Cloning of a candidate gene for ataxia-telangiectasia group D.
Cloning of a candidate gene for ataxia-telangiectasia group D.
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发表时间：
1992-07
影响因子：
9.8
通讯作者：
Leon N. Kapp;Robert B. Painter;Yu Lc;N. vanLoon;Richard Cw rd;Michael R. James;David Cox;J. Murnane
中科院分区：
文献类型：
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作者：
Leon N. Kapp;Robert B. Painter;Yu Lc;N. vanLoon;Richard Cw rd;Michael R. James;David Cox;J. Murnane
用人粘粒克隆文库转染来自互补组 D 的共济失调毛细血管扩张 (AT) 细胞系 (AT5BIVA)，先前导致分离出部分恢复对电离辐射抗性的细胞系 (1B3)。我们拯救了1B3内的整合粘粒序列，并获得了两个含有来自染色体区域11q23的重叠DNA的粘粒克隆，之前显示该区域含有来自三个互补组的AT基因。从 HeLa 细胞文库中分离出明显全长的 3.0-kb cDNA，证明这些粘粒克隆中存在先前未识别的基因 (ATDC)。 1B3 中 ATDC 基因的转染副本在 3' 端被截短，但由于相邻粘粒 DNA 内存在 SV40 终止序列，因此它是一个完整的转录单位。从 11 号染色体文库中进一步筛选粘粒克隆后，我们鉴定出了包含该基因缺失部分的连续 DNA。 Southern blot分析表明ATDC基因在人类基因组中以单拷贝形式存在；然而，RNA 印迹分析显示，不同细胞系中存在不同大小的 mRNA（1.8、2.6、3.0、4.7 和 5.7 kb）。由于通过 Southern 或 RNA 印迹分析未在 AT5BIVA 细胞中检测到大的重排，因此该细胞系中 ATDC 基因的任何改变都将涉及点突变或小重排。用其中一种粘粒转染 AT5BIVA 细胞系可部分恢复放射抗性。对 100 个含有 11q23 染色体区域不同片段的 X 射线杂交细胞系的分析表明，ATDC 基因与 THY1 紧密连锁。因此，ATDC 基因位于预计包含互补组 A 和 C 的 AT 基因的连锁区域之外，表明 AT 互补组 D 基因有一个单独的基因座。
Transfection, with a human cosmid clone library, of an ataxia-telangiectasia (AT) cell line (AT5BIVA) from complementation group D previously resulted in the isolation of a cell line (1B3) with partially restored resistance to ionizing radiation. We rescued the integrated cosmid sequences within 1B3 and obtained two cosmid clones that contained overlapping DNA from chromosomal region 11q23, previously shown to be the region containing the AT gene(s) from three complementation groups. Isolation of an apparently full-length 3.0-kb cDNA from a HeLa cell library demonstrated a previously unidentified gene (ATDC) within these cosmid clones. The transfected copy of the ATDC gene in 1B3 is truncated at the 3' end but is a complete transcription unit, because of the presence of SV40 termination sequences within the adjacent cosmid DNA. After further screening of cosmid clones from a chromosome 11 library, we identified contiguous DNA that contained the missing portion of the gene. Southern blot analysis indicated that the ATDC gene is present in a single copy in the human genome; however, RNA blot analysis revealed mRNA of several sizes (1.8, 2.6, 3.0, 4.7, and 5.7 kb) that varied among different cell lines. Because no large rearrangements were detected in AT5BIVA cells by Southern or RNA blot analysis, any alteration in the ATDC gene in this cell line would involve a point mutation or a small rearrangement. Transfection of the AT5BIVA cell line with one of the cosmids partially restored radioresistance. Analysis of 100 X-radiation hybrid cell lines containing various fragments from the chromosomal region 11q23 showed that the ATDC gene is closely linked to THY1. The ATDC gene therefore lies outside the linkage region predicted to contain the AT gene(s) for complementation groups A and C, indicating a separate locus for the AT complementation group D gene.