Pulmonary surfactant stabilizes alveoli by reducing surface tension at the alveolar air-liquid interface. Deficient production of surfactant is the major cause of respiratory distress syndrome (RDS) of the newborn. Surfactant protein B(SP-B) plays a major role in prevention of alveolar collapse by enhancing the surface active properties of surfactant phospholipids. It is the objective of this proposal to elucidate molecular mechanisms that mediate cell-specific, developmental, and hormonal regulation of expression of rabbit SP-B gene.
The specific aims of the proposal are 1. Characterization of SP-B cDNA and investigation of molecular mechanisms that mediate tissue-specific and developmental regulation of SP-B gene expression. 2. Investigation of molecular mechanisms that mediate cAMP and glucocorticoid dependent induction of SP-B gene expression in fetal lung in vitro. 3. Isolation and characterization of SP-B gene and identification of cis-acting DNA sequences required for cell-specific and cAMP and glucocorticoid regulation. 4. Identification of putative trans-acting factors of SP-B gene and purification of trans-acting factor(s) required for cell- specific expression. Northern hybridization analysis of RNA and transcription run-on assays will be utilized to define the role of transcriptional and posttranscriptional mechanisms in the developmental, and cAMP and glucocorticoid regulation of SP-B gene expression. The role of chromatin structure in mediating cell-specific, and developmental regulation of SP- B gene expression will be analyzed by DNase I hypersensitivity studies of chromatin. Cis-acting DNA regulatory elements of SP-B gene will be identified by transfection and transient expression analysis of chimeric genes that contain 5' flanking DNA sequences of SP-B gene fused to CAT gene. Trans-acting factors of SP-B gene will be identified by DNase I footprinting, gel retardation and Southwestern blotting analysis of nuclear proteins of lung tissues. Cell specific trans-acting factor(s) will be purified by means of DNA-sequence specific affinity chromatography and their role in the transcriptional regulation of SP-B gene will be investigated using an in vitro reconstituted transcription assay system.
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