Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone inhibits gonadotropin-releasing hormone pulse frequency in the male sheep

The objective was to determine the effect of chronic testosterone (T) treatment on GnRH and LH secretion in wethers. Rams were either castrated only or castrated and immediately treated with Silastic implants containing T. Several weeks later, a device for collecting hypophyseal-portal blood was surgically implanted. Six to seven days later, blood samples were collected simultaneously and continuously from the portal vessels and jugular vein of pairs of conscious animals. Samples were divided at 10-min intervals for 6-12 h. One hour before the end of collection, all animals received i.v. injections of 250 ng of GnRH. In samples collected simultaneously from 6 pairs of animals, T reduced the frequency of both GnRH pulses (1.8 +/- 0.2 vs. 0.9 +/- 0.3/h, p less than 0.03) and LH pulses (1.6 +/- 0.1 vs. 0.8 +/- 0.3/h, p less than 0.03). T did not alter amplitude of either GnRH or LH pulses. Testosterone reduced mean GnRH (9.7 +/- 0.6 vs. 7.9 +/- 0.5 pg/ml, p less than 0.05), whereas mean LH was not significantly reduced (9.6 +/- 1.4 vs. 6.1 +/- 1.8 ng/ml, p = 0.16). These results support the hypothesis that T reduces GnRH pulse frequency.     

Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation.

It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.