Preterm delivery continues as the leading of cause of neonatal morbidity and mortality in the United States. With a more complete understanding of the basic possible to devise effective strategies for preventing preterm delivery. Nitric oxide is a naturally occurring smooth muscle relaxant and regulator molecule which we have shown can be produced by the decidua(the inner lining of the uterus) in rabbits during pregnancy. The enzyme which produces nitric oxide- Nitric Oxide Synthase(NOS)- is active in late pregnancy and decreases 80% on the last day of gestation; it is insensitive to calcium/calmodulin concentrations as is the inducible isoform of the enzyme(iNOS) described macrophages; but decidual NOS is found in the particulate(membrane bound) fraction similar to constitutive NOS(cNOS) found in the vascular endothelium. We b(hypothesize) that decidual NOS is a unique isoform of the enzyme. Our b(specific aim) is to clone the cDNA coding for the enzyme so as to determine the deduced amino acid sequence and to discover clues to the regulation of decidual NOS. This will also make possible the construction of specific probes and antibodies to further study the functions and regulation of nitric oxide in the uterus during pregnancy. Our b(experimental design) is to construct a 27 day gestation rabbit decidual cDNA library in the phage g{lambda} Uni-ZAP II. The library will be screened with a rabbit decidual NOS probe constructed by polymerase chain reaction amplification. By selecting primers based upon conserved regions in the published sequences of NOS isoforms from other tissues, and primers degenerate at unconserved bases, all NOS transcripts in rabbit uterine cDNA can be amplified, whatever the isoform. The product of the predicted size is purified by gel electrophoresis and cloned into a Bluescript vector. A potentially unique uterine NOS partial clone is selected by dual screening with 32P labeled brain cNOS and hepatocyte iNOS probes. iNOS positive and cNOS negative clones will be sequenced by the dideoxynucleotide chain termination method. The partial clone with sufficient homology to iNOS will serve as a probe for screening the rabbit decidual cDNA library. The longest clones selected from the cDN library will be analyzed by Northern analysis and primer extension analysis to determine whether or not they are full length. Finally, the full length clone will be verified by transfection into kidney 293 cells and assaying for the acquisition of iNOS activity. The uniqueness of the uterine NOS gene will be investigated by performing Southern Blot analysis of rabbit genomic DNA using the full length uterine cDNA clone. The isolation and characterization of the uterine nitric oxide gene will further Magee-Womens Hospital's position as a preeminent perinatology research center and may lay the groundwork for entirely new treatments for preventing preterm delivery.
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