MOLECULAR DISSECTION OF AN EXTRACHROMOSOMAL AMPLICON REVEALS A CIRCULAR STRUCTURE CONSISTING OF AN IMPERFECT INVERTED DUPLICATION
MOLECULAR DISSECTION OF AN EXTRACHROMOSOMAL AMPLICON REVEALS A CIRCULAR STRUCTURE CONSISTING OF AN IMPERFECT INVERTED DUPLICATION
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DOI：
10.1006/geno.1993.1107
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发表时间：
1993-03-01
期刊：
影响因子：
4.4
通讯作者：
WAHL, GM
中科院分区：
文献类型：
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作者：
NONET, GH;CARROLL, SM;WAHL, GM
小鼠成纤维细胞系B-1/50具有4300倍扩增的腺苷脱氨酶基因座（Yeunget等人，1983，J.Biol.Chem.258：8338-8345），通过原位杂交显示在不同大小的染色体外元件上具有扩增的序列。我们在这里表明，最小的圆大约是500 kb。我们描述了一种简便的筛选技术，用于确定粘粒和酵母人工染色体（YAC）克隆来自扩增子。一个封闭的分子图谱，通过安排cosmetry和YAC到一个重叠群跨越超过250 kb的腺苷脱氨酶基因座。来自该重叠群两端的YAC显示出界定了250-kb的反向重复。B-1/50 DNA的aSalI部分消化的长程作图也与B-1/50细胞中500-kb腺苷脱氨酶扩增子是反向重复的解释一致。该扩增子是B-1/50细胞系中含有扩增序列的唯一或主要结构，这一发现表明，即使以如此高的拷贝数存在，这些结构也不固有地倾向于高频重排。这项研究提供了第一个参与哺乳动物基因扩增的附加体结构的分子描述。这一发现的基因扩增和附加体形成的模型的影响进行了讨论。
A mouse fibroblast line, B-1/50, with a 4300-fold amplification of the adenosine deaminase gene locus (Yeunget al., 1983,J. Biol. Chem.258: 8338-8345), was shown byin situhybridization to harbor the amplified sequences on variously sized extrachromosomal elements. We show here that the smallest circle is approximately 500 kb. We describe a facile screening technique for identifying cosmid and yeast artificial chromosome (YAC) clones derived from the amplicon. A closed molecular map was generated by arranging the cosmids and YACs into a contig spanning over 250 kb of the adenosine deaminase gene locus. YACs from the two ends of this contig were shown to delimit a 250-kb inverted duplication. Long-range mapping of aSalI partial digest of B-1/50 DNA is also consistent with the interpretation that the 500-kb adenosine deaminase amplicon in B-1/50 cells is an inverted duplication. The finding that this amplicon is the only or predominant structure containing amplified sequences in the B-1/50 cell line suggests that such structures are not inherently prone to high frequency rearrangement, even when present at such high copy number. This study provides the first molecular description of the structure of an episome involved in mammalian gene amplification. The implications of this finding for models of gene amplification and episome formation are discussed.