We similarly found that mouse Otolin is expressed by support cells in the utricle and saccule and is not produced by vestibular hair cells.
The mouse otolin gene is ∼21 Kb in size, located on chromosome 3E12, and consists of 5 exons and 4 introns (Fig. 1B).
Based on EST clones and genomic sequences corresponding to mouse otolin, a nested PCR approach was used to clone the entire coding region from 17-day mouse embryo cDNA (Clontech).
Using mass spectrometry, we demonstrated that nine out of the ten proline residues (within the sequence Gly-X-Pro) of the mouse Otolin protein are hydroxylated, consistent with similar modifications seen in other collagen proteins.
Of all the C1q-domain containing proteins, mouse Otolin shares the highest degree of amino acid identity (52%) in the globular domain with fish sacullar collagen [47], a protein found only in the inner ear of fish (Fig. S2).
The mouse otolin mRNA is 2157 bp in size, and consists of a 129 bp 5'UTR, a 1449 bp coding region, and 579 bp of 3'UTR sequences.
Further, the time course of otolin expression during inner ear development was similar to genes encoding otoconial membrane constituents, including Oc90, otogelin, otoancorin, α-tectorin, β-tectorin, and sparc (Fig. 3B).
An in silico search of putative O-glycosylation sites (http://www.cbs.dtu.dk/services/NetOGlyc/) [48] in the mouse Otolin protein predicted ten putative residues (Ser-72 and Thr-60, 62, 69, 70, 81, 135, 138, 146, and 147) that can be potentially modified with the attachment of O-linked glycans.
Mouse gene expression was directly derived from ImmGen (Heng and Painter, 2008).
Mouse snoRNA135 expression was assayed for normalization.
