Corneal transparency depends on the small uniform diameter and the regular spacing of collagen fibrils. The """"""""fibrillar"""""""" collagens are involved in determining fibril diameter; the """"""""fibril-associated"""""""" collagens, a new class, may be responsible for interfibrillar relationships, such as spacings. We have isolated cDNAs for two putative """"""""fibril associated"""""""" collagens expressed in cornea. One is for type XIV collagen, a known member of a fibril-associated family; the other potentially encodes a cornea-specific molecule with multiple, small collagenous domains. The genes for these collagens will be characterized and their exon/intron structures defined. Cis-acting elements (DNA sequences) that are important for transcriptional regulation of the genes will be identified. The region 5' to the transcriptional start site (containing putative promoters/enhancers/silencers) will be examined, as well as the first intron, which may .also be involved in transcriptional regulation. These regions will be ligated to a reporter gene and the constructs transfected into skin and corneal fibroblasts. To precisely identify the regulatory sequences within these regions and to determine whether the sequences function as promoters, enhancers, or silencers, systematic nested deletions of portions of the regulatory regions of these constructs will be made and evaluated by transfection. The presence of potential cis-elements unique to cornea will be examined by in vivo footprinting, a method which allows identification and sequencing of regions of genes to which DNA-binding proteins (trans-acting factors) are bound. The technique employs the polymerase chain reaction for amplification, and thus can be performed on the amounts of tissues available from embryos. Once obtained, the DNA sequences of these cis-acting regions will be utilized to screen a corneal cDNA expression library to isolate cDNAs expressing DNA binding proteins (trans-acting factors). If trans-acting factors unique to cornea are found they will be tested for functionality in co-transfection experiments. For this, non-corneal cells, such as skin fibroblasts will be simultaneously co-transfected with a construct encoding the transacting factor driven by a constitutive promoter, and a construct of the promoter/enhancer regions of the cornea-specific collagen gene promoter linked to a reporter gene. If transcription of the reporter gene is observed, it will indicate that a trans=acting factor unique to cornea is able to effect directly the regulation of a cornea-specific gene in a non-corneal cell.
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