Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening

Journal Article · Thu Sep 01 00:00:00 EDT 1994 · Genomics

DOI:https://doi.org/10.1006/geno.1994.1457· OSTI ID:186053

Robertson, N G ^[1]; Gutierrez-Espeleta, G A ^[2]; Bieber, F R ^[1]

---

1. Brigham and Women`s Hospital, Boston, MA (United States)
2. Univ. of Costa Rica, San Jose (Costa Rica)

We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with {sup 32}P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than 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{sup 1}-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones 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 subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like 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, showing differential expression of this novel gene in the cochlea. 68 refs., 3 figs.

• OSTI ID: 186053
• Journal Information: Genomics, Vol. 23, Issue 1; Other Information: PBD: 1 Sep 1994
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
GENES
DNA HYBRIDIZATION
GENE MUTATIONS
TRANSCRIPTION
STRUCTURE-ACTIVITY RELATIONSHIPS
GENE REGULATION
PATIENTS
SENSE ORGANS DISEASES
EVALUATION
ETIOLOGY
RNA
PROBES
DNA SEQUENCING
COLLAGEN
AUDITORY ORGANS
FETUSES
POLYMERASE CHAIN REACTION
NUCLEOTIDES
ELECTROPHORESIS