Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.
Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.
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酵母人工染色体之间的减数分裂重组产生包含整个 BCL2 原癌基因的单个克隆。
DOI：
10.1073/pnas.87.24.9913
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发表时间：
1990
影响因子：
11.1
通讯作者：
Korsmeyer,SJ
中科院分区：
文献类型：
--
作者：
Silverman,GA;Green,ED;Young,RL;Jockel,JI;Domer,PH;Korsmeyer,SJ
人类滤泡性淋巴瘤中常见的易位 t(14;18)(q32;q21) 会导致 BCL2 原癌基因失调。完整基因的分离将为分析这种恶性肿瘤的分子基础提供重要的底物。脉冲场凝胶电泳表明该三外显子基因有数百千碱基 (kb) 长。因此，筛选了酵母人工染色体 (YAC) 克隆文库以分离完整的 BCL2 基因。两个克隆，yA85B6 (200 kb) 和 yB206A6 (700 kb)，分别通过使用外显子 I/II 和外显子 III 特异性的聚合酶链式反应 (PCR) 测定来分离。然而，两个 YAC 都不包含整个 BCL2 基因座。由于发现两个 YAC 重叠 60 kb，我们试图利用酵母中的高重组频率来诱导两个克隆之间的物理重组。含有每种 YAC 的细胞交配并诱导进行减数分裂和孢子形成。对所得四分体的分析揭示了含有 800 kb 的单一重组 YAC 的孢子。 PCR 测定和 Southern 印迹证明该重组 YAC 包含完整的约 230 kb BCL2 基因。此外，探针顺序是保守的，没有证据表明存在明显的重排或删除。这些结果表明用重叠的 YAC 克隆重建大型基因组片段来研究跨越数百个碱基的基因的可行性。
The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protoonocogene. The isolation of the intact gene would provide an essential substrate to analyze the molecular basis of this malignancy. Pulsed-field gel electrophoresis suggested that this three-exon gene was several hundred kilobases (kb) long. Therefore, a library of yeast artificial chromosome (YAC) clones was screened to isolate the intact BCL2 gene. Two clones, yA85B6 (200 kb) and yB206A6 (700 kb), were isolated by using polymerase chain reaction (PCR) assays specific for exon I/II and exon III, respectively. However, neither YAC contained the entire BCL2 locus. Since the two YACs were found to overlap by 60 kb, we sought to take advantage of the high recombination frequency in yeast and induce physical recombination between the two clones. Cells containing each YAC were mated and induced to undergo meiotic division and sporulation. Analysis of the resulting tetrads revealed a spore containing a single recombinant YAC of 800 kb. PCR assays and Southern blotting demonstrated that this recombined YAC contained the entire approximately 230-kb BCL2 gene. Furthermore, probe order was conserved and there was no evidence of overt rearrangements or deletions. These results indicate the feasibility of reconstructing large genomic segments with overlapping YAC clones to study genes spanning hundreds of kilobases.
酵母人工染色体克隆染色体带 18q21 内的 2 兆碱基大小的重叠群在 BCL2 和纤溶酶原激活剂抑制剂 2 型之间建立了物理联系。
影响因子：
4.4
作者：
Silverman,GA;Jockel,JI;Domer,PH;Mohr,RM;Taillon-Miller,P;Korsmeyer,SJ
通讯作者：
Korsmeyer,SJ