The transparent human ocular lens is the molecular and morphological epitome of temporally precise and spatially directed gene activity, which commences in the 5/6th week of gestation and continues throughout life. In the recent past, work from our laboratory and other investigators has been instrumental in bringing about a remarkable revision of our understanding of alpha-crystallins, the predominant proteins of the mammalian lens. We showed that both alphaA and alphaB are expressed in extralenticular tissues and that they exist as independent proteins. We suggested that these proteins may perform similar, non-structural (non- crystallin) functions both inside and outside of the lens. Data from other laboratories have shown that alphaA and alphaB, like other heat shock proteins (with whom they share primary sequence homologies), have chaperone-like activities, a catalytic aspect of these proteins which may be fundamental to their role in the lens. In this application we propose to study the transcriptional regulation of alphaB-crystallin gene in the lens, with particular emphasis on the delineation of the role of heat shock factors. We suggest two simultaneous approaches: (1) Develop an in vitro transcription system from the rat lens, which will allow characterization of lens-specific promoter elements and identification of interacting factors; and (2) Use probes from known transcription factors (e.g., HSF and C/EBP) to isolate their homologues to investigate tissue-specific heterogeneity with relevance to the expression of the alphaB gene. Both these approaches will utilize (a) a highly sensitive in vivo footprinting of the rat and human alphaB genes with emphasis on conserved DNA sequences and (b) functional assays in in vitro and in transient expression systems. Characterization of transcription factors will involve cloning, isolation (from """"""""normalized"""""""" and regular rat and human lens cDNA libraries), sequence analysis and expression in bacterial cells. We also propose to initiate investigations into the very likely possibility of autoregulation of the alphaB gene. We are conceptually and technically well poised to exploit the opportunity that the alphaB gene offers for elucidation of the mechanistic aspects of the activation of a crystallin promoter at the epithelium/fiber cell interface - an innate genetic mechanism which must remain fundamental to our understanding of lens growth and its attendant pathology, cataractogenesis.
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