Gu J, Guan X Y, Ashlock M A
Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-4442 USA.
Genome Res. 1999 Feb;9(2):182-8.
Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA, NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.
定位克隆过程中使用的基因分离方法依赖于由细菌人工染色体、P1或黏粒克隆组成的物理重叠群。然而，在大多数情况下，物理图谱构建的初始框架由酵母人工染色体（YAC）重叠群组成，YAC是用于基因分离的次优载体。在此，我们报告一种策略，通过使用cDNA代表性差异分析（RDA）直接分离在哺乳动物细胞中从YAC表达的转录本，来鉴定YAC中包含的基因序列。RDA测试cDNA是从先前报道的仓鼠细胞系中产生的，该细胞系通过稳定转移一个表达NPC1（负责尼曼-匹克C型病（NP-C）的人类基因）的590-kb YAC（911D5）而获得。驱动cDNA是从不含表达NPC1的YAC的对照仓鼠细胞系中产生的。在通过RDA获得的基因片段中，NPC1是最丰富的产物。此外，还分离出两个非NPC1片段，它们定位于911D5并从其表达。这些RDA基因片段之一（7-R）跨越多个外显子，与先前作为表达序列标签（EST）报道的人类cDNA克隆具有98%的序列同一性，但未定位到染色体区域。另一个片段（2-R）与已知的哺乳动物基因或EST没有明显的序列相似性，进一步定位于YAC 911D5和844E3之间的重叠区域。后一个YAC是NP-C候选区域连续重叠群的一部分，但不包含NPC1。当在候选区间有YAC重叠群时，这种先进行稳定YAC转移然后进行cDNA RDA的两部分方法应该是加速人类基因克隆的有用辅助策略。
