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Gonadotropin-releasing hormone (GnRH) activates release of hypothalamic GnRH into the hypothalamospinothalamic tract. Recent evidence suggests that GnRH acts directly on the pituitary gland, with the resultant secretion of gonadotropins. A number of GnRH effects, including release of gonadotropins, require calcium and cAMP. We investigated the mechanisms by which GnRH activates intracellular cAMP and calcium in a clonal pituitary tumor cell line (GH3 cells). In the absence of extracellular calcium, GnRH caused a transient, large rise in cytoplasmic calcium (0.8-1.3 muM) followed by sustained, oscillatory increases in calcium (0.3-0.4 muM). Treatment of cells with 2.5 mM EGTA in the presence of calcium resulted in the disappearance of GnRH-induced calcium oscillations. Transient increases in cytosolic cAMP following addition of GnRH were blocked by cadmium, indicating that GnRH activates adenyl cyclase. In contrast, Ca(2+)-dependent cAMP elevations in response to GnRH were not blocked by extracellular cadmium. These data suggest that GnRH activates calcium and cAMP through different pathways in GH3 cells.Insertion mutagenesis in vivo using chimeric selectable genes.
Insertion mutagenesis was generated in vivo in Saccharomyces cerevisiae by using unamplified sequences as selectable markers. This approach resulted in the recovery of a mutant allele encoding valine at position 312 of the major surface mannoprotein, cell wall mannoprotein II. Approximately 1% of the recovered colonies carried a PCR-amplified version of the mutagenic sequence. Although the unamplified sequences could not self-amplify, they could mediate mutagenesis following transformation with a PCR-amplified DNA. you can
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