Sequence of a 42-kb mouse region containing the imprinted H19 locus:: identification of a novel muscle-specific transcription unit showing biallelic expression
Sequence of a 42-kb mouse region containing the imprinted H19 locus:: identification of a novel muscle-specific transcription unit showing biallelic expression
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DOI：
10.1007/s003359900863
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发表时间：
1998-09-01
期刊：
影响因子：
2.5
通讯作者：
Sasaki, H
中科院分区：
文献类型：
--
作者：
Ishihara, K;Kato, R;Sasaki, H
小鼠H19基因编码一种功能未知的未翻译的RNA，受到基因组印记的影响，并仅从母系等位基因表达(Bartolomei等人。1991年)。该基因位于G-带F4/F5中染色体(Chr)7的远端区域，其中至少包含5个其他印记基因(Beeceh等人)。1997年)。其中，胰岛素样生长因子II(IGF2)和胰岛素II(Ins2)基因位于H19的上游70-100kb，并以父系方式表达。证据表明，H19和Igf2/Ins2的相互印迹是通过它们竞争一组内胚层特异性增强剂而发生的(Leighton等人。1995年a、1995年b)。在这个模型中，位于H19下游5-7kb的共同增强子与母亲染色体上的H19和父亲染色体上的Igf2/Ins2特异地相互作用。我们先前在H19下游35kb的区域发现了一个名为L23(线粒体)相关蛋白(L23mrp或Rp123)的非印记基因(Zubair等人)。1997年)。结果表明，靶向删除上述增强子不会干扰L23mrp的表达(Zubair等人。1997年)。因此，L23 MRP在功能上与Igf2/Ins2/H19结构域在印迹和增强子作用方面都是隔绝的。为了更详细地研究全域的调控机制，并了解印迹结构域是如何界定的，我们测定了H19基因座及其3-侧翼区的序列，一直到相邻的L23mrp基因座。两个粘粒克隆CDH1和CDH2，先前从129/SVJ文库中分离(Zubair等人1997)，用于制备适合测序的质粒亚克隆。获得的序列(GenBank登录号：AF049091)全长41680个碱基，包括5个侧翼的5517个碱基、H19结构基因的2615个碱基和3个侧翼的33548个碱基(图1)。将5个侧翼和结构基因的序列与已发表的数据进行比较(Pachnis等人)。1988年；Tremblay et al.1995年)，有许多差异，包括在先前的数据中，内含子2和4内含子分别有28和27个碱基的大缺失，这可能是由于多态所致。增强子序列(Yoo-Warren等人位于12676-12942和14330-14625号阵地。在369-502位置发现了散布重复家族B1家族的成员，在两端有17个碱基的重复序列。3个(CA)n重复序列分别位于22696-22984、26002-26047和39960-39995位。该区域的平均GC含量为52.5%，与附近的Igf2相似(Sasaki等人)。1996)和L23mrp(Zubair等人1997年)。当用BLAST搜索同源序列时，从性交后19.5天的小鼠胎儿(DPC)中鉴定出一个表达序列标签(EST)(GenBank登录号：AA062189)。该EST全长493bp，其5部分与我们的序列(位置25066-25259)相同，但有一个错配(图2A)。东部夏令时的其余部分与
The mouse H19 gene, encoding an untranslated RNA with unknown function, is subject to genomic imprinting and expressed exclusively from the maternal allele (Bartolomei et al. 1991). The gene is located in the distal region of Chromosome (Chr) 7 within G-bands F4/F5, which contains at least five other imprinted genes (Beechey et al. 1997). Among these, the insulin-like growth factor II (Igf2) and insulin II (Ins2) genes are located 70–100 kb upstream from H19 and expressed paternally. Evidence suggests that the reciprocal imprinting of H19 and Igf2/Ins2 occurs through their competition for a set of endoderm-specific enhancers (Leighton et al. 1995a, 1995b). In this model, the common enhancers located 5–7 kb downstream from H19 specifically interact with H19 on the maternal chromosome and with Igf2/Ins2 on the paternal chromosome. We previously identified a non-imprinted gene called L23 (mitochondrial)-related protein (L23mrp or Rp123) in a region 35 kb downstream from H19 (Zubair et al. 1997). It was shown that a targeted deletion of the above enhancers did not disrupt L23mrp expression (Zubair et al. 1997). Thus, L23 mrp is functionally insulated from the Igf2/Ins2/H19 domain in terms of both imprinting and enhancer action.To study the domain-wide regulatory mechanisms in more detail and to know how the imprinted domain is delimited, we have determined the sequence of the H19 locus and its 3-flanking region, all the way down to the adjacent L23mrp locus. Two cosmid clones cDH1 and cDH2, isolated previously from a 129/SVJ library (Zubair et al. 1997), were used to make plasmid subclones appropriate for sequencing. The obtained sequence (GenBank accession: AF049091) covered a total of 41680 bp and included 5517 bp of 5 flank, 2615 bp of the H19 structural gene, and 33548 bp of 3 flank (Fig. 1). When the sequences of the 5 flank and structural gene were compared with the published data (Pachnis et al. 1988; Tremblay et al. 1995), there were numerous differences, including two large deletions of 28 and 27 bp within introns 2 and 4, respectively, in the previous data, possibly owing to polymorphisms. The enhancer sequences (Yoo-Warren et al. 1988) were located at positions 12676–12942 and 14330–14625. A member of an interspersed repetitive family, the B1 family, was found at position 369–502 with a duplication of a 17-bp sequence at both ends. Three (CA) n repeats were located at positions 22696–22984, 26002–26047, and 39960–39995. The average GC content of the region was 52.5%, which was similar to that of nearby Igf2 (Sasaki et al. 1996) and L23mrp (Zubair et al. 1997). When databases were screened for homologous sequences with BLAST, an expressed sequence tag (EST) from mouse fetus of 19.5 days postcoitum (dpc) was identified (GenBank accession: AA062189). This EST was 493 bp in size and its 5 part was identical with our sequence (position 25066–25259) with one mismatch (Fig. 2A). The rest of the EST was identical with position