Molecular characterization of the human trk oncogene.
Molecular characterization of the human trk oncogene.
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人类 trk 癌基因的分子特征。
DOI：
10.1101/sqb.1986.051.01.112
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Barbacid,M
中科院分区：
文献类型：
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作者：
Martin-Zanca,D;Mitra,G;Long,LK;Barbacid,M
trk致癌基因是利用从结肠癌活检中分离的DNA进行基因转移试验确定的（Pulciani et al. 1982）。在NIH-3T3细胞中转染3个周期后，发现两个人类（alu阳性）EcoRI DNA片段分别为25 kbp和4 kbp，与转化的表型共分离。这些结果表明，trk致癌基因的大小将至少为30 kbp，这表明克隆其eDNA将比克隆其整个基因组序列更省力，而且可能提供更多信息。为此，我们首先从106-63个细胞制备的sau3ai部分消化的基因组文库中分离出包含约20 kbp trk基因组序列的大alu阳性DNA片段，这是一个第三周期的NIH-3T3转化。在pUCI8载体中亚克隆了几个小的alu阴性DNA片段，并测试了它们检测trk特异性转录物的能力。其中一个克隆pDM6含有一个209 bp的BamHI-NcoI DNA片段，在106-63细胞中检测到一个2.5 kbp的含多聚(a)的RNA，这在正常的NIH-3T3细胞或ras衍生的NIH-3T3转化体中是不存在的（Martin-Zanca et al. 1986）。接下来，我们从106-63个trk转化的NIH-3T3细胞的poly (a)-containing RNA中制备了一个包含106个克隆的eDNA文库（Helfman et al. 1983）。在与209 bp的BamHI-NcoI trk特异性探针特异性杂交的11个菌落中，一个（pDMI0-1）包含一个2.3 kbp的DNA插入，其大小与106-63细胞中鉴定的trk特异性mRNA的估计大小相似。通过将pDM10-1的2.3 kbp salii - ecori插入片段的5‘端连接到Moloney小鼠肉瘤病毒（Mo-MSV）长末端重复序列（LTR），并将其3’端连接到SV40多聚腺苷化信号，证实pDM10-1含有trk致癌基因的功能cDNA克隆。由此产生的质粒，命名为pDM-16，能够诱导NIH-3T3细胞的形态转化，其比活性为每微克DNA 2 × 104个灶形成单位，转化效率与ras癌基因相当（Martin-Zanca et al. 1986）。
The trk oncogene was identified in gene transfer assays utilizing DNA isolated from a colon carcinoma biopsy (Pulciani et al. 1982). Two human (Alu-posi-tive) EcoRI DNA fragments of 25 kbp and 4 kbp were found to cosegregate with the transformed phenotype after three cycles of transfection in NIH-3T3 cells. These results indicated that the size of the trk oncogene would be at least 30 kbp, suggesting that cloning its eDNA would be less laborious and perhaps more informative than cloning its entire genomic sequence. For this purpose, we first isolated large Alu-positive DNA fragments encompassing about 20 kbp of trk genomic sequences from a Sau3AI-partially digested genomic library prepared from 106-63 cells, a third-cycle NIH-3T3 transformant. Several small Alu-negative DNA fragments were subcloned in the pUCI8 vector and tested for their ability to detect trk-specific transcripts. One such clone, pDM6, contained a 209-bp BamHI-NcoI DNA fragment that detected in the 106-63 cells a 2.5-kbp poly (A)-containing RNA not present in normal NIH-3T3 cells or in ras-derived NIH-3T3 transformants (Martin-Zanca et al. 1986). We next prepared a eDNA library consisting of 106 clones from poly (A)-containing RNA of 106-63 trk-transformed NIH-3T3 cells (Helfman et al. 1983). Of 11 colonies that specifically hybridized to the 209-bp BamHI-NcoI trk-specific probe, one (pDMI0-1) contained a 2.3-kbp DNA insert that resembled the size estimated for the trk-specific mRNA identified in 106-63 cells. Demonstration that pDM10-1 contained a functional cDNA clone of the trk oncogene was obtained by linking the 5'end of its 2.3-kbp SalI-EcoRI insert to a Moloney murine sarcoma virus (Mo-MSV) long terminal repeat (LTR) and its 3'end to a SV40 polyadenylation signal. The resulting plasmid, designated pDM-16, was capable of inducing morphological transformation of NIH-3T3 cells with a specific activity of 2 x 104 focus-forming units per microgram of DNA, a transforming efficiency comparable to those of ras oncogenes (Martin-Zanca et al. 1986).