A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22.
A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22.
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基于 YAC 的分箱策略促进粘粒重叠群的快速组装：Xp22 中 1.6 Mb 的重叠粘粒。
DOI：
10.1093/hmg/3.7.1155
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发表时间：
1994
影响因子：
3.5
通讯作者：
Ballabio,A
中科院分区：
文献类型：
--
作者：
Wapenaar,MC;Schiaffino,MV;Bassi,MT;Schaefer,L;Chinault,AC;Zoghbi,HY;Ballabio,A
我们已经应用了酵母人工染色体（YAC）为基础的粘粒分离和分仓策略，将Xp22中的YAC重叠群转换为1.6 Mb的重叠cosmos。这种策略是基于筛选高密度阵列X染色体特异性粘粒库与大YAC衍生的限制性片段和整个YAC探针。以这种方式选择的Cosmos在斑点印迹上网格化，并进一步映射到由YAC和YAC片段的重叠间隔定义的箱中。这种快速分箱的cosmos简化了随后的组装粘粒重叠群的限制性指纹杂交。总之，我们确定了139 cosestrin跨越整个1.6 Mb区域的最小重叠集的53个克隆。这些重叠群被分配到17个箱和9个重叠群。其中一个重叠群长度为665 kb，是迄今为止报道的人类中最大的不间断粘粒重叠群之一。重叠群之间的间隙很小，它们共同代表不到7%的覆盖区域。两个先前确定的基因包含在这些cosmetry，基因的釉原蛋白（AMG）和最近分离的推定氯离子通道基因CICN4。此外，两个疾病位点已被映射到该区域：X连锁眼白化病1型（OA1）和小眼伴线性皮肤缺损（MLS）综合征。粘粒图谱的组装使我们能够确定这两个位点的缺失间隔的大小，估计OA1为110 kb，MLS为570 kb。这些粘粒重叠群将极大地促进OA1和MLS疾病基因的定位克隆。与4号染色体上的亨廷顿病基因区域一起，Xp22中的该区域代表了人类基因组中最具特征的大区域之一。
We have applied a yeast artificial chromosome (YAC)-based cosmid isolation and binning strategy to convert a YAC contig in Xp22 into 1.6 Mb of overlapping cosmids. This strategy is based on the screening of a high-density arrayed X chromosome-specific cosmid library with large YAC-derived restriction fragments and entire YAC probes. Cosmids selected in this way were gridded on dot blots and further mapped into bins defined by the overlap intervals of the YACs and YAC fragments. This rapid binning of cosmids simplified the subsequent assembly of cosmid contigs by restriction fingerprint hybridization. In total, we identified 139 cosmids spanning the entire 1.6 Mb region with a minimal overlap set of 53 clones. These cosmids were assigned to 17 bins and 9 contigs. One of the contigs is 665 kb in length and is one of the largest uninterrupted cosmid contigs in humans reported to date. The gaps between the contigs are minor and, together, they represent less than 7% of the region covered. Two previously identified genes are contained in these cosmids, the gene for amelogenin (AMG) and the recently isolated putative chloride channel gene CICN4. In addition, two disease loci have been mapped to this region: X-linked ocular albinism type 1 (OA1) and the microphthalmia with linear skin defects (MLS) syndrome. The assembly of the cosmid maps allowed us to determine the size of the deletion intervals for these two loci, which were estimated to be 110 kb for OA1 and 570 kb for MLS. These cosmid contigs will greatly facilitate the positional cloning of the OA1 and MLS disease genes. Together with the Huntington disease gene region on chromosome 4, this region in Xp22 represents one of the best characterized large regions in the human genome.