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The goal of this work was to determine how polyunsaturated fatty acids (PUFAs) affect gene expression at the molecular level. Glucose-6-phosphate dehydrogenase (G6PD) is a member of the family of lipogenic enzymes known to be inhibited by dietary PUFAs. Addition of PUFAs to diet causes a 70-80% decrease in G6PD enzyme activity and mRNA accumulation in liver with no change in transcriptional activity of the gene, indicating that regulation occurs at a posttranscriptional step. Similarly, regulation of G6PD by starvation and refeeding results in a 12- to 15-fold increase in G6PD activity and mRNA accumulation but no change in transcriptional activity of the gene. Lack of transcriptional activity in response to positive and negative effectors is consistent with lack of change in chromatin structure as detected by nuclease hypersensitivity (HS) analysis. Two HS were detected in the 5{dollar}\\sp\\prime{dollar}-flanking DNA at {dollar}-{dollar}1000 bp and {dollar}-{dollar}400 bp relative to the transcriptional start site. Two additional HS were detected in the first and second introns at positions +600 bp and +1500 bp, respectively. The occurrence of these sites did not change with respect to dietary state. In contrast, only sites at {dollar}-{dollar}1000 and {dollar}-{dollar}400 bp were detected in nonhepatic tissues. These HS are most probably involved in basal transcription or developmentally-regulated transcriptional activity of the gene. I next determined the posttranscriptional step at which accumulation of G6PD mRNA was being regulated. Ribonuclease protection assays were used to measure G6PD mRNA accumulation in the nucleus versus the cytoplasm. Precursor RNA for G6PD in the nucleus was increased 15-fold by refeeding starved mice and was inhibited 75% by dietary fat. Kinetic analysis of the G6PD precursor mRNA from livers of mice fed low-fat and high-fat diets demonstrated that no change in half-life of the message occurred in response to additional fat in the diet. From these results we conclude that expression of the G6PD gene is regulated by changes in the accumulation of precursor RNA for G6PD in the nucleus and this regulation does not involve changes in the stability of this mRNA.