This proposal seeks funding for the application of molecular biological techniques to the study of the hair cell epithelia of the peripheral vestibular system of vertebrates. It proposes the study of the proteins of the macular otoconia to determine the interactions of the hair cell containing epithelia with the apical, extracellular otoconial membrane. This membrane is a necessary component for the normal function of the vestibular end organs. In aging, disintegration of otoconia can cause vertigo.
The specific aims are to: isolate cDNA clones coding for these proteins from inner ear libraries I have constructed; use these clones to determine the developmental expression of otoconial proteins from the rat and Xenopus laevis and identify the specific site of the protein expression; use the clones to make constructs which will maintain the fidelity of protein modifications for expression of the protein in amounts suitable for protein structure determination using x-ray crystallography. The approach is to use existing antibodies raised against the otoconial proteins of rat and X. laevis to screen the cDNA libraries constructed in the expression vector lambda ZAP II. Protein sequence will also be determined using a combination of micro Edman sequencing and mass spec sequencing. This sequence information will be used to synthesize oligonucleotide probes to screen the cDNA libraries. These proteins will be used to model the interactions of proteins and calcium carbonate in biominerals. Their study will elucidate the interactions of the hair cell epithelium with its extracellular environment and specifically with the accessory sensory structure, the otoconial membrane.
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