The overall objective is to determine the various molecular mechanisms by which the amount and activity of a specific non- secreted protein with a known function, the enzyme glucose-6- phosphate dehydrogenase (G6PD), is regulated in uterus by ovarian hormones. Estradiol (E2) induces G6PD in uterus by: a) increasing transcription, b)increasing translation (elongation rate), c) enhancing processing of a pre-G6PD to G6PD, and d) inhibiting degradation.
SPECIFIC AIMS; 1) To determine whether demethylation, E2-receptor binding or binding of an E2-induced protein to sequences in the G6PD gene are involved in regulation of transcription of the gene in uterus by E2, 2) To determine whether E2 activation of transcription of the G6PD gene in uterus increases the number of polymerase molecules engaged in G6PDmRNA synthesis or the rate of elongation of the G6PDmRNA by a constant number of polymerases, 3) To determine whether the amount of prolyl-tRNA and glycyl-RNA present in vivo is E2 regulated and whether they are rate-limiting in the translation of G6PDmRNA, 4) To verify that the E2-dependent activation of tRNApro and tRNAgly occurs by activation of the repair of the 3'- (CCA) amino acid acceptor terminus of a reserve pool of inactive tRNA's and to determine the mechanism by which E2 induces the synthesis of CTP, which is the rate-limiting step in the repair of the 3'-(CCA) terminus, 5) To prepare a cDNA probe to the 5'- terminus of rat uterine G6PDmRNA and to determine the amino acid sequence at the site on the 64,000d pre-G6PD that is clipped to produce the 57,000d mature G6PD monomer, and 6) To determine whether uterine G6PD undergoes partial destablization and/or lipophile inhibition in vivo at a time after E2-treatment (36 h) when in vivo degradation of the enzyme is resumed. METHODS: Methylation of G6PD gene will be determined by differentiated MspI/HpaII cleavage of the gene. Nuclear run-off assays will be used to study transcription rate/amount. Synthesis and sequencing of G6PD will be by the primed synthesis an ddNTP/primer extension method. Pro-tRNA and gly-tRNA will be measured by HPLC Translation will be measured in vivo and in a tRNA-dependent reticulocyte lysate translation system. SIGNIFICANCE: E2 regulates the functional levels of specific uterine proteins by modifying the rates of multiple molecular events which are either specific for the individual protein, or general in action on multiple proteins with similar properties which must act in concert to regulate E2-dependent tissues.
| Young, A J; Barker, K L (1991) Effect of estradiol and progesterone on long chain fatty acyl-coenzyme A levels in the rat uterus. Biochim Biophys Acta 1092:211-7 |
| Rasmussen, K R; Whelly, S M; Barker, K L (1988) Estradiol regulation of the synthesis of uterine proteins with clusters of proline- and glycine-rich peptide sequences. Biochim Biophys Acta 970:177-86 |
| Rasmussen, K; Whelly, S; Barker, K (1988) Estradiol regulation of reactions involved in turnover of the amino acid acceptor terminus of tRNA in the rat uterus. Biochim Biophys Acta 972:179-91 |
| Cummings, A M; Barker, K L (1986) Isolation of a precursor and a nascent chain form of glucose-6-phosphate dehydrogenase from rat uterus and regulation of precursor processing by estradiol. Biochim Biophys Acta 880:226-41 |
| Lutz, W H; Barker, K L (1986) Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis. J Biol Chem 261:11230-5 |
