ISOLATION OF NOVEL AND KNOWS GENES FROM A HUMAN FETAL COCHLEAR CDNA LIBRARY USING SUBTRACTIVE HYBRIDIZATION AND DIFFERENTIAL SCREENING

We used a combination of subtractive hybridization and differential sc reening strategies to identify genes that may function normally in hea ring and, when mutated, result in deafness. A human fetal cochlear (me mbranous labyrinth) cDNA library was subtracted against total human fe tal brain RNAs by an avidin-biotin-based procedure to enrich for cochl ear transcripts. Subtracted cochlear clones were differentially screen ed with P-32-labeled total cochlear and total brain cDNA probes. Seque nce analysis of clones that hybridized more intensely with cochlear th an with brain cDNA probes revealed some previously characterized genes , including mitochondrial sequences, collagen type I alpha-2 (COL1A2), collagen type II alpha-1 (COL2A1), collagen type III alpha-1 (COL3A1) , spermidine/ spermine N-1-acetyltransferase (SAT), osteonectin (SPARC ), and peripheral myelin protein 22 (PMP22). Also identified were clon es that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the sub tractive procedure by showing preferential cochlear expression. A numb er of these genes serve structural or regulatory functions in extracel lular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence ana lysis of Coch-5B2 reveals a von Willebrand factor type Alike domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, sh owing differential expression of this novel gene in the cochlea. (C) 1 994 Academic Press, Inc.