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In vitro Expression and Mutagenesis of a Gene for Corticotropin Releasing Factor
Annual rept. Nov 1988-Oct 1989
WEST VIRGINIA UNIV MORGANTOWN DEPT OF BIOCHEMISTRY
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The specific goals of this proposal are to 1 create a recombinant gene for corticotropin releasing factor CRF, 2 express that gene by in vitro transcription and translation, 3 test the function of this recombinant protein by receptor binding assay and agonist-induced release of ACTH from cultured pituitary cells and 4 create and test mutants of the CRF molecule starting at the level of the DNA. We have accomplished the first two of these goals and partially completed the third. We have synthesized the CRF gene, expressed it and characterized the recombinant protein. This protein is active when applied to pituitary cells, but the in vitro translation extract contains substances which partially interfere with that activity. We are presently purifying the recombinant protein from the translation extract. In a related area, we are conducting experiments to characterize the stress non-responsive period SNRP in the neonatal rat. We find that the spontaneously hypertensive rat SHR is not entirely subject to this quiescent adrenocortical period during the first two weeks of neonatal life when compared with the normotensive control animal. This difference is not caused by alterations in the levels of circulating or stored ACTH, implying that there are differences in the responsiveness of the adrenal cortex. kt
APPROVED FOR PUBLIC RELEASE