Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG.
Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG.
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发表时间：
1996-04
期刊：
影响因子：
11.2
通讯作者：
M. Wei;F. Latif;S. Bader;V. Kashuba;J. Chen;F. Duh;Y. Sekido;Cheng Chi Lee;L. Geil;I. Kuz
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文献类型：
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作者：
M. Wei;F. Latif;S. Bader;V. Kashuba;J. Chen;F. Duh;Y. Sekido;Cheng Chi Lee;L. Geil;I. Kuz
人类染色体3p21.3上含有可能的肺癌肿瘤抑制基因(TSG)的关键区域以前是通过等位基因分型定义的，最近通过重叠的纯合缺失进行了精炼。我们报道了一个700 kb(粘粒和一个P1噬菌体)克隆重叠群的构建，覆盖了缺失重叠及其侧翼。最小的23个宇宙体集包括600kb，并被一个P1噬菌体延伸到700kb，以覆盖重叠的远端断裂点。克隆的重叠群通过限制性内切酶和表达图谱进行了广泛的鉴定，以获得克隆区域的高分辨率物理和转录图谱。通过与聚合酶链式反应扩增的cDNA文库杂交、直接选择“Zoo”杂交、筛选和鉴定24个CpG岛来检测潜在的转录片段。到目前为止，由部分或全长cDNA代表的15个新基因被分离、鉴定并精确定位在重叠群上。两个先前克隆的基因，即GNAI-2和GNAT-1也被定位。此外，还发现并定位了NCI H740缺失的端粒断裂点和重叠的GLC20缺失的着丝粒断裂点，以精确定义候选TSG区域。这个大的粘粒克隆重叠群和高分辨率图谱将被证明在肺癌TSG(S)的鉴定中至关重要。
The critical region on human chromosome 3p21.3 harboring a putative lung cancer tumor suppressor gene (TSG) was previously defined by allelotyping and recently refined by overlapping homozygous deletions. We report the construction of a 700-kb (cosmid and one P1 phage) clone contig covering the deletion overlap and its flanks. The minimal set of 23 cosmids comprises 600 kb and is extended by one P1 phage to 700 kb to cover the distal breakpoint of the overlap. The clone contig was extensively characterized by restriction and expression mapping to produce high resolution physical and transcription maps of the cloned region. Potential transcribed fragments were detected by hybridization with PCR-amplified cDNA libraries, direct cDNA selection "zoo" blotting, cDNA screening, and identification of 24 CpG islands. Thus far, 15 new genes represented by partial or full-length cDNAs were isolated, characterized, and precisely positioned on the contig. Two previously cloned genes, namely GNAI-2 and GNAT-1, were also positioned. In addition, the telomeric breakpoint of the NCI H740 deletion and centromeric breakpoint of the overlapping GLC20 deletion were discovered and mapped to define precisely the candidate TSG region. This large cosmid clone contig and high resolution maps will prove crucial in the identification of the lung cancer TSG(s).