Isolation of a gene encoding a novel member of the nuclear receptor superfamily from the critical region of usher syndrome type IIa at 1q41
Isolation of a gene encoding a novel member of the nuclear receptor superfamily from the critical region of usher syndrome type IIa at 1q41
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DOI：
10.1006/geno.1998.5345
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发表时间：
1998-06-15
期刊：
影响因子：
4.4
通讯作者：
Sumegi, J
中科院分区：
文献类型：
--
作者：
Eudy, JD;Yao, SF;Sumegi, J
Usher综合征是由一组异质性常染色体隐性遗传疾病组成，其特征是视网膜色素变性和中度至重度先天性耳聋。Usher IIa型（USH 2a）疾病基因座已通过连锁分析分配到1 q41（3）。Sumegi等人在1 q41上D1 S474和AFM 144 XF 2之间USH 2a基因座的关键区域生成了酵母人工染色体（YAC）重叠群（9）。使用Gibco-BRL外显子扩增试剂盒从延伸到USH 2a关键区域的YAC 762 a6粘粒亚克隆中分离候选外显子。用外显子E2筛选胎脑cDNA文库，分离出920 bp的cDNA Fbrn-1（图1A）。BLAST（n）检索GenBank，发现3！cDNA片段的末端与5！来自胎脑cDNA文库（GenBank登录号H 08990）的1612-bp表达序列标签（EST）的末端。一个5！cDNA末端快速扩增（RACE）程序导致分离1435-bp cDNA序列（图1A）。三个重叠的克隆，920 bp的cDNA，EST 45826，和RACE产品，分别产生2964 bp的cDNA序列。使用RACE产物作为探针分析人和小鼠多组织北方（MTN）印迹（Clontech）（图1B）。一个约5.5 kb的单转录本可在多种成人组织中检测到，包括脑、肺、骨髓、肾上腺、气管、脊髓和甲状腺。在测试的人类胎儿组织中，该基因在肾脏、肺和肝脏中表达，在发育中的大脑中表达水平极高。小鼠印迹还在脑、脾、肺、肾和睾丸中产生约5.5 kb的转录物，表明该基因在小鼠和人之间高度保守。由于在北方印迹中未检测到除单个5.5 kb mRNA以外的转录物，因此数据表明存在约3.0 kb的额外cDNA。测序一个10-kb的基因组DNA片段含有3！2964-bp cDNA的末端鉴定出与另外两个EST相同的序列。EST克隆254158（GenBank登录号N22470）分离自人胚胎耳蜗迷路文库，EST克隆362492（GenBank登录号AA 018085）分离自视网膜cDNA文库。使用EST 254158作为探针的北方印迹分析检测到与RACE产物相同的5.5 kb转录物（数据未显示）。5号！EST 362492的结尾与3！2964-bp cDNA重叠群的末端。重叠cDNA产生5221个核苷酸的序列，其与通过北方印迹分析鉴定的转录物的大小一致。该cDNA序列（GenBank登录号AF 058291）含有位于bp位置219处的ATG起始位点和延伸至1526的1308-bp开放阅读框（ORF），随后是异常大的3！非翻译区3695 bp（图1A）。ORF预测了由436个氨基酸组成的48.7-kDa蛋白。经BLAST（p）同源性检索，该蛋白为类固醇/甲状腺/维甲酸受体超家族的新成员。它与孤儿受体雌激素相关受体（ESRRB）有76%相同，与雌激素相关受体（ESRRA）有63%相同（2）。由于该受体与ESRRB和ESRRA的广泛同源性，我们将该受体称为雌激素相关受体-ESRRG。用ESRRG和多态性微卫星标记AFM 144 XF 2的引物进行Southern杂交和PCR分析的组合显示，ESRRG基因的跨度约为100 kb，并以AFM 144 XF 2标记为中心。
Usher syndrome is composed of a group of heterogeneous autosomal recessive disorders characterized by retinitis pigmentosa and moderate to severe congenital deafness. The Usher type IIa (USH2a) disease locus has been assigned by linkage analysis to 1q41 (3). Sumegi et al. generated a yeast artificial chromosome (YAC) contig across the critical region of the USH2a locus between D1S474 and AFM144XF2 on 1q41 (9). Candidate exons, from cosmid subclones of YAC762a6, which extends into the USH2a critical region, were isolated using the Gibco-BRL exon amplification kit. Screening of a fetal brain cDNA library with exon E2 resulted in the isolation of a cDNA, Fbrn-1, of 920 bp (Fig. 1A). A BLAST (n) search of GenBank revealed that the 3! end of the cDNA fragment overlapped with the 5! end of a 1612-bp expressed sequence tag (EST) from a fetal brain cDNA library (GenBank Accession No. H08990). A 5! rapid amplification of cDNA ends (RACE) procedure resulted in the isolation of a 1435-bp cDNA sequence (Fig. 1A). The three overlapping clones, the 920-bp cDNA, EST 45826, and the RACE product, respectively, generated 2964 bp of cDNA sequence. Both human and mouse multiple tissue Northern (MTN) blots (Clontech) were analyzed using the RACE product as a probe (Fig. 1B). A single transcript of approximately 5.5 kb was detectable in a variety of adult human tissues including brain, lung, bone marrow, adrenal gland, trachea, spinal cord, and thyroid gland. In the human fetal tissues tested, the gene was expressed in the kidney, lung, and liver and at extremely high levels in the developing brain. The mouse blot also yielded transcripts of approximately 5.5 kb in brain, spleen, lung, kidney, and testis, suggesting that the gene is highly conserved between mouse and human. As no transcript other than the single 5.5-kb mRNA was detected in Northern blots, the data suggested that approximately 3.0 kb of additional cDNA existed. Sequencing of a 10-kb genomic DNA fragment containing the 3! end of the 2964-bp cDNA identified sequences identical to two additional ESTs. EST clone 254158 (GenBank Accession No. N22470) was isolated from a human embryonic cochlear labyrinth library, and EST clone 362492 (GenBank Accession No. AA018085) was isolated from a retina cDNA library. Northern blot analysis using EST 254158 as the probe detected the same 5.5-kb transcripts as the RACE product (data not shown). The 5! end of EST 362492 overlapped with the 3! end of the 2964-bp cDNA contig. The overlapping cDNAs generated a 5221-nucleotide sequence that was in agreement with the size of the transcript identified by Northern blot analysis. The cDNA sequence (GenBank Accession No. AF058291) contains an ATG start site at bp position 219 and a 1308-bp open reading frame (ORF) extending to 1526, followed by an unusually large 3! untranslated region of 3695 bp (Fig. 1A). The ORF predicts a 48.7-kDa protein consisting of 436 amino acids. A BLAST (p) similarity search of GenBank revealed that the predicted protein is a novel member of the steroid/thyroid/retinoid receptor superfamily. It is 76% identical to the orphan receptor estrogen-related receptor-(ESRRB) and 63% identical to the estrogen-related receptor-(ESRRA)(2). Due to the extensive homology of the receptor to ESRRB and ESRRA, we will refer to this receptor as estrogen-related receptor-ESRRG. A combination of Southern hybridization and PCR analysis with primers derived from ESRRG and the polymorphic microsatellite marker AFM144XF2 revealed that the ESRRG gene spans approximately 100 kb and is centered upon the AFM144XF2 marker …