficiency is an autosomal recessive disorder that is characterized by chronic hemolytic anemia and neuromuscular dysfunction. Defective TPI alleles that do not produce detectable TPI enzyme activity or immunologically crossreacting material will be analyzed. Diseaseassociated mutations will be localized by DNA sequencing, and the effect of these mutations on TPI gene transcription, RNA metabolism, protein metabolism, and enzyme activity will be assessed. The regulatory mechanism of housekeeping gene transcription as exemplified by the TPI gene will be studied. Cis-acting DNA sequences that specify the efficiency and accuracy of TPI gene transcription will be localized by introducing specific deletion, linker scanning, point, and inversion mutatioma into the potative TPI promoter region of hybrid TPI promoterbacterial chloramphemicol acetyl transferase (CAT0 genes, and determining the quantitative and qualitative effects of these mutations on transient TPICAT gene expression in L and HeLa cells. TPICAT gene expression will be carefully measured by both CAT enzyme assays and TPI-CAT mRNA structural analyses. The cis-acting regulatory elements of a transcription unit that lies upstream of the TPI gene and is transcribed in a direction opposite to that of TPI gene transcripiton will be localized by characterizing the expression of hybrid upstream DNACAT genes in HeLa cells. These regulatory elements will be compared with those identified for the TPI gene to determine how trasncription of the upstream unit mechanistically relates to TPI gene transcription. To identify basepairs upstream unit TPI gene that function in serumstimulation of TPI gene transcription, the effect of specific promoter mutations on TPI promoterCAT gene expression will be quantitated in HeLa cells undergoing the seruminduced transition form a quiescent to a growing state. In complementary studies, the metabolism of TPI RNA in resting and proliferating HeLa cells will be analyzed to determine if posttranscriptional changes contribute to seruminduced TPI gene expression. To define sequences responsible for the differential expression of TPI and Beta-globin genes in non-erythroid cells, a concerted series of hybride 5' TPI/3' beta-globin and 5' beta- globin/3' TPI genes will be constructed and assayed for RNA synthesis in HeLa cells.
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