Silverman G A, Yang E, Proffitt J H, Zutter M, Korsmeyer S J
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115.
Mol Cell Biol. 1993 Sep;13(9):5469-78. doi: 10.1128/mcb.13.9.5469-5478.1993.
The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.
本研究的目的是确定通过DNA转染来研究大型人类基因（如BCL2原癌基因）的表达是否可行。BCL2原癌基因大小为230 kb，在肿瘤细胞中通过易位至免疫球蛋白重链基因座而失调。含有人类BCL2基因的酵母人工染色体（YAC）在酿酒酵母中通过同源重组进行改造，以产生正常和易位等位基因的复制品。含有任一等位基因且大小在360至800 kb之间的构建体被稳定整合到小鼠肿瘤细胞系中。58%的克隆含有整个YAC插入片段的一个拷贝。这些克隆中有超过50%表达了适当水平的人类BCL2 RNA和蛋白质。这些研究表明，利用在酿酒酵母中繁殖的YAC的转染技术可以研究大型人类基因的表达及其病理重排。
