An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.
An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.
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发表时间：
2003-01
期刊：
影响因子：
11.2
通讯作者：
A. Protopopov;V. Kashuba;V. Zabarovska;O. Muravenko;M. Lerman;G. Klein;E. Zabarovsky
中科院分区：
文献类型：
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作者：
A. Protopopov;V. Kashuba;V. Zabarovska;O. Muravenko;M. Lerman;G. Klein;E. Zabarovsky
为了便于鉴定染色体3p21.3-p22 AP 20亚区中的肿瘤抑制基因，我们构建了该区段（标记D3 S4285和D3 S3873之间）的3.5 Mb物理和基因图谱，其跨越GB 4遗传图谱的124.4cR3000至133.5 cR 3000的距离。我们使用NotI-连接和跳跃克隆，序列标记位点PCR标记分析，和DNA和纤维荧光原位杂交，以确认序列的顺序和地图的方向。由5条酵母人工染色体、15条细菌人工染色体、5条P1人工染色体和8个NotI连锁克隆组成的克隆重叠群为该图谱的构建提供了物理基础。我们明确地确定了该区域28个序列标记位点和35个基因的顺序。已发表的细菌人工染色体重叠群之间的间隙由我们自己的序列数据确定和覆盖。此外，分离出三个新基因，即大鼠高尔基体外周膜蛋白p65的人类同源物GOLPH 5（GORASP 1）、应激诱导蛋白基因STI 2和AP 20区基因1 APRG 1。肿瘤抑制基因候选者APRG 1位于小细胞肺癌细胞系ACC-LC 5中纯合缺失的边界附近。用组织特异性cDNA组的表达分析揭示了该信息的七种不同的组织特异性剪接变体（A-G）（大小范围，1.0- 1.8kb）。尽管该基因在所有测试组织中以低水平表达，但在胰腺（剪接形式B和D）、肾（A）和胎盘（B和C）中检测到相对较高的表达。APRG 1基因编码一个170个氨基酸的蛋白质（亚型B），其具有在真核翻译因子6基因家族成员中保守的NH 2-末端部分。APRG 1B蛋白还具有一个与细胞粘附序列Arg-Gly-Asp相对应的Prosite结构域，该结构域的存在提示APRG 1B可能直接参与细胞膜相互作用和细胞粘附。我们发现，在哺乳动物的进化过程中，AP 20区域是重复的，同源基因簇存在于人类2号染色体和小鼠1号、2号和9号染色体上的同线区域。有趣的是，HYA 22基因（酵母YA 22基因的人类直向同源物）位于两个断点的边界，进化上保守的基因簇和纯合缺失检测在肺，肾和其他癌症。NotI酶切显示，在肾癌细胞系和肿瘤活检组织中，AP 20区域频繁且广泛地甲基化。
To facilitate the identification of tumor suppressor genes in the chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb physical and gene map of this segment (between markers D3S4285 and D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of the GB4 genetic map. We used NotI-linking and -jumping clones, sequence-tagged site PCR marker analysis, and multicolor and fiber fluorescence in situ hybridization to confirm the sequence order and map orientation. An integrated clone contig composed of 5 yeast artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artificial chromosome, and 8 NotI-linking clones provided the physical base of the map. We unequivocally established the order of 28 sequence-tagged sites and 35 genes in the region. Gaps between published bacterial artificial chromosome contigs were determined and covered by our own sequence data. Furthermore, three new genes were isolated, namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible protein, STI2, and the AP20-region gene 1, APRG1. The tumor suppressor gene candidate APRG1 was positioned close to the border of the homozygous deletion in a small cell lung cancer cell line ACC-LC5. Expression analysis with a tissue-specific panel of cDNA revealed seven distinct tissue-specific splice variants (A-G) of the message (size range, 1.0-1.8 kb). Although the gene was expressed at a low level in all tested tissues, comparatively higher expression was detected in pancreas (splice forms B and D), kidney (A) and placenta (B and C). The APRG1 gene encoded a predicted protein of 170 amino acids (isoform B), which had an NH2-terminal part conserved among members of the eukaryotic translation factor 6 gene family. A Prosite pattern corresponding to the cell attachment sequence Arg-Gly-Asp was also found. The presence of this domain raised the intriguing possibility that APRG1B may be directly involved in membrane interactions and cell adhesion. We showed that the AP20 region was duplicated during mammalian evolution and homologous gene clusters were present in human chromosome 2 and syntenic mouse regions on chromosomes 1, 2, and 9. Interestingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was located at the borders of both breakpoints, evolutionarily conserved gene cluster and homozygous deletions detected in lung, kidney and other cancers. NotI digestion revealed that the AP20 region was frequently and extensively methylated in renal carcinoma cell lines and tumor biopsies.