ISOLATION OF NOVEL AND KNOWS GENES FROM A HUMAN FETAL COCHLEAR CDNA LIBRARY USING SUBTRACTIVE HYBRIDIZATION AND DIFFERENTIAL SCREENING
ISOLATION OF NOVEL AND KNOWS GENES FROM A HUMAN FETAL COCHLEAR CDNA LIBRARY USING SUBTRACTIVE HYBRIDIZATION AND DIFFERENTIAL SCREENING
复制标题
DOI：
10.1006/geno.1994.1457
复制
发表时间：
1994-09-01
期刊：
影响因子：
4.4
通讯作者：
MORTON, CC
中科院分区：
文献类型：
--
作者：
ROBERTSON, NG;KHETARPAL, U;MORTON, CC
我们使用减法杂交和差异筛选策略的组合来识别可能在听力中正常工作的基因，当突变时，导致耳聋。通过基于亲和素的程序将人胎儿耳蜗（膜迷路）cDNA文库与总人胎儿脑rna相减去，以丰富耳蜗转录本。用p -32标记的全耳蜗和全脑cDNA探针对缺失的耳蜗克隆进行差异筛选。与人工耳蜗杂交的克隆序列分析显示，与人工耳蜗杂交的克隆比与脑cDNA探针杂交的克隆更强烈，揭示了一些先前表征的基因，包括线粒体序列、I型胶原α -2 （COL1A2）、II型胶原α -1 （COL2A1）、III型胶原α -1 （COL3A1）、亚精胺/精胺n -1乙酰转移酶（SAT）、骨连接素（SPARC）和外周髓鞘蛋白22 （PMP22）。还发现了潜在的新型耳蜗基因克隆。用COL1A2， COL2A1, COL3A1， SAT， SPARC， PMP22和一个新的序列Coch-5B2探测耳蜗和脑rna的Northern blots，通过显示耳蜗优先表达证实了减法程序的结果。这些基因中的许多在细胞外基质或神经传导中起结构或调节作用；其中一些基因的缺陷与听力丧失等疾病有关。Coch-5B2的部分序列分析显示，该cDNA中存在一个von Willebrand因子类型相似结构域。为了评估Coch-5B2在耳蜗中的特异性，我们用Coch-5B2对14个人胎儿组织rna进行了Northern blot检测，结果显示该新基因在耳蜗中的表达存在差异。(C) 1994学术出版社，Inc.
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 P-32-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-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 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, showing differential expression of this novel gene in the cochlea. (C) 1994 Academic Press, Inc.