Protopopov Alexei, Kashuba Vladimir, Zabarovska Veronika I, Muravenko Olga V, Lerman Michael I, Klein George, Zabarovsky Eugene R
Microbiology and Tumor Biology Center, Karolinska Institute, 171 77 Stockholm, Sweden.
Cancer Res. 2003 Jan 15;63(2):404-12.
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.
为便于鉴定位于染色体3p21.3 - p22 AP20亚区域的肿瘤抑制基因，我们构建了该片段（在标记D3S4285和D3S3873之间）的3.5兆碱基物理图谱和基因图谱，该片段跨越GB4遗传图谱上从124.4 cR3000到133.5 cR3000的距离。我们使用NotI连接和跳跃克隆、序列标签位点PCR标记分析以及多色和纤维荧光原位杂交来确认序列顺序和图谱方向。由5个酵母人工染色体、15个细菌人工染色体、5个P1人工染色体和8个NotI连接克隆组成的整合克隆重叠群为该图谱提供了物理基础。我们明确确定了该区域中28个序列标签位点和35个基因的顺序。已确定已发表的细菌人工染色体重叠群之间的间隙，并由我们自己的序列数据覆盖。此外，还分离出三个新基因，即大鼠高尔基体周边膜蛋白p65的人类同源物GOLPH5（GORASP1）、应激诱导蛋白基因STI2和AP20区域基因1（APRG1）。肿瘤抑制基因候选物APRG1位于小细胞肺癌细胞系ACC - LC5纯合缺失边界附近。用组织特异性cDNA面板进行的表达分析揭示了该信使RNA的七个不同的组织特异性剪接变体（A - G）（大小范围为1.0 - 1.8 kb）。尽管该基因在所有测试组织中表达水平较低，但在胰腺（剪接形式B和D）、肾脏（A）和胎盘（B和C）中检测到相对较高的表达。APRG1基因编码一个预测的170个氨基酸的蛋白质（异构体B），其NH2末端部分在真核翻译因子6基因家族成员中保守。还发现了一个与细胞附着序列Arg - Gly - Asp相对应的Prosite模式。该结构域的存在引发了一个有趣的可能性，即APRG1B可能直接参与膜相互作用和细胞粘附。我们发现AP20区域在哺乳动物进化过程中发生了复制，人类染色体2以及小鼠染色体1、2和9上的同线区域中存在同源基因簇。有趣的是，HYA22基因（酵母YA22基因的人类直系同源物）位于进化保守基因簇和在肺癌、肾癌及其他癌症中检测到的纯合缺失的两个断点边界处。NotI消化显示AP20区域在肾癌细胞系和肿瘤活检组织中经常且广泛地发生甲基化。
